Cells and organisms must adapt to a variety of conditions to ensure survival. These adaptations have led to the evolution of a set of genetic stress response pathways, many of which have co-evolved to affect longevity. One of these pathways is the hypoxic response pathway, which is important for the response to diminished oxygen and is controlled primarily by the hypoxia inducible factor-1 (HIF-1). HIF-1 is a highly conserved transcription factor whose stabilization promotes increased lifespan and healthspan in C. elegans. Despite HIF-1 being highly conserved, similar stabilization of HIF-1 in humans leads to von Hippel-Lindau (VHL) syndrome, a disease characterized by increased tumor formation. Although the effects of global activation of the hypoxic response are well-documented, the tissue specificity and important downstream targets necessary for benefits or disease remain largely uncharacterized. My project focuses on the proteins downstream of HIF-1 that act to affect health and longevity. Work in our lab shows that multiple genes downstream of HIF-1 in C. elegans are sufficient to increase worm longevity when overexpressed in the absence of HIF-1 stabilization. One of these downstream genes that modulate worm aging is flavin-containing monooxygenase 2 (FMO-2). FMO-2 is a phase 1 xenobiotic metabolizing enzyme that may also play a role in protein homeostasis. We have found that the expression of FMO-2 either ubiquitously or in the intestine and head excretory glands of C. elegans increases lifespan and healthspan. My project involves further characterizing the FMO-2 protein by testing its role in resistance to stress and improving healthspan. In addition, I am contributing to epistasis experiments to determine whether FMO-2 acts in any other aging-related pathways. In characterizing FMO-2 in this way, my project seeks to better understand this protein in the context of health and longevity.

Biomarkers capable of diagnosing and tracking the progression of Parkinson’s disease (PD) are urgently needed. Saliva has been explored as an alternative to a more traditional sample source such as cerebral spinal fluid (CSF), which involves painful and invasive collection processes, and blood. Indeed, PD patients typically have dysfunction of salivary production, indicating that the disease involves this organ system. In order to increase sample availability, many protocols use cotton or plastic to stimulate saliva production. However, this raises the question of how these materials affect salivary protein composition, specifically in regards to proteins of interest as biomarkers. In order to explore these effects I incubated various materials potentially capable of stimulating increased saliva production in saliva samples and measured the concentration of alpha-synuclein (α-syn), a protein believed to be important in PD pathogenesis and a possible biomarker, and total protein. By comparing the concentrations of α-syn, total protein, and the α-syn:total protein ratio between the various material groups and control (no material) groups I am able to determine whether the materials increased, decreased, or had no effect on protein concentration. It was hypothesized that all tested materials would decrease both α-syn and total protein concentrations by some degree due to protein attraction to the materials. The results of this study contribute to a clearer basis for salivary biomarker studies involving induction techniques, facilitating collection techniques in a clinical setting, particularly in older patients with decreased saliva production.

Age-related cardiac pathologies are currently endemic in most developed nations and account for a large number of premature deaths and huge medical expenditures. Although lifestyle-associated risk factors have frequently been correlated with risk of cardiac impairment, it is also clear that aging impairs heart function even in the absence of these risk factors. Caloric restriction (CR) is known to extend lifespan and offset age-related impairment in diastolic function in murine models. Rapamycin is a CR mimetic that also extends lifespan (when given throughout life) and is protective in models of cardiac hypertrophy and failure. However, the potential role of Rapamycin for treatment of impaired heart function in aging has not been established. To determine the effect of short-term Rapamycin treatment on cardiac aging, we monitored cardiac function in 25 months old C57BL6/CR mice fed for 10 weeks with a control diet or a diet containing 14ppm encapsulated Rapamycin. Analysis by tissue dropper imaging showed improvement in diastolic function in the treatment group as early as 4 weeks. Normalized heart weights were also lower in the treatment group compared to old control mice, suggesting that short-term sub-acute Rapamycin treatment can reverse cardiac aging and regress age-related cardiac hypertrophy. Proteomic analysis showed increased stability (half-life) of the cardiac global proteome, increased abundance of mitochondrial proteins, and decreased oxidative damage of proteins in the treatment group. Further analysis revealed metabolic remodeling in the glycolytic and TCA pathways that improve energy metabolism and cardiac function in 10 weeks Rapamycin treated mice. Data from murine models offer potent approaches that may be replicable in humans and may prove effective in management of cardiac and perhaps other age-related ailments. In this project, we both assist in preparing the control and treatment diets, mice handling, imaging preparation, tissues harvesting, data analysis, and experimental design for future experiments.

Barth Syndrome is a rare X-linked genetic disorder that can cause cardiac myopathy, muscle weakness, fatigue, immune dysfunction, and childhood mortality. Like many such diseases, Barth Syndrome is the product of a mutation encoding for a single protein, Tafazzin, a phospholipid-lysophospholipidtransacylase found primarily in cardiac and skeletal muscle, where it plays an integral role in modifying cardiolipin, a component of the mitochondria, or energy production organelles of the body. When Tafazzin, and consequently the modified form of cardiolipin are absent, the function of the mitochondrialelectron transport chain, the primary pathway by which the body converts food into useable energy, is reduced. We hope to rescue defective cardiolipin modification through Tafazzin protein therapy and learn more about tafazzin structure and function. To this end we used X-Ray Crystallography to obtain a three-dimensional electron density map corresponding to the diffraction pattern of the protein, based on the distance and angle between each atom. The determination of structural properties illuminated binding sites and chemical interactions in both functional and mutant Tafazzin. This was supplemented by the synthesis of enzymatically active and stable human and mouse Tafazzin using E. coli (BL21(DE3)pLysS). Overexpression of Tafazzin results in relatively pure inclusion bodies which we we isolated through protein metal affinity chromatography. We produced several protein coding variants of Tafazzin in order to study the enzymatic activity of different mutants. Understanding of structure-activity relationships in Tafazzin will direct future work in Barth Syndrome treatment by facilitating  in vivo studies of Tafazzin protein delivery.

