A decline in effective mitochondrial functioning of cardiac cells is one the major underlying factors of aging. Many pathological conditions have shown to result in the peroxidation of the final electron carrier in the electron transport chain (ETC) known as cytochrome c. The ETC is constructed of a series of complexes responsible for the transfer of electrons from electron donors such as NADH through a continuity of redox reactions while coupling the electron transfer energy to the transfer of protons across the inner mitochondrial membrane, constructing a proton gradient to stimulate the production of ATP (Adenosine triphosphate). Peroxidation is known as oxidative degradation and causes cyctochrome c to lose its ability to act as an electron carrier through free radicals that steal the electrons from the complex. The SS-31 peptide works to bind to the inner mitochondrial membrane consisting of cardiolipin and prohibits the cardiolipin from converting cytochrome c into a peroxidase, an enzyme that catalyzes oxidation, and continue its function as an electron carrier. Additionally, administration of NMN (Nicotinamide mononucleotide) can help improve the overall bioenergenetics of the mitochondria through an enhanced production of NAD+, therefore increasing the amount of electrons donated into the ETC once NAD+ is reduced, leading to an increased production of ATP. Three different mice treatment groups were constructed for this experiment; the first group was treated with surgical incisions of pumps containing the SS-31 peptide, the second group was provided with the NMN drug administered through water pumps, while the third group was left as the control. Detection of bioenergenetic changes between treatment groups was evaluated through changes in the NAD+ stoichiometric peak evident through 31Phosphorous magnetic resonance spectroscopy. Administration of the SS-31 peptide and NMN through the mouse model provides a potential advantage in the treatment of age-related complications for human patients.

Age-associated diseases, like neurodegenerative disease, cancer, heart disease, and metabolic dysregulation limit healthy human lifespan. In recent years, biologists researching aging and longevity have turned their attention towards maximizing healthspan, the healthy portion of one’s life before the onset of age-related disease. By delaying age-associated diseases, we can fundamentally improve quality of life globally. Natural products and other pharmacological interventions hold particular promise as interventions to extend healthspan and lifespan. We seek to identify novel compounds that extend lifespan using the invertebrate model system Drosophila melanogaster (fruit fly). We have tested an extract made from Pterocarpus marsupium (PME), a tree native to India and Sri Lanka with uses in Ayurvedic medicine. PME extends cellular lifespan in budding yeast, another invertebrate model system. We also tested pterostilbene, a compound found in Pterocarpus marsupium extract. As a positive control for lifespan extension, we are treating other cohorts of flies with rapamycin. Rapamycin is a specific inhibitor of the nutrient sensing mechanistic Target Of Rapamycin (mTOR) pathway, a known longevity regulating cellular pathway. Using multiple fly genetic backgrounds, performed a dose response to identify concentrations of PME and pterostilbene that extend Drosophila lifespan. Through pharmacological methods, we seek to delay aging and minimize human vulnerability to age-induced diseases. Discovery of specific compounds that prolong lifespan is a first step in developing therapeutic methods to delay human aging and health decline.

DNA polymerase (Pol) proofreading and mismatch repair (MMR) cooperatively guard against DNA replication errors and cancer. Defects in these activities produce “mutator” phenotypes characterized by elevated levels of base-substitutions and frameshifts. Haploid yeast with combined defects in these activities rapidly go extinct, in a process termed error-induced extinction (EEX). Organisms adapt by duplicating their genome without dividing (polyploidization) or by lowering their mutation rate via antimutator mutations. In evolution experiments with haploid mutators that display a synthetic-sick phenotype due to Pol ε proofreading and MMR defects we found that polyploids routinely beat out antimutator mutants. To investigate whether polyploids arise during the evolution of mutator diploid strains, we propagated diploids defective in Pol δ proofreading and base-base MMR, which have a mutation rate an order of magnitude below the diploid error threshold. Our mathematical modeling predicts that EEX mutants will overtake mutator cultures by 250 generations. We found only a single tetraploid in 89 independent cultures evolved for 300 generations. We then measured mutation rates of isolates from cultures that remained diploid and repeatedly found antimutator phenotypes. Whole genome sequencing of independent isolates from each culture revealed a single dominant antimutator clone that caused significant changes in mutation spectra. Thus, spontaneous antimutator alleles and polyploidization rescues haploid and diploid cells from EEX with markedly different efficiencies. Differences in the relative frequency of each escape mechanism may reflect the nature of the mutator alleles, the starting ploidy of the cells, or the magnitude of the initial mutation rate. Our findings in diploids suggest that mutator cancer cells near the edge of error-induced extinction may similarly be under selection for spontaneous antimutator mutations and polyploidization. 

Aging is characterized by the loss of physiological and cellular functionality, however the mechanisms that underlie this deterioration are still unclear. Emerging evidence indicates that aging is associated with increased cell-to-cell variation in gene expression within tissues: homologous cells within tissues start expressing the same gene at varying levels. The causes of this age-related variation of gene expression are not known. Here, we aim to investigate this age-related dissimilarity in gene expression using C. elegans as a model system. We hypothesize that increase of gene expression variation is an early event during aging that may therefore underlie subsequent deterioration of tissues functionality. By characterizing aging in C. elegans, we hope to provide further insight into the molecular characteristics of aging in humans, and possible points of intervention. Previously, we have found that young C. elegans animals exhibit nearly identical stoichiometry of independent genes expression with very little difference between individual animals of the same genetic background. Our initial results support the idea of increased cell-to-cell and animal-to-animal variation of gene expression with age in C. elegans. Here, I use quantitative microscopy to measure animal-to-animal and cell-to-cell variation of genes expression in middle aged C. elegans using fluorescently-tagged proteins and quantitative microscopy. In addition, I use the methodology of molecular cloning and transgenics developed by the Mendenhall lab to create new transgenic strains of C. elegans. These transgenic strains will be also be used for quantitative microscopic analysis. By examining existing strains and developing new ones, I will determine if increase of gene expression variation represents early event in the aging of C. elegans.

The nematode C. elegans is a common model organism for investigating cellular aging mechanisms and there is an extensive database of known genes which influence their lifespan. Protein kinases are regulatory enzymes which function in the cell as molecular switches. Here we use RNA interference to disrupt 11 classes of protein kinases to determine their role in 10 different C. elegans mutant backgrounds known to affect aging. With the included controls this involved over 120 individual lifespan experiments. In order to perform this many experiments we used a custom robotics platform called the Wormbot, a novel, high-throughput technique for measuring worm lifespans that our laboratory developed. We present here the results from 120 combinations of longevity mutants and inhibited protein kinases. The findings of this study may identify biochemical pathways or interactions which may play a role in the regulation of human aging or development of age related disease.

