mportance of...

The purpose of a problem statement is to:

1. Introduce the reader to the importance of the topic being studied. The reader is oriented to the significance of the study.
2. Anchors the research questions, hypotheses, or assumptions to follow. It offers a concise statement about the purpose of
your paper.
3. Place the topic into a particular context that defines the parameters of what is to be investigated.
4. Provide the framework for reporting the results and indicates what is probably necessary to conduct the study and explain
how the findings will present this information.

So What!
In the social sciences, the research problem establishes the means by which you must answer the "So What" question. The "So What"
question refers to a research problem surviving the relevancy test [the quality of a measurement procedure that provides repeatability and
accuracy]. Note that answering the "So What" question requires a commitment on your part to not only show that you have reviewed the
literature, but that you have thoroughly considered its significance and its implications applied to obtaining new knowledge or understanding.
To survive the "So What" question, problem statements should possess the following attributes:

 Clarity and precision [a well-written statement does not make sweeping generalizations and irresponsible pronouncements; it also
does include unspecific determinates like "very" or "giant"],
 Demonstrate a researchable topic or issue [i.e., feasibility of conducting the study is based upon access to information that can be
effectively acquired, gathered, interpreted, synthesized, and understood],
 Identification of what would be studied, while avoiding the use of value-laden words and terms,
 Identification of an overarching question or small set of questions accompanied by key factors or variables,
 Identification of key concepts and terms,
 Articulation of the study's boundaries or parameters or limitations,
 Some generalizability in regards to applicability and bringing results into general use,
 Conveyance of the study's importance, benefits, and justification [i.e., regardless of the type of research, it is important to
demonstrate that the research is not trivial],
 Does not have unnecessary jargon or overly complex sentence constructions; and,
 Conveyance of more than the mere gathering of descriptive data providing only a snapshot of the issue or phenomenon under
investigation.

Bryman, Alan. “The Research Question in Social Research: What is its Role?” International Journal of
Social Research Methodology 10 (2007): 5-20; Castellanos, Susie. Critical Writing and Thinking. The
Writing Center. Dean of the College. Brown University; Ellis, Timothy J. and Yair Levy Nova Framework of
Problem-Based Research: A Guide for Novice Researchers on the Development of a Research-Worthy
Problem. Informing Science: the International Journal of an Emerging Transdiscipline 11 (2008); Thesis
and Purpose Statements. The Writer’s Handbook. Writing Center. University of Wisconsin,
Madison; Thesis Statements. The Writing Center. University of North Carolina; Tips and Examples for
Writing Thesis Statements. The Writing Lab and The OWL. Purdue University.

Structure and Writing Style

I. Types and Content
There are four general conceptualizations of a research problem in the social sciences:

1. Casuist Research Problem -- this type of problem relates to the determination of right and wrong in questions of conduct or
conscience by analyzing moral dilemmas through the application of general rules and the careful distinction of special cases.
2. Difference Research Problem -- typically asks the question, “Is there a difference between two or more groups or treatments?”
This type of problem statement is used when the researcher compares or contrasts two or more phenomena. This a common
approach to defining a problem in the clinical social sciences or behavioral sciences.
3. Descriptive Research Problem -- typically asks the question, "what is...?" with the underlying purpose to describe the
significance of a situation, state, or existence of a specific phenomenon. This problem is often associated with revealing hidden or
understudied issues.
4. Relational Research Problem -- suggests a relationship of some sort between two or more variables to be investigated. The
underlying purpose is to investigate specific qualities or characteristics that may be connected in some way.

A problem statement in the social sciences should contain:

 A lead-in that helps ensure the reader will maintain interest over the study,
  A declaration of originality [e.g., mentioning a knowledge void or a lack of clarity about a topic that will be revealed in the literature
review],
 An indication of the central focus of the study [establishing the boundaries of analysis], and
 An explanation of the study's significance or the benefits to be derived from investigating the research problem.

II. Sources of Problems for Investigation

The identification of a problem to study can be challenging, not because there's a lack of issues that could be investigated, but due to the
challenge of formulating an academically relevant and researchable problem which is unique and does not simply duplicate the work of
others. To facilitate how you might select a problem from which to build a research study, consider these sources of inspiration:
Deductions from Theory
This relates to deductions made from social philosophy or generalizations embodied in life and in society that the researcher is familiar with.
These deductions from human behavior are then placed within an empirical frame of reference through research. From a theory, the
researcher can formulate a research problem or hypothesis stating the expected findings in certain empirical situations. The research asks
the question: “What relationship between variables will be observed if theory aptly summarizes the state of affairs?” One can then design and
carry out a systematic investigation to assess whether empirical data confirm or reject the hypothesis, and hence, the theory.
Interdisciplinary Perspectives
Identifying a problem that forms the basis for a research study can come from academic movements and scholarship originating in
disciplines outside of your primary area of study. This can be an intellectually stimulating exercise. A review of pertinent literature should
include examining research from related disciplines that can reveal new avenues of exploration and analysis. An interdisciplinary approach to
selecting a research problem offers an opportunity to construct a more comprehensive understanding of a very complex issue that any single
discipline may be able to provide.
Interviewing Practitioners
The identification of research problems about particular topics can arise from formal interviews or informal discussions with practitioners who
provide insight into new directions for future research and how to make research findings more relevant to practice. Discussions with experts
in the field, such as, teachers, social workers, health care providers, lawyers, business leaders, etc., offers the chance to identify practical,
“real world” problems that may be understudied or ignored within academic circles. This approach also provides some practical knowledge
which may help in the process of designing and conducting your study.
Personal Experience
Don't undervalue your everyday experiences or encounters as worthwhile problems for investigation. Think critically about your own
experiences and/or frustrations with an issue facing society, your community, your neighborhood, your family, or your personal life. This can
be derived, for example, from deliberate observations of certain relationships for which there is no clear explanation or witnessing an event
that appears harmful to a person or group or that is out of the ordinary.
Relevant Literature
The selection of a research problem can be derived from a thorough review of pertinent research associated with your overall area of
interest. This may reveal where gaps exist in understanding a topic or where an issue has been understudied. Research may be conducted
to: 1) fill such gaps in knowledge; 2) evaluate if the methodologies employed in prior studies can be adapted to solve other problems; or, 3)
determine if a similar study could be conducted in a different subject area or applied in a different context or to different study sample [i.e.,
different setting or different group of people].Also, authors frequently conclude their studies by noting implications for further research; read
the conclusion of pertinent studies because statements about further research can be a valuable source for identifying new problems to
investigate. The fact that a researcher has identified a topic worthy of further exploration validates the fact it is worth pursuing.

III. What Makes a Good Research Statement?

A good problem statement begins by introducing the broad area in which your research is centered, gradually leading the reader
to the more specific issues you are investigating. The statement need not be lengthy, but a good research problem should incorporate
the following features:
1. Compelling Topic
Simple curiosity is not a good enough reason to pursue a research study because it does not indicate significance. The problem that you
choose to explore must be important to you, your readers, and to a the larger academic and/or social community that could be impacted by
the results of your study. The problem chosen must be one that motivates you to address it.

2. Supports Multiple Perspectives

The problem must be phrased in a way that avoids dichotomies and instead supports the generation and exploration of multiple perspectives.
A general rule of thumb in the social sciences is that a good research problem is one that would generate a variety of viewpoints from a
composite audience made up of reasonable people.

3. Researchability
This isn't a real word but it represents an important aspect of creating a good research statement. It seems a bit obvious, but you don't want
to find yourself in the midst of investigating a complex research project and realize that you don't have enough prior research to draw from for
your analysis. There's nothing inherently wrong with original research, but you must choose research problems that can be supported, in
some way, by the resources available to you. If you are not sure if something is researchable, don't assume that it isn't if you don't
find information right away--seek help from a librarian!
NOTE: Do not confuse a research problem with a research topic. A topic is something to read and obtain information about, whereas a
problem is something to be solved or framed as a question raised for inquiry, consideration, or solution, or explained as a source of
perplexity, distress, or vexation.
IV. Asking Analytical Questions about the Research Problem
Research problems in the social and behavioral sciences are often analyzed around critical questions that must be investigated. These
questions can be explicitly listed in the introduction [i.e., "This study addresses three research questions about women's psychological
recovery from domestic abuse in multi-generational home settings..."], or, the questions are implied in the text as specific areas of study
related to the research problem. Explicitly listing your research questions at the end of your introduction can help in designing a clear
roadmap of what you plan to address in your study, whereas, implicitly integrating them into the text of the introduction allows you to create a
more compelling narrative around the key issues under investigation. Either approach is appropriate.
The number of questions you attempt to address should be based on the complexity of the problem you are investigating and what areas of
inquiry you find most critical to study. Practical considerations, such as, the length of the paper you are writing or the availability of resources
to analyze the issue can also factor in how many questions to ask. In general, however, there should be no more than four research
questions underpinning a single research problem.
Given this, well-developed analytical questions can focus on any of the following:

 Highlights a genuine dilemma, area of ambiguity, or point of confusion about a topic open to interpretation by your readers;
 Yields an answer that is unexpected and not obvious rather than inevitable and self-evident;
 Provokes meaningful thought or discussion;
 Raises the visibility of the key ideas or concepts that may be understudied or hidden;
 Suggests the need for complex analysis or argument rather than a basic description or summary; and,
 Offers a specific path of inquiry that avoids eliciting generalizations about the problem.

NOTE: Questions of how and why about a research problem often require more analysis than questions about who, what, where, and when.
You should still ask yourself these latter questions, however. Thinking introspectively about the who, what, where, and when of a research
problem can help ensure that you have thoroughly considered all aspects of the problem under investigation.

V. Mistakes to Avoid
Beware of circular reasoning! Do not state that the research problem as simply the absence of the thing you are suggesting. For example,
if you propose the following, "The problem in this community is that there is no hospital," this only leads to a research problem where:

 The need is for a hospital

 The objective is to create a hospital
 The method is to plan for building a hospital, and
 The evaluation is to measure if there is a hospital or not.

This is an example of a research problem that fails the "So What?" test. In this example, the problem does not reveal the relevance of
why you are investigating the fact there is no hospital in the community [e.g., there's a hospital in the community ten miles away]; it does not
elucidate the significance of why one should study the fact there is no hospital in the community [e.g., that hospital in the community ten
miles away has no emergency room]; the research problem does not offer an intellectual pathway towards adding new knowledge or
clarifying prior knowledge [e.g., the county in which there is no hospital already conducted a study about the need for a hospital]; and, the
problem does not offer meaningful outcomes that lead to recommendations that can be generalized for other situations or that could
suggest areas for further research [e.g., the challenges of building a new hospital serves as a case study for other communities].

Alvesson, Mats and Jörgen Sandberg. “Generating Research Questions Through Problematization.” Academy of Management Review 36 (April 2011): 247-271;
Choosing and Refining
Topics. Writing@CSU. Colorado State University; Ellis, Timothy J. and Yair Levy Nova. "Framework of
Problem-Based Research: A Guide for Novice Researchers on the Development of a Research-Worthy
Problem." Informing Science: the International Journal of an Emerging Transdiscipline 11 (2008); How to
Write a Research Question. The Writing Center. George Mason University; Invention: Developing a
Thesis Statement. The Reading/Writing Center. Hunter College; Problem Statements PowerPoint
Presentation. The Writing Lab and The OWL. Purdue University; Procter, Margaret. Using Thesis
Statements. University College Writing Centre. University of Toronto; Trochim, William M.K. Problem
Formulation. Research Methods Knowledge Base. 2006; Thesis and Purpose Statements. The Writer’s
Handbook. Writing Center. University of Wisconsin, Madison; Thesis Statements. The Writing Center.
University of North Carolina; Tips and Examples for Writing Thesis Statements. The Writing Lab and The
OWL. Purdue University; Walk, Kerry. Asking an Analytical Question. [Class handout or worksheet].
Princeton University; White, Patrick. Developing Research Questions: A Guide for Social

efinition
A research problem is a statement about an area of concern, a condition to be improved, a difficulty to be eliminated, or a troubling question that
exists in scholarly literature, in theory, or in practice that points to the need for meaningful understanding and deliberate investigation. In some social
science disciplines the research problem is typically posed in the form of a question. A research problem does not state how to do something, offer a
vague or broad proposition, or present a value question.
Importance of...
The purpose of a problem statement is to:

1. Introduce the reader to the importance of the topic being studied. The reader is oriented to the significance of the study and the
research questions or hypotheses to follow.
2. Places the problem into a particular context that defines the parameters of what is to be investigated.
3. Provides the framework for reporting the results and indicates what is probably necessary to conduct the study and explain how the
findings will present this information.

So What!
In the social sciences, the research problem establishes the means by which you must answer the "So What?" question. The "So What?" question
refers to a research problem surviving the relevancy test [the quality of a measurement procedure that provides repeatability and accuracy]. Note that
answering the "So What" question requires a commitment on your part to not only show that you have researched the material, but that you have
thought about its significance.
To survive the "So What" question, problem statements should possess the following attributes:

 Clarity and precision [a well-written statement does not make sweeping generalizations and irresponsible statements],
 Identification of what would be studied, while avoiding the use of value-laden words and terms,
 Identification of an overarching question and key factors or variables,
 Identification of key concepts and terms,
 Articulation of the study's boundaries or parameters,
 Some generalizability in regards to applicability and bringing results into general use,
 Conveyance of the study's importance, benefits, and justification [regardless of the type of research, it is important to address the “so what”
question by demonstrating that the research is not trivial],
 Does not have unnecessary jargon; and,
 Conveyance of more than the mere gathering of descriptive data providing only a snapshot of the issue or phenomenon under
investigation.

Castellanos, Susie. Critical Writing and Thinking. The Writing Center. Dean of the College. Brown
University; Ellis, Timothy J. and Yair Levy Nova Framework of Problem-Based Research: A Guide for
Novice Researchers on the Development of a Research-Worthy Problem. Informing Science: the
International Journal of an Emerging Transdiscipline 11 (2008); Thesis and Purpose Statements. The
Writer’s Handbook. Writing Center. University of Wisconsin, Madison; Thesis Statements. The Writing
Center. University of North Carolina; Tips and Examples for Writing Thesis Statements. The Writing Lab
and The OWL. Purdue University.

Structure and Writing Style

I. Types and Content

There are four general conceptualizations of a research problem in the social sciences:

1. Casuist Research Problem -- this type of problem relates to the determination of right and wrong in questions of conduct or conscience by
analyzing moral dilemmas through the application of general rules and the careful distinction of special cases.
2. Difference Research Problem -- typically asks the question, “Is there a difference between two or more groups or treatments?” This type
of problem statement is used when the researcher compares or contrasts two or more phenomena.
3. Descriptive Research Problem -- typically asks the question, "what is...?" with the underlying purpose to describe a situation, state, or
existence of a specific phenomenon.
4. Relational Research Problem -- suggests a relationship of some sort between two or more variables to be investigated. The underlying
purpose is to investigate qualities/characteristics that are connected in some way.