The somatic mutation theory of aging states that aging is the result of accumulated DNA mutations in somatic cells, which lead to loss of physiological integrity through tissue and organ level dysfunction. In mouse model systems, decreases in lifespan through high mutation rates have been documented by creating mutator phenotypes in these organisms. Mutator phenotypes are marked by increased cellular mutation rates, and are known drivers of tumor development in human cancers. Mutator-driven adaptations occur in natural microbial populations. Mutators represent a unique opportunity to test the somatic mutation accumulation theory of aging due to their sustained high mutation rates. Nutritional status, particularly carbohydrate metabolism, influences lifespan in numerous organisms. In yeast, which preferentially ferments glucose to ethanol before switching to respiratory metabolism upon glucose depletion, the induction of respiratory metabolism is associated with increased lifespan. This study examines the effects of high mutation rate on yeast replicative lifespan and whether induction of respiratory metabolism is sufficient to change lifespan in organisms with increased mutation load. To do this, yeast defective for DNA replication proofreading, mismatch repair, and base-excision repair were grown under dietary restriction (reduced glucose) and in the presence of non-fermentable carbon sources. Then, mutation rates and replicative lifespan were assayed. Our results show that increased mutation rates correlate with decreased lifespan in strong mutators and that this decrease in lifespan can be partially suppressed through growth on limited glucose and non-fermentable media. These findings suggest that the metabolic state of the cell modulates the severity of mutator phenotypes, which has important implications for the role that mutators play in carcinogenesis.

Pericytes are mesenchymal cells that are attached to peritubular capillaries. The activation and differentiation of progenitor pericytes to myofibroblasts leads to matrix deposition, renal scarring, and is a major contributing factor leading to chronic kidney disease after injury. Connective tissue growth factor (CTGF) plays an important role in fibrogenesis by promoting the production and secretion of extracellular matrix components. The goal of this project is to dissect the contribution of CTGF/Wnt signaling pathways in pericytes during the development of kidney fibrosis and to examine the effect of using the canonical Wnt extracellular regulator Dickkopf related protein-1 (DKK-1) on CTGF/Wnt signaling. In-vitro signaling studies on purified kidney pericytes, migration assays, and immunological staining were used to characterize the roles of DKK-1 and CTGF in the regulation of renal fibrosis. Results show that CTGF induces Wnt/β-catenin signaling in pericytes. DKK-1 inhibits CTGF induced fibrotic gene activation, changes the morphology, and the capability of migration of kidney pericytes through JNK MAP kinase, and canonical WNT independent pathway.

Aging research depends on the use of model organisms to test hypotheses about the biological mechanisms common to many organisms. One commonly used model organism is the roundworm Caenorhabditis elegans. Unlike homeothermic mammals, C. elegans are poikilotherms, and as a result of a variable internal temperature, they also exhibit variable longevity based on the ambient temperature. Under standard laboratory conditions, C. elegans are commonly grown in incubators ranging between 15°C and 25°C, and lifespan varies inversely with temperature; worms live longer at colder temperatures. Our recent data show that the relationship between temperature and aging has a genetic component. By measuring the lifespan of worm strains with mutations in genes found in established aging pathways, we have identified mutations that have opposing effects on lifespan at different temperatures. For example, utilizing lifespan assays I have conducted, we have found worm strains that live longer than controls at high or low temperature, but not both, as well as strains that are short-lived at specific temperatures. These experiments suggest there may be overlapping but distinct mechanisms driving aging at different temperatures, and we are developing hypotheses to explain the temperature-dependent mechanisms of aging. Thus far, our data are consistent with the hypothesis that genes that contribute to protein homeostasis promote longevity at higher temperatures, whereas genes that limit effects of toxic compounds (both internally and externally produced) promote longevity at low temperatures. Understanding the temperature-dependent mechanisms of aging in C. elegans will advance the aging research field by extending knowledge on a widely used model organism and by testing how the mechanisms of aging and disease may be conserved in homeotherms. We hope our results will suggest how and why many genetic longevity pathways are conserved from worms to mammals despite drastically different life histories.

Tyrosinemia Type II is a genetic disorder caused by a mutation in the enzyme tyrosine aminotransferase (TAT), which leads to an elevated level of tyrosine in the blood. If untreated, individuals with tyrosinemia develop life-threatening clinical symptoms that include an increased tendency to bleed, damage to the nervous system, damage to the eyes, and organ failure. Unexpectedly, mutation of TAT in the nematode Caenorhabditis elegans results in animals that are long lived in comparison with wild-type (WT) worms. This study aims to elucidate the cellular mechanism and workings of TAT in the mitochondria, its role in the pathology of the disease, and its possible links with aging in C. elegans. Thus far, we have developed a simple method to measure the CO2 output of the worms and shown that the TAT mutant’s metabolism is about 50% compared to WT. This significant decrease in CO2 output was matched comparable to a strain deficient for mitochondrial Cytochrome C Oxidase, which served as a positive control. Cytochrome C Oxidase, or Electron Transport Chain Complex IV, is one of the three complexes that create the proton gradient inside the mitochondrial matrix upon accepting electrons. Notably, mutation of the gene acting downstream to TAT in the tyrosine degradation pathway, which causes Tyrosinemia Type III in people, or any other mutation in tyrosine metabolism that we have tested, causes no significant change in CO2 output of C. elegans. Furthermore, through our preliminary metabolomics assay we have identified three metabolites that are accumulated to extremely high levels in comparison to positive and negative controls. The future steps will include determining whether these metabolites play a direct role in the effects of TAT mutation on mitochondrial function and aging.