In microbiology, matrix-assisted laser desorption/ionization time of flight (MALDI/TOF) is a method used for the identification of a wide variety of micro-organisms such as bacteria or fungi directly from the selective medium used to isolate them. The technique uses laser energy absorbing matrix to create ions from proteins with minimal fragmentation. It is a three-step process where first, the microbial sample is mixed with suitable matrix material and applied to a metal plate. Second, a pulsed laser irradiates the sample, triggering ablation and desorption of the sample and matrix material. Finally, the analyte molecules are ionized in the hot plume of ablated gases and accelerated into the mass spectrometer. The mass spectra generated are analyzed and compared to stored profile using a dedicated software. Species diagnosis by this procedure is much faster, more accurate and cheaper than other procedures based on immunological or biochemical tests. Consequently, MALDI/TOF is becoming a standard method for species identification in medical microbiological laboratories. Bacteroides fragilis and Escherichia coli are two bacterial species that MALDI/TOF can accurately identify directly from the media they are growing on. Certain strains of enteropathogenic B. fragilis and E. coli ETBF and EPEC respectively, cause severe enteric disease manifesting by watery diarrhea and colic. On the other hand, non enterogenic strains of B. fragilis and E. coli such as NTBF and DH5 alpha are completely benign. Using MALDI/TOF technology and virulent and non-virulent strains of B. fragilis (BFT and BFT) and E. coli (EPEC and DH5 alpha), we hypothesize that a correlation between peaks and virulence proteins can be made conferring to MALDI/TOF the potential to predict virulence of bacteria based on the expression of proteins mediating virulence.

Different phenotypes of normal cells may influence genetic and epigenetic profiles and tumorigenicities of their transformed cells. The current study presents a comprehensive analysis of mutations in the whole mitochondrial genome for different immortalized (pre-neoplastic) cell types, which were derived from normal breast epithelial cells with two different phenotypes (stem vs non-stem). Duplex Sequencing, which shows the lowest error rates among currently available DNA sequencing methods, was applied to investigate rare and subclonal (low-frequency) mutations as well as homoplasmic (high-frequency) mutations. We found that the vast majority of mutations in the cells occur at low-frequency and are not detectable by conventional DNA sequencing methods. The most prevalent mutation types are C>T/G>A and A>G/T>C transitions. The overall frequencies of rare and subclonal mutations are significantly lower in stem cell-derived immortalized human breast epithelial cells (SV1) than in non-stem cell-derived immortalized human breast epithelial cells (SV22). The decreased mitochondrial mutagenesis of SV1 cells occurred mainly in the noncoding RNA region (rRNA and tRNA) of the mitochondrial genome. In addition, the predicted pathogenicity for rare and subclonal mutations in the tRNA region was significantly lower in SV1 cells than in SV22 cells. Our findings suggest that different phenotypes of normal cells lead to distinctive mitochondrial mutation profiles of immortalized cells. The identified mutation spectra and mutations specific to stem (versus non-stem)-derived immortalized cells have implications in characterizing heterogeneity of tumors and understanding the role of mitochondrial mutations in immortalization and transformation of human cells.

A person’s age is one of the greatest risk factors for many age-related diseases, such as Alzheimer’s disease, heart disease, and cancer. Developing interventions that target this common risk factor may allow us to slow down the progression of all age-associated diseases. One promising target for aging research is the mTOR (mechanistic Target of Rapamycin) signaling pathway, which is a master regulator of cell growth that allows the cell to fine tune its response to various environmental conditions. Inhibition of mTOR pathway has been shown to extend lifespan in yeasts, worms, fruit flies, and mice. In addition of being a target in aging research, the mTOR pathway is also a well-known effective target in some human cancers to prevent cancerous cells from dividing uncontrollably. We have previously developed a yeast-based system to identify mTOR inhibitors by comparing the growth of yeast strains that are differentially sensitive to mTOR inhibitors; a wild type strain, a tor1Δ mutant, and an fpr1Δ mutant. However, the system is not able to identify a few known ATP competitive inhibitors that have been shown to inhibit mTOR in human cells. This could be due to dissimilarities between yeast and human mTOR protein kinase domains. To address this issue, we are developing a humanized strain of yeast that should allow for identification of additional mTOR inhibitors that are effective in human cells. I am developing a novel hybrid gene containing the human mTOR kinase domain fused with the N-terminus of the yeast TOR2 gene. This new humanized strain will be utilized in our yeast-based assay with the goal of first detecting mTOR inhibitors known to be effective in human cells but unable to inhibit yeast mTOR. Successful completion of this goal will facilitate high-throughput screening of novel compounds that may have mTOR inhibitory and cancer fighting effects.

Methods to differentiate stem cells into cardiomyocytes have been well established. However, a limitation for the successful application of these cells for research and medicine has been their fetal-like phenotype with respect to cell size, contractility, calcium handling, metabolism, and electrophysiology. We sought to increase the maturity of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) through metabolic pathway regulation. We hypothesized that switching the main metabolic substrates from glucose to fatty acids, mimicking the switch from placental to breast milk nutrient consumption that occurs during development, will increase hPSC-CM maturation. RUES2 embryonic stem cells were differentiated into cardiomyocytes and then treated with base media with varying glucose and calcium concentrations and fatty acid supplementation. Using quantitative PCR to measure gene expression, we measured an inverse relationship between glucose levels and markers of cardiomyocyte maturation: increased expression of cardiac troponin I relative to skeletal troponin I isoforms (TNNI3:TNNI1), increased expression of metabolic (CPT1B, PPARGC1A) and electrical maturity markers (KCNJ2, RYR2). These maturation markers were not influenced with fatty acid supplementation but were enhanced upon the addition of thyroid hormone and dexamethasone. We further quantified nucleation and sarcomere spacing to assess structural features of maturation using confocal microscopy. To understand the mechanisms by which media nutrients and signaling molecules cause phenotypic changes we investigated the relationship between maturation and global epigenetic state. Western blot revealed increases in global acetylation levels, measured by histone H3 acetylation, linked to maturation signaling. These findings depict a direct relationship between glucose metabolism and the development of a mature phenotype in hPSC-CMs, mediated by epigenetic mechanisms.