A problem statement in the social sciences should contain:

 A lead-in that helps ensure the reader will maintain interest over the study
 A declaration of originality [e.g., mentioning a knowledge void, which would be supported by the literature review]
 An indication of the central focus of the study, and
 An explanation of the study's significance or the benefits to be derived from an investigating the problem.

II. Sources of Problems for Investigation

 Identifying a problem to study can be challenging, not because there is a lack of issues that could be investigated, but due to pursuing a goal of
formulating a socially relevant and researchable problem statement that is unique and does not simply duplicate the work of others. To facilitate how
you might select a problem from which to build a research study, consider these three broad sources of inspiration:
Deductions from Theory
This relates to deductions made from social philosophy or generalizations embodied in life in society that the researcher is familiar with. These
deductions from human behavior are then fitted within an empirical frame of reference through research. From a theory, the research can formulate a
research problem or hypothesis stating the expected findings in certain empirical situations. The research asks the question: “What relationship
between variables will be observed if theory aptly summarizes the state of affairs?” One can then design and carry out a systematic investigation to
assess whether empirical data confirm or reject the hypothesis and hence the theory.
Interdisciplinary Perspectives
Identifying a problem that forms the basis for a research study can come from academic movements and scholarship originating in disciplines outside
of your primary area of study. A review of pertinent literature should include examining research from related disciplines, which can expose you to new
avenues of exploration and analysis. An interdisciplinary approach to selecting a research problem offers an opportunity to construct a more
comprehensive understanding of a very complex issue than any single discipline might provide.
Interviewing Practitioners
The identification of research problems about particular topics can arise from formal or informal discussions with practitioners who provide insight into
new directions for future research and how to make research findings increasingly relevant to practice. Discussions with experts in the field, such as,
teachers, social workers, health care providers, etc., offers the chance to identify practical, “real worl” problems that may be understudied or ignored
within academic circles. This approach also provides some practical knowledge which may help in the process of designing and conducting your study.
Personal Experience
Your everyday experiences can give rise to worthwhile problems for investigation. Think critically about your own experiences and/or frustrations with
an issue facing society, your community, or in your neighborhood. This can be derived, for example, from deliberate observations of certain
relationships for which there is no clear explanation or witnessing an event that appears harmful to a person or group or that is out of the ordinary.
Relevant Literature
The selection of a research problem can often be derived from an extensive and thorough review of pertinent research associated with your overall
area of interest. This may reveal where gaps remain in our understanding of a topic. Research may be conducted to: 1) fill such gaps in knowledge; 2)
evaluate if the methodologies employed in prior studies can be adapted to solve other problems; or, 3) determine if a similar study could be conducted
in a different subject area or applied to different study sample [i.e., different groups of people]. Also, authors frequently conclude their studies by noting
implications for further research; this can also be a valuable source of problems to investigate.

III. What Makes a Good Research Statement?

A good problem statement begins by introducing the broad area in which your research is centered and then gradually leads the reader to the more
narrow questions you are posing. The statement need not be lengthy but a good research problem should incorporate the following features:
Compelling topic
Simple curiosity is not a good enough reason to pursue a research study. The problem that you choose to explore must be important to you and to a
larger community you share. The problem chosen must be one that motivates you to address it.

Supports multiple perspectives

The problem most be phrased in a way that avoids dichotomies and instead supports the generation and exploration of multiple perspectives. A
general rule of thumb is that a good research problem is one that would generate a variety of viewpoints from a composite audience made up of
reasonable people.

Researchable
It seems a bit obvious, but you don't want to find yourself in the midst of investigating a complex research project and realize that you don't have much
to draw on for your research. Choose research problems that can be supported by the resources available to you. Not sure? Seek out help from a
librarian!
NOTE: Do not confuse a research problem with a research topic. A topic is something to read and obtain information about whereas a problem is
something to solve or framed as a question that must be answered.

IV. Mistakes to Avoid

Beware of circular reasoning. Don’t state that the research problem as simply the absence of the thing you are suggesting. For example, if you
propose, "The problem in this community is that it has no hospital."
This only leads to a research problem where:

 The need is for a hospital

 The objective is to create a hospital
 The method is to plan for building a hospital, and
 The evaluation is to measure if there is a hospital or not.

This is an example of a research problem that fails the "so what?" test because it does not reveal the relevance of why you are investigating the
problem of having no hospital in the community [e.g., there's a hospital in the community ten miles away] and because the research problem does not
elucidate the significance of why one should study the fact that no hospital exists in the community [e.g., that hospital in the community ten miles
away has no emergency room].

Defining a Research Problem

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 Defining a Research Problem

Martyn Shuttleworth 519.9K reads

Defining a research problem is the fuel that drives the scientific process, and is the foundation of any research
method and experimental design, from true experiment to case study.
It is one of the first statements made in any research paper and, as well as defining the research area, should include a quick synopsis of how
the hypothesis was arrived at.
Operationalization is then used to give some indication of the exact definitions of the variables, and the type of scientific measurements used.
This will lead to the proposal of a viable hypothesis. As an aside, when scientists are putting forward proposals for research funds, the quality of their
research problem often makes the difference between success and failure.

Structuring the Research Problem

Look at any scientific paper, and you will see the research problem, written almost like a statement of intent.

Defining a research problem is crucial in defining the quality of the answers, and determines the exact research method used.
A quantitative experimental design uses deductive reasoning to arrive at a testable hypothesis.
Qualitative research designs use inductive reasoning to propose a research statement.
Defining a Research Problem

Formulating the research problem begins during the first steps of the scientific process.
As an example, a literature review and a study of previous experiments, and research, might throw up some vague areas of interest.
Many scientific researchers look at an area where a previous researcher generated some interesting results, but never followed up. It could be an
interesting area of research, which nobody else has fully explored.

A scientist may even review a successful experiment, disagree with the results, the tests used, or the methodology, and decide to refine the research
process, retesting the hypothesis.
This is called the conceptual definition, and is an overall view of the problem. A science report will generally begin with an overview of the previous
research and real-world observations. The researcher will then state how this led to defining a research problem.
The Operational Definitions

The operational definition is the determining the scalar properties of the variables.
For example, temperature, weight and time are usually well known and defined, with only the exact scale used needing definition. If a researcher
is measuring abstract concepts, such as intelligence, emotions, and subjective responses, then a system of measuring numerically needs to be
established, allowing statistical analysis and replication.
For example, intelligence may be measured with IQ and human responses could be measured with a questionnaire from ‘1- strongly disagree’, to ‘5 -
strongly agree’.
Behavioral biologists and social scientists might design an ordinal scale for measuring and rating behavior. These measurements are always
subjective, but allow statistics and replication of the whole research method. This is all an essential part of defining a research problem.
Examples of Defining a Research Problem

An anthropologist might find references to a relatively unknown tribe in Papua New Guinea. Through inductive reasoning, she arrives at the research
problem and asks,

‘How do these people live and how does their culture relate to nearby tribes?’

She has found a gap in knowledge, and she seeks to fill it, using a qualitative case study, without a hypothesis.
The Bandura Bobo Doll Experiment is a good example of using deductive reasoning to arrive at a research problem and hypothesis.
Anecdotal evidence showed that violent behavior amongst children was increasing. Bandura believed that higher levels of violent adult role models on
television, was a contributor to this rise. This was expanded into a hypothesis, and operationalization of the variables, and scientific measurement
scale, led to a robust experimental design.

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Full reference:
Martyn Shuttleworth (Oct 2, 2008). Defining a Research Problem. Retrieved Apr 17, 2018 from Explorable.com: https://explorable.com/defining-a-
research-problem

3.2 Formulating the research problem

Once the general topic or problem has been identified, this should then be stated as a clear research problem, that is, taken from just a statement

about a problematic situation to a clearly defined researchable problem that identifies the issues you are trying to address.
It is not always easy to formulate the research problem simply and clearly. In some areas of scientific research the investigator might spend years

exploring, thinking, and researching before they are clear about what research questions they are seeking to answer. Many topics may prove too wide-

ranging to provide a researchable problem. Choosing to study, for instance a social issue such as child poverty, does not in itself provide a researchable

problem. The problem is too wide-ranging for one researcher to address. Time and resources would make this unfeasible and the results from such a

study would consequently lack depth and focus.

Statement of research problem

An adequate statement of the research problem is one of the most important parts of the research. Different researchers are likely to generate a variety

of researchable problems from the same situation since there are many research issues that can arise out of a general problem situation. Your research

will be able to pursue only one in depth.

For a problem statement to be effective in the planning of applied research it should have the following characteristics (Andrew and Hildebrand 1982).

1. The problem reflects felt needs

2. The problem is non-hypothetical, ie it must be based on factual evidence

3. It should suggest meaningful and testable hypotheses - to avoid answers that are of little or no use to the alleviation of the

problem

4. The problems should be relevant and manageable

Formulating the research problem allows you to make clear, both to yourself and the reader, what the purpose of your research is. Subsequent

elaboration of method should be oriented to providing information to address that problem. The problem statement is therefore a very important device

for keeping you on track with your research. It is also one means by which your research will be evaluated - does the research address the problem as

stated.

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In all research projects, on whatever subject, there is a need to define and delineate the research problem clearly. The research problem is a general statement of an
issue meriting research. Its nature will suggest appropriate forms for its investigation. Here are several forms in which the research problem can be expressed to
indicate the method of investigation.

The research problem in some social science research projects using the hypothetico-deductive method is expressed in terms of the testing of a
particular hypothesis. It is therefore important to know what makes good hypotheses and how they can be formulated. However, it is not appropriate to use the
hypothetico-deductive method, or even scientific method, in every research study. Much research into society, design, history, philosophy ..
Spectrophotometry
From Wikipedia, the free encyclopedia

Table top spectrophotometer

Beckman IR-1 Spectrophotometer, ca. 1941

Handheld spectrophotometer used in graphic industry. [1]

External video

Jacqueline Boytim discusses the

development and early use of

spectrophotometers, Chemical
 Heritage Foundation

In chemistry, spectrophotometry is the quantitative measurement of the reflection or transmission

properties of a material as a function of wavelength. It is more specific than the general
[2]

term electromagnetic spectroscopy in that spectrophotometry deals with visible light, near-ultraviolet,
and near-infrared, but does not cover time-resolved spectroscopic techniques.
Contents
[hide]

 1Overview
 2History
 3Design
o 3.1Applications in biochemistry
 4UV-visible spectrophotometry
o 4.1Applications
o 4.2Experimental Application
 5IR spectrophotometry
 6Spectroradiometers
 7See also
 8References
 9External links

Overview[edit]
Spectrophotometry is a tool that hinges on the quantitative analysis of molecules depending on how
much light is absorbed by colored compounds. Spectrophotometry uses photometers, known as
spectrophotometers, that can measure a light beam's intensity as a function of its color (wavelength).
Important features of spectrophotometers are spectral bandwidth (the range of colors it can transmit
through the test sample), the percentage of sample-transmission, the logarithmic range of sample-
absorption, and sometimes a percentage of reflectance measurement.
A spectrophotometer is commonly used for the measurement of transmittance or reflectance of
solutions, transparent or opaque solids, such as polished glass, or gases. Although many
biochemicals are colored, as in, they absorb visible light and therefore can be measured by
colorimetric procedures, even colorless biochemicals can often be converted to colored compounds
suitable for chromogenic color-forming reactions to yield compounds suitable for colorimetric
analysis. However, they can also be designed to measure the diffusivity on any of the listed light
[3]

ranges that usually cover around 200 nm - 2500 nm using different controls and calibrations. Within
[2]

these ranges of light, calibrations are needed on the machine using standards that vary in type
depending on the wavelength of the photometric determination. [4]

An example of an experiment in which spectrophotometry is used is the determination of the

equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a
forward and reverse direction, where reactants form products and products break down into
reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium
point. In order to determine the respective concentrations of reactants and products at this point, the
light transmittance of the solution can be tested using spectrophotometry. The amount of light that
passes through the solution is indicative of the concentration of certain chemicals that do not allow
light to pass through.
The absorption of light is due to the interaction of light with the electronic and vibrational modes of
molecules. Each type of molecule has an individual set of energy levels associated with the makeup
of its chemical bonds and nuclei, and thus will absorb light of specific wavelengths, or energies,
resulting in unique spectral properties. This is based upon its specific and distinct makeup.
[5]
The use of spectrophotometers spans various scientific fields, such as physics, materials
science, chemistry, biochemistry, and molecular biology. They are widely used in many industries
[6]

including semiconductors, laser and optical manufacturing, printing and forensic examination, as well
in laboratories for the study of chemical substances. Spectrophotometry is often used in
measurements of enzyme activities, determinations of protein concentrations, determinations of
enzymatic kinetic constants, and measurements of ligand binding reactions. Ultimately, a
[3]

spectrophotometer is able to determine, depending on the control or calibration, what substances

are present in a target and exactly how much through calculations of observed wavelengths.
In astronomy, the term spectrophotometry refers to the measurement of the spectrum of a celestial
object in which the flux scale of the spectrum is calibrated as a function of wavelength, usually by
comparison with an observation of a spectrophotometric standard star, and corrected for the
absorption of light by the Earth's atmosphere. [7]

History[edit]
Invented by Arnold O. Beckman in 1940, the spectrophotometer was created with the aid of his
colleagues at his company National Technical Laboratories founded in 1935 which would become
Beckman Instrument Company and ultimately Beckman Coulter. This would come as a solution to
the previously created spectrophotometers which were unable to absorb the ultraviolet correctly. He
would start with the invention of Model A where a glass prism was used to absorb the UV light. It
would be found that this did not give satisfactory results, therefore in Model B, there was a shift from
a glass to a quartz prism which allowed for better absorbance results. From there, Model C was born
with an adjustment to the wavelength resolution which ended up having three units of it produced.
The last and most popular model became Model D which is better recognized now as DU which
contained the instrument case, hydrogen lamp with ultraviolent continuum and a better
monochromator. It was produced from 1941 to 1976 where the price for it in 1941 was US$723 (far-
[8]

UV accessories were an option at additional cost). In the words of Nobel chemistry laureate Nobel
chemistry laureate Bruce Merrifield said it was "probably the most important instrument ever
developed towards the advancement of bioscience." [9]

Once it became discontinued, another company known as Hewlett-Packard created the first
commercially available diode-assay spectrophotometer in 1979 known as the HP 8450A. Diode- [10]

assay spectrophotometers differed from the original spectrophotometer created by Beckman

because it was the first single-beam microprocessor-controlled spectrophotometer that scanned
multiple wavelengths at a time in seconds. It irradiates the sample with polychromatic light which the
sample absorbs depending on its properties. Then it is transmitted back by grating the photodiode
array which detects the wavelength region of the spectrum. Since then, the creation and
[11]

implementation of spectrophotometry devices has increased immensely and has become one of the
most innovative instruments of our time.