Affecting millions of people across the globe, the WHO deems myocardial infarctions (heart attacks) as the largest cause of death worldwide. The heart’s weak regenerative capacity leads to scarring of the dead tissue and a reduction in cardiac output that presents numerous health consequences. Tissue engineering is a promising outlook in restoring the structural integrity and contractile force of the damaged area. Recent findings have demonstrated that engineered heart tissues grown from stem cells can display significantly improved characteristics when subjected to mechanical stimuli. Inspired by these results, this project seeks to improve tissue performance even further by investigating a novel concept where stem cell-derived cardiac tissues are treated with the same mechanical profile experienced by native heart tissue. In order to simulate and implement these forces, tissues are grown and then anchored onto two posts that can vary in distance and stiffness. Upon contracting, post stiffness is increased providing a resistance to each beat. This resistance simulates the afterload stress that the ventricular wall contracts against to eject blood into the aorta. Next, the tissues are stretched by increasing the distance between each post. The stress produced by elongating the tissues simulates the preload stress exhibited by the ventricular wall as blood is pumped into the chamber prior to the next contraction. Finally, combining the stiffness (afterload) and stretching (preload) schemes allows the tissues to experience the entire force profile exhibited by the native heart. Simulating the cardiac mechanical environment gives the engineered tissues the stimulus to yield contractile forces and a cellular structure at par with their native counterparts. This investigation thus aims to advance regenerative medicine by bringing stem-cell derived cardiac tissues one step closer towards curing myocardial infarctions.

Clonal evolution is a key feature of cancer progression and relapse. As cancers progress they accumulate large numbers of mutations. Some of these mutations are present in all of cells (clonal mutations) and drive the malignant progress. We will ask if there are other mutations that are present in only a subset of cells within tumors; as the environment changes during therapy these could become new driver mutations and could be the source of resistance to therapy. The subclones with the best evolutionary fitness will, in time, come to dominate the cancer cell population. Studying the number and identity of subclonal mutations may help us to predict whether or not a tumor has potential resistance to different drugs. Our hypothesis is that the large amount of subclonal mutations within a cancer will increase the overall fitness of the tumor, by providing a greater diversity from which new phenotypes can arise. We will modify the newly developed sequencing methodology, termed Duplex Sequencing, to identify specific subclonal mutations present in colorectal cancers. By adding a barcoded, partially non-complementary adapters to both ends of an individual DNA molecules which allow mutations in both strands of a DNA duplex to be identified, this approach greatly reduces sequencing errors. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be identified as technical error. We believe that most colorectal cancers will have a complex subclonal structure and may show clusters of subclonal variants, including subclonal driver mutations. Furthermore, presence of a subclonal driver mutation may be an independent risk factor for disease progression. Our study will thus uncover patterns of mutational evolution in colorectal cancer, providing insights into its stepwise clonal evolution.

The mammalian Target of Rapamycin (mTOR) is a protein kinase that integrates signaling from growth factors and nutrient availability to regulate metabolism and cell growth. Reducing mTOR signaling with rapamycin extends lifespan in several model organisms such as yeast, flies, and mice, and rescues virtually all symptoms in a mouse model of Leigh syndrome, a neurodegenerative disorder that causes loss of motor function and early death. Previous observations from our laboratory suggest that rapamycin induces changes in the metabolism of mice modeling Leigh syndrome, favoring fatty and amino acid catabolism over glycolysis. We believe this shift in metabolism is, at least in part, responsible for the beneficial effects of rapamycin in normative aging. In order to test this hypothesis, we are examining the effects of daily rapamycin injections in 4, 12, and 22 months old mice. We performed baseline measurements of serum metabolites, forelimb strength, voluntary running, gait and balance, body composition, blood glucose, and hemoglobin A1c, and are currently administering 8mg/kg of rapamycin daily via intra-peritoneal injections for 60 days. During this period we are monitoring body weight and food intake daily, as well as blood glucose every 15 days. At the end of this period we will measure body composition, gait and balance, forelimb strength, and voluntary running again, while maintaining the mice under treatment with rapamycin for an additional 30 days. Finally, we will sacrifice the mice and analyze serum, liver, brain, adipose, and other tissues for the activation of metabolic pathways, metabolite composition, and signs of pathology. We expect rapamycin to improve most parameters studied and profoundly alter the metabolic profile of mice at all three ages. These results will ultimately allow us to investigate how modulating metabolic pathways can increase healthspan in mice and provide insight on how to promote healthy aging in humans.

The Rieske iron-sulfur protein (Rip1) is a nuclear encoded protein that assembles into the cytochrome bc1 complex of the electron transport chain in budding yeast, Saccharomyces cerevisiae. The iron-sulfur cluster bound by this protein facilitates the flow of electrons during cellular respiration in the inner membrane of the mitochondrial matrix. In the nematode worm, Caenorhabditis elegans, a mutation in a conserved residue of the Rieske iron-sulfur protein has been found that delays development and extends the lifespan of this organism. Mutagenesis experiments from our laboratory have identified suppressors of this C. elegans developmental phenotype that are due to secondary mutations in conserved residues within a flexible hinge region of the Rieske iron-sulfur protein. The primary mutation and secondary suppressing mutations were introduced to the yeast Rip1 gene through site directed PCR mutagenesis. Transgenic yeast have been created expressing the mutant forms of Rip1 homologous to those identified in C. elegans. The next stages of my project will entail the characterization of these mutations in yeast. I plan to assay the replicative lifespan, growth rate, respiratory efficiency, as well as sensitivity to variance in temperature, reactive oxygen species and carbon source in an effort to understand how this mutant’s lifespan extension fits into the larger picture of known aging pathways. The extrapolation of the mechanisms found in these model organisms serve as the basis for understanding our own complex aging process.

