KatzClass

Exploring the Validity of Using the Free Radical Theory of Aging as

a Model for Senescence
Nikolay Avramov

Introduction
Why aging?

Aging is a cruel process. Slowly and surely, it causes the human body to degrade and

wither away, leaving the body open for a myriad of pathogenic attacks and systemic failures.

Ultimately, the result of this slow and painful process is death. For as long as civilization has

existed, aging has not been a matter of choice it has been a fact of life.

Fortunately, winds of change are sweeping over our society. With todays technology, we

can create and utilize groundbreaking new tools and knowledge in an effort to understand and

reverse engineer our bodies. The intimate knowledge of what makes us 'tick' metabolically and

what makes us age might soon be in our grasp. We are poised for a revolution, the likes of which

have never been seen before. If we gained a coherent understanding of how we age, we could

then begin to develop ways that can help slow and reverse the pathology caused by aging. One's

chronological age would no longer be tied to how likely they were to die. It is my fascination

with the idea of extending the human lifespan that has led me to pursue this course of research.

Imagine a world where people remain young and healthy indefinitely; a society where

death and disease are truly optional. Such prospects lead us to ask, Will we be the last

generation to die from aging and disease, or will we be the first generation to achieve everlasting

youth?
Asking the big question

There are many theories of aging and much debate about which one correctly describes

the aging process. The problem with finding a theory of aging is that it must account for a wide

variety of pathologies and describe them accurately on a molecular level; more broad and vague

explanations of aging do little to advance our knowledge of biology. Some researchers have

opted for an approach that holds the DNA responsible for causing aging. According to this

theory of programmed aging, senescence is caused by the execution of a genetic program inside

each cell, which subsequently causes the cell to decay. This is unlikely, though, because

evolution does not select explicitly for immortality or death - it simply advances the passing on

of genes from one generation to the next. Hence, evolution and by extension our biology should

not have selected for pre-programmed death.1 Others argue that random errors in the cell build

up to the point where the cell can no longer survive, eventually resulting in the death of the entire

organisms when vital organ cease to function. Yet this theory fails to explain why the cellular

repair mechanisms inherent within every organism do not manage to adequately repair cellular

and tissue damage. Even if one considers that the repair mechanisms might themselves fail, the

theory does not offer insight into how degradation of repair mechanisms comes about.

Another theory, elegant in the mechanism it uses to account for cellular senescence, deals

with small, damaging intracellular particles called free radicals. The free radical theory of aging

is significant because there has been an enormous amount of research done that has shown that

free radicals are important players in agings pathophysiology.2 Thus far, it has been shown

that free radicals cause damage closely associated with the aging process. The free radical theory

of aging is highly attractive because it points to a single root cause of aging; the intracellular free
radical interactions proposed by the free radical theory of aging (FTA) can be extrapolated to

many types of damage, which could also play key roles in many types of disease.

This paper will attempt to highlight the intricate workings of free radical theory, and its

strengths and shortcomings. Ultimately, the goal of this analysis is to determine the viability of

using free radical theory as a model for aging.

What is the free radical theory of aging?

The free radical theory of aging states that free radicals, which interact with the cell, will cause

cellular damage; it is the accumulation of this damage that is responsible for the aging process.3

Free radicals are molecules or atoms that have an unpaired electron in their valence shell. The

reactivity of an atom is determined by the number of electrons in its valence. Usually, electrons

that are paired together in an orbital contain much less energy than an electron that sits in an

orbital alone. For an atom, having less energy means being more stable, and since atoms are

constantly trying to achieve greater stability, the unpaired electrons will try to pair themselves up

with other free electrons. Hence, atoms or molecules which have unpaired electrons will react

readily with other atoms and molecules.4 For the cell, a free radicals high degree of reactivity is

problematic. Free radicals will react with lipids, nucleic acids, and proteins in the cell, causing

mutations in the vital cellular organelles that are made of these components. Alternatively, free

radical reactions with substrates within the cell could generate toxic compounds.5

Free Radicals: Their Chemistry and Creation

What forms do free radicals take?

Free radicals can come in various forms, but the three main ones are O2-, NO, and OH.

Oxygen contains unpaired electrons in its outermost shells, but because of the way that these
unpaired electrons spin, oxygens separate electrons behave as if they were paired hence, O2

molecules (atmospheric oxygen) do not have same reactivity that free radicals do.6 However,

because of its electron configuration, O2 is especially prone to becoming a free radical (Whereby

the double bond shared between the two oxygen atoms becomes a single bond, causing a valence

electron to be exposed. Or, the double bond between the oxygen atoms remains and an electron

simply tacks itself onto O2 and becomes the unpaired electron itself). When oxygen is reduced by

1 electron, it becomes a reactive oxygen species otherwise known as a free radical (FR), and it

can take one of the three forms shown below:

7
Fig 1: Several types of reactive oxygen species

Superoxide anions can form through catalysis of the enzymes NAD(P)H oxidase and xanthine

oxidase, although the reaction can occur without enzymes, as well. Alternatively, oxygen can

also become hydrogen peroxide or a hydroxyl radical. Trace amounts of transition metals in cells

can convert H2O2 into the hydroxyl radical, OH.8

NO can form several types of reactive nitrogen species, but the significant type is NO

formed by the oxidation (Removal of an electron) of the nitrogen atom at the terminal end of the

amino acid argenine.9

 Fig 2: Scheme of free radical interactions within the cell, including signaling, types of damage, and defenses. ROS
10
in the diagram refers to Reactive Oxygen Species.

Free radical genesis from the environment

Free radicals can be generated in one of two ways: either by the environment or by the

bodys own cells.11 This section will discuss the former. The former can be the result of exposure

to ionizing radiation, a type of high energy radiation which has the capability to knock electrons

off atoms, thereby creating an ion. Ionizing radiation can come in the form of gamma rays, x-

rays, and ultraviolet rays. Waves that that have longer wavelengths (radio waves and light waves,

for example) than these do not contain sufficient energy to ionize an atom but can instead excite

electrons to a higher state of energy, inducing small amounts of harmless heat when the electrons

return to their former, stable shell. When an electron is kicked out from an atom, the atom that

lost the electron becomes highly reactive because it will have one unpaired electron left in its

valence shell (Given that it was stable in the first place). Hence, ionizing radiation can be
harmful or even lethal because of its potential to excite the electrons in many atoms, thereby

generating large amounts of free radicals. To put this in perspective, 450 rads (radiation

absorption dose) are lethal to humans, but only 100 rads are necessary to kill a cell. This means

that free radical damage can progressively accumulate, below a fatal threshold, in cells in the

body. These stealthy and subtle changes to cells and tissues can lead to cancer which can indeed

be life threatening much later in life.12 For instance, causing mutations in the part of the genome

responsible for control of mitosis, and particularly S phase, can lead to such uncontrolled

replication.

The primary source of free radicals is metabolism, which will be discussed in the next

section. The four main regions where free radicals are generated are the electron transport chain,

the paroxysmal fatty acid metabolism, cytochrome P-450 reactions, and phagocyte cells.13

Free radicals genesis in the electron transport chain (ETC)

The electron transport chain is part of a larger mechanism used for ATP synthesis. ATP is

the battery of the cell, where energy that can later be used to power cellular activity is stored.