Design[edit]

Single beam spectrophotometer

There are two major classes of devices: single beam and double beam. A double beam
spectrophotometer compares the light intensity between two light paths, one path containing a
[12]

reference sample and the other the test sample. A single-beam spectrophotometer measures the
relative light intensity of the beam before and after a test sample is inserted. Although comparison
measurements from double-beam instruments are easier and more stable, single-beam instruments
can have a larger dynamic range and are optically simpler and more compact. Additionally, some
specialized instruments, such as spectrophotometers built onto microscopes or telescopes, are
single-beam instruments due to practicality.
Historically, spectrophotometers use a monochromator containing a diffraction grating to produce the
analytical spectrum. The grating can either be movable or fixed. If a single detector, such as
a photomultiplier tube or photodiode is used, the grating can be scanned stepwise so that the
detector can measure the light intensity at each wavelength (which will correspond to each "step").
Arrays of detectors, such as charge coupled devices (CCD) or photodiode arrays (PDA) can also be
used. In such systems, the grating is fixed and the intensity of each wavelength of light is measured
by a different detector in the array. Additionally, most modern mid-infrared spectrophotometers use
a Fourier transform technique to acquire the spectral information. This technique is called Fourier
transform infrared spectroscopy.
When making transmission measurements, the spectrophotometer quantitatively compares the
fraction of light that passes through a reference solution and a test solution, then electronically
compares the intensities of the two signals and computes the percentage of transmission of the
sample compared to the reference standard. For reflectance measurements, the spectrophotometer
quantitatively compares the fraction of light that reflects from the reference and test samples. Light
from the source lamp is passed through a monochromator, which diffracts the light into a "rainbow"
of wavelengths through a rotating prism and outputs narrow bandwidths of this diffracted spectrum
through a mechanical slit on the output side of the monochromator. These bandwidths are
transmitted through the test sample. Then the photon flux density (watts per metre squared usually)
of the transmitted or reflected light is measured with a photodiode, charge coupled device or
other light sensor. The transmittance or reflectance value for each wavelength of the test sample is
then compared with the transmission or reflectance values from the reference sample. Most
instruments will apply a logarithmic function to the linear transmittance ratio to calculate the
'absorbency' of the sample, a value which is proportional to the 'concentration' of the chemical being
measured.
In short, the sequence of events in a modern spectrophotometer is as follows:

1. The light source is shone into a monochromator, diffracted into a rainbow, and split into two
beams. It is then scanned through the sample and the reference solutions.
2. Fractions of the incident wavelengths are transmitted through, or reflected from, the sample
and the reference.
3. The resultant light strikes the photodetector device, which compares the relative intensity of
the two beams.
4. Electronic circuits convert the relative currents into linear transmission percentages and/or
absorbance/concentration values.
Many older spectrophotometers must be calibrated by a procedure known as "zeroing", to balance
the null current output of the two beams at the detector. The transmission of a reference substance
is set as a baseline (datum) value, so the transmission of all other substances are recorded relative
to the initial "zeroed" substance. The spectrophotometer then converts the transmission ratio into
'absorbency', the concentration of specific components of the test sample relative to the initial
[13]

substance. [6]

Applications in biochemistry[edit]
Spectrophotometry is an important technique used in many biochemical experiments that involve
DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses. A brief explanation of
[14]
the procedure of spectrophotometry includes comparing the absorbency of a blank sample that does
not contain a colored compound to a sample that contains a colored compound. This coloring can be
accomplished by either a dye such as Coomasie Brilliant Blue G-250 dye measured at 595 nm or by
an enzymatic reaction as seen between β-galactosidase and ONPG (turns sample yellow) measured
at 420 nm. The spectrophotometer is used to measure colored compounds in the visible region of
[15]

light (between 350 nm and 800 nm), thus it can be used to find more information about the
[3]

substance being studied. In biochemical experiments, a chemical and/or physical property is chosen
and the procedure that is used is specific to that property in order to derive more information about
the sample, such as the quantity, purity, enzyme activity, etc. Spectrophotometry can be used for a
number of techniques such as determining optimal wavelength absorbance of samples, determining
optimal pH for absorbance of samples, determining concentrations of unknown samples, and
determining the pKa of various samples. Spectrophotometry is also a helpful process for protein
[15]

purification and can also be used as a method to create optical assays of a compound.
[16]

Spectrophotometric data can also be used in conjunction with the Beer-Lambert Equation, A= -
log T=εcl=OD, in order to determine various relationships between transmittance and concentration,
10

and absorbance and concentration. Because a spectrophotometer measures the wavelength of a

[15]

compound through its color, a dye binding substance can be added so that it can undergo a color
change and be measured. It is possible to know the concentrations of a two component mixture
[17]

using the absorption spectra of the standard solutions of each component. To do this, it is necessary
to know the extinction coefficient of this mixture at two wave lengths and the extinction coefficients of
solutions that contain the known weights of the two components. Spectrophotometers have been
[18]

developed and improved over decades and have been widely used among chemists. Additionally,
Spectrophotometers are specialized to measure either UV or Visible light wavelength absorbance
values. It is considered to be a highly accurate instrument that is also very sensitive and therefore
[15]

extremely precise, especially in determining color change. This method is also convenient for use
[19]

in laboratory experiments because it is an inexpensive and relatively simple process.

UV-visible spectrophotometry[edit]
Main article: Ultraviolet-visible spectroscopy

Most spectrophotometers are used in the UV and visible regions of the spectrum, and some of these
instruments also operate into the near-infrared region as well. The concentration of a protein can be
estimated by measuring the OD at 280 nm due to the presence of tryptophan, tyrosine and
phenylalanine. This method is not very accurate since the composition of proteins varies greatly and
proteins with none of these amino acids do not have maximum absorption at 280 nm. Nucleic acid
contamination can also interfere. This method requires a spectrophotometer capable of measuring in
the UV region with quartz cuvettes. [20]

Ultraviolet-visible (UV-vis) spectroscopy involves energy levels that excite electronic transitions.
Absorption of UV-vis light excites molecules that are in ground-states to their excited-states [5]

Visible region 400–700 nm spectrophotometry is used extensively in colorimetry science. It is a

known fact that it operates best at the range of 0.2-0.8 O.D. Ink manufacturers, printing companies,
textiles vendors, and many more, need the data provided through colorimetry. They take readings in
the region of every 5–20 nanometers along the visible region, and produce a spectral
reflectance curve or a data stream for alternative presentations. These curves can be used to test a
new batch of colorant to check if it makes a match to specifications, e.g., ISO printing standards.
Traditional visible region spectrophotometers cannot detect if a colorant or the base material has
fluorescence. This can make it difficult to manage color issues if for example one or more of the
printing inks is fluorescent. Where a colorant contains fluorescence, a bi-spectral fluorescent
spectrophotometer is used. There are two major setups for visual spectrum spectrophotometers, d/8
(spherical) and 0/45. The names are due to the geometry of the light source, observer and interior of
the measurement chamber. Scientists use this instrument to measure the amount of compounds in a
sample. If the compound is more concentrated more light will be absorbed by the sample; within
small ranges, the Beer-Lambert law holds and the absorbance between samples vary with
concentration linearly. In the case of printing measurements two alternative settings are commonly
used- without/with uv filter to control better the effect of uv brighteners within the paper stock.
Samples are usually prepared in cuvettes; depending on the region of interest, they may be
constructed of glass, plastic (visible spectrum region of interest), or quartz (Far UV spectrum region
of interest).
Applications[edit]

 Estimating dissolved organic carbon concentration

 Specific ultraviolet absorbance for metric of aromaticity
 Bial's test for concentration of pentoses
Experimental Application[edit]
As described in the applications section, spectrophotometry can be used in both qualitative and
quantitative analysis of DNA, RNA, and proteins. Qualitative analysis can be used and
spectrophotometers are used to record spectra of compounds by scanning broad wavelength
regions to determine the absorbance properties (the intensity of the color) of the compound at each
wavelength. One experiment that can demonstrate the various uses that visible spectrophotometry
[5]

can have is the separation of β-galactosidase from a mixture of various proteins. Largely,
spectrophotometry is best used to help quantify the amount of purification your sample has
undergone relative to total protein concentration. By running an affinity chromatography, B-
Galactosidase can be isolated and tested by reacting collected samples with ONPG and determining
if the sample turns yellow. Following this testing the sample at 420 nm for specific interaction with
[15]

ONPG and at 595 for a Bradford Assay the amount of purification can be assessed
quantitatively. In addition to this spectrophotometry can be used in tandem with other techniques
[15]

such as SDS-Page electrophoresis in order to purify and isolate various protein samples.

IR spectrophotometry[edit]
Main article: Infrared spectroscopy

Spectrophotometers designed for the infrared region are quite different because of the technical
requirements of measurement in that region. One major factor is the type of photosensors that are
available for different spectral regions, but infrared measurement is also challenging because
virtually everything emits IR light as thermal radiation, especially at wavelengths beyond about 5 μm.
Another complication is that quite a few materials such as glass and plastic absorb infrared light,
making it incompatible as an optical medium. Ideal optical materials are salts, which do not absorb
strongly. Samples for IR spectrophotometry may be smeared between two discs of potassium
bromide or ground with potassium bromide and pressed into a pellet. Where aqueous solutions are
to be measured, insoluble silver chloride is used to construct the cell.

Spectroradiometers[edit]
Spectroradiometers, which operate almost like the visible region spectrophotometers, are designed
to measure the spectral density of illuminants. Applications may include evaluation and
categorization of lighting for sales by the manufacturer, or for the customers to confirm the lamp they
decided to purchase is within their specifications. Components:

1. The light source shines onto or through the sample.

2. The sample transmits or reflects light.
3. The detector detects how much light was reflected from or transmitted through the sample.
 4. The detector then converts how much light the sample transmitted or reflected into a
number.

See also[edit]
 Atomic absorption spectrophotometry
 Atomic emission spectroscopy
 Inductively coupled plasma atomic emission spectroscopy
 Inductively coupled plasma mass spectrometry
 Spectroradiometry
 Slope spectroscopy
 Microspectrophotometry

References[edit]
1. Jump up^ ISO 12647-2: Graphic technology — Process control for the production of halftone colour separations, proof and production prints — Part
2: Offset lithographic processes. Geneva: International Organization for Standardization. 2013. p. 13.
2. ^ Jump up to:a b Allen, D., Cooksey, C., & Tsai, B. (2010, October 5). Spectrophotometry. Retrieved
from https://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm
3. ^ Jump up to:a b c Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2010). Fundamental Laboratory Approaches for Biochemistry and
Biotechnology (2nd ed.). p. 65.
4. Jump up^ Schwedt, Georg. (1997). The Essential Guide to Analytical Chemistry. (Brooks Haderlie, trans.). Chichester, NY: Wiley. (Original Work
Published 1943). pp. 16-17
5. ^ Jump up to:a b c J., Ninfa, Alexander; P., Ballou, David (2004). Fundamental laboratory approaches for biochemistry and biotechnology. Wiley.
p. 66. ISBN 9781891786006. OCLC 633862582.
6. ^ Jump up to:a b Rendina, George. Experimental Methods in Modern Biochemistry W. B. Saunders Company: Philadelphia, PA. 1976. pp. 46-55
7. Jump up^ Oke, J. B.; Gunn, J. E. (1983). "Secondary standard stars for absolute spectrophotometry". The Astrophysical Journal. 266:
713. Bibcode:1983ApJ...266..713O. doi:10.1086/160817.
8. Jump up^ Ganguli, Ishani. "The first commercial UV-vis spectrophotometer." The Scientist, Mar. 2006, p. 100. Science In
Context, http://link.galegroup.com/apps/doc/A143579063/SCIC?u=cuny_hunter&sid=SCIC&xid=910964e6.
9. Jump up^ Robert D. Simoni, Robert L. Hill, Martha Vaughan and Herbert Tabor (5 December 2003). "A Classic Instrument: The Beckman DU
Spectrophotometer and Its Inventor, Arnold O. Beckman". THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 49. Retrieved 20
January 2016.
10. Jump up^ “Hewlett Packard: Compound Identification with HP 8450 A UV Visible Spectrophotometer.” Analytical Chemistry, vol. 51, no. 12, 1 Oct.
1979
11. Jump up^ Ninfa, Alexander J., and David P. Ballou. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. 2nd ed., John Wiley
& Sons, 2015. p. 77
12. Jump up^ "Fully Automatic Double Beam - Atomic Absorption Spectrophotometer (AA 8000)". Laboratory Equipment.
13. Jump up^ name=Meece
14. Jump up^ Trumbo, Toni A.; Schultz, Emeric; Borland, Michael G.; Pugh, Michael Eugene (April 27, 2013). "Applied Spectrophotometry: Analysis of a
Biochemical Mixture". Biochemistry and Molecular Biology Education: 243. doi:10.1002/bmb.20694 (inactive 2017-06-21).
15. ^ Jump up to:a b c d e f Ninfa, Alexander; Ballou, David; Benore, Marilee (2009). Fundamental Laboratory Approaches for Biochemistry and
Biotechnology. United Kingdom: John WIley and Sons Ltd. pp. 21–119. ISBN 0470087668.
16. Jump up^ Cortez, C.; Szepaniuk, A.; Gomes da Silva, L. (May 1, 2010). "Exploring Proteins Purification Techniques Animations as Tools for the
Biochemistry Teaching". Journal of Biochemistry Education.
17. Jump up^ Garrett, Reginald H.; Grisham, Charles M. Biochemistry. Cengage. p. 106.
18. Jump up^ Holiday, Ensor Roslyn (May 27, 1936). "Spectrophotometry of proteins" (PDF). Biochemical Journal. 30: 1795–
1803. doi:10.1042/bj0301795. PMC 1263262  . PMID 16746224 – via NCBI.
19. Jump up^ Mavrodineanu, Radu; Schultz, J. I.; Menis, Oscar (1973). Accuracy in Spectrophotometry and Luminescence Measurements:
Proceedings. Washington, D.C.: U.S. National Bureau of Standards. p. 2.
20. Jump up^ Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2010). Fundamental Laboratory Approaches for Biochemistry and Biotechnology
(2nd ed.). p. 135.