Leprosy, also known as Hansen’s disease, is a deteriorative skin condition caused by chronic infections of the bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Leprosy was once believed to be highly contagious, so patients with leprosy were quarantined and isolated to prevent further contamination within the population. In Korea, patients with leprosy were quarantined on Sorok Island where some patients received treatment for leprosy with the antibiotic, 4,4'-diaminodiphenylsulfone (DDS). Data collected on patients receiving DDS indicated that the average lifespans of the patients were longer than the average lifespans of the general Korean population suggesting that DDS may impact aging. Consistent with this idea, DDS has been reported to extend lifespan in the nematode Caenorhabditis elegans. We have confirmed these results in C. elegans, and the goal of this project is to identify the specific mechanisms and pathways through which DDS is able to extend lifespan. Our unpublished data support the model that one of the mechanisms underlying DDS’s effect on aging in C. elegans is activation of AMP- activated protein kinase (AMPK). We are currently working to understand how DDS activates AMPK, as well as the interaction between DDS and other known longevity pathways in C. elegans, such as dietary restriction and the hypoxic response. I am analyzing the mechanisms through which DDS affects aging by performing lifespan analysis on C. elegans and its mutants. By analyzing the effects of DDS on aging, we can get a better understanding of the mechanisms that control aging to eventually comprehend the pathways through which certain aging diseases, like Alzheimer's act.

Parkinson’s Disease (PD) is the second most common neurodegenerative disorder in the world, affecting nearly 7 million people globally and more than 1 million people in the U.S. Symptoms of PD typically include tremors, rigidity, postural instability and slowness of movement. These motor control deficiencies can be attributed to a decrease in the expression of the neurotransmitter dopamine and the loss of many dopaminergic neurons in the substantia nigra of the brain. To study PD a mouse model is used in which C57BL/6 mice are administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is a neurotoxin that inhibits complex I activity in the mitochondrial electron transport chain. Mice treated with MPTP exhibit PD-like symptoms, including the loss of dopamine producing neurons and physical motor control deficiencies analogous to PD. Specifically, we are interested in utilizing the MPTP mouse model to understand a mechanism by which the drug rapamycin might prevent or treat symptoms of PD. Rapamycin inhibits signaling of the mechanistic target of rapamycin (mTOR) pathway, which is a key regulator of many functions, including cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. Previous research by our lab has shown that rapamycin dramatically attenuates disease progression in a mouse model of mitochondrial complex I deficiency. Because PD patients also exhibit defects in complex I, we believe that rapamycin may alleviate Parkinson’s disease progression by a mechanism similar to the effects we have seen. Currently we are administering high doses of encapsulated rapamycin in mouse diet during the three weeks prior to MPTP exposure. The expression of PD-like biomarkers in rapamycin fed mice will be measured through immunohistochemistry and western blot assays and compared to a control fed group. We hypothesize that pre-treatment with high doses of rapamycin in the diet will have robustly beneficial effects in MPTP mice.

Polymerases epsilon and delta are responsible for replicating the leading and lagging strands of DNA, respectively. Accurate DNA replication occurs through Watson-Crick base pairing catalyzed by the enzymes’ polymerase domains, and also through the action of the polymerases’ exonuclease (proofreading) domains. The exonuclease domain of polymerase epsilon (POLE) has recently been reported to be mutated in ~4% of sporadic colorectal cancers and is associated with a high frequency of clonal base substitutions throughout the tumor genome. We hypothesize that the extent of mutational heterogeneity within a tumor will be predictive of the likelihood of resistance to any given chemotherapeutic agent and that the mutational heterogeneity due to mutations in POLE may also allow us to clinically stratify patents.The ultimate goal of the project is to identify POLE mutations in a tumor tissue, if applicable, and to find the quantity of mutations to compare with normal tissue and tumor tissue without POLE. I will utilize the newly developed Duplex Sequencing methodology to identify mutations that are present in a small number of cells within a tumor. By independently tagging and sequencing each of the two strands of individual DNA duplexes, this approach greatly reduces the number of errors. As the two strands of DNA are complementary, true mutations should be found at the same position in both strands. I will first identify tumors carrying somatic POLE mutations and use Duplex Sequencing to quantify the subclonal mutational load. My research team and I expect that the extent of mutational heterogeneity will be greatly increased in tumors bearing POLE mutations due to its impairment of proofreading capacity. For the symposium, we hope to present the quantity of sequencing errors in each of the tissues listed above or at least explain the process if we fail to collect the results in time.

The budding yeast, Saccharomyces cerevisiae is a single-celled eukaryote that provides many advantages for use as a model organism, including the ease of genetic and environmental manipulation. The replicative lifespan assay is a commonly used method for quantifying the lifespan of yeast by measuring the number of times an individual mother cell can divide. My laboratory has found that yeast lacking genes encoding subunits of the vacuolar ATPase (V-ATPase) have an extremely short replicative lifespan. The V-ATPase is a multisubunit protein complex that pumps protons across membranes and acidifies intracellular organelles, such as the lysosome-like vacuole in yeast. Lysosomes are organelles in eukaryotic cells containing digestive enzymes that break down and digest macromolecules and waste products. Yeast provides an ideal model for the study of disruption of the V-ATPase as they are able to survive loss of this complex, which is essential in most other eukaryotes. Vma21 is a protein required for the assembly of the V0 subunit of the V-ATPase. I have observed that vma21 mutants are short-lived, have a respiratory growth defect, and are sensitive to low iron levels. My experimental model demonstrates that supplemental iron and antioxidants, such as sodium ascorbate (vitamin C), dramatically increase the shortened lifespan of vma21 mutants. Supplemental iron also rescues the respiratory defect of vma21 mutants. My data supports a model where loss of V-ATPase function results in disrupted iron homeostasis leading to loss of mitochondrial function and shortened lifepsan, which can be rescued by supplemental iron and antioxidants.