ATP synthesis involves breaking down molecules of glucose (or other molecules that similarly

have high energy bonds). The first two steps in generating ATP are glycolysis and the citric acid

cycle. Glycolysis breaks down sugars by using an enzyme called dehydrogenase, which steals

two electrons from the sugar molecules and uses them to create energy bearing molecules called

NADH. Glycolysis does not utilize oxygen and thus cannot create reactive oxygen species. The

citric acid cycle, which accepts the pyruvate molecules from the glycolysis step, also does not

utilize oxygen. It is however considered aerobic because it creates reduced electron carriers that

are later completely oxidized in oxidative phosphorylation within the mitochondria. The focus

will be on the third step, oxidative phosphorylation (OP). OP consists of a journey through the
electron transport chain located in the mitochondria (the powerhouse of the cell). Proteins in the

mitochondria (Complexes I-IV) facilitate the oxidation of NADH and FADH in order to power

ATP synthesis. First, dehydrogenase enzymes catalyze a reaction that removes two hydrogen

atoms (and thereby two electrons) and then binds them to NAD+, a molecule that accepts two of

the electrons and one hydrogen atom. NAD+ loses its positive charge and then becomes NADH,

and carries the hydrogen and electrons to the first in a series of proteins that oxidizes the NADH

molecule, which is where the electron transport chain begins.14 The electrons pass from the

NADH molecule to the five protein complexes within the mitochondria. One of molecules in the

ETC that is not a protein, Coenzyme Q10, otherwise known as ubiquinone, accepts two electrons

individually and then passes them down to the rest of the protein components in the electron

transport chain. Each subsequent complex reduces the next, until the energy originally stored in

the 2 electrons at the beginning of the cycle is captured, and the electrons rejoin with two protons

(H+) and one oxygen atom, forming water.

 Electron Transport Chain

Fig 3: A diagram of the electron transport chain, from complex I-IV. A free energy
15
scale is included on the right.

Part of the reason that oxidative phosphorylation generates free radicals is because there are

many steps where individual electrons are transferred, as demonstrated by the individual transfer

of electrons to and from ubiquinone. If an electron goes astray at any one point in the ETC, the

final oxygen may as a result be reduced by one electron, forming the free radical O2-.16 As with

all biological systems, there is no such thing as perfection. This means that electrons can escape
the ETC, form superoxide or other types of free radicals, and finally react with other compounds

within the cell.

It should also be noted that free radical reactions with substrates can result in chain

reactions - these reactions are able to oxidize multiple substrates. When a free radical approaches

an atom, it will have a tendency to pull electrons off in order to pair. This means that the stable

atom will subsequently lack paired electrons and also become highly reactive, thus spawning

more free radical reactions. It is this property that makes free radicals so inherently deleterious to

cellular mechanisms.

Fig 4: A schematic of a chain oxidation reaction occurring between multiple atoms. 17

Free Radical Formation in Phagocytes

The mammalian immune system utilizes phagocytes (white blood cells) in order to

combat pathogens. Phagocytes typically engulf the target pathogen, such as a bacterium or virus,

and then release a barrage of highly reactive free radicals in an oxidative burst. However, the

free radicals that are released in the attempt to kill the pathogen also come in contact with the

organelles in the surroundings, causing both tissue damage and inflammation. Mydel et al

showed that bacteria equipped with rubrerythrin, a protein shield against oxidative damage,

could withstand the oxidative burst generated by phagocytes in mice. It was found that not only

was this immune response ineffective in destroying the bacteria, but it also caused tissue damage

and further weakened the mouse host's cells, which in turn increased the bacterias chances for

survival.18 This suggests that pathogens that are protected against oxidative bursts can provoke
especially damaging oxidative immune responses that hurt rather than help the host organism.

One might speculate that this phenomenon might be prevalent even in successful elimination of a

pathogen, whereby the pathogen is destroyed but there is collateral damage in the process.

Damage caused by reactive oxygen species and oxidation

There are many types of damage caused by free radicals within the cell. Nucleic acids,

lipids, and proteins are all subject to oxidation. There is growing evidence which suggests that

oxidative damage to each of these cellular components is linked to corresponding diseases in

both mammals and bacteria. Equally important is the recently established link between oxidative

stress and cancer, linked by free radicals potential to act mutagenically on DNA.19

Carmella S. Moody et al demonstrated the mutagenicity of oxygen free radicals to E. Coli.

They showed the effect of paraquat (PQ2+), a common herbicide that creates large quantities of

reactive oxygen species (ROS) in biological systems, on the DNA of E. coli. Their results

demonstrated that exposure to PQ2+ can cause base pair substitutions and frameshift mutations in

DNA. Furthermore, they demonstrated that of the two media that were used for E. Coli cultures,

the TSY media that contained high concentrations of SOD, the enzyme that scavenges for free

radicals, greatly reduced the mutagenicity of PQ2+.20

There is evidence that one of the radicals generated by PQ2+, H2O2 (hydrogen peroxide is

technically a precursor to OH, the actual free radical), causes both breakages in DNA strands

and detachments of nucleobases, and that another free radical, O2-, can cause single stand breaks

in DNA, as well.21

DNA damage in mitochondria is particularly problematic because mitochondria lack

histones (protective substrates around which DNA wraps itself), and other DNA repair

mechanisms. Also, because mitochondria produce a large percentage of intracellular free radicals
via oxidative phosphorylation, mtDNA is especially vulnerable due to its close proximity to free

radicals. Recent evidence points toward an increase in mtDNA damage as age increases. 22

Cell membranes can also suffer greatly from oxidative damage. The proteins that are

imbedded in the phospholipid bilayer can be denatured by redox reactions from ROS, and the

fluidity of the membrane can also be compromised. This can in turn make the cell less permeable

to life-sustaining components such as a proteins, water, and sugars via the denaturation of

receptor and transport proteins on the cell surface.23

24
Fig 5: Membrane erosion by free radicals and anti-oxidant FR scavenging.

Proteins are one of the primary targets for oxidative damage. Michael Davies et al

estimate that 50-70% of free radicals are scavenged via reacting with protein substrates.25

Oxidation can cause structural changes that can impair protein function (proteins are reliant on

shape for their function). Protein reactions with toxic compounds produced by free radicals or
even ROS or RNS themselves (Reactive Nitrogen Species) can lead to peptide backbone

cleavage, cross-linking, and/or modification of the side chain of virtually every amino acid.26

Protein damage can indirectly affect other molecules in the cell, including DNA. For instance,

the repair mechanisms used by DNA can themselves be oxidized. This makes free radicals

especially dangerous on two fronts: 1) ROS/RNS can directly do damage to DNA molecules

(Which are in limited supply), and 2) DNA repair mechanism which maintain the integrity of the

DNA blueprints are comprised. Because DNA maps out the structure for all other proteins within

the cell, even a small but permanent change in DNA structure could have a significant impact.

Once a protein has been damaged, a proteosome will clear it from the cell by breaking its peptide

bonds. However, proteins that are not full degraded can occasionally build up in and around the

cell, going untouched by proteosomes. Nicole Sitte et al exposed cells to lipofuscin, the

undegradeable proteins within the cell, and discovered that proteosome activity is inhibited by

25% in the presence of lipofuscin.27 This is important because, as Isabella Dalle-donne states,

lipofuscin accumulation accelerates, and a cycle of attrition is formed, ultimately leading to cell

death through apoptosis.