External links

Problem area

Initially, it is useful to define no more than a problem area, rather than a specific research problem,
within the general body of knowledge which interests you, e.g. housing and homelessness, parks in
cities, building regulations and historic conservation. Your aim should be to subsequently narrow down
the scope of the idea or problem until it becomes a highly specific research problem. This narrowing
process will require a lot of background reading in order to discover what has been written about the
subject already, what research has been carried out, where further work needs to be done and where
controversial issues still remain. You should keep in mind three questions when engaged in the
preliminary exploratory work. The first is, what is your motivation for doing the research? A major
motivation should be a curiosity about the research results. Another will undoubtedly be the fulfilment
of the requirements of a research degree. Learning about the process of research – practical knowledge
which can be used in the future – is also likely to be a motivation. The choice of problem is likely to be
influenced by these motivational factors. The second question is, what relevant interest, experience or
expertise do you bring to bear on the subject? Obviously, interest in a subject is essential if you are to
concentrate happily on it for a year or more. Although experience or expertise in a subject is not a
prerequisite to doing research in that field, it does have an effect on the preliminary and information
gathering stage of the work, as you will be familiar with the literature and the potential problem areas.
However, a ‘new light’ may be cast on a subject by someone looking at it with ‘fresh eyes’. The third
question is, what are you going to produce? As a researcher, your priority will be to produce a
defendable thesis or useful research report within your time limit. If you are a research student, you
should check the requirements of your university or college in the regulations issued about the nature of
suitable research topics. (It might be a good idea to do that now. Problem You will find the information
in the latest university research degree regulations kept in the library. You should also be issued with
your own copy.) If you are doing a dissertation as part of a course, check the course notes for guidance.
If you are doing a funded research project, then you will need to know the requirements of the likely
funders or of the policy of the organization for which you work. Initial literature review, and defining the
problem area The objective of the initial review of the literature is to discover relevant material
published in the chosen field of study and to search for a suitable problem area. Fox (1969) mentions
two kinds of literature which should be reviewed. The first is ‘conceptual literature’. This is written by
authorities on the subject you have in mind, giving opinions, ideas, theories or experiences, and
published in the form of books, articles and papers. The second is ‘research literature’ which gives
accounts and results of research which has been undertaken in the subject, often presented in the form
of papers and reports. Chapter 2 in this book tells you how you can effectively carry out this search
through the literature. As every piece of research contributes only a small part to a greater body of
knowledge or understanding, researchers must be aware of the context within which their research
work is to be carried out. At this stage it is important to get an overview of the subject, rather than
knowledge in depth. This will provide you with an understanding of the principal issues and problems or
controversies, and the opportunity to select a problem area within a frame of reference. Within this
problem area, it is important that you familiarize yourself with those aspects which have already been
well established by previous research, and are generally accepted as true. These ‘truths’ can then be
assumed to need no further proof, and the research problem simply uses them. It is not possible for a
researcher to question absolutely everything in his/her investigations. Alternatively the research
problem can be in the form of a challenge to veracity of one or more of these ‘truths’. Advances in
wisdom are only made by building on the solid foundations of previous knowledge. Obviously, someone
who is already familiar with the subject investigated will tend to be quicker to advance through this
stage. At this early stage in your research programme you are exploring your subject field only to
identify a problem area, and do not need to try to define your research problem in any detail. All the
same, I think it is useful to know what the next steps will be so that you can see the direction in which
you will be moving. This might well help you to choose a problem area. The knowledge and techniques
which you will require for defining your specific research problem in detail are explained in Chapters 2–7
of this book. Research problem definition From the interest in the wider issues of the chosen subject,
and after the selection of a problem area, the next step is to define the research problem more closely
so that it becomes a specific research problem, with all the characteristics already discussed. This stage
requires an enquiring mind, an eye for inconsistencies and inadequacies in current theory and a
measure of imagination. It is often useful in identifying a specific problem to pose a simple question, for
example, ‘Does the presence of indoor plants affect people’s frame of mind?’ or ‘How can prevention
measures reduce vandalism?’ or ‘Can planning and building regulations prevent the destruction of
indigenous architecture?’ Such a question can provide a starting point for the formulation of a specific
research problem, whose conclusion should aim to answer the question. At this stage, the nature of the
question will give some indication of the type of research approach (or approaches) which could be
appropriate. Will it be a historical study, or a descriptive inquiry, an analysis of correlations or an
experimental exercise, or a combination of more than one of them? (More about this in Chapter 3.)
Seemingly simple questions are riddled with ambiguities, which must be cleared up by careful definition:
for example, in the above questions, what does ‘frame of mind’ mean, what sort of ‘prevention
measures’ are envisaged, and does the question embrace all types of ‘indigenous architecture’
everywhere? It is likely that the problem is too broad if you can state it in less than half a dozen words. A
few additional questions posed against each word can help to delineate the problem – where, who,
what, which, when? Break the problem down into short sentences, not worrying at this stage about the
overall length of the problem statement. It is a useful trick to put each sentence on a separate slip of
paper, so that they can be put into order in different sequences. When the best logical progression from
sentence to sentence is achieved, the statement can be edited into a more elegant form (Chapter 4
deals in more detail with the techniques of problem statement). While developing a specific research
problem, keep in mind the skills which you will require to carry out the research posed by the problem.
Fox (1969, p. 39) defines five types of skill which are essential: research design, instrument
development, data collection, data analysis and research writing. Designing research can be learned, in
consultation with your tutor or supervisor (just wait till Chapters 5 and 6). Instrument development is,
however, a highly specialized skill, so it is advisable to formulate the problem so that you can use
standardized or previously developed instruments. The skills required by data collection techniques are
generally readily acquired (introduced in Chapter 7), though consideration must be given to the extent
of data needed. Data analysis does require specialist skills, which can be of a highly sophisticated nature
(specialist help is on hand when you get that far). It will definitely be worth your while to consult your
tutor or supervisor on the implications for data analysis that the research problem might have. Skills in
research writing will be developed in Chapter 8, and by consultation with your tutors or supervisors over
the next months (or years). Careful consideration of these points will ensure that the planned research is
practicable and has a good chance of success. The sub-problems Most research problems are difficult, or
even impossible, to solve without breaking them down into smaller problems. The short sentences
devised during the problem formulation period can give a clue to presence of subproblems. Does one
aspect have to be researched before another aspect can be begun? For example, in one of the research
questions asked above, the kinds of prevention measures that can be used against vandalism, how they
can be employed and for what types of vandalism they are suitable, will have to be examined. The sub-
problems should delineate the scope of the work and, taken together, should define the entire problem
to be tackled as summarized in the main problem. According to Booth et al. (1995, p. 40) you can
organize your questions to define the sub-problems by looking at your topic from these four
perspectives: 1 What are the parts of your topic and what larger whole is it a part of? 2 What is its
history and what larger history is it a part of? 3 What kind of categories can you find in it, and to what
larger categories of things does it belong? 4 What good is it? What can you use it for? Second review of
literature A more focused review of literature follows the formulation of the research problem. The
purpose of this review is to learn about research already carried out into one or more of the aspects of
the research problem, in order to: 1 summarize the results of previous research to form a foundation on
which to build your own research 2 collect ideas on how to gather data 3 investigate methods of data
analysis Sub-problems RESEARCH AND THE RESEARCH PROBLEM 27 RP01 16/10/00 3:07 pm Page 27 4
study instrumentation which has been used 5 assess the success of the various research designs of the
studies already undertaken.
Mitochondrion
The mitochondrion (plural mitochondria) is a double-membrane-bound organelle found in
most eukaryotic organisms. Some cells in some multicellular organisms may however lack them (for
example, mature mammalian red blood cells). A number of unicellular organisms, such
as microsporidia, parabasalids, and diplomonads, have also reduced or transformed their
mitochondria into other structures. To date, only one eukaryote, Monocercomonoides, is known to
[1]

have completely lost its mitochondria. The word mitochondrion comes from the Greek μίτος, mitos,
[2]

"thread", and χονδρίον, chondrion, "granule" or "grain-like". Mitochondria generate most of the cell's
[3]

supply of adenosine triphosphate (ATP), used as a source of chemical energy. [4]

Mitochondria are commonly between 0.75 and 3 μm in diameter but vary considerably in size and
[5]

structure. Unless specifically stained, they are not visible. In addition to supplying cellular energy,
mitochondria are involved in other tasks, such as signaling, cellular differentiation, and cell death, as
well as maintaining control of the cell cycle and cell growth. Mitochondrial biogenesisis in turn
[6]

temporally coordinated with these cellular processes. Mitochondria have been implicated in several
[7][8]

human diseases, including mitochondrial disorders, cardiac dysfunction, heart failure and
[9] [10] [11]

autism.[12]

The number of mitochondria in a cell can vary widely by organism, tissue, and cell type. For
instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000. The [13][14]

organelle is composed of compartments that carry out specialized functions. These compartments or
regions include the outer membrane, the intermembrane space, the inner membrane, and
the cristae and matrix.
Although most of a cell's DNA is contained in the cell nucleus, the mitochondrion has its own
independent genome that shows substantial similarity to bacterial genomes. Mitochondrial proteins [15]

(proteins transcribed from mitochondrial DNA) vary depending on the tissue and the species. In
humans, 615 distinct types of protein have been identified from cardiacmitochondria, whereas [16]

in rats, 940 proteins have been reported. The mitochondrial proteome is thought to be dynamically
[17]

regulated.[18

History
The first observations of intracellular structures that probably represented mitochondria were
published in the 1840s. Richard Altmann, in 1890, established them as cell organelles and called
[19]

them "bioblasts". The term "mitochondria" was coined by Carl Benda in 1898. Leonor
[19][20] [19][21]

Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900. In
1904, Friedrich Meves, made the first recorded observation of mitochondria in plants in cells of the
white waterlily, Nymphaea alba and in 1908, along with Claudius Regaud, suggested that they
[19][22]

contain proteins and lipids. Benjamin F. Kingsbury, in 1912, first related them with cell respiration,
but almost exclusively based on morphological observations. In 1913, particles from extracts of
[19]

guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called "grana".
Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism,
disagreed on the chemical nature of the respiration. It was not until 1925, when David
Keilindiscovered cytochromes, that the respiratory chain was described. [19]

In 1939, experiments using minced muscle cells demonstrated that cellular respiration using one
oxygen atom can form two adenosine triphosphate (ATP) molecules, and, in 1941, the concept of
the phosphate bonds of ATP being a form of energy in cellular metabolism was developed by Fritz
Albert Lipmann. In the following years, the mechanism behind cellular respiration was further
elaborated, although its link to the mitochondria was not known. The introduction of tissue
[19]

fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and
biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome
oxidase and other enzymes responsible for the respiratory chain were isolated to the
mitochondria. Eugene Kennedy and Albert Lehninger discovered in 1948 that mitochondria are the
site of oxidative phosphorylation in eukaryotes. Over time, the fractionation method was further
developed, improving the quality of the mitochondria isolated, and other elements of cell respiration
were determined to occur in the mitochondria. [19]

The first high-resolution electron micrographs appeared in 1952, replacing the Janus Green stains
as the preferred way of visualising the mitochondria. This led to a more detailed analysis of the
[19]

structure of the mitochondria, including confirmation that they were surrounded by a membrane. It
also showed a second membrane inside the mitochondria that folded up in ridges dividing up the
inner chamber and that the size and shape of the mitochondria varied from cell to cell.
The popular term "powerhouse of the cell" was coined by Philip Siekevitz in 1957. [23]

In 1967, it was discovered that mitochondria contained ribosomes. In 1968, methods were
[24]

developed for mapping the mitochondrial genes, with the genetic and physical map of yeast
mitochondrial DNA being completed in 1976. [19]

Origin and evolution

Main article: Endosymbiotic theory

There are two hypotheses about the origin of mitochondria: endosymbiotic and autogenous. The
endosymbiotic hypothesis suggests that mitochondria were originally prokaryoticcells, capable of
implementing oxidative mechanisms that were not possible for eukaryotic cells; they
became endosymbionts living inside the eukaryote. In the autogenous hypothesis, mitochondria [25]

were born by splitting off a portion of DNA from the nucleus of the eukaryotic cell at the time of
divergence with the prokaryotes; this DNA portion would have been enclosed by membranes, which
could not be crossed by proteins. Since mitochondria have many features in common with bacteria,
the endosymbiotic hypothesis is more widely accepted. [25][26]

A mitochondrion contains DNA, which is organized as several copies of a single, usually

circular, chromosome. This mitochondrial chromosome contains genes for redox proteins, such as
those of the respiratory chain. The CoRR hypothesis proposes that this co-location is required for
redox regulation. The mitochondrial genome codes for some RNAs of ribosomes, and the
22 tRNAs necessary for the translation of mRNAs into protein. The circular structure is also found in
prokaryotes. The proto-mitochondrion was probably closely related to the Rickettsia. However, the [27][28]

exact relationship of the ancestor of mitochondria to the alphaproteobacteria and whether the
mitochondrion was formed at the same time or after the nucleus, remains controversial. [29]

A recent study by researchers of the University of Hawaii at Manoa and the Oregon State
[30]

University indicates that the SAR11 clade of bacteria shares a relatively recent common ancestor
with the mitochondria existing in most eukaryotic cells.
‹ The template below (Cladogram) is being considered for merging. See templates for discussion to help reach a consensus. ›

Schematic ribosomal RNA phylogeny of Alphaproteobacteria

Magnetococcidae
Magnetococcus marinus

Caulobacteridae
Rhodospirillales, Sphingomonadales,
Rhodobacteraceae, Rhizobiales, etc.

Holosporales

Rickettsidae Pelagibacterales Pelagibacteraceae

Pelagibacter

Subgroups Ib, II, IIIa, IIIb, IV and V

Proto-mitochondria

Anaplasmataceae
Ehrlichia

Anaplasma

Wolbachia

Neorickettsia

Midichloriaceae
Midichloria

Rickettsiaceae
Rickettsia

The cladogram of Rickettsidae has been inferred by Ferla et al. [31] from the comparison of 16S + 23S ribosomal RNA sequences.

The ribosomes coded for by the mitochondrial DNA are similar to those from bacteria in size and
structure. They closely resemble the bacterial 70S ribosome and not
[32]

the 80Scytoplasmic ribosomes, which are coded for by nuclear DNA.

The endosymbiotic relationship of mitochondria with their host cells was popularized by Lynn
Margulis. The endosymbiotic hypothesis suggests that mitochondria descended from bacteria that
[33]

somehow survived endocytosis by another cell, and became incorporated into the cytoplasm. The
ability of these bacteria to conduct respiration in host cells that had relied
on glycolysis and fermentation would have provided a considerable evolutionary advantage. This
symbiotic relationship probably developed 1.7 to 2 billion years ago. A few groups of unicellular [34][35]
eukaryotes have only vestigial mitochondria or derived structures:
the microsporidians, metamonads, and archamoebae. These groups appear as the most primitive [36]

eukaryotes on phylogenetic trees constructed using rRNA information, which once suggested that
they appeared before the origin of mitochondria. However, this is now known to be an artifact
of long-branch attraction—they are derived groups and retain genes or organelles derived from
mitochondria (e.g., mitosomes and hydrogenosomes). [1]

Monocercomonoides appear to have lost their mitochondria completely and at least some of the
mitochondrial functions seem to be carried out by cytoplasmic proteins now. [37]

Structure

Mitochondrion ultrastructure (interactive diagram) A mitochondrion has a double membrane; the inner one contains its chemiosmotic apparatus and has deep grooves which

increase its surface area. While commonly depicted as an "orange sausage with a blob inside of it" (like it is here), mitochondria can take many shapes[38] and their

intermembrane space is quite thin.