Mitochondria, the energy producing organelles in eukaryotic cells, play a critical role in cell metabolism and regulation. Interestingly, mitochondrial dysfunction promotes lifespan extension in Caenorhabditis elegans. One proposed mechanism is that the induction of mitochondrial unfolded protein response (UPR^mt) increases the expression of mitochondrial chaperones, nuclear-encoded enzymes that assist in folding of misfolded or aggregated proteins, to maintain mitochondrial protein homeostasis. In an effort to elucidate genes involved in the UPR^mt pathway, we performed a genome-wide RNAi screen in transgenic animals expressing mitochondrial chaperone GFP reporter hsp-6_pr::gfp. Unexpectedly, we found that knockdown of genes involved in the pentose phosphate pathway (PPP) activates hsp-6_pr::gfp expression. The PPP is a metabolic shunt off of glycolysis that produces NADPH, a reducing equivalent used for glutathione reduction and lipid metabolism, and ribose 5-phosphate for nucleotide synthesis. In addition, knockdown of transaldolase, a gene in the reductive phase of the PPP, increases the lifespan of C. elegans. Through lifespan epistasis analysis, we found that transaldolase deficiency promotes longevity by a mechanism that is genetically distinct from known longevity factors such as the insulin-like/IGF-1 target (DAF-16), the hypoxia inducible factor-1 (HIF-1), AMP kinase (AAK-2), the UPR^mt transcription factor (ATFS-1), or the germline signaling (GLP-1). This suggests that the PPP inhibition promotes longevity through an uncharacterized pathway that may respond to mitochondrial dysfunction. In addition, we found that transaldolase knockdown alters expression of fatty acid desaturase, an enzyme that oxidizes fatty acid chains. We hypothesize that dysregulation of lipid metabolism by inhibition of the PPP may cause a defect in mitochondrial lipid composition, which may increase mitochondrial proteotoxic stress, inducing the UPR^mt. Understanding the genetics behind the UPR^mt and the PPP’s role in aging has enormous benefits to human health as both the PPP and mitochondrial dysfunction are highly correlated with many age-related diseases including cancer and neurodegenerative diseases.

 The mitochondria are cell organelles that perform important cellular functions such as energy production, regulation of the cell cycle, and cell death. Therefore, proper mitochondrial function is critical for healthy aging of an organism. Paradoxically, attenuation of mitochondrial function in the nematode worm Caenorhabditis elegans has been observed to extend lifespan. It has been proposed that induction of the mitochondrial unfolded protein response (UPRmt) is responsible for this enhanced longevity. The UPRmt is a response to proteotoxic stress in the mitochondria that results in induction of nuclear-encoded mitochondrial chaperones, proteins that stabilize and fold misfolded and aggregated proteins. However, the mechanism(s) by which proteotoxic stress is recognized in the mitochondria and signaled to the nucleus, resulting in the UPRmt response, remain largely unknown. To further understand the UPRmt pathway, we carried out a genome-wide RNAi screen to search for RNAi clones that specifically reduce UPRmt signaling under conditions of mitochondrial stress. To do this, we used a mutation in the electron transport chain (ETC) gene isp-1 to cause constitutive activation of the UPRmt GFP reporter, hsp-6p::gfp and screened for RNAi clones that reduced GFP fluorescence. We expect that positive hits in our screen will target signaling components of the UPRmt. In total, we identified approximately 60 RNAi clones that reduce the UPRmt response in isp-1(qm150) worms. Currently, we are re-testing positive hits after sequence validation and determining if the targeted genes specifically signal the UPRmt or non-specifically affect expression of GFP reporters. Once we identify genes that are specific to the UPRmt, we plan to test the role of these genes in mitochondrial function and lifespan. A better understanding of the UPRmt pathway could provide significant insight into mitochondrial biology, which impacts healthy aging and several age-related diseases.

One of the goals of modern longevity research is to find pharmacological means to increase lifespan and/or healthspan without deleterious physiological effects. Berberine is a yellow, bitter-tasting natural alkaloid with a long history in Eastern Asian medicinal use in treatments of a variety of age-associated diseases and infections. In human cell culture models, berberine treatment increases AMPK activity, resulting in delayed senescence through inhibition of the mTOR/S6 pathway. Here, we use the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans to investigate the effects of berberine on healthspan and lifespan. For the yeast S. cerevisiae, growth rates of haploid and diploid yeast cells, as well as chronological (a measure of population viability through time) and replicative (a measure of mitotic potential) yeast aging paradigms were assayed in the presence of concentrations of berberine ranging from 0.1 - 100.0 ng/uL. For C. elegans, organismal lifespan as well as various healthspan measures are being analyzed. Currently, we have found that concentrations of berberine greater than 10 ng/uL in haploids are sufficient to inhibit cell growth and lower concentrations (0.1-10 ng/uL) result in increased yeast viability when shifted to respiratory metabolism, a means of extending lifespan in yeast. Our lifespan studies are ongoing. Our experiments will provide valuable insights into the role of this long-used medicinal substance on cell aging and health using two important longevity models.

