Fluorodeoxyuridine, FUDR, is a widely used treatment for many different types of cancer. It starves cells of deoxythymidine triphosphate, dTTP, which causes an imbalance in the deoxyribonucleotide, dNTP, pool sizes in the cell during DNA replication. We are predicting that with a lack of dTTP, the DNA polymerase sometimes will substitute an incorrect nucleotide in place of dTTP causing mutations, and we believe that this will affect the number of mutations in the mitochondrial genome. To study this, we are using combinations of FUDR and O4-ethyldeoxythymidine (O4-EtdT). This second mutagen, a modification of Thymidine, is used during the salvage pathway for the synthesis of dTTP. It can compete with dTTP and causes a loss in specificity for the DNA polymerase during further rounds of replication. We are treating colon cancer cells with combinations of these mutagens and using the random mutation capture (RMC) assay to quantify the mutations in the mitochondrial genome. The mutations are enriched with a restriction enzyme that cleaves the wild type DNA leaving only mutated DNA to be amplified using qPCR. We are then using TOPO cloning to sequence the mutations. Many believe that uracil will be used as substitute when the cell is starved of thymine because of their similar structures. However, incorporation of uracil will not result in mutagenesis, while incorporation of the other three nucleotides will. With the results from TOPO cloning, we will be able to take a closer look at this hypothesis. Further on, results will be analyzed using the Student T-Test and One Way ANOVA. So far, the data shows a change in the quantity of mutations in mitochondrial DNA when either O4-EtdT or FUDR are added. Results can help us to understand and respond to post-chemotherapy health problems that may be due to mutations in the mitochondrial genome.

Humans are exposed to environmental toxins which can contribute to acute or chronic diseases, such as Parkinson's disease. Different people, however, have variable susceptibility to these toxins due to genetic variability within the population. In this project, we will utilize the model organism Saccharomyces cerevisiae(yeast) to investigate the molecular mechanisms of differential responses to environmental toxins that target the mitochondria. Many yeast single-gene deletion mutants will be exposed to mitochondrial toxins using a high-throughput Biosreen C instrument(combined shaker, incubator and plate reader, allowing for growth rate determination of yeast cells) and subsequently, bioinformatic methods will be exploited to identify genes involved in the response to mitochondrial stress and mechanisms of toxicity. We explored optimum concentration of different toxins for mutant yeast, expecting that different mutations will response genotype-dependently to different compounds. Identifying genotype-dependent responses to the environmental mitochondrial toxins may illuminate how mitochondria toxins contribute to disease and identify genetic factors invovled in susceptibility.

The hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcription factor that plays a crucial role in an animal’s response to changing oxygen availability. When oxygen is plentiful, HIF-1 is ubiquitinated and targeted for degradation by the von Hippel Lindau protein (VHL-1), which inactivates HIF-1. Recent studies have shown that HIF-1 stabilization through mutation of vhl-1 increases longevity in the model organism Caenorhabditis elegans. This is contrary to humans, where VHL-1 mutations cause a disease characterized by angiomas, renal carcinomas and various other tumors as a direct result of aberrant HIF-1 activation. Since nematodes are post-mitotic organisms in adulthood, constitutive activation of HIF-1 does not cause tumor formation and is able to increase lifespan. In order to better understand how HIF-1 increases longevity and to relate these findings to mammals, our project aims to find the specific genes and tissues downstream of HIF-1 that benefit worm longevity without the consequences of VHL disease. To identify these genes, we conducted an RNAi screen for genes that are necessary for HIF-1-mediated longevity. Using the results of this screen, we are creating transgenic worms that overexpress these genes under specific nematode promoters. To test for the tissue specificity of HIF-1-mediated longevity, we are stabilizing HIF-1 in different tissues using tissue specific promoters. Our initial data are promising; we have found two genes downstream of HIF-1 that when overexpressed increase nematode longevity. At least one of these genes, flavin-monooxygenase-2, is a well-conserved protein known to be involved in xenobiotic metabolism. Additionally, we have found that expressing stabilized HIF-1 in neurons is sufficient to increase nematode lifespan. Our continuing studies will identify additional target genes and tissues important for HIF-1 mediated longevity and will attempt to elucidate the mechanism by which they increase lifespan.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction (DR) is one promising pro-longevity intervention, though its mechanism is poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species, including yeast, flies, mice, and primates. However this extension is highly dependent on genetic background. To investigate this, we are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We have screened over 1500 genes; 61 of which are short-lived and 67 of which are long-lived. We are currently following up on our collected data by measuring the full lifespans using the RNAi clones that significantly shortened or extended lifespans in our screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

 Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has been used extensively to study the biology of aging, and several determinants of C. elegans longevity are conserved in higher order organisms. The specific mechanisms of longevity in C. elegans have not been completely identified and many pathways are still poorly understood. Dietary restriction is one promising pro-longevity intervention, though its mechanism is poorly defined. DR, a reduction in caloric intake in the absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species, including yeast, flies, mice, and primates. However, this extension is highly dependent on genetic background. To investigate this, we are performing a genome-wide screen of the Vidal RNA interference (RNAi) Library, which has a catalog of 11,511 clones. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe an extension or reduction in longevity in response to DR. We have screened over 1500 genes, 61 of which are short-lived and 67 of which are long-lived. We are currently following up on our collected data by measuring the full lifespans using the RNAi clones that significantly shortened or extended lifespans in our screen. By screening such a large number of genes, the widespread effect, and hopefully mechanism, of DR will be elucidated. We hope to see genes significantly affected by DR cluster into groups with common functionality, or along the same pathway, and then follow up on those specific pathways to clarify the mechanisms underlying DR. If these pathways are conserved, research findings relating to C. elegans may be applicable to other organisms. We hope to confirm these results and further investigate the interaction between DR and other potential longevity pathways.

Leigh syndrome is a clinically defined collection of diseases comprised of multiple genetic defects related to mitochondrial function. This syndrome results in early death in children typically due to respiratory failure as a consequence of progressive neuropathy, and currently no viable treatment options exist. Leigh syndrome can result from mutations in several different mitochondrial genes, one of which encodes the NDUFS4 subunit of complex I in the mitochondrial electron transport chain. As the NDUFS4 knockout mice phenocopy human patients with Leigh syndrome remarkably well, this model was utilized for the purpose of this study. Dietary restriction (DR) was identified as a possible treatment for mitochondrial deficiencies through a yeast genetic screen, which found that mitochondrial gene mutants generally received a robust lifespan benefit from DR. Thus, the primary focus of our research is to determine the efficacy of rapamycin (a DR mimetic) as an intervention in the NDUFS4 -/- mitochondrial disease mouse model. Rapamycin functions as a DR mimetic through its inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway, which is responsible for modulating a variety of metabolic and cellular processes. Our model suggests that the increased NADH/NAD ratio resulting from a complex I deficiency in these knockout mice is sensed as energy abundance and results in systemic metabolic derailment. We propose that inhibiting mTOR signaling addresses this defect by altering intracellular energy sensing and modifying metabolic status. In order to test the efficacy of rapamycin treatment, NDUFS4 -/- mice were treated with daily injections of rapamycin or vehicle. Lifespan data for each treatment and additional assays for health related parameters were conducted to determine the response to the treatment regime. Investigating Leigh syndrome in this context will allow us to better understand the underlying mechanisms of pathogenesis, and directly test novel interventions aimed at attenuating this disease.

Ethosuximide is a succinimide anticonvulsant used in the clinical treatment of petit mal epileptic seizures in humans. Although its exact mechanism is not fully elucidated, it is believed to inhibit T-type calcium channels. In addition to its medical use, ethosuximide has been found to increase lifespan in the model organism, the nematode C. elegans. Nematodes exhibit lifespan extension when treated with ethosuximide at concentrations below a toxic threshold. While the published experimental results on ethosuximide-treated nematodes are promising, we have discovered that ethosuximide breaks down in the presence of 254 nm ultra-violet (UV) light, forming a toxic by-product. This change in ethosuximide that results in toxicity is specifically caused by irradiation, not heat. Acute exposure of C. elegans to ethosuximide treated with UV light results in death at lower dosages compared to nematodes treated with the control, native compound. UV-treated ethosuximide toxicity is independent of chemosensation (i.e. the sensing of chemical stimuli) as evidenced by the fact that chemosensory mutants do not show enhanced resistance to the UV-treated drug. Some human patients taking ethosuximide experience a butterfly rash under the eyes following exposure to sunlight. We speculate that this rash may be indicative of a similar mechanism of UV-induced cytotoxicity occurring in these patients. Taken together, these studies will help explicate the mechanism of toxicity resulting from exposure of ethosuximide to UV light, as well as the mechanism of action of native ethosuximide.