A mitochondrion contains outer and inner membranes composed of phospholipid

bilayers and proteins. The two membranes have different properties. Because of this double-
[13]

membraned organization, there are five distinct parts to a mitochondrion. They are:

1. the outer mitochondrial membrane,

2. the intermembrane space (the space between the outer and inner membranes),
3. the inner mitochondrial membrane,
4. the cristae space (formed by infoldings of the inner membrane), and
5. the matrix (space within the inner membrane).
Mitochondria stripped of their outer membrane are called mitoplasts.
Outer membrane
The outer mitochondrial membrane, which encloses the entire organelle, is 60 to 75 angstroms(Å)
thick. It has a protein-to-phospholipid ratio similar to that of the eukaryotic plasma membrane (about
1:1 by weight). It contains large numbers of integral membrane proteins called porins. These porins
form channels that allow molecules of 5000 daltons or less in molecular weight to freely diffuse from
one side of the membrane to the other. Larger proteins can enter the mitochondrion if a signaling
[13]

sequence at their N-terminus binds to a large multisubunit proteincalled translocase of the outer
membrane, which then actively moves them across the membrane. Mitochondrial pro-proteins are [39]

imported through specialised translocation complexes. The outer membrane also

contains enzymes involved in such diverse activities as the elongation of fatty
acids, oxidation of epinephrine, and the degradation of tryptophan. These enzymes
include monoamine oxidase, rotenone-insensitive NADH-cytochrome c-
reductase, kynurenine hydroxylase and fatty acid Co-A ligase. Disruption of the outer membrane
permits proteins in the intermembrane space to leak into the cytosol, leading to certain cell
death. The mitochondrial outer membrane can associate with the endoplasmic reticulum (ER)
[40]

membrane, in a structure called MAM (mitochondria-associated ER-membrane). This is important in

the ER-mitochondria calcium signaling and is involved in the transfer of lipids between the ER and
mitochondria. Outside the outer membrane there are small (diameter: 60Å) particles named sub-
[41]

units of Parson.
Intermembrane space
The intermembrane space is the space between the outer membrane and the inner membrane. It is
also known as perimitochondrial space. Because the outer membrane is freely permeable to small
molecules, the concentrations of small molecules, such as ions and sugars, in the intermembrane
space is the same as in the cytosol. However, large proteins must have a specific signaling
[13]

sequence to be transported across the outer membrane, so the protein composition of this space is
different from the protein composition of the cytosol. One protein that is localized to the
intermembrane space in this way is cytochrome c. [40]

Inner membrane
Main article: Inner mitochondrial membrane

The inner mitochondrial membrane contains proteins with five types of functions: [13]

1. Those that perform the redox reactions of oxidative phosphorylation

2. ATP synthase, which generates ATP in the matrix
3. Specific transport proteins that regulate metabolite passage into and out of the matrix
4. Protein import machinery
5. Mitochondrial fusion and fission protein
It contains more than 151 different polypeptides, and has a very high protein-to-phospholipid ratio
(more than 3:1 by weight, which is about 1 protein for 15 phospholipids). The inner membrane is
home to around 1/5 of the total protein in a mitochondrion. In addition, the inner membrane is rich [13]

in an unusual phospholipid, cardiolipin. This phospholipid was originally discovered in cow hearts in
1942, and is usually characteristic of mitochondrial and bacterial plasma membranes. Cardiolipin [42]

contains four fatty acids rather than two, and may help to make the inner membrane
impermeable. Unlike the outer membrane, the inner membrane doesn't contain porins, and is highly
[13]

impermeable to all molecules. Almost all ions and molecules require special membrane transporters
to enter or exit the matrix. Proteins are ferried into the matrix via the translocase of the inner
membrane (TIM) complex or via Oxa1. In addition, there is a membrane potential across the inner
[39]

membrane, formed by the action of the enzymes of the electron transport chain.
Cristae

Cross-sectional image of cristae in rat liver mitochondrion to demonstrate the likely 3D structure and relationship to the inner membrane

Main article: Cristae

The inner mitochondrial membrane is compartmentalized into numerous cristae, which expand the
surface area of the inner mitochondrial membrane, enhancing its ability to produce ATP. For typical
liver mitochondria, the area of the inner membrane is about five times as large as the outer
membrane. This ratio is variable and mitochondria from cells that have a greater demand for ATP,
such as muscle cells, contain even more cristae. These folds are studded with small round bodies
known as F particles or oxysomes. These are not simple random folds but rather invaginations of
1

the inner membrane, which can affect overall chemiosmotic function. [43]

One recent mathematical modeling study has suggested that the optical properties of the cristae in
filamentous mitochondria may affect the generation and propagation of light within the tissue. [44]
Matrix
Main article: Mitochondrial matrix

The matrix is the space enclosed by the inner membrane. It contains about 2/3 of the total protein in
a mitochondrion. The matrix is important in the production of ATP with the aid of the ATP synthase
[13]

contained in the inner membrane. The matrix contains a highly concentrated mixture of hundreds of
enzymes, special mitochondrial ribosomes, tRNA, and several copies of the mitochondrial
DNA genome. Of the enzymes, the major functions include oxidation of pyruvate and fatty acids, and
the citric acid cycle. [13]

Mitochondria have their own genetic material, and the machinery to manufacture their
own RNAs and proteins (see: protein biosynthesis). A published human mitochondrial DNA
sequence revealed 16,569 base pairs encoding 37 genes: 22 tRNA, 2 rRNA, and
13 peptide genes. The 13 mitochondrial peptides in humans are integrated into the inner
[45]

mitochondrial membrane, along with proteins encoded by genes that reside in the host
cell's nucleus.
Mitochondria-associated ER membrane (MAM)
Main article: Mitochondria associated membranes (MAM)

The mitochondria-associated ER membrane (MAM) is another structural element that is increasingly

recognized for its critical role in cellular physiology and homeostasis. Once considered a technical
snag in cell fractionation techniques, the alleged ER vesicle contaminants that invariably appeared in
the mitochondrial fraction have been re-identified as membranous structures derived from the
MAM—the interface between mitochondria and the ER. Physical coupling between these two
[46]

organelles had previously been observed in electron micrographs and has more recently been
probed with fluorescence microscopy. Such studies estimate that at the MAM, which may comprise
[46]

up to 20% of the mitochondrial outer membrane, the ER and mitochondria are separated by a mere
10–25 nm and held together by protein tethering complexes. [46][47][48]

Purified MAM from subcellular fractionation has been shown to be enriched in enzymes involved in
phospholipid exchange, in addition to channels associated with Ca signaling. These hints of a
2+ [46][48]

prominent role for the MAM in the regulation of cellular lipid stores and signal transduction have
been borne out, with significant implications for mitochondrial-associated cellular phenomena, as
discussed below. Not only has the MAM provided insight into the mechanistic basis underlying such
physiological processes as intrinsic apoptosis and the propagation of calcium signaling, but it also
favors a more refined view of the mitochondria. Though often seen as static, isolated 'powerhouses'
hijacked for cellular metabolism through an ancient endosymbiotic event, the evolution of the MAM
underscores the extent to which mitochondria have been integrated into overall cellular physiology,
with intimate physical and functional coupling to the endomembrane system.
Phospholipid transfer

The MAM is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase
on the ER face and phosphatidylserine decarboxylase on the mitochondrial face. Because [49][50]

mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a
constant and well-regulated supply of phospholipids for membrane integrity. But mitochondria are
[51][52]

not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a
role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic
pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolism. [50][52]

Such trafficking capacity depends on the MAM, which has been shown to facilitate transfer of lipid
intermediates between organelles. In contrast to the standard vesicular mechanism of lipid transfer,
[49]

evidence indicates that the physical proximity of the ER and mitochondrial membranes at the MAM
allows for lipid flipping between opposed bilayers. Despite this unusual and seemingly energetically
[52]

unfavorable mechanism, such transport does not require ATP. Instead, in yeast, it has been shown
[52]

to be dependent on a multiprotein tethering structure termed the ER-mitochondria encounter

structure, or ERMES, although it remains unclear whether this structure directly mediates lipid
transfer or is required to keep the membranes in sufficiently close proximity to lower the energy
barrier for lipid flipping. [52][53]

The MAM may also be part of the secretory pathway, in addition to its role in intracellular lipid
trafficking. In particular, the MAM appears to be an intermediate destination between the rough ER
and the Golgi in the pathway that leads to very-low-density lipoprotein, or VLDL, assembly and
secretion. The MAM thus serves as a critical metabolic and trafficking hub in lipid metabolism.
[50][54]

Calcium signaling

A critical role for the ER in calcium signaling was acknowledged before such a role for the
mitochondria was widely accepted, in part because the low affinity of Ca channels localized to the 2+

outer mitochondrial membrane seemed to fly in the face of this organelle's purported responsiveness
to changes in intracellular Ca flux. But the presence of the MAM resolves this apparent
2+ [46][55]

contradiction: the close physical association between the two organelles results in Ca microdomains 2+

at contact points that facilitate efficient Ca transmission from the ER to the 2+

mitochondria. Transmission occurs in response to so-called "Ca puffs" generated by spontaneous

[46] 2+

clustering and activation of IP3R, a canonical ER membrane Ca channel. 2+ [46][47]

The fate of these puffs—in particular, whether they remain restricted to isolated locales or integrated
into Ca waves for propagation throughout the cell—is determined in large part by MAM dynamics.
2+

Although reuptake of Ca by the ER (concomitant with its release) modulates the intensity of the
2+

puffs, thus insulating mitochondria to a certain degree from high Ca exposure, the MAM often 2+

serves as a firewall that essentially buffers Ca puffs by acting as a sink into which free ions released 2+

into the cytosol can be funneled. This Ca tunneling occurs through the low-affinity Ca receptor
[46][56][57] 2+ 2+

VDAC1, which recently has been shown to be physically tethered to the IP3R clusters on the ER
membrane and enriched at the MAM. The ability of mitochondria to serve as a Ca sink is a
[46][47][58] 2+

result of the electrochemical gradient generated during oxidative phosphorylation, which makes
tunneling of the cation an exergonic process. Normally, mild calcium influx from cytosol into the [58]

mitochondrial matrix causes transient depolarization that is corrected by pumping out protons.
But transmission of Ca is not unidirectional; rather, it is a two-way street. The properties of the
2+ [55]

Ca pump SERCA and the channel IP3R present on the ER membrane facilitate feedback regulation
2+

coordinated by MAM function. In particular, the clearance of Ca by the MAM allows for spatio- 2+

temporal patterning of Ca signaling because Ca alters IP3R activity in a biphasic

2+ 2+

manner. SERCA is likewise affected by mitochondrial feedback: uptake of Ca by the MAM

[46] 2+

stimulates ATP production, thus providing energy that enables SERCA to reload the ER with Ca for 2+

continued Ca efflux at the MAM. Thus, the MAM is not a passive buffer for Ca puffs; rather it
2+ [56][58] 2+

helps modulate further Ca signaling through feedback loops that affect ER dynamics.
2+

Regulating ER release of Ca at the MAM is especially critical because only a certain window of
2+

Ca uptake sustains the mitochondria, and consequently the cell, at homeostasis. Sufficient
2+

intraorganelle Ca signaling is required to stimulate metabolism by activating dehydrogenase

2+

enzymes critical to flux through the citric acid cycle. However, once Ca signaling in the [59] 2+

mitochondria passes a certain threshold, it stimulates the intrinsic pathway of apoptosis in part by
collapsing the mitochondrial membrane potential required for metabolism. Studies examining the [46]

role of pro- and anti-apoptotic factors support this model; for example, the anti-apoptotic factor Bcl-2
has been shown to interact with IP3Rs to reduce Ca filling of the ER, leading to reduced efflux at 2+

the MAM and preventing collapse of the mitochondrial membrane potential post-apoptotic
stimuli. Given the need for such fine regulation of Ca signaling, it is perhaps unsurprising that
[46] 2+

dysregulated mitochondrial Ca has been implicated in several neurodegenerative diseases, while

2+

the catalogue of tumor suppressors includes a few that are enriched at the MAM. [58]

Molecular basis for tethering

Recent advances in the identification of the tethers between the mitochondrial and ER membranes
suggest that the scaffolding function of the molecular elements involved is secondary to other, non-
structural functions. In yeast, ERMES, a multiprotein complex of interacting ER- and mitochondrial-
resident membrane proteins, is required for lipid transfer at the MAM and exemplifies this principle.
One of its components, for example, is also a constituent of the protein complex required for
insertion of transmembrane beta-barrel proteins into the lipid bilayer. However, a homologue of the
[52]

ERMES complex has not yet been identified in mammalian cells. Other proteins implicated in
scaffolding likewise have functions independent of structural tethering at the MAM; for example, ER-
resident and mitochondrial-resident mitofusins form heterocomplexes that regulate the number of
inter-organelle contact sites, although mitofusins were first identified for their role
in fission and fusion events between individual mitochondria. Glucose-related protein 75 (grp75) is
[46]

another dual-function protein. In addition to the matrix pool of grp75, a portion serves as a
chaperone that physically links the mitochondrial and ER Ca channels VDAC and IP3R for efficient
2+

Ca transmission at the MAM. Another potential tether is Sigma-1R, a non-opioid receptor whose
2+ [46][47]

stabilization of ER-resident IP3R may preserve communication at the MAM during the metabolic
stress response. [60][61]

Model of the yeast multimeric tethering complex, ERMES

Perspective

The MAM is a critical signaling, metabolic, and trafficking hub in the cell that allows for the
integration of ER and mitochondrial physiology. Coupling between these organelles is not simply
structural but functional as well and critical for overall cellular physiology and homeostasis. The
MAM thus offers a perspective on mitochondria that diverges from the traditional view of this
organelle as a static, isolated unit appropriated for its metabolic capacity by the cell. Instead, this
mitochondrial-ER interface emphasizes the integration of the mitochondria, the product of an
endosymbiotic event, into diverse cellular processes.