Astrocytes are a type of glial cells in the central nervous system, play a critical role in protecting neuronal signaling by regulating brain homeostasis, synaptic plasticity and transmission, and blood brain barrier functioning in central nervous system. Accumulating evidence has indicated that abnormal behaviors of astrocytic functions, including astrodegeneration and astrogliosis, are implicated as the primary factors contributing to a number of chronic neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Kir4.1 is an inwardly rectifying K+ channel expressed on the projections of astrocytes, which serve important roles in the neuroprotective function of astrocytes, such as maintaining K+ homeostasis and regulating extracellular glutamate. Abnormal expression of Kir4.1 has been reported in certain neurodegenerative diseases, including Amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD), suggesting a vital role in the development of pathophysiology. However, the association between the molecular mechanism and expression of Kir4.1 and the underlying pathogenesis of AD and PD has been largely uninvestigated. In this study, we have had the critical opportunity to access human post-mortem brain tissue, provided by the University of Washington Alzheimer’s Disease Research Center, and conducted confocal microscopy studies. Through a quantitative immunofluorescense staining approach, we expect to demonstrate a distinct expression pattern of Kir4.1 in various brain regions of AD and PD post-mortem tissues when compared to control subjects. Determining the role this protein has in neurodegeneration may provide new insight into the development of therapeutic targets to ameliorate the progression of AD and PD.

Alzheimer’s disease (AD) is characterized as an irreversible, progressive brain disorder that gradually destroys memory and thinking skills, and eventually the ability to perform simple everyday tasks. Despite many decades of research focus on AD, the cause and mechanistic understanding of the progression remains enigmatic. Identification of amyloid beta plaques and hyperphosphorylated Tau in post-mortem analysis of brain biopsy is viewed as definitive for diagnosis of AD. Dr. Su-in Lee’s lab has developed a machine learning method that integrates brain tissue pathology metrics with gene expression analysis from the same patient samples. Their analysis identifies genes and pathways that are statistically-associated with the concordance of pathological AD phenotypes. Working with researchers in the Kaeberlein lab, we are using the nematode C. elegans to directly test the impact that these genes have on the progression of human tau-induced neuronal dysfunction and death. The Kaeberlein lab previously identified a subset of genes in complex I of the electron transport chain (ETC) that are highly correlated with human amyloid beta-induced paralysis. We are now extending that work by examining the relationship of these ETC genes in the lifespan and healthspan of human Tau-model nematodes. Our preliminary results suggest that reduction of Complex I activity increases the lifespan of human tau-models nematodes. We are conducting further studies to determine the replicability of these findings as well as disrupting ETC function with RNAi from other genes. Through directly testing nematode orthologs of human genes that are statistically-associated with AD neuropathology we will generate a better understanding of the cellular mechanisms that influence normative aging and Alzheimer’s disease.

Myocardial infarction (MI) is the leading cause of death globally. Methods to regenerate cardiac tissue after MI has focused on inducing proliferation in adult cardiomyocytes near infarcted tissue or injecting stem cell-derived cardiomyocytes with proliferative capacity into the infarcted tissue. However, optimal regeneration has not been achieved with these methods, as the mechanism behind adult cardiomyocyte proliferation is not well understood and proliferative stem cell-derived cardiomyocytes are phenotypically and functionally immature. Exploration of the mechanism of cardiomyocyte proliferation is therefore necessary to enable optimal regeneration of cardiac tissue and function and MI. We hypothesize that the sarcomere structure, the basic muscle unit of the cardiomyocyte, is the limiting factor in proliferation of cardiomyocytes. To investigate this hypothesis, we have performed a thorough characterization and comparison of stem cell-derived wild type cardiomyocytes (WTC-CMs) and troponin I double knock out cardiomyocytes (TNNIDKO-CMs) which have an incomplete sarcomere structure due to the lack of troponin I. After confirming TNNIDKO-CMs and WT-CMs only vary in their sarcomere structure, we developed a coculture platform to demonstrate the mechanical weakness of TNNIDKO-CMs sarcomere structure. We then performed proliferation assays utilizing multiple proliferation markers to observe if proliferation was higher in the TNNIDKO-CMs with the incomplete sarcomere structures. Preliminary results have shown that TNNIDKO-CMs are more proliferative than WTC-CMs, thus implicating that sarcomere structure plays a role in controlling cardiomyocyte proliferation. Successful characterization of TNNDKIO-CMs and their increased proliferative capacity will elucidate the sarcomere structure’s role in proliferation as well as develop a more comprehensive understanding of the underlying mechanism behind proliferation to help progress therapies for regeneration of cardiac tissue after MI.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The survival outcomes are poor for patients with relapsed and metastatic disease. Embryonal RMS (ERMS), a major subtype of RMS, is driven by genetic mutations in the RAS pathway. Cancer stem cells (CSCs) drive the process of self-renewal to recapitulate the heterogeneity of the cancer and are thought to be responsible for cancer relapse due to their resistance to conventional chemotherapy. In ERMS cells, a population of CSCs has been identified and is believed to play a critical role in tumor relapse and metastasis. A previous screen of all known human kinases identified candidates that played a role in regulating self-renewal of ERMS CSCs. I have prioritized two most promising candidates, ERN1 and MAPK10, for further characterization. I hypothesize that these kinases regulate the self-renewal capacity of CSCs but do not affect growth of non-CSC tumor cells. Using the gene-editing tool, CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9, I knocked out ERN1 and MAPK10 in ERMS cells and assessed their role in regulating ERMS CSC cell growth and self-renewal. To assess whether the effects of gene disruption are specific to the changes in the CSC population in vitro and in vivo, I used an ERMS CSC reporter cell line that specifically expresses Green Fluorescent Protein (GFP) in CSCs. The ERMS CSC reporter cell lines harboring ERN1 or MAPK10 gene disruption are subjected to self-renewal assays in cultured ERMS cells in vitro and ERMS xenograft model in vivo. My findings will provide new insights into the biological mechanism underlying as well as new potential targets against relapse and metastasis of ERMS.

Progeroid syndrome is a group of genetic disorders characterized by accelerated aging. Molecular mechanisms of progeroid syndrome include abnormalities in genomic stability, nuclear structure, lipid and carbohydrate metabolisms as well as mitochondrial functions. Genomic instability is a major category of progeroid syndromes in humans. Prototypical example is the Werner syndrome caused by the mutations in the WRN gene which encodes a DNA helicase. Cells derived from progeroid syndrome patients generally exhibit phenotypes of accelerated cellular senescence with a very limited replicative lifespan. International Registry of Werner Syndrome recruits progeroid syndrome patients from all over the world for the genetic study. Among the subset of those patients, a recurrent disease mutation has been identified in the POLD1 gene. The POLD1 gene encodes a catalytic subunit of polymerase delta, a major replication polymerase which is also involved in translesion DNA synthesis. The POLD1 mutation is known to cause MDPL (mandibular hypoplasia, deafness, progeroid features, and lipodystrophy) syndrome. In our study, cellular model of MDPL syndrome was generated by expressing mutant POLD1 cDNA in human fibroblasts via lentiviral system. Consequences of the POLD1 mutation are determined by various assays including cell growth, accumulation of DNA damage and sensitivity to genotoxic agents. The eventual goal is to develop therapeutic approaches that ameliorate cellular disease phenotypes. We plan to test candidate therapeutic approaches using established POLD1 cell lines and assay methods. More recently, a mutation in the SMAD4 gene, was identified in one progeroid patient. SMAD4 gene encodes a crucial member of the cellular transduction pathway. The wildtype and mutant SMAD4 are being expressed in human fibroblasts to determine whether cells expressing mutant SMAD4 exhibit phenotypes of accelerated senescence. This project would provide us with an understanding of recently identified progeroid loci and likely sheds a light on the previously unknown mechanisms of normal aging and age-related disorders.