The hypoxia-inducible factor (HIF-1) is an oxygen-dependent transcription factor that plays a crucial role in an animal’s response to changing oxygen availability. When oxygen is plentiful, HIF-1 is ubiquitinated and targeted for degradation by the von Hippel Lindau protein (VHL-1). Recent studies have shown that HIF-1 stabilization through mutation of vhl-1 increases longevity in the model organism Caenorhabditis elegans. This is contrary to humans, where VHL-1 mutations cause a disease characterized by angiomas, renal carcinomas and various other tumors as a direct result of aberrant HIF-1 activation. Since nematodes are post-mitotic organisms in adulthood, constitutive activation of HIF-1 does not cause tumor formation and is able to increase lifespan. In order to better understand the how HIF-1 increases longevity and to relate these findings to mammals, our project aims to find the specific genes and tissues downstream of HIF-1 necessary for its effect on worm longevity. Our goal is to find genes downstream of HIF-1 that benefit longevity without the consequences of VHL disease. To identify these genes, we conducted an RNAi screen for genes that are necessary for HIF-1-mediated longevity. Using the results of this screen, we are creating transgenic worms that overexpress these genes under specific nematode promoters. To test for the tissue specificity of HIF-1-mediated longevity, we are stabilizing HIF-1 in different tissues using tissue specific promoters. Our initial data are promising; we have found two genes downstream of HIF-1 that when overexpressed, increase nematode longevity. At least one of these genes, flavin-monooxygenase-2, is a well-conserved protein known to be involved in xenobiotic metabolism. Additionally, we have found that expressing stabilized HIF-1 in neurons is sufficient to increase nematode lifespan. Our continuing studies will identify additional target genes and tissues important for HIF-1 mediated longevity and will attempt to elucidate the mechanism for their action.

Organisms have evolved specific mechanisms to respond to molecular and environmental stress. These stress-response pathways are evolutionarily conserved and can modify lifespan in model organisms, making them popular targets of aging research. Using the nematode Caenorhabditis elegans (C. elegans), we have found the loss of important genes in stress-response pathways can cause a phenomenon called age-related vulval integrity defects (Avid). Worms with Avid show a small protrusion near their vulva that can eventually lead to expulsion of the intestine and premature death. While many labs have noted this phenotype, it is unknown if Avid is a natural part of C. elegans aging or if genetic and environmental factors induce this phenotype. Because of this, there is confusion as to whether worms with Avid should be censored in aging experiments. The goal of our project is to determine the mechanism of Avid and whether it is part of normal worm aging. Our initial work has identified several conditions and genetic pathways important for Avid, including temperature, food availability, the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. We have also identified that the reproductive period of the C. elegans lifecycle is influential on Avid frequency, despite the phenotype occurring post-reproductively. We are currently testing the hypothesis that Avid is caused by secretions from the bacteria worms eat that influence nematode physiology and lead to Avid. We hypothesize that the pathways that influence Avid frequency do so by altering the digestion of bacteria in the intestine or by modifying the worm’s immune response. To test our hypothesis we measured the quantity of bacteria ingested and digested by various worm strains at different temperatures and tested whether bacterial metabolites can cause Avid. We intend to combine our results and develop a more complete model of Avid.