Oxidative Regulatory Mechanisms and Importance of Free Radicals to Cells

Free radicals help to regulate many functions in the cell. Some of these include regulation

of vascular tone (Extent of constriction of blood vessels28), sensing levels of oxygen tension,

increased sensitivity to signal transduction of receptor proteins, and oxidative stress responses

that ensure maintenance of redox homeostasis.29 The concentration of free radicals within

biological tissues is relatively low in most instances. This concentration, or point of balance, is

dictated by the balance between the generation and the destruction of free radicals.30 Redox (The
short name for Reduction and Oxidation reactions) signaling is a mechanism that enables the cell

to ellicit cellular responses to oxidative damage, and to rebalance the creation and the destruction

of free radicals. Anti-oxidants play an important role in minimizing the amounts of free radicals

that are present in cells by acting as oxidizable substrates (whereby electrons will be ripped off

those substrates in place of more important proteins or molecules). Vitamin C, Glutathione

peroxidase, and superoxide dismutase (SOD) can act as anti-oxidants by either catalyzing ROS

into more harmless substances or by reacting with free radicals and nullifying their chemical

reactivity through oxidation.

One example of intracellular ROS regulation is the oxidation of proteins. Proteins that are

structurally intact are more resistant to oxidation than ones that are already denatured. Thus, the

more oxidized proteins are produced via oxidative stress within the cell, the more likely it is that

those same proteins will increasingly attain anti-oxidant properties that would inhibit further

oxidation of cellular substrates.31 Another example of oxidative regulation is elevated gene

expression of anti-oxidants in response to high levels of oxidative stress.

Fig 6: This is a diagram that illustrates ROS homeostasis in terms of the

 32
production and elimination of ROS.

Macrophages (white blood cells) use free radicals as weapons in order to fight pathogens. Since

the oxidative burst from lymphocytes poses a threat to other benign cellular components, one

can see that an increase in SOD and anti-oxidants via oxidative regulation is crucial to cell

function. Furthermore, redox regulation acts as a cellular precaution, helping to illicit a large

immune response from lymphocytes, such as T-cells, even when very modest amounts of

pathogen are present in an organism.33

Evidence for and against the free radical theory of aging

Connecting the macro and micro: A link between free radicals and age related pathology

It is important to consider the ways in which free radical damage could potentially

manifest itself as age related pathology. Establishing a link between the affects of free radicals at

the small scale and the diseases which occur at a large scale as a result of FR damage elucidates

the potency of FTA as a model for aging. Of course, analyzing all types of pathologies that free

radicals can be applied to would be out of the scope of this paper, so instead a more narrow look

must suffice.

Free radicals can easily be applied to one of the most common phenomena that is

associated with the aging process: Wrinkles. On the surface, healthy skin appears elastic and

resilient. The molecule that is responsible for these properties is collagen. Collagen has 3 side

chains that compose a triple helix. This rope-like structure is woven into sheets, helping to

support membranes such as the skin.34 Peter schoeder et al showed in vivo that

mellanoproteinases were upregulated in 80% of individuals who were exposed to infrared

radiation. Anti-oxidant content within the skin was also reduced in the presence of infrared
radiation.35 This is significant because mellanoproteinases are enzymes which break down

collagen. Schroeder et al report that mellanoproteinases are activated in the presence of

oxidative stress. These key facts link free radicals to the pathology seen with wrinkled skin.

Mellanoproteinases effectively break down collagen in the skin, reducing its elastic and supple

nature, and causing wrinkles. Thus, indirectly, it is postulated that free radicals will lead to a

deterioration of the collagen and an increase in wrinkles.36

Parkinson's disease is also closely associated with the aging process. Parkinson's occurs

when a crucial neurotransmitter, dopamine, is not generated by the substantia nigra par compacta

and is not able to exert its function. Parkinson's disease primarily affect one's motor skills, which

progressively deteriorate over time. Paula Keeney et al show that complex one, the largest of the

protein complexes within the mitochondria (Part of the ETC), is oxidatively damaged in

Parkinson's patients. Complex I is especially vulnerable to the affects of mutagenesis because it

has subunits which are derived from both nDNA and mtDNA. Thus, if either set of DNA is

mutated and codes for the incorrect proteins/mishapen proteins, the entire Complex I

morphology is degraded. Although they state that they do not know the reason why PD brain

tissues are oxidatively damaged, their experiment demonstrates that oxidative stress is a

prevailing phenomenon within one of the most commonly encountered age-related diseases. 37

Furthermore, dopamine in the brain is highly sensitive to oxidation. Iron, an agent capable of

causing oxidative damage to dopaminergic neurons, has been found in higher concentrations in

PD patients. Feritin, a protein which encapsulates iron in order to protect the host from iron

overload, is found in decreased concentrations in PD patients as well.38

It should be noted that many other disorders, such as Alzheimer's and cancer, have been

linked to free radicals. As previously mentioned, part of what makes the free radical theory of

aging so appealing is that it can be linked to such a large variety of disorders and provide a
singular and common biochemical reason for why our bodies 'mess up', seemingly producing

dramatic pathologies spontaneously. While it may seem like scientists could simply point fingers

at free radicals when addressing every disease, it is important to remember that there is evidence

which contradicts FTA as well. In other words, it is highly likely there are external disease

causing mechanisms which fall out of the 'jurisdiction' of the free radical theory of aging.

Mitochondrial free radical theory of aging

There is substantial criticism of FTA, and some of it begins with more specific criticism

toward the role of mitchondrial degradation (A subset of FTA called the mitochondrial free

radical theory of aging, MTA) in cells and its role in the aging process.

It was previously mentioned that histones allow nuclear DNA (nDNA) protection against

free radical damage; however, this has been called into question by the discovery that after a

histone has been oxidized (thus playing its protective role toward the nuclear DNA), subsequent

oxidative reactions between nDNA and the histone subtrate can occur. This ability to transfer

oxidative reactions means that oxidation could potentially inflict damage to nDNA, thus casting

doubt on a histones protection from oxidative stress.39

Mitochodria are almost universally present in the tissues of respirating organisms, and

there are many copies of mitochondria, and by extension mtDNA, per cell. Mitochondria

replicate independantly from the entire cell, so mtDNA damage might be considered neligible in

light of the fact that mitochondrial genesis can help replace damaged or defective mitochondria

with new copies.40 Aubrey de Grey, PhD, counters with the fact that, A small proportion of

spontaneous mutants will inevitably achieve homozygosity in one mitochondrion.41 Thus,

mutations via replicating mitochondria might spread until a substantial population of

mitochondria within the cell is affected by mtDNA mutations.

 As previously mentioned, mitochondria contain DNA (mtDNA) that is independent of

that which is found in the nucleus. H2O2, a precursor of OH, can freely diffuse through the

membrane of mitochondria, allowing it to come in contact with the mtDNA. Transition metals

within mitochondria, such as Fe2+, can incur the conversion of H2O2 into hydroxyl free

radicals.42 Within mitochondria, the enzymes glutathione peroxidase (GPx) and catalase,

catalyze hydrogen peroxide into water. It is evident that these mechanisms help to reduce the

burden of oxidative stress. This raises the question, How prevalent must free radicals be in

order to pose a significant threat so as to overwhelm the scavenging functions of GPx?