Organization and distribution

Typical mitochondrial network (green) in two human cells (HeLa cells)

Mitochondria (and related structures) are found in all eukaryotes (except one—
the Oxymonad Monocercomonoides sp.). Although commonly depicted as bean-like structures
[2][62]

they form a highly dynamic network in the majority of cells where they constantly
undergo fissionand fusion. Mitochondria vary in number and location according to cell type. A single
mitochondrion is often found in unicellular organisms. Conversely, numerous mitochondria are found
in human liver cells, with about 1000–2000 mitochondria per cell, making up 1/5 of the cell
volume. The population of all the mitochondria of a given cell constitutes the chondriome. The
[13]

mitochondrial content of otherwise similar cells can vary substantially in size and membrane
potential, with differences arising from sources including uneven partitioning at cell divisions,
[63]

leading to extrinsic differences in ATP levels and downstream cellular processes. The mitochondria [64]

can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum. Often, [13]

they form a complex 3D branching network inside the cell with the cytoskeleton. The association with
the cytoskeleton determines mitochondrial shape, which can affect the function as well: different [65]

structures of the mitochondrial network may afford the population a variety of physical, chemical, and
signalling advantages or disadvantages. Mitochondria in cells are always distributed along
[66]

microtubules and the distribution of these organelles is also correlated with the endoplasmic
reticulum. Recent evidence suggests that vimentin, one of the components of the cytoskeleton, is
[67]

also critical to the association with the cytoskeleton. [68]

Function
The most prominent roles of mitochondria are to produce the energy currency of the cell, ATP (i.e.,
phosphorylation of ADP), through respiration, and to regulate cellular metabolism. The central set [14]

of reactions involved in ATP production are collectively known as the citric acid cycle, or
the Krebs cycle. However, the mitochondrion has many other functions in addition to the production
of ATP.
Energy conversion
A dominant role for the mitochondria is the production of ATP, as reflected by the large number of
proteins in the inner membrane for this task. This is done by oxidizing the major products
of glucose: pyruvate, and NADH, which are produced in the cytosol. This type of cellular [14]

respiration known as aerobic respiration, is dependent on the presence of oxygen. When oxygen is
limited, the glycolytic products will be metabolized by anaerobic fermentation, a process that is
independent of the mitochondria. The production of ATP from glucose has an approximately 13-
[14]

times higher yield during aerobic respiration compared to fermentation. Plant mitochondria can also[69]

produce a limited amount of ATP without oxygen by using the alternate substrate nitrite. ATP [70]
crosses out through the inner membrane with the help of a specific protein, and across the outer
membrane via porins. ADP returns via the same route.
Pyruvate and the citric acid cycle
Main articles: Pyruvate dehydrogenase, Pyruvate carboxylase, and Citric acid cycle

Pyruvate molecules produced by glycolysis are actively transported across the inner mitochondrial
membrane, and into the matrix where they can either be oxidized and combined with coenzyme A to
form CO , acetyl-CoA, and NADH, or they can be carboxylated (by pyruvate carboxylase) to form
2
[14]

oxaloacetate. This latter reaction ”fills up” the amount of oxaloacetate in the citric acid cycle, and is
therefore an anaplerotic reaction, increasing the cycle’s capacity to metabolize acetyl-CoA when the
tissue's energy needs (e.g. in muscle) are suddenly increased by activity. [71]

In the citric acid cycle, all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate,
fumarate, malate and oxaloacetate) are regenerated during each turn of the cycle. Adding more of
any of these intermediates to the mitochondrion therefore means that the additional amount is
retained within the cycle, increasing all the other intermediates as one is converted into the other.
Hence, the addition of any one of them to the cycle has an anaplerotic effect, and its removal has a
cataplerotic effect. These anaplerotic and cataplerotic reactions will, during the course of the cycle,
increase or decrease the amount of oxaloacetate available to combine with acetyl-CoA to form citric
acid. This in turn increases or decreases the rate of ATP production by the mitochondrion, and thus
the availability of ATP to the cell. [71]

Acetyl-CoA, on the other hand, derived from pyruvate oxidation, or from the beta-oxidation of fatty
acids, is the only fuel to enter the citric acid cycle. With each turn of the cycle one molecule of acetyl-
CoA is consumed for every molecule of oxaloacetate present in the mitochondrial matrix, and is
never regenerated. It is the oxidation of the acetate portion of acetyl-CoA that produces CO and 2

water, with the energy thus released captured in the form of ATP. [71]

In the liver, the carboxylation of cytosolic pyruvate into intra-mitochondrial oxaloacetate is an early
step in the gluconeogenic pathway, which converts lactate and de-aminated alanine into
glucose, under the influence of high levels of glucagon and/or epinephrine in the blood. Here, the
[14][71] [71]

addition of oxaloacetate to the mitochondrion does not have a net anaplerotic effect, as another citric
acid cycle intermediate (malate) is immediately removed from the mitochondrion to be converted into
cytosolic oxaloacetate, which is ultimately converted into glucose, in a process that is almost the
reverse of glycolysis. [71]

The enzymes of the citric acid cycle are located in the mitochondrial matrix, with the exception
of succinate dehydrogenase, which is bound to the inner mitochondrial membrane as part of
Complex II. The citric acid cycle oxidizes the acetyl-CoA to carbon dioxide, and, in the process,
[72]

produces reduced cofactors (three molecules of NADH and one molecule of FADH ) that are a
2

source of electrons for the electron transport chain, and a molecule of GTP (that is readily converted
to an ATP). [14]

NADH and FADH2: the electron transport chain

Main articles: Electron transport chain and Oxidative phosphorylation
Electron transport chain in the mitochondrial intermembrane space

The redox energy from NADH and FADH is transferred to oxygen (O ) in several steps via the
2 2

electron transport chain. These energy-rich molecules are produced within the matrix via the citric
acid cycle but are also produced in the cytoplasm by glycolysis. Reducing equivalents from the
cytoplasm can be imported via the malate-aspartate shuttle system of antiporter proteins or feed into
the electron transport chain using a glycerol phosphate shuttle. Protein complexes in the inner
[14]

membrane (NADH dehydrogenase (ubiquinone), cytochrome c reductase, and cytochrome c

oxidase) perform the transfer and the incremental release of energy is used to pump protons (H ) +

into the intermembrane space. This process is efficient, but a small percentage of electrons may
prematurely reduce oxygen, forming reactive oxygen species such as superoxide. This can [14]

cause oxidative stress in the mitochondria and may contribute to the decline in mitochondrial
function associated with the aging process. [73]

As the proton concentration increases in the intermembrane space, a strong electrochemical

gradient is established across the inner membrane. The protons can return to the matrix through
the ATP synthasecomplex, and their potential energy is used to synthesize ATP from ADP and
inorganic phosphate (P ). This process is called chemiosmosis, and was first described by Peter
i
[14]

Mitchell who was awarded the 1978 Nobel Prize in Chemistry for his work. Later, part of the 1997
[74][75]

Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walkerfor their clarification of
the working mechanism of ATP synthase. [76]

Heat production

Under certain conditions, protons can re-enter the mitochondrial matrix without contributing to ATP
synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to
the facilitated diffusion of protons into the matrix. The process results in the unharnessed potential
energy of the proton electrochemical gradient being released as heat. The process is mediated by a [14]

proton channel called thermogenin, or UCP1. Thermogenin is a 33 kDa protein first discovered in
[77]

1973. Thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for
[78]

non-shivering thermogenesis. Brown adipose tissue is found in mammals, and is at its highest levels
in early life and in hibernating animals. In humans, brown adipose tissue is present at birth and
decreases with age. [77]
Storage of calcium ions

Transmission electron micrograph of a chondrocyte, stained for calcium, showing its nucleus (N) and mitochondria (M).

The concentrations of free calcium in the cell can regulate an array of reactions and is important
for signal transduction in the cell. Mitochondria can transiently store calcium, a contributing process
for the cell's homeostasis of calcium. In fact, their ability to rapidly take in calcium for later release
[79] [80]

makes them very good "cytosolic buffers" for calcium. The endoplasmic reticulum (ER) is the
[81][82][83]

most significant storage site of calcium, and there is a significant interplay between the
[55]

mitochondrion and ER with regard to calcium. The calcium is taken up into the matrix by[84]

the mitochondrial calcium uniporter on the inner mitochondrial membrane. It is primarily driven by [85]

the mitochondrial membrane potential. Release of this calcium back into the cell's interior can occur
[80]

via a sodium-calcium exchange protein or via "calcium-induced-calcium-release" pathways. This [85]

can initiate calcium spikes or calcium waves with large changes in the membrane potential. These
can activate a series of second messenger system proteins that can coordinate processes such
as neurotransmitter release in nerve cells and release of hormones in endocrine cells. [86]

Ca influx to the mitochondrial matrix has recently been implicated as a mechanism to regulate
2+

respiratory bioenergetics by allowing the electrochemical potential across the membrane to

transiently "pulse" from ΔΨ-dominated to pH-dominated, facilitating a reduction of oxidative
stress. In neurons, concomitant increases in cytosolic and mitochondrial calcium act to synchronize
[87]

neuronal activity with mitochondrial energy metabolism. Mitochondrial matrix calcium levels can
reach the tens of micromolar levels, which is necessary for the activation of isocitrate
dehydrogenase, one of the key regulatory enzymes of the Krebs cycle. [88]

Additional functions
Mitochondria play a central role in many other metabolic tasks, such as:

 Signaling through mitochondrial reactive oxygen species [89]

 Regulation of the membrane potential [14]

 Apoptosis-programmed cell death [90]

 Calcium signaling (including calcium-evoked apoptosis) [91]

 Regulation of cellular metabolism [92]

 Certain heme synthesis reactions (see also: porphyrin) [93]

 Steroid synthesis. [81]

 Hormonal signaling Mitochondria are sensitive and responsive to hormones, in part by the
[94]

action of mitochondrial estrogen receptors (mtERs). These receptors have been found in various
tissues and cell types, including brain and heart [95] [96]

Some mitochondrial functions are performed only in specific types of cells. For example,
mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of
protein metabolism. A mutation in the genes regulating any of these functions can result
in mitochondrial diseases.

Cellular proliferation regulation

The relationship between cellular proliferation and mitochondria has been investigated using
cervical cancer HeLa cells. Tumor cells require an ample amount of ATP (Adenosine triphosphate)
in order to synthesize bioactive compounds such as lipids, proteins, and nucleotides for rapid cell
proliferation. The majority of ATP in tumor cells is generated via the oxidative
[97]

phosphorylation pathway (OxPhos). Interference with OxPhos have shown to cause cell cycle
[98]

arrest suggesting that mitochondria play a role in cell proliferation. Mitochondrial ATP production is [98]

also vital for cell division in addition to other basic functions in the cell including the regulation of cell
volume, solute concentration, and cellular architecture. ATP levels differ at various stages of the
[99][100][101]

cell cycle suggesting that there is a relationship between the abundance of ATP and the cell's ability
to enter a new cell cycle. ATP's role in the basic functions of the cell make the cell cycle sensitive
[102]

to changes in the availability of mitochondrial derived ATP. The variation in ATP levels at different [102]

stages of the cell cycle support the hypothesis that mitochondria play an important role in cell cycle
regulation. Although the specific mechanisms between mitochondria and the cell cycle regulation is
[102]

not well understood, studies have shown that low energy cell cycle checkpoints monitor the energy
capability before committing to another round of cell division. [103]

Genome

The circular 16,569 bp human mitochondrial genome encoding 37 genes, i.e., 28 on the H-strand and 9 on the L-strand.

Main article: Mitochondrial DNA

Mitochondria contain their own genome, an indication that they are derived from bacteria
through endosymbiosis. However, the ancestral endosymbiont genome has lost most of its genes so
that the mitochondrial genome is one of the most reduced genomes across organisms.
The human mitochondrial genome is a circular DNA molecule of about 16 kilobases. It encodes 37 [104]

genes: 13 for subunits of respiratory complexes I, III, IV and V, 22 for mitochondrial tRNA (for the 20
standard amino acids, plus an extra gene for leucine and serine), and 2 for rRNA. One [104]

mitochondrion can contain two to ten copies of its DNA. [105]

As in prokaryotes, there is a very high proportion of coding DNA and an absence of repeats.
Mitochondrial genes are transcribed as multigenic transcripts, which are cleaved
and polyadenylated to yield mature mRNAs. Not all proteins necessary for mitochondrial function are
encoded by the mitochondrial genome; most are coded by genes in the cell nucleus and the
corresponding proteins are imported into the mitochondrion. The exact number of genes encoded [45]

by the nucleus and the mitochondrial genome differs between species. Most mitochondrial genomes
are circular, although exceptions have been reported. In general, mitochondrial DNA lacks introns,
[106]

as is the case in the human mitochondrial genome; however, introns have been observed in some
[45]

eukaryotic mitochondrial DNA, such as that [107]

of yeast and protists, including Dictyostelium discoideum. Between protein-coding regions,

[108] [109] [110]

tRNAs are present. During transcription, the tRNAs acquire their characteristic L-shape that gets
recognized and cleaved by specific enzymes. Mitochondrial tRNA genes have different sequences
from the nuclear tRNAs but lookalikes of mitochondrial tRNAs have been found in the nuclear
chromosomes with high sequence similarity. [111]

In animals, the mitochondrial genome is typically a single circular chromosome that is approximately
16 kb long and has 37 genes. The genes, while highly conserved, may vary in location. Curiously,
this pattern is not found in the human body louse (Pediculus humanus). Instead, this mitochondrial
genome is arranged in 18 minicircular chromosomes, each of which is 3–4 kb long and has one to
three genes. This pattern is also found in other sucking lice, but not in chewing lice. Recombination
[112]

has been shown to occur between the minichromosomes. The reason for this difference is not
known.
Alternative genetic code
While slight variations on the standard genetic code had been predicted earlier, none was [113]

discovered until 1979, when researchers studying human mitochondrial genesdetermined that they
used an alternative code. However, the mitochondria of many other eukaryotes, including most
[114]

plants, use the standard code. Many slight variants have been discovered since, including
[115] [116]

various alternative mitochondrial codes. Further, the AUA, AUC, and AUU codons are all allowable
[117]

start codons.