 Alzheimer’s disease (AD) is a severe neurodegenerative disease and the most common cause of senile dementia in the United States. AD affects approximately 5.7 million Americans. Presently, there is no treatment for the disease and the mechanisms underlying the development and progression of the disease are not well understood. Human induced pluripotent stem cells (hiPSCs) are a promising technology for the study of Alzheimer’s disease pathogenesis, but current stem cell maturation methods, used to produce functionally mature neurons for accurate disease models, are too time-consuming to allow for the efficient study of disease mechanisms. Therefore, there is a need for a more rapid maturation method to produce cultures of functional neurons without the extended time commitment that is standard for common differentiation protocols. To achieve accelerated neuron maturation, optogenetic stimulation will be used to periodically stimulate neural stem cells according to an optimized stimulation protocol. To accomplish this, we have created a lentivirus using a construct that expresses the channelrhodopsin, Chronos, under an EF1α promoter, designed RT PCR primers to measure Chronos expression, and collected RT PCR data on transduced cells. This virus will then be used to transiently transduce hiPSC derived neurons that have previously been gene edited to express a NeuN (marker of maturity) RFP (Red Fluorescent Protein) tagged fluorescent reporter that will function as a cell maturity readout. This stem cell line will be used to develop an optimized protocol for the light driven maturation of stem cells in vitro. The maturity of the neurons will be measured using electrophysiology to evaluate cell function and RT PCR to monitor gene expression. The success of this project will allow for more efficient study of Alzheimer’s disease in stem cell based disease models, which could lead to the discovery of disease mechanisms, revolutionizing the understanding of this burdensome disease.

Alzheimer's disease (AD) is the most common cause of dementia, a general term for memory loss and other cognitive abilities. Although this disease has been a major research focus since the 1980s the pathologic mechanisms are still not understood, and therapeutic interventions have been ineffective. The most definitive method for classifying AD is through identifying accumulations of toxic proteins amyloid-beta and tau proteins in post-mortem brain tissue. Dr. Su-in Lee’s lab has developed a machine learning method that integrates the pathological tau phenotypes with gene expression levels in the same brain tissue. This analysis highlights the genes with expression level changes that correlate with the pathological protein aggregation phenotypes. For this proposal I will directly test the impact of these candidate genes on cellular pathologies resulting from aggregating human tau protein with a new C. elegans AD model in which human tau is expressed in the worm’s muscle. This tau expression will likely result in premature paralysis because previous nematode AD models with human amyloid-beta have shown this phenotype. The results of my genetic screening will lead to a better understanding of the human genes that are dysregulated in human AD brains and provide a basis for genetically-dissecting the pathways that influence the mechanisms of tau toxicity.

Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric cancer that affects soft tissue such as skeletal muscle and connective tissue. Currently, there is no effective treatment for patients with ERMS. Mutations in the gene PTPN11 are found to have cancer-promoting roles in leukemia, lung, and breast cancers, but its role in ERMS is virtually unknown. PTPN11 codes for the SHP-2 protein, which is a component of the RAS/MAPK (mitogen-activated protein kinase) signaling pathway. Abnormalities in this pathway are known to transform normal cells into cancer cells when certain proteins are upregulated. My central hypothesis is that PTPN11 promotes RMS tumor growth by allowing cells to proliferate and differentiate out of control. To test the hypothesis, I used CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas gene editing technology to disrupt PTPN11 gene function in two models. I designed constructs that express guide RNAs that target the PTPN11 locus in the zebrafish or human genome. If PTPN11 has a tumor-promoting role, I expect targeted disruption of zebrafish PTPN11, delivered via microinjection, to result in reduced RMS tumor formation and growth compared to zebrafish tumor with no PTPN11 gene disruption. In the human ERMS cell lines, I will use virus-mediated transfer to introduce the CRISPR DNA construct in vitro. My hypothesis will be supported if the cells harboring targeted disruption of PTPN11 have reduced growth and less self-renewal capacity compared to the control cells. My findings will elucidate the role of PTPN11 in RMS, which will allow further research into the potential therapeutic benefit of targeting PTPN11 in pre-clinical RMS models.

Neurodegenerative diseases, such as Alzheimer’s (AD), Parkinson's, and Huntington’s, affect millions of people. In AD, prior studies indicate the formation and accumulation of amyloid-beta proteins may play a crucial role in the pathology of the disease. The Herpes Simplex Virus (HSV-1) encodes an alkaline nuclease (UL12.5) known to cause degradation of the mitochondrial genome. HSV-1 infection has been previously associated with AD brain pathology. We hypothesize that UL12.5 activity in the brain may predispose an individual to amyloid-beta aggregation and AD neuropathology. Here, we controlledl the amyloid-beta protein aggregation using a degron attached UL12.5, which is induced by the plant hormone auxin through a molecular signaling pathway known as auxin-inducible degron. We have engineered an auxin UL12.5-degron construct in order to precisely control the temporal and cell type expression of UL12.5 in Caenorhabditis elegans (C.elegans). This construct was microinjected into the worms and by using auxin, we controleld the expression of UL12.5 and tested its effects on amyloid-beta and Huntington protein aggregation. Here, we have elucidated the relationship between HSV-1 infection, UL12.5 expression, and neurodegenerative disease which may form the basis of novel treatments.