Moreoever, "What is the metabolic pathway that triggers this change in redox homeostasis,

especially in aged individuals?" These answers to these questions remain unclear.

It has also been speculated that mtDNA mutation leads to production of mishapen or

defective proteins which then become part of one of the complexes in the ETC within

mitochondria. These defective proteins would in turn give rise to more free radicals which would

lead to more uncontrolled oxidation and damage. However, Trifunovic et al demonstrated, using

a modified mtDNA polymerase to cause mtDNA mutations in mice, that accumulation of

mtDNA mutations does not lead to an increase in free radicals generated by the ETC.43 44 This

could signify that the vicious cycle theory of progressively increasing ROS generation via

mutated mitochondrial complexes may be wrong. Albeit the hurdle this finding poses in positing

that MTA is valid, it is of some consolation that the mice did exhibit premature signs of aging,

such as osteoporosis and weight loss, at only 25 weeks of age.

Furthermore, FTA predicts that an increase in free radicals, and thus an increase in

cellular damage, should facilitate the aging process. SOD scavenging through catalysis of ROS,

as previously mentioned, plays a vital role in redox regulation. However, Jeremy Raamsdonk et

al experimented with SOD inhibition in C. Elegans and found that this notion may not be
correct.45 In their experiment, they inhibited genes which coded for five different types of SOD.

Their results showed that the lack of SOD increased sensitivity to oxidative stress, but it did not

decrease lifespan. Additionally, they found that deletion of one of the five types of SOD, Sod-2

(also known as mitochondrial MnSOD, which relies on magnesium for its function), actually

increases the lifespan of C. Elegans, quite the opposite of what FTA would conclude. The impact

of this finding is bolstered by the fact that, SOD-2 is the primary SOD present in the

mitochondrial matrix and the mitochondria is a major site of superoxide production in the cell.46

It would seem reasonable to assume that a lack of such a crucial oxidative defense would cause

significant cellular attrition, and perhaps aging, but it is evident that there is a more complex set

of processes that governs senescence within cells.

A possible way to explain this may lie with the fact that the cellular defense system is

highly dependent on oxidative regulation. Dr. Zhongtao Zhang of New York Medical College

proposes that experiments which tamper with the amounts of ROS scavengers within cells

disrupt the delicate balance maintained by oxidative homeostatic regulation. Thus, even if one

were to increase the amounts of free radical scavengers within cells, one might not see an

increase in life span. Likewise, in the Raamsdonk experiment, a decrease in SOD might cause a

shift in the amount of ROS produced by the cell, and not affect it at all (Or, in this case, even

shift the balance in a way that extends life). This experiment is especially interesting in light of

the fact that media everywhere advertises anti-oxidant supplements as a means to achieve better

health. In fact, taking large quantities of anti-oxidants could disrupt oxidative homeostasis of

one's cells, as was possible with this experiment, and actually make one's health worse.47

ROS Presence and Defense in Varying Species

Experiments which utilize a range of different species as investigative models into the

relationship between lifespan and free radical production (and damage) have also given
considerable weight to the arguments against FTA. It would stand to reason that species which

produce more free radicals would live shorter lives than species which produced less. This

assertion was tested when sets of heart cells from bats, mice, and shrews were examined for free

radical concentration. Because bats can live up to 30 years, it came as a surprise that bat heart

cells produced roughly 50% more free radicals than those of mice and shrews.48 However, one

might argue that it is not simply the amounts of FR that are produced that are important in

assessing the relationship between lifespan and FR, but also how well the defense mechanisms,

such as SOD and anti-oxidants, cope with FR.

Fig 7: Chart A (below) illustrates the catalase levels in several species, along with respective lifespans. Chart B
shows the levels of oxidative damage to proteins, measured by concentrations of carbonyl, on the y-axis, along with
49
numerous species (with their respective lifespans included)

At first glance, it may seem that this chart is in support of FTA, due to the fact that vampire bats

live a significant amount of time, and have high levels of catalase functioning within their cells to

help reduce damage caused by oxidative stress. However, little brown bats (MN) do not fit this trend; they

have low levels of catalase, indicating that they do much less in terms of ROS scavenging, yet they live

an impressive 34 years. Equally astonishing is the fact that laboratory mice and naked mole rats (NMR)
have roughly equal amounts of catalase activity and levels of protein damage, yet Naked Mole Rats live

24 years longer, a 7 fold lifespan difference, than Lab mice do.

Finally, there is also evidence that might argue against the conclusions that the free radical theory

of aging makes in terms of the link between cancer and oxidative stress. It follows, according to FTA, that

free radical output would be significantly higher in aged individuals. Therefore, cancers that emerge as a

result of aging should also exhibit an increase in ROS, according to FTA. It has been found that, through

studies done on mice with induced breast cancer, that mice which developed cancer when they were older

had a lower expression of oxidant-estrogen+ER signature (the compound that was used to detect levels of

oxidative stress) within their tumors. Likewise, breast cancer that occurred in younger individuals

contained a marked increase in oxidative stress.50 However, one might criticize this study because of the

fact that the Warburg Effect (Whereby cancer cells rely on glycolysis for energy production, and not on

oxidative phosphorylation which can potentially produce FR/ROS) exists in cancer cells and prevents

them from producing large quantities of ROS (cancer switches to glycolysis to produce energy, perhaps in

an attempt to reduce free radical damage to themselves). Since the main source of ROS is inhibited in

cancer cells, it makes sense to assume that the remaining FR presence is largely inconsequential and can

be overlooked.

Final thoughts

There is much evidence for and against the free radical theory of aging. It is clear that

there are substantial questions that must be answered before FTA can be accepted. Why doesnt

SOD inhibition shorten lifespan? How do free radicals manage to overwhelm the cellular

defenses organisms have in place? How does damage on the cellular level translate to the

pathphysiology of aging? What causes a shift in redox homeostasis that is visible in aged

organisms? Why dont levels of ROS in different species correlate with with one specific aging

trend?
 Is free radical production causative or correlative to the aging process? Due to our

fragmented understanding, we still do not understand all of the key players in what causes us to

age. It is likely that aging has different facets which contribute to the overal process that

manifests itself in organisms. The damage that is caused by each of these yet unknown factors is

likely crucial to understanding why we age. This damage could perhaps be in part due to free

radicals, or free radicals themselves could be a byproduct of some unseen damage that

accumulates within the cell. The evidence is mixed and the seemingly contradictory results,

especially in animals models where SOD quantities are inhibited or overexpressed, must be

resolved in order to allow for a conclusive judgement of whether not free radical theory is valid.

I find that I still stand by the sentiment that I shared at the beginning of the paper: Free radicals

are an important part of metabolism, capable of accomplishing regulative and destructive

functions. Whether or not free radicals are the sole cause of aging is something that needs to be

researched more thoroughly.

It has been shown that free radicals are indeed crucial to maintenance of homeostasis

within the body. Given the fact that free radicals play a central role in homeostatic regulation

within organisms, the future approaches that might tackle aging could focus on repairing damage

left behind by free radicals instead of destroying free radicals themselves. Regardless of the type

of damage and havoc free radicals may potentially wreak on our tissues, they remain key to our

survival. They symbolize a force which helps and hurts, gives and takes, all at once.