Cardiovascular disease is an epidemic, prevailing as the leading cause of death worldwide. One consequence of cardiovascular disease is a myocardial infarction (MI), or heart attack. An MI is caused by a blockage in the coronary arteries which cuts off the blood supply to a portion of the heart wall, forming nonfunctioning scar tissue. We are aiming to improve heart recovery post-MI by replacing scar tissue with functioning cardiac tissue through a cell engraftment therapy. Cardiac cell engraftment is difficult because cardiac cells die quickly due to lack of vascularization of the graft. We believe that by promoting vessel growth in grafts, we will improve therapeutic cardiac cell survival for better heart function during recovery. To study the coronary network’s ability to vascularize cell grafts, we inject skeletal muscle cells, which survive better than cardiac cells, into the heart wall post-MI. We observed vessel development in mice 14 days after MI and engraftment, and compared it to vessel development at other time points to better understand how the coronaries can vascularize cell grafts. To characterize the graft vasculature, I used histology to mark vessels and quantify vessel growth in the graft. My results shows that the coronary vasculature is able to expand and vascularize newly implanted tissue grafts post-MI. I detected capillaries as well as smooth muscle coated vessels in the graft suggesting vascular remodeling has occurred by 14 days post MI. My work establishes a baseline comparison for future research that will identify factors to increase the vascularization of graft tissue. We hope that promoting vessel growth will improve cell therapy methods for heart recovery to become applicable to clinical practice in the future.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Soft tissue sarcoma is a cancer that develops in the muscle, fat, fibrous tissue, blood vessels, or other supporting tissue. RMS is categorized into two major subtypes: alveolar RMS (ARMS) and embryonal RMS (ERMS). Most ARMS express the PAX3-FOXO1 fusion oncoprotein generated by the 2;13 chromosomal translocation. Among RMS tumors, PAX3-FOXO1-positive ARMS commonly gives the worst prognosis. Sphingolipids are defined by a common structural feature (a long-chain (sphingoid) base). Sphingosine is the most common sphingoid base composed of an 18 carbon-chain. The overall goal of this study is to investigate roles of sphingosine on growth, apoptosis (programmed cell death), and MYCN oncogene expression in PAX3-FOXO1-positive ARMS cells (RH30 cell line). The specific aims of this study are to determine: 1) effects of sphingosine on apoptosis; 2) effects of sphingosine on MYCN expression; 3) effects of MYCN overexpression on growth inhibitory and pro-apoptotic activities of sphingosine. Our previous results have indicated that sphingosine caused time and dose-dependent inhibition of the growth and death of RH30 cells, suggesting sphingosine as a potential chemotherapeutic agent against ARMS. Sphingosine, at a concentration that can induce apoptosis, down-regulates the MYCN expression as early as two hours. In the present study, the data from flow cytometry demonstrates that sphingosine induces apoptosis identified by a well-known apoptosis marker, AnnexinV+/PI+. Experiments are in progress to determine whether the growth inhibitory effects of sphingosine are decreased in RH30 cells overexpressing MYCN. This will test a hypothesis that MYCN is a mechanism which mediates the growth inhibitory and pro-apoptotic effects of sphingosine on PAX3-FOXO1-positive ARMS cells. Thus, this experiment will determine if MYCN is a possible target for chemotherapeutic agents in ARMS.

Cancers contain large numbers of mutations. This reservoir of genetic variation can be used by cancer to produce new phenotypes in response to environmental barriers. In order to accumulate these mutations we have proposed that cancers express a mutator phenotype, i.e. an increased rate of mutation. Previous results in the lab demonstrate that sequential selection of cancer cell lines (colon cancer cells HCT116 and SW620) for novel phenotypes enriches for cell lineages with elevated mutation rates. After three rounds of sequential selection for resistance to non-mutagenic drugs (diphtheria toxin, puromycin, staurosporine), both cancer cell lines exhibit an increase in mutation rate. Specifically a 3-fold for HCT116 and 6-fold for SW620 was measured. I will extend these experiments using additional selection conditions (e.g., elevated temperature, hypoxia and nutrient deprivation) and will determine mutation rate of the resulting cell lines using the PIGA gene mutation assay. The random mutation load will be characterized using Duplex Sequencing. We believe the process of sequential selection favors mutator lineages as has been observed in bacteria. However, because an increased rate of mutation is potentially deleterious to a cell, we propose the existence of a mutation limit beyond which cells are no longer viable, i.e. the cells undergo apoptosis. I will screen mutagenic nucleosides for their potential to increase the mutation rate of cancer cells beyond this threshold. Targeting cancer’s error threshold may represent a novel and innovative therapeutic strategy for the treatment of human malignancies.

A replicative lifespan (RLS) screen in yeast (Saccharomyces cerevisiae) was recently performed in the Kaeberlein lab to determine how different mutants respond to caloric restriction (CR). The results of this screen indicated that many mutants lacking genes encoding proteins related to mitochondrial function were rescued to wild-type lifespan by CR. The beneficial effects of CR in both yeast and mammals are mediated by reduced signaling through the mechanistic target of rapamycin (mTOR) pathway and can be mimicked by the drug rapamycin, a specific inhibitor of mTOR. Based on the findings from the yeast screen, we decided to investigate the effects of rapamycin on mitochondrial disease in a mammal. Leigh syndrome is clinically defined as a collection of genetically heterogeneous human mitochondrial diseases. These diseases result from mutations in genes encoding mitochondrial proteins and are characterized by progressive neurological degeneration resulting in early childhood mortality. To test rapamycin as a therapeutic intervention for Leigh syndrome, we are using a mouse line carrying a heterozygous loss of the electron transport chain complex I subunit Ndufs4, a direct homolog of the human NDUFS4 gene whose mutation causes Leigh syndrome. Mice homozygous for loss of NDUFS4 provide a robust phenocopy of the human disease, including neurological degeneration and early life mortality. Our results so far show that rapamycin dramatically increases lifespan and rescues the Ndufs4-/- mice for several health-related parameters, such as general mobility, visual ability, and grip strength. We are working towards determining the mechanistic basis for these effects, and we hypothesize that they may result from a metabolic shift toward increase fat metabolism in the liver. Understanding how rapamycin rescues Leigh Syndrome mice has clear translational implications, as rapamycin derivatives are FDA approved and patients with Leigh syndrome and other human mitochondrial diseases currently have limited treatment options.