Mitochondrial dysfunction, characterized by decreased efficiency of the Electron Transport Chain (ETC) and loss of structural integrity, is linked to cellular senescence, an irreversible end to the cell-division cycle that contributes to aging. Minimizing senescence through late age intervention may prevent aging tissue dysfunction. Systematic treatment of old mice with a tetrapeptide, SS-31 reduced mitochondrial dysfunction and senescence in kidneys. We hypothesized that a similar effect would occur in other organs, and with other mitochondrial targeting interventions. These interventions included the SS-31 tetrapeptide which interacts with mitochondrial cardiolipin to improve structure and function, and NMN (Nicotinamide Mononucleotide), which fuels the organelle for more efficient adenosine triphosphate generation through the ETC. We treated old mice at 24 months-of-age for 8 weeks with either SS-31, NMN, or both interventions combined. Control groups included young-untreated mice at 4 months-of-age and old-untreated mice sacrificed with the treatment groups at 26 months-of-age. By comparing tissues, including heart, kidney, liver, skeletal muscle, skin and brain, within individual mice, we were able to account for differing rates of aging between mice. To determine the relative levels of senescence and treatment response in each tissue, we used Immunohistochemistry to quantify known senescence markers p16 and HMGB1, and quantitative-PCR to measure p16 mRNA transcript levels. As expected, preliminary results from p16 staining showed higher overall senescence burden in old as compared to young mice. The staining patterns also revealed senescence susceptible cells, with majority of p16 positivity in liver satellite cells, glomeruli and tubules of kidney, fibrocytes of heart and myocytes of skeletal muscle. Overall, the kidney and liver had more p16 positive senescent cells than did heart and muscle. These tissues are currently under analysis to determine whether intervention treatments can reduce cellular senescence.

The human adaptive immune system is composed of B and T cell lymphocytes. Of the latter, these cells are further differentiated and identified by the presence of CD (cluster of differentiation) molecules. CD molecules are expressed on the surface of the cell and can range from receptors essential to the cell’s functionality to glycoproteins marking distinct stages in their maturation. For example, CD45RA is expressed on naïve T cells while the presence of CCR7 marks the change to effector memory cells. Flow cytometry can be used as a tool to aid in detecting multiple types of T cells in a peripheral blood sample both rapidly and accurately. The use of 10-color flow cytometry in the clinical laboratory can provide simultaneous detection of ten cell markers to help differentiate the populations of T cells in a patient. Seattle Children’s Hospital utilizes this technology to monitor immune status in patients with auto-immune disorders, immunodeficiencies and post transplantation. Extended T-cell immunophenotyping by flow cytometry is not a mainstream clinical test. Since only a few laboratories offer this service, samples are often received from sites outside the hospital. With distance comes the issue of peripheral blood stability, especially regarding the loss of CD markers among the T-cell population over time. This study will compare the stability of T cell subsets through peripheral blood samples collected in EDTA vs sodium heparin collection tubes through four days of daily T cell immunophenotyping testing, as well as to determine the last day of stability for each T cell subset. The results will help form the guidelines regarding specimen stability for transport and the availability of offering this test to both adult and pediatric patients across the globe and help aid the diagnosis and treatment of various diseases and conditions that these patients face.

Mutations in the ND2 gene (mitochondrially encoded NADH dehydrogenase 2) have been linked to a reduction in efficient energy production, shortened lifespan, progressive neurodegeneration, and mitochondrial diseases such as Leigh’s syndrome. The ND2 gene codes for the production of NADH, a crucial portion of the electron transport chain in cellular respiration. Previous studies have hypothesized that mutations within the ND2 gene are responsible for mitochondrial disease development. However, previous studies have not extensively investigated how genetic variation present in a population might affect the mutant ND2 phenotype. This study attempts to understand how nuclear genetic variation can ameliorate or exacerbate the effects of mutations in the mitochondrially encoded gene, ND2. To model genetic variation in a population, the project utilizes the Drosophila Genome Reference Panel (DGRP), a set of 200 fully sequenced, inbred lines with over 4 million single nucleotide polymorphisms (SNPs) throughout the genome. These SNPs are particularly useful for Genome Wide Association Studies (GWAS). To assess the effects other genes might have on the ND2 mutant phenotype, DGRP lines were crossed with a mutant ND2 line that expresses ‘bang sensitivity’, measured as recovery time following paralysis after being vigorously shaken in a test tube. Recorded progeny recovery times varied significantly among DGRP lines, suggesting that nuclear genetic variants influence the ND2 mutant phenotype. After conducting GWAS, a list of identified SNPs associated with varying recovery times was acquired. With further study, we hope to identify which specific genes interact with ND2, what function those genes have, and whether those genes might be part of a larger regulatory pathway involving the ND2 gene.

Alzheimer's Disease (AD) is the 6th leading cause of death in the United States. The rate of AD diagnoses has substantially increased in the last two decades. The direct causes behind the initiation of AD are still unknown; however, genetic heritability is known to be a significant risk factor. The goal of our research is to identify the genetic variants that directly influence resistance to the formation of amyloid plaques, which are believed to lead to the initiation of AD. To investigate this idea, we are performing a genome-wide association study (GWAS) using the model organism Drosophila melanogaster. Female flies expressing the human form of amyloid beta (Aβ) are crossed with male flies from each of the 200 lines that make up the Drosophila Genetic Reference Panel (DGRP), a collection of highly inbred lines. The complete genome sequence is known for each of these lines, enabling researchers to carry out GWAS to identify genes associated with variation in any trait of interest. In our case, the progeny of these crosses all express Aβ, but differ from each other with respect to their inherited DGRP genotype. These progeny undergo image analysis to measure the amount of degeneration caused by the aggregation of amyloid plaques in the eyes. Eye degeneration is scored on a five-point scale, from zero, indicating a wildtype phenotype, to four, indicating an extremely degenerated phenotype. Preliminary results show that there is variation in the level of resistance to the aggregation of amyloid plaques among the DGRP lines, which can be accounted for by the genetic variation among these lines. The ability to identify the single nucleotide polymorphisms and the resulting proteins that cause this resistance could be pivotal in identifying methods to prevent the initiation and progression of AD.

The lysosome is a critical organelle affected by the aging process. Changes to lysosomal physiology during aging and their effects on cellular function are poorly understood. Here, we study the lysosome-like vacuole of the budding yeast during replicative aging. Using a microfluidic device, we trap hundreds of mother cells and visualize them over the course of their replicative lifespans. Using strains with fluorescent protein tags we investigate protein abundance, intracellular physiological conditions, and organelle morphology. We find that during aging, the vacuole becomes progressively less acidic. Using a vma mutant as genetic model of vacuolar pH dysfunction, we find that loss of vacuolar acidity triggers a loss of respiratory capacity, loss of iron-sulfur cluster protein activity, and iron starvation response. We find that iron supplementation rescues both the loss of respiratory capacity and shortened replicative lifespan of vma mutant cells. We see that during aging, a similar iron starvation response occurs, correlated on a single-cell level with the loss of vacuolar acidity. We propose that the loss of iron-sulfur cluster synthesis capacity due to loss of vacuolar acidity is a conserved process underlying multiple aging phenotypes.