Patients with Alzheimer’s disease experience progressive loss of memory and thinking skills, gradual declines in the ability to carry out simple tasks and, in severe cases, full blown dementia. The brains of patients with Alzheimer’s accumulate extracellular plaques of a family of beta amyloid peptides (strings of amino acids cleaved from a larger protein called Amyloid Precursor Protein) in multiple regions of the cerebral cortex resulting in losses of synapses and neurons. Given the fact that beta amyloid peptides have been evolutionarily conserved, our group became interested in the potential normative roles of these peptides. Observations by a member of the Martin lab led to the hypothesis that small concentrations of beta amyloid may have a proliferative effect on cells. My project explores this idea with experiments that compare the proliferative effects of various members of the beta amyloid family and dose-response experiments that determine optimal concentrations for proliferative effects. In my experiments, various concentrations of beta amyloid 1-40 (and, as controls, comparable levels of a scrambled beta amyloid peptide 40-1) are added to a standard culture medium along with 10% embryonic serum (serum without detectible levels of beta amyloid peptides) and established required growth factors. The cells’ division rates within those two week periods are determined for individual clones though crystal violet staining. From these initial experiments, we have concluded that cells are indeed able to divide more rapidly in media that has beta amyloid 1-40 present and that further attempts to determine optimal concentrations should focus within the range of 1-10 nM and above 10 nM to the point at which we anticipate evidence of toxicity. Future experiments will probe changes in gene expression resulting from treatment with optimal concentrations of beta amyloid for the enhancement of cell proliferation.

After a myocardial infarction (heart attack), affected areas of the heart have limited function and capacity for regeneration. Injectable therapies with human embryonic stem cell-derived cardiomyocytes (immature heart cells derived from stem cells) can improve the function of damaged tissues, but graft-cell integration with the host is limited. Studying the migratory behavior patterns of these cells may provide insight to understand the basic biology of how the heart develops and may suggest new approaches to improve graft-cell integration. Toward this end, I have tested the hypothesis that a low-cost, high-throughput gap closure assay can be used to identify novel small-molecules that will promote the migratory behavior of human embryonic stem cell-derived cardiomyocytes. In particular, I developed a modified gap-closure assay that can be employed in the University of Washington's small-molecule screening core. Using a bottomless 384-well cell culture plate as the main platform, I used surface-modifying plasma lithography techniques to create a bottom surface that has alternating high- and low-affinity regions. Cells seeded into the multi-well plates with this patterned bottom initially adhered in the high-affinity regions and I used microscopy to monitor their migration into the low-affinity areas during exposure to various small-molecules introduced by robotics. To ensure these results were valid, I tested the assay using previously identified pro-migratory factors as a positive control. This assay can be a customizable and low-cost alternative to commercially available products, which is beneficial for studies requiring mass screening. Although this high-throughput assay was used to study the migratory behavior of stem cell-derived cardiomyocytes, it can be used for a variety of cell types.

The discipline of Pathology has an enormous scope and students are constantly challenged to determine which topics are essential for clinical patient care and for the National Medical Board exams. There is a significant gap between the material presented in pathology textbooks and the material outlined in medical board review books. In addition, much of the practical knowledge that is most useful for medical students and physicians falls in between this gap. To address this need, we created PocketPath, a web-based modular teaching system, to help students and physicians consume Pathology material more efficiently. PocketPath is designed in an easy to read format that delivers an optimal amount of high yield pathology content, which includes USMLE Step 1 Board Review-style questions and important research papers. Using student surveys, interviews and web usage data, we determined how students consumed PocketPath’s content and improved their pathology test scores. Initial data suggests that PocketPath may replace lecture time, promote active learning, and potentially change the way students prepare for Board exams.

Some symptoms of Parkinson’s disease, a movement disorder characterized by neuronal death in the basal ganglia, may result from or be exacerbated by low-level long-term exposure to manganese. Manganese-mediated impairment of normal glutamate uptake and metabolism may also contribute to abnormalities in the neuroprotective functioning of astrocytes, including manganese and other toxicant storage. Misfolding and aggregation of the protein α-synuclein, a product of the SNCA gene, has been implicated in neuronal degeneration in Parkinson’s disease, and various studies have proposed links between manganese exposure or defects in manganese metabolic systems and synuclein toxicity. Preliminary data from our laboratory suggest that knockout (KO) of SNCA in astrocytes attenuates some physiological effects of manganese exposure. The present study seeks to compare toxicant attenuation in SNCA KO and wild type (WT) astrocytes after increasing low-concentration 24-hour manganese pre-treatments. Imaging with fluo-4 dye will be used to observe glutamate-induced calcium waves in astrocyte cultures exposed to 1, 5 and 10 μM Mn2+. We predict significant deviations from a control waveform profile in both SNCA KO and WT cultures treated with higher concentrations of manganese. We further predict WT cultures to demonstrate significantly altered waveform profiles compared to SNCA KO cultures treated with equal concentrations of manganese.