Aging in the budding yeast Saccharomyces cerevisiae can be measured by its replicative life span, which is defined by the finite number of mitotic divisions of mother cells. The Kaeberlein lab measures the replicative life span of S. cerevisiae through the microdissection of dividing mother cells. This laborious process separates daughter cells from their mothers, which quantifies the replicative life span. We are assessing how various environmental factors affect the replicative life span of the cells. Our research project involves paraquat, a commonly used pesticide that produces reactive oxygen species within cells. This substance is normally considered to be toxic, and as expected, we have observed that the replicative lifespan of yeast is decreased in the presence of high concentrations of paraquat. However, we have also found that low concentrations of paraquat increased the replicative life span of the yeast. This observation is an example of hormesis- a phenomenon where beneficial effects are obtained at low doses of an agent that is otherwise toxic when given at higher doses. These findings are significant because a similar effect has been published with the nematode worm, Caenorhabditis elegans. C. elegans and S. cerevisiae are evolutionarily separated by approximately one billion years, suggesting that there is a conserved mechanism of life span extension influenced by paraquat. We are currently characterizing the effect of paraquat and other environmental conditions that will help us to find conserved pathways that modulate longevity.

The nematode Caenorhabditis elegans is a powerful model for the analysis of the conserved mechanisms that modulate healthy aging, from organismal to molecular levels. As animals age, tissues deteriorate at different rates. It has been shown previously in the Kaeberlein lab that deletion of the hypoxia-inducible factor, HIF-1, extended lifespan of worms at high temperature (25°C), but did not do so at low temperature (15°C). Accordingly, it was speculated that at low temperature, worms were not as healthy. At low temperature, we noticed an abnormal vulva phenotype and called it age-related vulva integrity defect (Avid). As worms age, their vulvae protrude and rupture, causing internal organs such as the intestine to erupt out of their bodies. Through investigation, we realized that animals lacking oocytes tend to show more Avid phenotype, compared with the animals that produce enough oocyte. It has been shown before that at 15°C worms produce around 300 fertilized and unfertilized oocytes, while at 25°C this number increases to 400. My preliminary results suggest that germline is involved in the progress of Avid. I studied animals with germline defects and observed 100% Avid phenotype in germless mutants. Avid is a sign of healthspan as animals age, and decline in germline fecundity due to aging can induce Avid. Healthspan is defined as the period of life spent in relatively good health, and maintenance of healthspan is an important component of aging. Our data indicate that Avid can be used as a measurement tool of the age-related healthspan of worms.

Manganism, a movement disorder characterized by neuronal death in the basal ganglia and symptomatology similar to Parkinson’s disease, has been shown to involve manganese-mediated increase of toxic reactive oxygen species (ROS) production in mitochondria. The protein alpha-synuclein, a product of the SNCA gene, has been implicated in glial and neuronal degeneration in Parkinson’s disease, and preliminary data from our laboratory suggests that removal of alpha-synuclein from astrocytes confers some protection against ROS toxicity. Astrocytic hydrogen peroxide metabolism has been shown to produce ROS capable of damaging a variety of cellular macromolecules. The present study seeks to compare the effectiveness of toxicant attenuation in SNCA knock-out and wild type astrocytes in response to hydrogen peroxide after gradated 24-hour manganese pre-treatments. Calcium imaging with fluo-4 dye will be performed to measure astrocytic calcium by quantifying the amplitudes and durations of induced calcium waves, known to form an important mechanism of astrocytic communication, after addition of hydrogen peroxide. Based on previous studies, we predict that increased manganese dosages will correlate with elevated calcium wave amplitudes in both SNCA knock-out and control cultures. We further predict significantly lower calcium wave amplitudes in wild type cultures after each manganese treatment.