1
Aubrey de Grey says we can avoid aging. Dir. TED. Aubrey de Grey. TED, October 2006.
2
Beckman, K. & Ames, B. (1998) "The free radical theory of aging matures" Physiol Rev 78: 548-81.
3
Beckman, Pg. 3.
4
Aubrey D.N.J de Grey, The Mitochondrial Free Radical Theory of Aging, Pg. 36, 1999
5
Beckman, Pg. 7
6
Beckman, pg. 4.
7
Image courtesy: http://www.vivo.colostate.edu/hbooks/pathphys/misc_topics/radicals.html
8
Wulf Drge, Free Radicals in the Physiological Control of Cell Function, Physiol. Rev. 82: 47-95, 2002.
9
Drge, pg. 5.

10
The oxidative stress theory of aging: embattled or invincible? Insights from non-traditional model organisms. by:
R. Buffenstein, Y. H. Edrey, T. Yang, J. Mele. Age (Dordrecht, Netherlands), Vol. 30, No. 2-3. (September 2008),
pp. 99-109.

11
"Conversation with Dr. Zhongtao Zhang, PhD, New York Medical College." Telephone interview. 6 Oct. 2009.
12
"Ionizing Radiation." Purdue University College of Science Welcome. Web. 12 Oct. 2009.
<http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch23/radiation.php#biology>.
13
Beckman, pg 6.
14
Campbell, Neil A. Biology AP. Eighth ed. San Francisco: Pearson Benjamin Cummings, 2008. Print.
15
Campbell, Pg. 173
16
Beckman, pg. 6.
17
"Select Full Spectrum Antioxidant Supplement, 60 tablets, 3g each at PetsTruly.com."PetsTruly.com - discount
pet health supplies for dogs, cats, birds and horses. Web. 10 Dec. 2009. <http://www.petstruly.com/1844.html>.
18
Mydel P, Takahashi Y, Yumoto H, Sztukowska M, Kubica M, et al. 2006 Roles of the Host Oxidative Immune
Response and Bacterial Antioxidant Rubrerythrin during Porphyromonas gingivalis Infection . PLoS Pathog 2(7):
e76. doi:10.1371/journal.ppat.0020076
19
Isabella Dalle-Donne, Ranieri Rossi, Roberto Colombo, Daniela Giustarini, and Aldo Milzani Biomarkers of
Oxidative Damage in Human Disease Clin Chem 2006 52: 601-623. First published as
10.1373/clinchem.2005.061408, pg
20
Carmella S Moody, Hosni M. Hassan, Mutagenicity of Oxygen Free Radicals, National Academy of Sciences,
USA, Volume 79, pp. 2855-2859, pg 6
21
Carmella S Moody, Hosni M. Hassan, Pg. 6
22
DNA damage, cellular senescence and organismal ageing: causal or correlative? Jian-Hua Chen, C. Nicholes
Hales, and Susan E. Ozanne Nucleic Acids Res. 2007 December; 35(22): 74177428. Published online 2007
December. doi: 10.1093/nar/gkm681, Page 7423
23
Isabella Dalle-Donne, Ranieri Rossi, Roberto Colombo, Daniela Giustarini, and Aldo Milzani, page 605
24
"Essential Nutrition Report." Essential Health. Web. 08 Dec. 2009.
<http://www.essentialhealthandwellnesscentre.com/Essential_Nutrition_Report.html>.
25
Davies M.J., Fu S., Wang H., Dean R.T. Stable markers of oxidant damage to proteins and their application in the
study of human disease(1999) Free Radical Biology and Medicine, 27 (11-12), pp. 1151-1163
26
Isabella Dalle-Donne, Ranieri Rossi, Roberto Colombo, Daniela Giustarini, and Aldo Milzani, page 604
27
NICOLLE SITTE, MICHAEL HUBER, TILMAN GRUNE, AXEL LADHOFF, WOLF-DIETRICH DOECKE,
THOMAS VON ZGLINICKI, and KELVIN J. A DAVIES Proteasome inhibition by lipofuscin/ceroid during
postmitotic aging of fibroblasts FASEB J. 14: 1490-1498.
28
"CV Physiology: Vascular Tone." CV Physiology: Home Page. Web. 18 Nov. 2009.
<http://www.cvphysiology.com/Blood%20Flow/BF002.htm>.
29
Wulf Drge Free Radicals in the Physiological Control of Cell Function
Physiol. Rev. 82: 47-95, 2002, Pg. 49.
30
Drge, pg. 50.
31
Drge, pg. 51.
32
Drge, pg. 51.
33
Drge, pg. 59.
34
"RCSB Protein Data Bank : Molecule of the Month - Collagen." Web. 12 Dec. 2009.
<http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb4_1.html>.
35
Schroeder P, Lademann J, Darvin ME, et al. Infrared radiation-induced matrix metalloproteinase in human skin:
implications for protection. J Invest Dermatol. 2008;128:24917.
36
Brannon, Heather. "What Causes Wrinkles." Dermatology - Guide to Skin Conditions and Skin Care. Web. 11
Dec. 2009. <http://dermatology.about.com/cs/beauty/a/wrinklecause_2.htm>.
37
Keeny PM, Xie J, Capaldi RA, Bennet JP. Parkinson's disease brain mitochondrial complex I has oxidatively
damaged subunits and is functionally impaired and misassembled. J Neurosci. 2006;26:52565264.
38
"Causes of Parkinson's Disease, Parkinson's Disease." Alternative Medicine, Complementary Medicine,
Integrative Medicine, Mind-Body Medicine, Herbs, Nutrition. Web. 04 Dec. 2009.
<http://www.holisticonline.com/Remedies/Parkinson/pd_causes.htm>.
39
Alexeyey, Mikhail. Is there more to aging than mitochondrial DNA and reactive oxygen species? FEBS Journal,
Vol. 276, No. 20., pp. 5768-5787. pg. 4.
40
Alexeyey, pg. 6
41
Aubrey D.N.J de Grey, pg. 125
42
Alexeyey, pg. 4.
43
Trifunovic A, Hansson A, Wredenberg A, et al. Somatic mtDNA mutations cause aging phenotypes without
affecting reactive oxygen species production. Proc Natl Acad Sci USA. 2005; pg. 1
44
Alexeyey, pg. 7.
45
Van Raamsdonk JM, Hekimi S, 2009 Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends
Lifespan in Caenorhabditis elegans. PLoS Genet 5(2): e1000361. doi:10.1371/journal.pgen.1000361; pg 2.
46
Raamsdonk, pg. 3.
47
Zhang, Zhongtao (2009) Telephone conversation with Nikolay Avramov, December 13th.
48
The oxidative stress theory of aging: embattled or invincible? Insights from non-traditional model organisms.
by: R. Buffenstein, Y. H. Edrey, T. Yang, J. Mele. Age (Dordrecht, Netherlands), Vol. 30, No. 2-3. (September
2008), pp. 99-109.
49
Buffenstein, pg. 5.
50
Christopher C. Benz & Christina Yau Ageing, oxidative stress and cancer: paradigms in parallax. Nature Reviews
Cancer 8, 875879 (November 2008); doi:10.1038/nrc2522. Pg. 5
 Annotated Bibliography:

1. Aubrey de Grey says we can avoid aging. Dir. TED. Aubrey de Grey. TED, October 2006.

This was a talk given by a biogerontologist Aubrey de Grey. I used the material from his lecture in my
paper because of what he said about nature's selection for death in organisms. Aubrey de Grey is renown
for his work on SENS, a plan to approach the problems of aging using a novel bioengineering approach.
His work is widely referenced and his name is widely recognized in many life extension communities.