Myocardial infarction and resulting heart failure continues to be the leading cause of death worldwide. The heart does not respond effectively to injury through tissue regeneration because cardiomyocyte proliferation after differentiation occurs at a greatly diminished rate. The goal of this research is to study stem cell-derived cardiomyocytes to better understand the mechanisms and possible controls of cardiomyocyte proliferation. Achieving this goal would allow for the intitiation of cardiomyocyte proliferation, which would better direct new technologies aimed at regenerating cardiac tissue after injury. RUES2 human embryonic stem cells are differentiated into cardiomyocytes using a directed differentiation protocol that manipulates the TGFβ and Wnt signaling pathways, by the controlled addition of Activin A, BMP4, chiron, and Xav signaling factors. After twelve to fifteen days, cells are harvested and stained for cardiac troponin T (cTnT), which is a protein present in the sarcomere of cardiomyocytes. Flow cytometery is used to identify cardiomyocytes expressing cTnT and indicate the purity of the differentiated culture. Cardiomyocyte proliferation is measured by exposing cells to Bromodeoxyuridine (BrdU), which is a thymidine analog that is incorporated into synthesized DNA. A double immunohistochemistry stain for Beta Myosin Heavy Chain (β-MHC) and BrdU is used to indicate proliferating cardiomyocytes. RNA is isolated from cardiomyocyte cultures and analyzed using quantitative real time PCR. Specific sequences of RNA present in cells can indicate relative gene expression, and allow for genes of interest to be monitored through stages of differentiation to determine peak expression. Ongoing experiments are investigating the activity of the Notch signaling pathway during directed differentiation of cardiomyocytes and its relation to cardiomyocyte proliferation. Results from these experiments will better indicate time points for enhancing cardiomyocyte proliferation using Notch signaling and help further characterize methods for induced proliferation as a potential therapeutic after cardiac injury.

Immunohistochemistry (IHC) is a valuable technique used to validate biomarkers, not only because it provides direct visualization of biomarker expression in histologically relevant regions of examined tissues, but also because validated IHC assays may be implemented readily into clinical practice. IHC assays have traditionally been semi-quantified by pathologists, but this method is significantly costly, inherently subjective, and produces ordinal rather than continuous variable data. Computer-aided analysis of digitized whole slide images may address these serious limitations. Using tissue microarrays (TMAs) representing 578 prostate cancer (PCa) specimens stained for estrogen receptor β2, I evaluated the degree to which data obtained using computer-aided methods correlated with data obtained by pathologist visual scoring. Digital images were obtained using a whole slide scanner. Under pathologist supervision I annotated TMA spots using annotation software. Images were preprocessed using the Genie Histology Pattern Recognition software suite to segment tissues into three user-defined Image Classes (tumor, stroma, glass). Malignant epithelial DAB staining was separately quantified within cytoplasmic and nuclear regions using the Cytoplasmic algorithm using a continuous variable metric that incorporates both the staining strength and the percentage of positive pixels in cytoplasmic/nuclear malignant epithelial areas. I compared the IHC intensity data derived from the Cytoplasmic algorithm with pathologist visual scores and identified high Spearman correlations. This study demonstrated that computer-aided methods to quantify IHC staining within carcinomatous areas of PCa tissue specimens can produce highly similar data to visual evaluation by a pathologist. This will allow further research studies to be performed using less time consuming and more technician based image anaylsis methods instead of costly pathologist consultations.

According to the World Health Organization, cardiovascular diseases are the leading cause of mortality worldwide. The low regenerative capacity of the heart renders it ineffective in healing itself after heart failure. Our lab has developed efficient methods to derive cardiomyocytes (cardiac muscle cells) from human embryonic stem cells (hESCs). However, it has become apparent that the cardiomyocytes derived using current differentiation methods exhibit immature morphological and functional properties. An important characteristic of mature cardiomyocytes is the presence of T-tubules, which are invaginations of the plasma membrane into the cell interior. These subcellular structures are critical for synchronized calcium release and contraction of mature cardiomyocytes. The protein caveolin-3 is a critical component of T-tubules, and thus its visualization can be used to assess the development of these important subcellular structures. I am currently evaluating the expression of caveolin-3 in hESC-derived cardiomyocytes using immunostaining techniques. For these studies, I expose the sample to anti-caveolin-3 as the primary antibody followed by a complementary secondary antibody conjugated to a fluorescent marker for visualizing the protein. Titration of several parameters, such as time of incubation and dilution of the antibodies allows for staining optimization. I have obtained detailed images of T-tubules aligning with striations in control heart tissue, but the staining is diffuse for the hESC-derived cardiomyocytes grown in standard 2D cultures. I am currently exploring whether this is a technical issue or the result of developmental immaturity. To assess the latter, I will stain hESC-derived cardiomyocytes that are more mature due to mechanical conditioning in 3D constructs. Optimization of this technique will facilitate our efforts to find novel agents that can accelerate the maturation of hESC cardiomyocytes. Finding ways to mature these cells will then provide a better platform for therapeutic applications such as drug screening, disease modeling and cardiac repair.

Aging is a degenerative process characterized by progressive deterioration of cellular components 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 mammals. The specific mechanisms of longevity in C. elegans have not been completely identified, and many pathways are poorly understood. Dietary restriction (DR) is one promising pro-longevity intervention, though its mechanism is still poorly defined. DR, a reduction in caloric intake in absence of malnutrition, extends lifespan in C. elegans and in a wide range of other species including yeast, flies, mice, and primates. To investigate the genetic determinants of the response to DR, we are performing a genome-wide screen using the Vidal RNA-interference (RNAi) Library, which contains 11,511 unique RNAi clones. The C. elegans are fed E. coli that express dsDNA that, when ingested, form ribonucleic-protein complexes that bind to and facilitate cleavage of target mRNAs; effectively preventing expression of specific genes. By knocking down specific genes within the C. elegans genome through RNAi, we are able to observe extension or reduction in longevity in response to DR. We have screened over 8,143 RNAi clones; 71 of which reduce lifespan under DR conditions and 85 of which increase lifespan. We are currently following up on our data by verifying the effect of these RNAi clones on lifespan under both control and DR conditions. We hope to verify how significantly these genes are affected by DR and follow up on those specific pathways to further investigate the interaction between DR and other potential longevity pathways. These findings will provide insight into fundamental mechanisms of aging and may facilitate the development of therapies to delay the onset and progression of age-related diseases in people. 