Exceptions to the standard genetic code in mitochondria[13]

Organism Codon Standard Mitochondria

Mammals AGA, AGG Arginine Stop codon

Invertebrates AGA, AGG Arginine Serine

Fungi CUA Leucine Threonine

All of the above AUA Isoleucine Methionine

UGA Stop codon Tryptophan

Some of these differences should be regarded as pseudo-changes in the genetic code due to the
phenomenon of RNA editing, which is common in mitochondria. In higher plants, it was thought that
CGG encoded for tryptophan and not arginine; however, the codon in the processed RNA was
discovered to be the UGG codon, consistent with the standard genetic code for tryptophan. Of [118]

note, the arthropod mitochondrial genetic code has undergone parallel evolution within a phylum,
with some organisms uniquely translating AGG to lysine. [119]

Evolution and diversity

Mitochondrial genomes have far fewer genes than the bacteria from which they are thought to be
descended. Although some have been lost altogether, many have been transferred to the nucleus,
such as the respiratory complex II protein subunits. This is thought to be relatively common over
[104]

evolutionary time. A few organisms, such as the Cryptosporidium, actually have mitochondria that
lack any DNA, presumably because all their genes have been lost or
transferred. In Cryptosporidium, the mitochondria have an altered ATPgeneration system that
[120]

renders the parasite resistant to many classical mitochondrial inhibitors such as cyanide, azide,
and atovaquone. [120]

Replication and inheritance

Mitochondria divide by binary fission, similar to bacterial cell division. The regulation of this division
[121]

differs between eukaryotes. In many single-celled eukaryotes, their growth and division is linked to
the cell cycle. For example, a single mitochondrion may divide synchronously with the nucleus. This
division and segregation process must be tightly controlled so that each daughter cell receives at
least one mitochondrion. In other eukaryotes (in mammals for example), mitochondria may replicate
their DNA and divide mainly in response to the energy needs of the cell, rather than in phase with
the cell cycle. When the energy needs of a cell are high, mitochondria grow and divide. When the
energy use is low, mitochondria are destroyed or become inactive. In such examples, and in contrast
to the situation in many single celled eukaryotes, mitochondria are apparently randomly distributed
to the daughter cells during the division of the cytoplasm. Understanding of mitochondrial dynamics,
which is described as the balance between mitochondrial fusion and fission, has revealed that
functional and structural alterations in mitochondrial morphology are important factors in pathologies
associated with several disease conditions. [122]

The hypothesis of mitochondrial binary fission has relied on the visualization by fluorescence
microscopy and conventional transmission electron microscopy (TEM). The resolution of
fluorescence microscopy(~200 nm) is insufficient to distinguish structural details, such as double
mitochondrial membrane in mitochondrial division or even to distinguish individual mitochondria
when several are close together. Conventional TEM has also some technical limitations in
[which?]

verifying mitochondrial division. Cryo-electron tomography was recently used to visualize

mitochondrial division in frozen hydrated intact cells. It revealed that mitochondria divide by
budding. [123]

An individual's mitochondrial genes are not inherited by the same mechanism as nuclear genes.
Typically, the mitochondria are inherited from one parent only. In humans, when an egg cell is
fertilized by a sperm, the egg nucleus and sperm nucleus each contribute equally to the genetic
makeup of the zygote nucleus. In contrast, the mitochondria, and therefore the mitochondrial DNA,
usually come from the egg only. The sperm's mitochondria enter the egg, but do not contribute
genetic information to the embryo. Instead, paternal mitochondria are marked with ubiquitin to
[124]

select them for later destruction inside the embryo. The egg cell contains relatively few
[125]

mitochondria, but it is these mitochondria that survive and divide to populate the cells of the adult
organism. Mitochondria are, therefore, in most cases inherited only from mothers, a pattern known
as maternal inheritance. This mode is seen in most organisms, including the majority of animals.
However, mitochondria in some species can sometimes be inherited paternally. This is the norm
among certain coniferous plants, although not in pine trees and yews. For Mytilids, paternal
[126]

inheritance only occurs within males of the species. It has been suggested that it occurs at a
[127][128][129]

very low level in humans. There is a recent suggestion that mitochondria that shorten male lifespan
[130]
stay in the system because they are inherited only through the mother. By contrast, natural
selection weeds out mitochondria that reduce female survival as such mitochondria are less likely to
be passed on to the next generation. Therefore, it is suggested that human females and female
animals tend to live longer than males. The authors claim that this is a partial explanation. [131]

Uniparental inheritance leads to little opportunity for genetic recombination between different
lineages of mitochondria, although a single mitochondrion can contain 2–10 copies of its DNA. For [105]

this reason, mitochondrial DNA is usually thought to reproduce by binary fission. What
recombination does take place maintains genetic integrity rather than maintaining diversity.
However, there are studies showing evidence of recombination in mitochondrial DNA. It is clear that
the enzymes necessary for recombination are present in mammalian cells. Further, evidence [132]

suggests that animal mitochondria can undergo recombination. The data are a bit more
[133]

controversial in humans, although indirect evidence of recombination exists. If recombination[134][135]

does not occur, the whole mitochondrial DNA sequence represents a single haplotype, which makes
it useful for studying the evolutionary history of populations.
Entities undergoing uniparental inheritance and with little to no recombination may be expected to be
subject to Muller's ratchet, the inexorable accumulation of deleterious mutations until functionality is
lost. Animal populations of mitochondria avoid this buildup through a developmental process known
as the mtDNA bottleneck. The bottleneck exploits stochastic processes in the cell to increase in the
cell-to-cell variability in mutant load as an organism develops: a single egg cell with some proportion
of mutant mtDNA thus produces an embryo where different cells have different mutant loads. Cell-
level selection may then act to remove those cells with more mutant mtDNA, leading to a
stabilisation or reduction in mutant load between generations. The mechanism underlying the
bottleneck is debated, with a recent mathematical and experimental metastudy providing
[136][137][138]

evidence for a combination of random partitioning of mtDNAs at cell divisions and random turnover
of mtDNA molecules within the cell. [139]

Population genetic studies

Main article: Human mitochondrial genetics

The near-absence of genetic recombination in mitochondrial DNA makes it a useful source of

information for scientists involved in population genetics and evolutionary biology. Because all the [140]

mitochondrial DNA is inherited as a single unit, or haplotype, the relationships between

mitochondrial DNA from different individuals can be represented as a gene tree. Patterns in these
gene trees can be used to infer the evolutionary history of populations. The classic example of this is
in human evolutionary genetics, where the molecular clock can be used to provide a recent date
for mitochondrial Eve. This is often interpreted as strong support for a recent modern human
[141][142]

expansion out of Africa. Another human example is the sequencing of mitochondrial DNA
[143]

from Neanderthal bones. The relatively large evolutionary distance between the mitochondrial DNA
sequences of Neanderthals and living humans has been interpreted as evidence for the lack of
interbreeding between Neanderthals and anatomically modern humans. [144]

However, mitochondrial DNA reflects only the history of the females in a population and so may not
represent the history of the population as a whole. This can be partially overcome by the use of
paternal genetic sequences, such as the non-recombining region of the Y-chromosome. In a [143]

broader sense, only studies that also include nuclear DNA can provide a comprehensive
evolutionary history of a population. [145]

Recent measurements of the molecular clock for mitochondrial DNA reported a value of 1 mutation
[146]

every 7884 years dating back to the most recent common ancestor of humans and apes, which is
consistent with estimates of mutation rates of autosomal DNA (10 per base per generation ).
−8 [147]

Dysfunction and disease

Mitochondrial diseases
Main article: Mitochondrial disease

Damage and subsequent dysfunction in mitochondria is an important factor in a range of human

diseases due to their influence in cell metabolism. Mitochondrial disorders often present themselves
as neurological disorders, including autism. They can also manifest as myopathy, diabetes,
[12]

multiple endocrinopathy, and a variety of other systemic disorders. Diseases caused by mutation in
[148]

the mtDNA include Kearns-Sayre syndrome, MELAS syndrome and Leber's hereditary optic
neuropathy. In the vast majority of cases, these diseases are transmitted by a female to her
[149]

children, as the zygote derives its mitochondria and hence its mtDNA from the ovum. Diseases such
as Kearns-Sayre syndrome, Pearson syndrome, and progressive external ophthalmoplegia are
thought to be due to large-scale mtDNA rearrangements, whereas other diseases such as MELAS
syndrome, Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibers (MERRF),
and others are due to point mutations in mtDNA. [148]

In other diseases, defects in nuclear genes lead to dysfunction of mitochondrial proteins. This is the
case in Friedreich's ataxia, hereditary spastic paraplegia, and Wilson's disease. These diseases [150]

are inherited in a dominance relationship, as applies to most other genetic diseases. A variety of
disorders can be caused by nuclear mutations of oxidative phosphorylation enzymes, such
as coenzyme Q10 deficiency and Barth syndrome. Environmental influences may interact with
[148]

hereditary predispositions and cause mitochondrial disease. For example, there may be a link
between pesticide exposure and the later onset of Parkinson's disease. Other pathologies with
[151][152]

etiology involving mitochondrial dysfunction include schizophrenia, bipolar

disorder, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, cardiovascular
[153]

disease, chronic fatigue syndrome, retinitis pigmentosa, and diabetes mellitus. [154][155]

Mitochondria-mediated oxidative stress plays a role in cardiomyopathy in Type 2 diabetics.

Increased fatty acid delivery to the heart increases fatty acid uptake by cardiomyocytes, resulting in
increased fatty acid oxidation in these cells. This process increases the reducing equivalents
available to the electron transport chain of the mitochondria, ultimately increasing reactive oxygen
species (ROS) production. ROS increases uncoupling proteins (UCPs) and potentiate proton
leakage through the adenine nucleotide translocator (ANT), the combination of which uncouples the
mitochondria. Uncoupling then increases oxygen consumption by the mitochondria, compounding
the increase in fatty acid oxidation. This creates a vicious cycle of uncoupling; furthermore, even
though oxygen consumption increases, ATP synthesis does not increase proportionally because the
mitochondria is uncoupled. Less ATP availability ultimately results in an energy deficit presenting as
reduced cardiac efficiency and contractile dysfunction. To compound the problem, impaired
sarcoplasmic reticulum calcium release and reduced mitochondrial reuptake limits peak cytosolic
levels of the important signaling ion during muscle contraction. The decreased intra-mitochondrial
calcium concentration increases dehydrogenase activation and ATP synthesis. So in addition to
lower ATP synthesis due to fatty acid oxidation, ATP synthesis is impaired by poor calcium signaling
as well, causing cardiac problems for diabetics. [156]

Possible relationships to aging

Given the role of mitochondria as the cell's powerhouse, there may be some leakage of the high-
energy electrons in the respiratory chain to form reactive oxygen species. This was thought to result
in significant oxidative stress in the mitochondria with high mutation rates of mitochondrial DNA
(mtDNA). Hypothesized links between aging and oxidative stress are not new and were proposed
[157]

in 1956, which was later refined into the mitochondrial free radical theory of aging. A vicious cycle
[158] [159]

was thought to occur, as oxidative stress leads to mitochondrial DNA mutations, which can lead to
enzymatic abnormalities and further oxidative stress.
A number of changes can occur to mitochondria during the aging process. Tissues from elderly[160]

patients show a decrease in enzymatic activity of the proteins of the respiratory chain. However, [161]

mutated mtDNA can only be found in about 0.2% of very old cells. Large deletions in the
[162]
 mitochondrial genome have been hypothesized to lead to high levels of oxidative stress and
neuronal death in Parkinson's disease. [163]

In popular culture
Madeleine L'Engle's 1973 science fantasy novel A Wind in the Door prominently features the
mitochondria of main character Charles Wallace Murry, as being inhabited by creatures known as
the farandolae. The novel also features other characters travelling inside one of Murry's
mitochondria.
The 1995 horror fiction novel Parasite Eve by Hideaki Sena depicts mitochondria as having
some consciousness and mind control abilities, attempting to use these to overtake eukaryotes as
the dominant life form. This text was adapted into an eponymous film, video game, and video game
sequel all involving a similar premise.
In the Star Wars franchise, microorganisms referred to as "midi-chlorians" give some characters the
ability to sense and use the Force. George Lucas, director of the 1999 film Star Wars: Episode I –
The Phantom Menace, in which midi-chlorians were introduced, described them as "a loose
depiction of mitochondria". The non-fictional bacteria genus Midichloria was later named after the
[164]

midi-chlorians of Star Wars.

As a result of the mitochondrion's prominence in modern science education, the phrase
"mitochondria is [sic] the powerhouse of the cell" became a popular Internet meme. The meme is [165]

used to imply that secondary education places an insufficient focus on life skills, compared
to academic knowledge such as the role of the mitochondrion, which has been considered
comparatively impractical. [166]

See also

 Mitochondria portal

 Anti-mitochondrial antibodies
 Chloroplast
 Hydrogenosome
 Inhibitor protein
 Midichloria bacterial genus
 Mitochondrial metabolic rates
 Mitochondrial permeability transition pore
 Nebenkern
 Oncocyte
 Oncocytoma
 Paternal mtDNA transmission
 Plastid
 Submitochondrial particle
 TIM/TOM complex
 TMEM143