In the United States prostate cancer (PCa) is one of the most commonly diagnosed cancers and estimated to affect 239,000 men in 2013. Although few prognostic biomarkers are currently used in the clinical setting, biomarkers that predict the risk of disease aggressiveness leading to prostate specific antigen (PSA) failure after prostatectomy may assist clinicians in selecting adjuvant therapies following prostatectomy. In studies utilizing co-registered pre-operative imaging data with post-operative pathology data, validated prognostic biomarkers may also be highly useful as “gold standards” against which to train imaging methods to assess disease aggressiveness pre-operatively. We previously identified 33 potentially prognostic biomarkers from the analysis of multiple gene expression datasets and current literature. Using tissue microarrays containing PCa tissue from 170 prostatectomy specimens from men with detailed PSA and follow up data, we quantified immunohistochemical (IHC) staining for these markers within selective tissue sub-regions (malignant epithelium) and identified 16 of the 33 biomarkers which were associated with shorter time to PSA failure. While our aim is to use validated biomarkers to pre-operatively train in vivo imaging methods, current imaging capabilities have lower resolution than digital histology and it is unclear whether biomarker expression in total tumor areas (encompassing malignant epithelium, benign epithelium, stroma, and other tissue structures) which are resolvable by imaging methods will also be prognostic. In this study, we have re-evaluated our list of 16 strong candidate biomarkers to determine if IHC expression within total tumor areas are associated with PCa aggressiveness (PSA failure following prostatectomy) using our TMA cohort.

Aging is a process characterized by the degeneration of bodily components and functions. Scientists are limited in their ability to study this process in humans, in part because a human lifespan is too long to conveniently study. Instead, model organisms are studied extensively in the hope that discoveries made will be applicable to another organism of interest. Model organisms are usually short-lived, easy to manipulate, and easy to handle. One of the best examples of a model organism is the budding yeast Saccharomyces cerevisiae. S. cerevisiae lifespans are easy to measure and reproduce, and since they have a short lifespan we can study a large amount at one time. Aging in Saccharomyces can be measured in two different ways. A yeast cell can be kept in a non-dividing, nutrient limited environment and tested to see if it can successfully re-enter the cell cycle, when it cannot, its lifespan has ended. This method is used to measure the chronological lifespan, and the model can be applied to human cells that are no longer replicating (post-mitotic cells). There is another model we can use to understand a different aging process, called the replicative lifespan. The replicative lifespan refers to the number of times a mother cell can divide to produce daughter cells, and is a much better representation of cells that are still dividing (mitotically active cells) in other organisms. As undergraduates, we help study this process by physcially separating daughter cells from the mothers using fiberoptic needles (dissecting) and counting them at regular intervals. When the mother cell no longer produces daughter cells, the replicative lifespan has ended. The data collected can be compared to past and expected results in order to help us gauge how well the cells are dividing. By manipulating the environment that each cell line is grown in, we can gain insight towards specific drugs and conditions that mother cells divide best in, and apply this to other mitotically active cells in other organisms. Both measurements of lifespan can be manipulated in similar ways, and many labs have started to establish a bank of genes and environmental factors that can be manipulated in order to change the outcomes.

My research focuses on mTOR (mammalian target of Rapamycin)-dependent signaling and its involvement in neurodegeneration and neurofibrillary degeneration. A lot of studies have shown that Rapamycin, which is an mTOR inhibitor, is able to provide protection in models of neurodegenerative diseases especially Alzheimer's and Parkinson's disease. Rapamycin is involved in a lot of cellular processes including cell growth, proliferation, and it accelerates clearance of these toxins. My project focuses on important neuronal proteins levels such as alpha synuclein, DJ1, Beta Amyloid, tau, and phosphorylated tau in Rapamycin treated control, old, and young cortex and cerebellum samples using the Luminex machine. Altered levels of the mentioned proteins have been associated with Alzheimer's and Parkinson’s disease. In the Luminex Instrument, cell lysates are combined with a biotinylated double stranded consensus binding sequence for the transcription factor family of interest. Fluorescently labeled magnetic beads, which have been conjugated to antibodies that target the individual transcription factor family members, are used to capture the transcription factor-bound DNA. At the end, a dye called Streptavidin-phycoerythrin is used to bind the biotinylated oligo and determine the magnitude of the signal. We hope to see a significant difference in the levels of the mentioned proteins in Rapamycin treated mice vs. the control group especially in the cerebrum group.