Understanding how genetic variation shapes phenotypic variation for complex quantitative traits is fundamental to developing more accurate disease prognoses and therapeutic interventions. Genes that are important in early development contribute to adult quantitative traits, such as height, vision, and health. The fruit fly Drosophila melanogaster is a model organism for studying complex traits, such as aging. Drosophila possesses many well-developed genetic tools and shares evolutionarily conserved age-regulating pathways with our species. One such conserved pathway is the mechanistic Target Of Rapamycin (mTOR) nutrient signaling pathway. Rapamycin is a specific allosteric inhibitor of mTOR signaling that extends lifespan in adult Drosophila melanogaster and delays development in larvae. However, the functional explanation for these effects is incomplete and a genetic association between development and lifespan is unknown. We used the Drosophila Genetic Reference Panel (DGRP), a highly inbred fruit fly population representing natural genetic variation, to measure rapamycin-mediated developmental delay. We used these data to carry out a Genome Wide Association Study (GWAS), and combined our data with data from a screen for the effects of rapamycin on lifespan in the DGRP, also carried out in our lab. GWAS analysis will help us to identify genetic variants associated with rapamycin efficacy and to discover novel variants associated with developmental timing. By connecting lifespan and development genetically, we identified shared candidate genes that modify these two very different molecular genetic programs. Accomplishing this is the first step towards identifying early life biomarkers that are predictive of rapamycin success as a longevity intervention in later years. These pharmacogenomic analyses advance a precision medicine approach where interventions are tailored towards genetic background to maximize human health.

This project examines reproductive output across different wildtype genotypes in the fruit fly, Drosophila melanogaster, and how it correlates with lifespan, activity and age-specific metabolite levels. To answer this question, we used the Drosophila Genome Reference Panel (DGRP), a collection of ~190 fully sequenced Drosophila strains that have been inbred to near homozygosity. Fecundity was measured as the per capita number of adult offspring produced by female flies over 48 hours when they are 4, 8, and 12 days old, using 3-5 replicate vials for each Drosophila strain. We have shown that there is highly significant genetic variation for reproductive output. Reproductive output declines with age in most but not all genotypes. The correlation between reproductive output and lifespan was not statistically significant, suggesting that there is not a direct trade-off between survival and reproduction in this population. A genome wide association study (GWAS) identified statistically significant single nucleotide polymorphisms correlated with variation in reproductive output, although most of them were located in genes of unknown function.

Numerous interventions and genetic modifications have been shown to extend lifespan across a diversity of species. However, these studies often assume that extended lifespan is synonymous with extended healthspan. Recent research in the nematode worm, Caenorhabditis elegans, has questioned this assumption, and suggests that increasing lifespan can prolong the frailty associated with old age. This is particularly important for humans, as increasing lifespan without a corresponding increase in healthspan could spell disaster. The majority of healthcare costs are associated with aging-related pathologies, and prolonging life without prolonging health could radically inflate these costs. To parse out the genetic relationship between healthspan and lifespan, we have turned to Drosophila melanogaster, a well characterized model organism for studies on the genetics of aging. We have collected lifespan data as well as multiple measures of healthspan across these genotypes, and found a strong positive correlation between lifespan and healthspan in these flies. This confirms the importance of lifespan as the primary parameter in aging studies, and suggests that genetic interventions increasing lifespan may generally be accompanied by an increase in healthspan.

High-density lipoprotein (HDL) plays an important protective role in development of atherosclerosis, one of the leading causes of cardiovascular disease (CVD) and death. Although HDL is generally believed to exert anti-inflammatory effects in cells, recent data have emerged showing that HDL can enhance the pro-inflammatory effects of inflammatory stimuli under some conditions. However, molecular mechanisms involved in the pro-inflammatory effects of HDL are not well understood. We hypothesized that HDL-mediated cholesterol depletion in macrophages drives the pro-inflammatory effect of HDL in an ADAM metallopeptidase domain 17 (ADAM17)-dependent manner. Bone marrow-derived macrophages (BMDMs) were collected from wild-type mice and from mice deficient in ADAM17 in hematopoietic cells. ADAM17 is a membrane-bound sheddase that cleaves extracellular parts of several membrane proteins. The BMDMs were pretreated with HDL for 18 hours before stimulation with lipopolysaccharide (LPS). At the end of the LPS treatment (10 ng/ml, 6 hours), BMDMs were collected and used for gene expression or protein analysis. We observed that HDL (100 μg/ml) exaggerated the response of LPS on tumor necrosis factor alpha (4-fold over LPS alone) and interleukin 1 beta (2.8-fold over LPS alone) gene expression in BMDMs. This effect was prevented by cholesterol loading of the macrophages. The pro-inflammatory effects of HDL were associated with increases ADAM17 gene expression (50% over LPS alone) and protein (~2.5-fold over LPS alone) levels. Moreover, deletion of ADAM17 in macrophages prevented the pro-inflammatory effects of HDL by inhibiting HDL-mediated cholesterol depletion. We also observed that BMDMs from ADAM17-deficient mice exhibit elevated expression of genes related to fatty acid and cholesterol synthesis. Overall, these data suggest that deficiency of ADAM17 alleviates the pro-inflammatory effects of HDL by preventing cholesterol depletion in macrophages. This research helps us to understand HDL’s functions in inflammatory cells, which become dysfunctional in several pathophysiological states, including diabetes and cardiovascular disease.

Sexual dimorphism is a characteristic of many organisms that is genetically encoded and typically studied as a binary trait. Biomedical research routinely categorizes subjects into “male” and “female”, and the resulting data are used to establish sex-specific measures of good health and disease. However, this practice overlooks the existence of intersexual phenotypes and oversimplifies the overlapping variation between sex-specific groups. The phenotypic expression of sex is directed by a complex orchestration of many genes. As such, it is reasonable to consider that there are more than just two discrete expressions of sex, and furthermore that the dimorphism of so-called sex-specific traits resides along a continuum. Metabolomics studies the small molecules that make up all the molecular building blocks in the body, and offers a unique opportunity to quantify sex variation that is morphologically invisible. Doublesex (dsx) is a gene that plays a pivotal role in the sex development of Drosophila melanogaster, and its absence results in an intersexual morphological phenotype. This study models sex in Drosophila melanogaster as a continuous trait by comparing metabolomic profiles for wildtype females, males and dsx null mutants using statistical analysis of metabolome data. While many studies have been conducted to understand the role of dsx in sex determination, to our knowledge no one has attempted to use this novel approach to quantify sex variation as a complex trait. Here we measure over 1000 metabolites in fly samples and test a hypothesis that some metabolites exhibit continuity between male, intersex and female phenotypes. Future work could explore the degree to which these metabolic sexual continuities exist in natural fly populations, and provide a powerful model to study factors that are influenced by sex differences more comprehensively.