____________________________________________________________________________

2. Beckman, K. & Ames, B. (1998) "The free radical theory of aging matures" Physiol Rev 78: 548-81.

This paper was extremely useful to me because it provided general background information and assumed
the reader was not highly versed in free radicals and FTA. Kenneth Beckman is the associate professor of
biochemistry and molecular biology in the University of Minnesota. Bruce Ames both work at the
Department of Molecular and Cell Biology in University of California, Berkeley, California. Mr. Ames
has published over 450 times and is one of the most cited scientists in the field. This paper in particular
was cited by 1638 other people.

____________________________________________________________________________

3. Aubrey D.N.J de Grey, The Mitochondrial Free Radical Theory of Aging, Pg. 36, 1999

This book was written by Aubrey de Grey, just previously mentioned. This book offered a wealth of
information on the specifics of a subset of the free radical theory of aging called the mitochondrial free
radical theory of aging. Some part of the book get highly technical, and although I wasn't able to get
through it all, it was an excellent source of information and gave me a starting point to go to for other
references.

____________________________________________________________________________

4. Wulf Drge, Free Radicals in the Physiological Control of Cell Function, Physiol. Rev. 82: 47-95, 2002.

Wulf Drge received his PhD at the Max Planck Institue of Immunobiology, where he worked on
bacterial Lypopolysaccharides. He is also a postdoctoral fellow at Harvard. he currently resides at the
German cancer research Center in the department of immunochemistry. His article, elucidated the
mechanisms inside the cell that were dependent on free radicals for their function. This was especially
instrumental in helping me demonstrate that free radicals are ncessary for life, and their destruction and
creation form a point of crucial redox homeostasis within cells.

____________________________________________________________________________

5. "Conversation with Dr. Zhongtao Zhang, PhD, New York Medical College." Telephone interview. 6
Oct. 2009.
Zhongtao Zhang, PhD, is a professor of biochemistry at New York Medical College. He has helped me
greatly throughout the project, and he was also my mentor during a summer internship. In this
conversation Dr. Zhang laid out a clear distinction between the sources of free radical genesis.

____________________________________________________________________________

6. "Ionizing Radiation." Purdue University College of Science Welcome. Web. 12 Oct. 2009.
<http://chemed.chem.purdue.edu/genchem/topicreview/bp/ch23/radiation.php#biology>.

This website provided information about the on Ionizing radiation, which composed the majority of my
section on environmental sources of free radicals. This link was part of the Purdue's main website.

____________________________________________________________________________

7. Campbell, Neil A. Biology AP. Eighth ed. San Francisco: Pearson Benjamin Cummings, 2008. Print.

This was another extremely valuable source. Biology AP is a high school biology textbook that has
general information on cells, compounds, and different mechanisms. The information in this book was
provided at a high school level, so it made it that much easier for me to dissect and expand on the
information already given in the text book. Biology AP was especially helpful in my section on oxidative
phosphorylation.

____________________________________________________________________________

8. "Select Full Spectrum Antioxidant Supplement, 60 tablets, 3g each at PetsTruly.com."PetsTruly.com -

discount pet health supplies for dogs, cats, birds and horses. Web. 10 Dec. 2009.
<http://www.petstruly.com/1844.html>.

This was a website that sold pet supplements. I only used this source because it contained an appropriate
picture of a chain oxidation reaction.

____________________________________________________________________________

9. Mydel P, Takahashi Y, Yumoto H, Sztukowska M, Kubica M, et al. 2006 Roles of the Host Oxidative
Immune Response and Bacterial Antioxidant Rubrerythrin during Porphyromonas gingivalis Infection .
PLoS Pathog 2(7): e76. doi:10.1371/journal.ppat.0020076

The article was part of the backbone that formed my section on phagocytic genesis of free radicals. Mydel
et al showed that bacteria equipped with rubrerythrin can withinstand the oxidative burst that is generated
by phagocytes in mice.

____________________________________________________________________________

10. Isabella Dalle-Donne, Ranieri Rossi, Roberto Colombo, Daniela Giustarini, and Aldo Milzani
Biomarkers of Oxidative Damage in Human Disease Clin Chem 2006 52: 601-623. First published as
10.1373/clinchem.2005.061408, pg 607
This article contained information on the types of free radical damage that can occur and the means by
which this damage can be detected. The mutagenicity of free radicals on mtDNA and nDNA was
explored in their paper, and they also explored the effects of denaturation of receptor proteins on the cell's
surface.

____________________________________________________________________________

11. Carmella S Moody, Hosni M. Hassan, Mutagenicity of Oxygen Free Radicals, National Academy of
Sciences, USA, Volume 79, pp. 2855-2859, pg 6

This article demonstrated the mutagencity of free radicals on E. Coli via an agent which triggers free
radical formation, paraquat (commonly used as a herbicide). Carmella S Moody and Hosni M. Hassan
work at the Departments of Microbiology and Food Science, North Carolina University, Raleigh, North
Carolina. This article was cited by 144 other people.

____________________________________________________________________________

12. "DNA damage, cellular senescence and organismal ageing: causal or correlative?" Jian-Hua Chen, C.
Nicholes Hales, and Susan E. Ozanne Nucleic Acids Res. 2007 December; 35(22): 74177428. Published
online 2007 December. doi: 10.1093/nar/gkm681, Page 7423

The article helped me ask an important question, named, are free radicals causative or correlative to the
aging process. Are they are result of some unseen damage or are they founding damage itself. This point
is what helped lend the first framework for a debate on whether or not FTA was a valid model for aging.
Jian-Hua chen is from the department of Clinical Biochemistry at the University of Cambridge. This
article was cited by 27 others.

____________________________________________________________________________

13. "Essential Nutrition Report." Essential Health. Web. 08 Dec. 2009.

<http://www.essentialhealthandwellnesscentre.com/Essential_Nutrition_Report.html>.

This website provided me with an image of oxidation of a cell membrane, as well as an oxidative chain
reaction.

____________________________________________________________________________

14. Davies M.J., Fu S., Wang H., Dean R.T. Stable markers of oxidant damage to proteins and their
application in the study of human disease(1999) Free Radical Biology and Medicine, 27 (11-12), pp.
1151-1163

Davies et al are from the Heart Research Institute in Sydney, Australia. This paper helped quantify the
amount of free radical scavenging that proteins do within the cell, utilizing novel mark techniques. This
paper contributed to my section on damage caused by reactive oxygen species.

____________________________________________________________________________
15. Nicolle Sitte, Michael Huber, Tilman Grune, Axel Ladhoff, Wolf-dietrich Doecke, Thomas von
Zglinicki, and Kelvin J. Davies. Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of
fibroblasts FASEB J. 14: 1490-1498.