References
1. ^ Jump up to:a b Henze K, Martin W; Martin, William (2003). "Evolutionary biology: essence of mitochondria". Nature. 426 (6963): 127–
8. doi:10.1038/426127a. PMID 14614484.
2. ^ Jump up to:a b Karnkowska, Anna; Vacek, Vojtěch; Zubáčová, Zuzana; Treitli, Sebastian; Petrželková, Romana; Eme, Laura; Novák, Lukáš; Žárský,
Vojtěch; Barlow, Lael; Herman, Emily; Soukal, Petr; Hroudová, Miluše; Doležal, Pavel; Stairs, Courtney; Roger, Andrew; Eliáš, Marek; Dacks, Joel;
 Vlček, Čestmír; Hampl, Vladimír (2016). "A Eukaryote without a Mitochondrial Organelle" (PDF). Current Biology. 26: 1–
11. doi:10.1016/j.cub.2016.03.053. Retrieved 16 May 2016.
3. Jump up^ "mitochondria". Online Etymology Dictionary.
4. Jump up^ Campbell, Neil A.; Brad Williamson; Robin J. Heyden (2006). Biology: Exploring Life. Boston, Massachusetts: Pearson Prentice
Hall. ISBN 0-13-250882-6.
5. Jump up^ Wiemerslage L, Lee D (2016). "Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple
parameters". J Neurosci Methods. 262: 56–65. doi:10.1016/j.jneumeth.2016.01.008. PMC 4775301  . PMID 26777473.
6. Jump up^ McBride HM, Neuspiel M, Wasiak S (July 25, 2006). "Mitochondria: more than just a powerhouse". Curr. Biol. 16 (14): R551–
60. doi:10.1016/j.cub.2006.06.054. PMID 16860735.
7. Jump up^ Valero T (2014). "Mitochondrial biogenesis: pharmacological approaches". Curr. Pharm. Des. 20 (35): 5507–
9. doi:10.2174/138161282035140911142118. PMID 24606795. Mitochondrial biogenesis is therefore defined as the process via which cells increase
their individual mitochondrial mass [3]. ... Mitochondrial biogenesis occurs by growth and division of pre-existing organelles and is temporally
coordinated with cell cycle events [1].
8. Jump up^ Sanchis-Gomar F, García-Giménez JL, Gómez-Cabrera MC, Pallardó FV (2014). "Mitochondrial biogenesis in health and disease.
Molecular and therapeutic approaches". Curr. Pharm. Des. 20 (35): 5619–
5633. doi:10.2174/1381612820666140306095106. PMID 24606801. Mitochondrial biogenesis (MB) is the essential mechanism by which cells control
the number of mitochondria.
9. Jump up^ Gardner A, Boles RG (2005). "Is a 'Mitochondrial Psychiatry' in the Future? A Review". Curr. Psychiatry Review. 1 (3): 255–
271. doi:10.2174/157340005774575064.
10. Jump up^ Lesnefsky EJ, Moghaddas S, Tandler B, Kerner B, Hoppel CL (June 2001). "Mitochondrial dysfunction in cardiac disease: ischemia—
reperfusion". Journal of Molecular and Cellular Cardiology. 33 (6): 1065–1089. doi:10.1006/jmcc.2001.1378. PMID 11444914.
11. Jump up^ Dorn GW, Vega RB, Kelly DP (2015). "Mitochondrial biogenesis and dynamics in the developing and diseased heart". Genes
Dev. 29 (19): 1981–91. doi:10.1101/gad.269894.115. PMC 4604339  . PMID 26443844.
12. ^ Jump up to:a b Study Confirms Mitochondrial Deficits in Children with Autism. biosciencetechnology.com. May 2014
13. ^ Jump up to:a b c d e f g h i j k l Alberts, Bruce; Alexander Johnson; Julian Lewis; Martin Raff; Keith Roberts; Peter Walter (1994). Molecular Biology of the
Cell. New York: Garland Publishing Inc. ISBN 0-8153-3218-1.
14. ^ Jump up to:a b c d e f g h i j k l Voet, Donald; Judith G. Voet; Charlotte W. Pratt (2006). Fundamentals of Biochemistry, 2nd Edition. John Wiley and
Sons, Inc. pp. 547, 556. ISBN 0-471-21495-7.
15. Jump up^ Andersson SG, Karlberg O, Canbäck B, Kurland CG (January 2003). "On the origin of mitochondria: a genomics
perspective". Philosophical Transactions of the Royal Society of London B. 358 (1429): 165–77; discussion 177–
9. doi:10.1098/rstb.2002.1193. PMC 1693097  . PMID 12594925.
16. Jump up^ Taylor SW, Fahy E, Zhang B, Glenn GM, Warnock DE, Wiley S, Murphy AN, Gaucher SP, Capaldi RA, Gibson BW, Ghosh SS (March
2003). "Characterization of the human heart mitochondrial proteome". Nat. Biotechnol. 21 (3): 281–6. doi:10.1038/nbt793. PMID 12592411.
17. Jump up^ Zhang J, Li X, Mueller M, Wang Y, Zong C, Deng N, Vondriska TM, Liem DA, Yang J, Korge P, Honda H, Weiss JN, Apweiler R, Ping P
(2008). "Systematic Characterization of the Murine Mitochondrial Proteome Using Functionally Validated Cardiac Mitochondria". Proteomics. 8 (8):
1564–1575. doi:10.1002/pmic.200700851. PMC 2799225  . PMID 18348319.
18. Jump up^ Zhang J, Liem DA, Mueller M, Wang Y, Zong C, Deng N, Vondriska TM, Yang J, Korge P, Drews O, Maclellan WR, Honda H, Weiss JN,
Apweiler R, Ping P (2008). "Altered Proteome Biology of Cardiac Mitochondria Under Stress Conditions". J. Proteome Res. 7 (6): 2204–
14. doi:10.1021/pr070371f. PMC 3805274  . PMID 18484766.
19. ^ Jump up to:a b c d e f g h i j Ernster, Lars; Schatz, Gottfried (December 1981). "Mitochondria: a historical review" (PDF). The Journal of Cell
Biology. 91 (3 Pt 2): 227s–255s. doi:10.1083/jcb.91.3.227s. PMC 2112799  . PMID 7033239.
20. Jump up^ Altmann, R. 1890 . Die Elementarorganismen und ihre Beziehungen zu den Zellen. Veit, Leipzig, [1].
21. Jump up^ Benda, C. 1898. Ueber die Spermatogenese der Vertebraten und höherer Evertebraten. II. Theil: Die Histiogenese der Spermien. Arch.
Anal. Physiol. 393-398, [2].
22. Jump up^ Ernster's citation Meves, Friedrich (May 1908). "Die Chondriosomen als Träger erblicher Anlagen. Cytologische Studien am
Hühnerembryo". Archiv für mikroskopische Anatomie. 72(1): 816–867. doi:10.1007/BF02982402. is wrong, correct citation is Meves, Friedrich (1904).
"Über das Vorkommen von Mitochondrien bezw. Chondromiten in Pflanzenzellen". Ber. D. Deutsch. Bot. Ges. 22: 284–286., cited in Meves' 1908
paper and in Schmidt, Ernst Willy (1913). "Pflanzliche Mitochondrien". Progressus rei botanicae. 4: 164–183. Retrieved 21 September 2012., with
confirmation of Nymphaea alba
23. Jump up^ Siekevitz P (1957). "Powerhouse of the cell". Scientific American. 197 (1): 131–140. doi:10.1038/scientificamerican0757-131.
24. Jump up^ Martin, William F.; Garg, Sriram; Zimorski, Verena (2015). "Endosymbiotic theories for eukaryote origin". Philosophical Transactions of the
Royal Society B. 370: 20140330. doi:10.1098/rstb.2014.0330. PMC 4571569  . PMID 26323761.
25. ^ Jump up to:a b Margulis, Lynn; Sagan, Dorion (1986). Origins of Sex. Three Billion Years of Genetic Recombination. New Haven: Yale University
Press. pp. 69–71, 87. ISBN 0 300 03340 0.
26. Jump up^ William F. Martin and Miklós Müller "Origin of mitochondria and hydrogenosomes", Springer Verlag, Heidelberg 2007.
27. Jump up^ Emelyanov VV (2003). "Mitochondrial connection to the origin of the eukaryotic cell". Eur. J. Biochem. 270 (8): 1599–
1618. doi:10.1046/j.1432-1033.2003.03499.x. PMID 12694174.
28. Jump up^ Muller, Miklos; Martin, William (1999). "The genome of Rickettsia prowazekii and some thoughts on the origin of mitochondria and
hydrogenosomes" (PDF). BioEssays. 21 (5): 377–381. doi:10.1002/(sici)1521-1878(199905)21:5<377::aid-bies4>3.0.co;2-w.
29. Jump up^ Gray MW, Burger G, Lang BF (March 1999). "Mitochondrial evolution". Science. 283(5407): 1476–
81. doi:10.1126/science.283.5407.1476. PMID 10066161.
30. Jump up^ Thrash, J. Cameron; et al. (2011). "Phylogenomic evidence for a common ancestor of mitochondria and the SAR11 clade". Scientific
Reports. 1: 13. doi:10.1038/srep00013. PMC 3216501  . PMID 22355532.
31. Jump up^ Ferla, M. P.; Thrash, J. C.; Giovannoni, S. J.; Patrick, W. M. (2013). "New rRNA gene-based phylogenies of the Alphaproteobacteria
provide perspective on major groups, mitochondrial ancestry and phylogenetic instability". PLoS ONE. 8 (12):
e83383. doi:10.1371/journal.pone.0083383. PMC 3859672  . PMID 24349502.
32. Jump up^ O'Brien TW (2003). "Properties of human mitochondrial ribosomes". IUBMB Life. 55 (9): 505–
13. doi:10.1080/15216540310001626610. PMID 14658756.
33. Jump up^ Lynn Sagan (1967). "On the origin of mitosing cells". J Theor Biol. 14 (3): 255–274. doi:10.1016/0022-5193(67)90079-3. PMID 11541392.
34. Jump up^ Emelyanov VV (2001). "Rickettsiaceae, rickettsia-like endosymbionts, and the origin of mitochondria". Biosci. Rep. 21 (1): 1–
17. doi:10.1023/A:1010409415723. PMID 11508688.
35. Jump up^ Feng DF, Cho G, Doolittle RF (1997). "Determining divergence times with a protein clock: Update and reevaluation". Proc. Natl. Acad.
Sci. 94 (24): 13028–13033. doi:10.1073/pnas.94.24.13028. PMC 24257  . PMID 9371794.
36. Jump up^ Cavalier-Smith T (1991). "Archamoebae: the ancestral eukaryotes?". Biosystems. 25 (1–2): 25–38. doi:10.1016/0303-2647(91)90010-
I. PMID 1854912.
w large the sample must be to find an effect of a given size with a given design at the desired
probability of making a Type I or Type II error.
Non-experimental research designs[edit]
Non-experimental research designs do not involve a manipulation of the situation,
circumstances or experience of the participants. Non-experimental research designs can be
broadly classified into three categories. First, in relational designs, a range of variables are
measured. These designs are also called correlation studies, because correlation data are
most often used in analysis. Since correlation does not imply causation, such studies simply
identify co-movements of variables. Correlational designs are helpful in identifying the
relation of one variable to another, and seeing the frequency of co-occurrence in two natural
groups (See
correlation and dependence). The second type is comparative research. These designs compare
two or more groups on one or more variable, such as the effect of gender on grades. The third type
of non-experimental research is a longitudinal design. A longitudinal design examines variables such
as performance exhibited by a group or groups over time. See Longitudinal study.

Examples of flexible research Research design

From Wikipedia, the free encyclopedia

A research design is the set of methods and procedures used in collecting and analysing measures
of the variables specified in the research problem research. The design of a study defines the study
type (descriptive, correlation, semi-experimental, experimental, review, meta-analytic) and sub-type
(e.g., descriptive-longitudinal case study), research problem, hypotheses, independent and
dependent variables, experimental design, and, if applicable, data collection methods and a
statistical analysis plan. Research design is the framework that has been created to find answers to
research questions.

Design types and sub-types[edit]

There are many ways to classify research designs, but sometimes the distinction is artificial and
other times different designs are combined. Nonetheless, the list below offers a number of useful
distinctions between possible research designs. A research design is an arrangement of conditions
or collections. [1]

 Descriptive (e.g., case-study, naturalistic observation, survey)

 Correlational (e.g., case-control study, observational study)
 Semi-experimental (e.g., field experiment, quasi-experiment)
 Experimental (experiment with random assignment)
 Review (literature review, systematic review)
 Meta-analytic (meta-analysis)
Sometimes a distinction is made between "fixed" and "flexible" designs. In some cases, these types
coincide with quantitative and qualitative research designs respectively, though this need not be the
[2]

case. In fixed designs, the design of the study is fixed before the main stage of data collection takes
place. Fixed designs are normally theory-driven; otherwise, it is impossible to know in advance
which variables need to be controlled and measured. Often, these variables are measured
quantitatively. Flexible designs allow for more freedom during the data collection process. One
reason for using a flexible research design can be that the variable of interest is not quantitatively
measurable, such as culture. In other cases, theory might not be available before one starts the
research.
Grouping[edit]
The choice of how to group participants depends on the research hypothesis and on how the
participants are sampled. In a typical experimental study, there will be at least one "experimental"
condition (e.g., "treatment") and one "control" condition ("no treatment"), but the appropriate method
of grouping may depend on factors such as the duration of measurement phase and participant
characteristics:

 Cohort study
 Cross-sectional study
 Cross-sequential study
 Longitudinal study

Confirmatory versus exploratory research[edit]

Confirmatory research tests a priori hypotheses — outcome predictions that are made before the
measurement phase begins. Such a priori hypotheses are usually derived from a theory or the
results of previous studies. The advantage of confirmatory research is that the result is more
meaningful, in the sense that it is much harder to claim that a certain result is generalizable beyond
the data set. The reason for this is that in confirmatory research, one ideally strives to reduce the
probability of falsely reporting a coincidental result as meaningful. This probability is known as α-
level or the probability of a type I error.
Exploratory research on the other hand seeks to generate a posteriori hypotheses by examining a
data-set and looking for potential relations between variables. It is also possible to have an idea
about a relation between variables but to lack knowledge of the direction and strength of the relation.
If the researcher does not have any specific hypotheses beforehand, the study is exploratory with
respect to the variables in question (although it might be confirmatory for others). The advantage of
exploratory research is that it is easier to make new discoveries due to the less stringent
methodological restrictions. Here, the researcher does not want to miss a potentially interesting
relation and therefore aims to minimize the probability of rejecting a real effect or relation; this
probability is sometimes referred to as β and the associated error is of type II. In other words, if the
researcher simply wants to see whether some measured variables could be related, he would want
to increase the chances of finding a significant result by lowering the threshold of what is deemed to
be significant.
Sometimes, a researcher may conduct exploratory research but report it as if it had been
confirmatory ('Hypothesizing After the Results are Known', HARKing—see Hypotheses suggested by
the data); this is a questionable research practice bordering on fraud.

State problems versus process problems[edit]

A distinction can be made between state problems and process problems. State problems aim to
answer what the state of a phenomenon is at a given time, while process problems deal with the
change of phenomena over time. Examples of state problems are the level of mathematical skills of
sixteen-year-old children or the level, computer skills of the elderly, the depression level of a person,
etc. Examples of process problems are the development of mathematical skills from puberty to
adulthood, the change in computer skills when people get older and how depression symptoms
change during therapy.
State problems are easier to measure than process problems. State problems just require one
measurement of the phenomena of interest, while process problems always require multiple
measurements. Research designs such as repeated measurements and longitudinal study are
needed to address process problems.
Examples of fixed designs[edit]
Experimental research designs[edit]
See also: Experiment

In an experimental design, the researcher actively tries to change the situation, circumstances, or
experience of participants (manipulation), which may lead to a change in behavior or outcomes for
the participants of the study. The researcher randomly assigns participants to different conditions,
measures the variables of interest and tries to control for confounding variables. Therefore,
experiments are often highly fixed even before the data collection starts.

In a good experimental design, a few things are of great importance. First of all, it is
necessary to think of the best way to operationalize the variables that will be measured, as
well as which statistical methods would be most appropriate to answer the research
question. Thus, the researcher should consider what the expectations of the study are as well
as how to analyse any potential results. Finally, in an experimental design the researcher
must think of the practical limitations including the availability of participants as well as how
representative the participants are to the target population. It is important to consider each of
these factors before beginning the experiment. Additionally, many researchers employ
[3]

power analysis before they conduct anexperiment, in order to determine hodesigns[edit]

Case study[edit]
See also: Case study

Famous case studies are for example the descriptions about the patients of Freud, who were
thoroughly analysed and described.
Bell (1999) states “a case study approach is particularly appropriate for individual researchers
because it gives an opportunity for one aspect of a problem to be studied in some depth within a
limited time scale”.
[4]

Ethnographic study[edit]
See also: Ethnography

This type of research is involved with a group, organization, culture, or community. Normally the
researcher shares a lot of time with the group.
Grounded theory study[edit]
Grounded theory research is a systematic research process that works to develop "a process, and
action or an interaction about a substantive topic".
[5]