Aging is the single largest risk factor for many diseases, including Alzheimer’s disease (AD). Therefore, many of the molecular mechanisms that cause aging may also contribute to the onset and progression of aging-related diseases. Our approach to tackling the progression of AD takes a biology of aging standpoint, where screening compounds that impact lifespan may help identify therapies for AD. For this project, we are focusing on compounds that are either FDA approved or naturopathic. This is not only a novel approach but also has the potential to accelerate clinical translation. This study uses a human Amyloid-beta (Aß) protein expressing C. elegans strain that becomes progressively paralyzed with age. We are also utilizing a novel robotic imaging system (the WormBot) to explore the impact of these compounds on the healthspan of the AD model strain. Recording motility behavior of the nematodes under each compound condition and translating the time-lapse image data into quantitative healthspan curves, we will determine how each compound impacts the progression of paralysis. These results will provide insight into mechanisms of Aß toxicity and could lead to promising treatments that increase the quality of life for Alzheimer’s disease patients.

Alzheimer’s Disease (AD) leads to a progressive loss of neuronal function and ultimately death. Age is the greatest risk factor for AD; thus, a compound that slows down the aging process can help delay AD. In the lab, we use a Caenorhabditis elegans (C. elegans) model of AD in which human amyloid beta (Abeta) is expressed in body wall muscle cells resulting in age-onset paralysis. When combined with a robotic image acquisition device (the WormBot), we can analyze 144 different treatments simultaneously. The drug Istudying is Alpha Lipoic Acid. It is an antioxidant and cofactor for metabolic reactions required for oxidative metabolism. It has been previously reported to increase lifespan in C. elegans. My project focus on determining if Alpha Lipoic Acid reduces paralysis in the Abeta model. I investigated the mechanism of action of Alpha Lipoic Acid using RNAi knockdown of specific genetic pathways that are known to modulate aging. Ultimately, I have determined the optimal dose and a mechanism of action.

Previous research has established extracellular vesicle (EV) signaling as a ubiquitous cell-signaling modality used by all cells. In this modality RNA, peptide, and protein signals are packaged together in small vesicles and directed to specific downstream cell targets. In addition to being necessary for healthy physiology, EVs can also contribute to pathological processes such as neurodegeneration in Alzheimer’s disease through carrying toxic proteins tau, Aβ and β-synuclein between cell types. Recent research in the Kaeberlein and Mendenhall labs has shown that when human tau is specifically expressed in neurons of C. elegans, there is a significant increase in HSP90 chaperone and proteostatic stress reporter expression in the intestine. I hypothesize that the induced human tau neuron-to intestine stress signal is conveyed by the extracellular vesicles. To determine the signal pathway, extracellular vesicle signaling was disrupted specifically in neurons of a transgenic nematode line with neuron-specific human tau and fluorescent intestinal proteostatic stress reporter (daf-21::GFP). RAL-1 is a small GTPase that has been shown to be necessary for EV signaling in the C. elegans hypodermis and intestine. Therefore, RAL-1 function is disrupted though knocking down neuron-specific expression with either a double-stranded RNA ral-1 construct or a dominant-negative form of RAL-1 The degree of daf-21p::GFP expression was compared between the reporter lines and those with disrupted ral-1 function. Many studies utilizing C. elegans have shown that neurons send cellular stress signals to other tissues which strongly affect lifespan, metabolism, and proteostatic stress. Therefore, the results from my experiments contribute to determining whether EV-signaling carries neuronal stress-signals induced by human tau and establish a methodology for using C. elegans as a model for studying this important signaling pathway.

Aging is driven by the time-dependent disruption of cellular processes.  Collectively these changes lower physiological resilience and increase the probabilitity of death.  As the population of elderly humans gets larger, studying pathways that mitigate age-dependent changes has taken on increasing importance.  The round worm Caenorhabditis elegans has proven to be a powerful model organism for studying the basic mechanisms of aging.  Here we have investigated a novel life-extending pathway acting in these animals that is triggered in response to mitochondrial electron transport chain disruption, a phenomemon that occurs naturally in humans with age.  The pathway is comprised of mitogen-activated protein kinase (MAPK) signaling cascade comprised of DLK-1, SEK-3, and PMK-3 and the downstream reporter gene tbb-6.  Our primary objective in this study has been to identify signaling factors triggered distal to PMK-3 activation that mediate life extension.  We have utilized several independant approaches including both targeted and unbiases genetic screens, as well as co-immunoprecipitation.  Among a family of 33 candidate bZIP transcription factors, we have identified six that are required for PMK-3 mediated life extension.  Using an ENU mutagenesis screen we have identified ten genetic mutations that trigger constitutive tbb-6 activation.  Sequencing tests suggest that these lines contain specifc allelic mutations that form on the X chromosome.   Finally we have generated transgenic strains containing tagged versions of PMK-3 and SEK-3.  We have succeeded in immunoprecipitating both proteins from whole worm lysates, in preparation for future mass spectral analyses.  Updated results from our studies will be presented.

Alzheimer’s is a progressive, age-associated, neurodegenerative disease characterized by memory loss, deterioration in thinking and reasoning skills and gradual loss of executive function. Two well-documented biochemical hallmarks associated with disease progression are the accumulation of extracellular beta-amyloid (Aβ) plaques and intracellular neurofibrillary tangles found in the brains of Alzheimer’s patients. One theory of the disease holds that aggregated proteins found in these two deposits inhibit protein degradation by the 26S proteasome, a large protein that is one of the central components of the ubiquitin-proteasome clearance system. This system, of which the proteasome is a key component, executes most of the controlled protein degradation in the cell. Importantly, impairments in proteasome function are associated with multiple neurodegenerative diseases. The overall goal of this project is to determine whether proteasome stimulators can delay toxicity associated with Aβ aggregation in a C. elegans Alzheimer’s disease model. C. elegans is a powerful model organism in biomedical research due to its ease of culturing, well-described genetics, gene homology and availability of transgenic tools. My project utilizes a C. elegans strain that expresses the full-length human amyloid beta peptide (Aβ_1-42) in body wall muscle cells. These animals become paralyzed when up-shifted to a restricted temperature due to aggregation of Aβ proteins in the muscle. To test the hypothesis that proteasome stimulators delay the progression of Alzheimer’s symptoms, I will treat animals with four drugs and measure the time of paralysis. Using a high-throughput drug screening system recently developed in our lab, I will treat animals with four proteasome stimulators or vehicle controls, in triplicate, and determine the onset of paralysis for each drug. These results will help shed light on the involvement of protein aggregation clearance in treating symptoms of Alzheimer’s in this model organism, and help inform future treatments for this and other neurodegenerative disorders.