This article helped me find out more about what happens to the substrates that come in contact with free
radicals, toxic compounds, or simply become denatured. The researchers are from many different
locations: Institute of Pathology, Clinics of Physical Medicine and Rehabilitation, and Institute of
Medical Immunology, Charite, Humboldt University, Berlin, Germany; and Ethel Percy Andrus
Gerontology Center and Division of Molecular Biology, the University of Southern California, Los
Angeles, California.

____________________________________________________________________________

16. "CV Physiology: Vascular Tone." CV Physiology: Home Page. Web. 18 Nov. 2009.
<http://www.cvphysiology.com/Blood%20Flow/BF002.htm>.

A cardiovascular physiology concepts website run by Richard E Klabunde, PhD, that helped me
understand the concept of oxidative regulation of vascular tone.

____________________________________________________________________________

17. "RCSB Protein Data Bank : Molecule of the Month - Collagen." Web. 12 Dec. 2009.
<http://www.rcsb.org/pdb/static.do?p=education_discussion/molecule_of_the_month/pdb4_1.html>.

A website that offered plenty of information on collagen, the molecule which helps maintain the elasticity
of the skin. This website is a resource for researchers who are looking to find information on biological
macromolecules.

____________________________________________________________________________

18. Schroeder P, Lademann J, Darvin ME, et al. Infrared radiation-induced matrix metalloproteinase in
human skin: implications for protection. J Invest Dermatol. 2008;128:24917.

Schroeder et al studied the effects of IR radiation on the expression of matrix mellanoproteinases within
the cell. This information was used in my section on the activation of mellanoproteinases via FR genesis.

____________________________________________________________________________

19. Brannon, Heather. "What Causes Wrinkles." Dermatology - Guide to Skin Conditions and Skin Care.
Web. 11 Dec. 2009. <http://dermatology.about.com/cs/beauty/a/wrinklecause_2.htm>.

This dermatology website provided me with a simple definition as to how free radicals could potentially
generate wrinkles within humans.

____________________________________________________________________________
20. Keeny PM, Xie J, Capaldi RA, Bennet JP. Parkinson's disease brain mitochondrial complex I has
oxidatively damaged subunits and is functionally impaired and misassembled. J Neurosci. 2006;26:5256
5264.

An article that described in great detail the mechanism of oxidative stress and its role in Parkinson's
disease. This article was cited by 110 other people. The researchers are from Center for the Study of
Neurodegenerative Diseases, University of Virginia, Charlottesville, Virginia 22908, 2Institute of
Molecular Biology, University of Oregon, Eugene, Oregon 97403, and 3MitoSciences, Inc.,

____________________________________________________________________________

21. "Causes of Parkinson's Disease, Parkinson's Disease." Alternative Medicine, Complementary

Medicine, Integrative Medicine, Mind-Body Medicine, Herbs, Nutrition. Web. 04 Dec. 2009.
<http://www.holisticonline.com/Remedies/Parkinson/pd_causes.htm>.

A website that provided information on iron concentrations in the brains of PD patients, as well as feretin,
an iron encapsulating molecules which acts as a neuronal defense against oxidation.

____________________________________________________________________________

22. Alexeyey, Mikhail. Is there more to aging than mitochondrial DNA and reactive oxygen species?
FEBS Journal, Vol. 276, No. 20., pp. 5768-5787. pg. 4.

A paper highlighted various studies which provided evidence against the free radical theory of aging. This
article was extremely helpful to me because it illuminated many of holes in FTA, which I later expanded
upon in my paper by going directly to the sources listed in the paper. Mikhail Alexeyev is an assistant
professor from the department of Cell Biology and Neuroscience at the University of South Alabama.

____________________________________________________________________________

23. Trifunovic A, Hansson A, Wredenberg A, et al. Somatic mtDNA mutations cause aging phenotypes
without affecting reactive oxygen species production. Proc Natl Acad Sci USA. 2005; pg. 1.

A paper which offered two interesting sides to an experiment involving induced mutagensis in mice using
modified DNA polymerase. The researchers debunked the theory that mitochondrial mutations would
lead to increasingly more rapid FR genesis. This article was cited by 125 others. Trifunovic et al
performed their research at the Department of Laboratory Medicine, Karolinska Institute, Stockholm,
Sweden.

____________________________________________________________________________

24. Raamsdonk JM, Hekimi S, 2009 Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends
Lifespan in Caenorhabditis elegans. PLoS Genet 5(2): e1000361. doi:10.1371/journal.pgen.1000361; pg
2.

An article which provided good evidence against FTA. Raamsdonk and Hekimi demonstrated that an
increase of superoxide dismutase actually increases life span of C. elegans, completely contrary to what
FTA would predict. Jeremy Raamsdonk and Siegfried Hekimi collaborated at the Department of Biology
at McGill University, Montreal, Quebec, Canada. This article was cited by 9 other people.

____________________________________________________________________________

25. Zhang, Zhongtao (2009) Telephone conversation with Nikolay Avramov, December 13th.

A subsequent conversation with my mentor was very helpful in helping me address the criticism of the
free radical theory of aging. The information that Dr. Zhang gave me helped to balance the amount of
evidence I had for and against FTA. We discussed topics such as controlled release of free radicals and
homeostatic oxidative regulation and its relationship with cellular defense mechanisms.

____________________________________________________________________________

26. The oxidative stress theory of aging: embattled or invincible? Insights from non-traditional model
organisms. by: R. Buffenstein, Y. H. Edrey, T. Yang, J. Mele. Age (Dordrecht, Netherlands), Vol. 30, No.
2-3. (September 2008), pp. 99-109.

The paper by Buffenstein et al provided yet another strong piece of evidence against the free radical
theory of aging. It described the levels of oxidative defense, quantified results for free radical genesis, and
their relationship to lifespan in several animal models. Buffenstein et al did their research at Barshop
Institute for Aging and Longevity Studies and Department of Physiology, University of Texas Health
Science Center at San Antonio.
 Second Semester Fieldwork Questions

Topic statement: Does photodynamic therapy (PDT) enhanced by gold nanoparticles present an

effective way to kill breast cancer cells, and does it hold potential as a future cancer therapy?

Sub-questions:

1. What degree of MCF-7 cell mortality is induced by PDT using gold nanoparticles as a

vehicle for 5-ALA?

2. How much do nanoparticles contribute to the effectiveness of PDT?

3. How important is light to photodynamic therapy?

4. How cytotoxic are gold nanoparticles to healthy keratinocytes and MCF-7 breast cancer

cells?

5. Do nanoparticles aggregate in MCF-7 cancer cells selectively in comparison to healthy

tissues (keratinocytes)?
Below you will find a time log detailing my activities during my second semester work at Stevens Institute of
Technology. I ask that you please take note of that fact that the three hour commute is not included in these hours,
and neither are lunch breaks. Any long wait times (for certain experimental procedures) are spent doing work that is
directly relevant to Focus, such as journals and portfolio writing.

Thank you for your consideration.

Day Fieldwork hours Activities

03/09/10 1:00pm - 5:00pm Performed cell culture training procedure.
03/16/10 2:00pm - 4:30pm Performed cell culture training procedure.
03/24/10 1:00pm - 4:00pm Performed cell culture training procedure.
03/26/10 3:30pm - 5:30pm Performed cell culture training procedure.
03/29/10 4:00pm - 6:00pm Performed cell culture training procedure. Experienced contamination, had to
throw away Flask of Movas cells.
04/01/10 4:30pm - 7:30pm MTT assay retry - started growing new MOVAS cells and placed 6 sets of
control cells in 24-well plate. Talked with Yang further about experiment.
04/03/10 3:30pm - 6:30pm Observed and counted cells in light microscope and hemocytometer. Cells
seemed fairly confluent in 24-well plate.
04/09/10 3:00pm - 6:00pm MTT assay successful, dye was turned into formazan and subsequently analyzed
using Biotek (c) microplate reader. Transfer of DMSO into 96-well plate was
done.
04/13/10 2:00pm - 5:30pm MOVAS cells were discarded; Yang showed me the seeding of the MCF-7
(confirm the name) breast cancer cells that I will be using for the PDT
treatment. Spoke with Malcolm - questions about paper and question about GNP
use for next session.
04/14/10 2:30pm - 5:30pm Seeded MCF-7 cells into 24 well plate.
04/15/10 3:00pm - 10:00pm Performed GNP conjugation procedure along with cell irradiation. Replaced
GNP-5-ALA medium with MCF-7 (HG) cell medium.
04/16/10 4:00pm - 9:40pm Attempted to assay cells treated with PDT. Utter failure to produce formazan
crystals, and thus no absorbance was detected (No sign of appreciable purple
dye, despite the presence of cells).
04/20/10 1:30pm - 5:00pm Seeded cells in 24-well plate and counted cells with hemocytometer to
determine cell density (used 10,000 cell/ml). Changed the medium of the cancer
cells in the flask (DMEM 5% FBS, High Glucose).
04/21/10 2:30pm - 9:30pm Performed GNP conjugation procedure along with treatment of cells with the
GNP-5-ALA medium. Also irradiated the cells and replaced PDT-5-ALA
conjugate medium.
04/22/10 4:30pm - 8:30pm Performed MTT assay on cells, transferred DMSO to 96-well plate, and
measured absorbance in microplate reader.
04/27/10 2:00pm - 5:00pm Seeded breast cancer cells into (12 wells in) 24-well plate at 10,000 cell/ml.
Continued discussion with Yang on making changes to the experiment.
Attended Malcolm's thesis defense!
04/28/10 3:00pm - 8:45pm Performed GNP conjugation procedure along with treatment of cells with the
GNP-5-ALA medium. Also irradiated the cells and replaced PDT-5-ALA
conjugate with DMEM (5% FBS, HG.)
04/29/10 3:00pm - 8:00pm Performed MTT assay on cells, transferred DMSO to 96-well plate, and
measured absorbance in microplate reader. Spoke with Wayne about his
experience doing MTT.
05/03/10 - I e-mailed Yang to ask him if he could seed 12 well at a higher seeding density.
I wouldn't be able to go on Wednesday.
05/05/10 3:00pm - 9:00pm Performed GNP conjugation procedure with modified concentration of 5-ALA
implemented, along with treatment of cells with the GNP-5-ALA 10x medium
and 1x medium. Also irradiated the cells and replaced GNP-5-ALA conjugate
with DMEM (5% FBS, HG.)
05/06/10 3:30pm - 7:30pm Performed MTT assay on cells, transferred DMSO to 96-well plate, measured
absorbance in microplate reader. Finally got a great reading with a clear
difference between the PDT group and control group. Increased formazan
detected.
05/11/10 1:00 pm - 4:30pm Counted cells in flask to determine cell density. Seeded breast cancer cells into
two separate well plates. 1st group was 5-ALA alone, GNP-5-ALA, and a
control. Second group for dark results was GNP-5-ALA and control.
05/12/10 3:00 to 11:30 Performed GNP conjugation procedure with 5-ALA (10x). Also created
suspension with mixture of 5-ALA alone. Distributed GNP-5-ALA to cells in
dark and light 24-well plates. Replaced medium of controls. Replaced medium
of flask. Performed 4 hour incubation (for both groups) and subsequent
irradiation of the light exposed 24-well plate.
05/13/10 4:00 to 8:30 Performed MTT assay on both groups of cells, making sure to keep dark group
away from light. Analyzed the results and created graphs of the findings.
Continued working on focus portfolio.
 Second Semester Bibliography

1. Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold
nanoparticles in cancer diagnostics: applications in oral cancer. El-Sayed Ivan H; Huang
Xiaohua; El-Sayed Mostafa. Department of Otolaryngology-Head and Neck Surgery,
Comprehensive Cancer Center, University of California at San Francisco, San Francisco,
California 94143, USA. ielsayed@ohns.ucsf.edu, Nano letters 2005;5(5):829-34.

2. 5-aminolevulinic acid-conjugated gold nanoparticles for photodynamic therapy of cancer.

Maung Kyaw Khaing Oo, Xiaochuan Yang, Henry Du, Hongjun Wang. Nanomedicine,
December 2008, Vol. 3, No. 6, Pages 777-786.

3. Ito, S., Miyoshi, N., Degraff, W. G., Nagashima, K., Kirschenbaum, L. J. & Riesz, P. (2009).
Enhancement of 5-Aminolevulinic acid-induced oxidative stress on two cancer cell lines by gold
nanoparticles. Free Radical Research, doi:10.1080/10715760903271249

4. Intracellular photodynamic therapy with photosensitizer-nanoparticle conjugates: cancer

therapy using a Trojan horse . Wieder M E, Hone D C, Cook M J, Handsley M M, Gavrilovic J
and Russell D A 2006 Photobiol. Sci. 5 727-34

5. "Students Win Big at Intel ISEF 2010." Science News. 14 May 2010. Web. 15 May 2010.
<http://www.sciencenews.org/view/generic/id/59221/title/Students_win_big_at_Intel_ISEF_201
0>.

6. J. C. Y. Kah, K. W. Kho, C. G. L. Lee, C. J. R. Sheppard, Z. X. S., K. C. Soo, M. C. Olivo.,

"Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles,"
Int. J. Nanomedicine, Vol. 2(4), 114 (2007)

7. Photothermal Nanotherapeutics and Nanodiagnostics for Selective Killing of Bacteria

Targeted with Gold Nanoparticles. Vladimir P. Zharov, Kelly E. Mercer, Elena N. Galitovskaya,
Mark S. Smeltzer, Biophysical Journal - 15 January 2006 (Vol. 90, Issue 2, pp. 619-627)

8. W. H. De Jong and P. J. Borm, Drug delivery and nanoparticles: Applications and hazards, Int
J Nanomedicine 3 (2008), no. 2, 133-149.

9. Multifunctional nanoparticles as biocompatible targeted probes for human cancer diagnosis

and therapy. Yong, Ken-Tye; Roy, Indrajit; Swihart, Mark T.; Prasad, Paras N. Journal of
Materials Chemistry (2009), 19(27), 4655-4672.

10. Cheng Y, C Samia, Meyers JD, Panagopoulos I, B Fei (co-corresponding author), Burda C.
"Highly efficient drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy
of cancer". Journal of American Chemical Society (JACS), 30(32):10643-7. 2008.
 Course Drops (Red crosses and blocks denote free chuncks of
time.)

Courses dropped: English, Gym.

Courses Partially dropped: Practice of statistics on Tuesday.