As we look forward to a global increase in our aging population and rising burden of noncommunicable diseases worldwide, exploring future treatments for age-associated diseases such as cardiovascular diseases, neurodegenerative diseases, cancers, diabetes, and chronic respiratory disease are of increasing importance to public health. Some studies credit the increasing burden of age-associated disease, especially that in younger populations, to the abandoning of traditional cultural practices and their replacement by Westernized diet, lifestyle, and medicine. This study explores conserved pathways of aging in response to compounds and extracts recognized by indigenous and ancestral medicine from various cultures. Employing a novel image acquisition robot, lifespan and age-associated disease phenotypes of wild type C. elegans nematodes exposed to selected compounds and extracts are observed and compared to identify potential treatments for age-associated diseases. Through conversations with traditional healers, review of the literature, and exploration of historical texts 4 major medicinal plants of interest were identified. Bitter Melon (Momordica charantia), vijayasar (Pterocarpus marsupium), rooibos (Aspalathus linearis), and Alaskan blueberries (Vaccinium ovalifolium) are recognized in East Asian, Indian, South African, and Native American traditional medicine, respectively, as treatments for slowing aging and age-associated diseases. Our research investigates how these compounds affect the longevity of C. elegans, with a focus on identifying their interactions with conserved longevity pathways. Our results contribute independent data and analysis for underrepresented compounds in aging and longevity literature, inform future research on more complex organisms, and increase recognition of indigenous and ancestral knowledge in the field of aging and longevity research. In our evermore connected global society, recognizing and respecting knowledge from diverse and often marginalized cultures and sources is critical to progressing longevity research, promoting equity, and improving health in our communities.

The tumor microenvironment (TME) dictates the outcome of many immuno-oncology therapies for solid tumors. To better understand the dynamic milieu of the TME, new multiplexed, spatially-resolved histologic techniques are being developed. A key limitation to evolving these techniques is identifying specific and selective antibodies that perform well in an immunohistochemistry (IHC) platform. The over-arching goal of this project is to develop a flexible, high-throughput platform to empirically test the IHC staining characteristics of antibodies in formalin-fixed paraffin-embedded (FFPE) tissues with improved throughput and lower test volumes. Essential elements in our platform design include: 1) flexibility to test a variety of biologic materials, which is dependent on the test antigen, 2) compatibility with manual and semi-automatic tissue microarray (TMA) builder, 3) easy use for pathologic assessment, and 4) future compatibility with fully-automated tissue staining instrumentation. Using computer-aided design software, and a stereolithography printer, we prototyped a guide template to build recipient TMA FFPE blocks. To screen for a variety of antigens in TMA "cores", we prepared cell lines, and acquired mouse tumor xenografts and human tissues. Standard IHC techniques were used to screen hybridoma supernatants generated from mouse immunizations. We have designed and tested six different multi-well slide-based IHC screening platforms. The current format consists of a six by four array of 2 mm cores. We have screened antibodies from two different mouse hybridoma campaigns. Thus far, we have identified candidate IHC antibodies for exogenous epitopes to identify chimeric antigen receptor T cells used to treat solid tumors, and a fusion protein hypothesized to be an oncogene in pediatric liver cancer. This project developed a single-slide antibody screening prototype for IHC. The device offers the flexibility to test multitude of tissues, and is built with design considerations for future automated tissue staining compatibility.

The DNA damage checkpoint (DDC) is critical for survival as it responds to damage such as mutations or chromosomal rearrangement, and prohibits the cell cycle from proceeding through a failed replication. One of the many responses DDC activates is histone degradation. When wrapped around histone proteins, DNA is blocked from repair mechanisms. By reducing histone and nucleosome density, the cell increases DNA mobility and allows the DNA to be quickly repaired via homologous recombination. The cell does this by uncoupling histone transcription from the S-phase of the cell cycle, which is normally confined to this phase. When DDC is activated outside of S-phase, the cell can no longer produce histones to replace those it consumed and ensure proper chromatin packing after the DNA is repaired. According to published literature, uncoupling histone transcription from S-phase increases lifespan but should compromise the DNA damage response. However, it is largely unknown how this uncoupling affects the DNA damage response in different mutants. Our goal is to explore how this affects mutants with reduced DNA damage responses. Our mutants of interest are 1) rad52Δ, which reduces homologous recombination, and 2) lif1Δ, which reduces non-homologous end joining (NHEJ). Our protocol involves yeast (Saccharomyces cerevisiae) spotting and exposing yeast to different durations of high-intensity UV energy. We will compare colony sizes across different strains, dilution factors, and UV-exposure degrees. We expect to see strains with rad52 or lif1 deletion to have a lower growth rate compared to the wild type. Following this experiment, we will explore how altering histone transcription interacts with, and may enhance rad52 and lif1 in DNA repair. We hope to apply our findings to mammalian and human genes, and help to decrease the onset frequency of age-related diseases such as cancer due to genomic instability.

Cellular growth and aging requires constant replication of the genome. The processes of genome replication and segregation must be carefully regulated to prevent DNA damage which can cause cell death or oncogenic transformation. Damage can arise from errors in cellular processes, as well as environmental factors such as UV radiation or chemical agents. In mammals, the protein Retinoblastoma (Rb) prevents cells from beginning the process of DNA replication for cell division if internal conditions are not suitable for replication. This function is disrupted in virtually all cancers, and mutations in Rb have been widely studied as a result. Whi5 is the functional analog to Rb in S. cerevisiae. Whi5 activity is modulated by phosphorylation at 4 different sites, triggering Whi5 export from the nucleus and allowing transcription of DNA replication genes. Previous research suggested Whi5 export initiates irreversible progression through cell division. However, using single-cell observation technology, we show that Whi5 can re-enter the nucleus prior to cellular division, arresting the cell cycle. Cells that undergo Whi5 re-entry events exhibit a longer average replicative life span, counted in number of divisions. Additionally, cells without a functional copy of Whi5 have a reduced replicative lifespan. Our results suggest Whi5 may play a role in arresting the cell cycle in response to DNA damage to prevent the cell from making fatal errors during replication and ensure healthy aging. To investigate the role of Whi5 in response to DNA damage, I am using CRISPR gene editing technology to modify the phosphorylation sites on the Whi5 gene, modulating Whi5 activity. The ability of modified Whi5 strains to recover from UV induced DNA damage and their relative replicative life spans will clarify the role of Whi5 in DNA damage response. Our results may help us better understand the interactions governing Rb dysfunction in cancer cells.