Chronic heart failure as a result of damaged myocardium affects millions of individuals around the world. Our laboratory has shown that human embryonic stem cell derived cardiomyocytes (hESC-CMs) are capable of improving contractile function and decreasing arrythmias in a guinea pig model of myocardial infarction (Shiba et al. Nature, 2012). We have also shown that hESC-CMs migrate in response to gradients of the soluble extracellular matrix protein Wnt5a in vitro (Moyes et al. Stem Cells Dev, 2013). We hypothesize that a gradient of Wnt5a, delivered by sustained release from gelatin microspheres, will drive the migration graft hESC-CMs towards healthy host tissue and increase the physical contact between graft and host cardiomyocytes. We fabricated gelatin microspheres by an oil/aqueous phase emulsion and controlled their diameter to range from 4-6 um in order to prevent capillary obstruction. We loaded Wnt5a into the microspheres overnight and characterized the release kinetics over 14 days through an ELISA assay of samples taken throughout that time. We verified the bioactivity of the eluted protein with a cell-based fluorescent response to a sample at a late time point. The migration-inducing effects of the sustained Wnt5a gradient on hESC-CMs can be modeled in future via in vitro, three dimensional collagen gel seeding experiments. The resultant in vitro data will inform future in vivo transplantation studies in which we will test the ability of Wnt-5a loaded microspheres to direct the migration of grafted hESC-CMs to locations where they can couple with host myocardium and more effectively contribute force generating units to a failing heart.

With ‘baby boomers’ reaching late adulthood, it is growing increasingly important for the scientific community to work toward treatments and potential cures for the diseases that affect this age group most often. Included in this category are the myelodysplastic syndromes (MDS), a group of related disorders involving the production of abnormal or insufficient quantities of myeloid blood cells. MDS is relatively prevalent in the older American population, yet much remains unknown about it. One treatment option is hematopoietic cell transplantation (HCT), in an effort to “reprogram” the body to produce healthy blood cells. The focus of this research study relates to the subgroup of MDS patients who undergo an HCT as part of their treatment but relapse 18 or more months later. The hypothesis was that there exist particular chromosomal abnormalities that manifest after transplantation which contribute to the propensity of MDS patients to late-relapse.
To test this, I worked with the Fang Laboratory at the Fred Hutchinson Cancer Research Center to optimize the process of extracting DNA from frozen patient cell pellets by manual and automated methods. We then analyzed paired patient DNA samples from before and after transplantation to look for signs of genomic evolution that could help explain why these patients exhibited such a long delay before relapsing. Chromosome genomic array testing (CGAT) was conducted on two patients’ paired samples. This method of analysis uses molecular probes to identify chromosomal abnormalities that may be causing disease. Both patients’ relapse samples showed clonal evolution, suggestive of disease progression and poor prognosis. These findings are promising evidence that the continuation of this research could help contribute to the discovery of MDS late-relapse markers and a more comprehensive understanding of these conditions.

5-fluorodeoxyuridine (FUDR), hydroxyurea (HU) and thymidine are all mutagenic agents that have been shown to alter cytosolic deoxyribonucleotide triphosphate (dNTP) pool levels in human cells. FUDR and HU affect the synthesis of dNTPs, whereas thymidine directly affects deoxythymidine triphosphate levels, one of the four types of dNTPs. Starving cells of dNTPs has a particularly large effect on cancerous cells, killing them more specifically than healthy cells since they multiply at a faster rate, making FUDR, HU and thymidine popular chemotherapy agents. Inhibiting dNTP synthesis therefore plays an important role in starving tumor cells of the proper resources they need to multiply. Studies have suggested that imbalanced cytosolic dNTP pool levels additionally play a role in the generation and accumulation of mutations in aging and large numbers of mutations found in human cancers. However, there have not been any concrete links between imbalanced mitochondrial dNTP pools and increased mutation frequency in mitochondrial DNA (mtDNA). The objective of this study is to determine whether FUDR, HU and thymidine alter mitochondrial dNTP pools, and whether the resulting imbalance affects mutation frequency in mtDNA. The method used is an extension assay with a single nucleotide overhang: a DNA duplex containing one 18-nucleotide strand tagged with Phosphorous-32, a radioisotope, and one 19-nucleotide strand. The samples are run through gel electrophoresis, which separates DNA molecules by size and allows us to determine the concentration of each dNTP in the treated cells. So far, I have found that FUDR and HU do in fact perturb mitochondrial dNTP pools and cause mtDNA mutations. My findings will help improve our understanding of the mechanisms of DNA mutagenesis and how mitochondrial dNTP pools contribute to the mutator phenotype in cancer and aging.

Aging is associated with increasing degrees of drifts in gene expression, thought to be epigenetic in origin. The resulting variegated gene expression would have the potential to lead to a loss of proliferative homeostasis, resulting in multifocal tissue atrophies and hyperplasias. Because of enhanced rates of DNA replication, the latter could lead to neoplasms via mutations in oncogenes and tumor suppressor genes. As an initial test of this hypothesis, I immunostained leiomyomas (a benign uterine tumor)of six patients for nuclear proteins, including p53, sirt 1, Cdk4 (Cyclin-dependent kinase 4) and p16/Ink4a (CDKN2A), and examined the degrees of variegated gene expression at the three sites: cells within benign leiomyomas of the uterus and of smooth muscle immediately contiguous to and distal to these lesions. The preliminary results showed that the majority of specimens gave results consistent with the hypothesis of clonal expansions derived from precursor populations of cells with more extensive variegation. There were several exceptional results, however, perhaps related to technical issues. These pilot studies encourage additional studies, including the use of alternative methodologies for the detection of neutral mutations associated with the predicted clonal expansions. In addition, these studies may provide new prospectives for diagnostic approaches to tumor formation in uterine.

The Kaeberlein laboratory uses the budding yeast Saccharomyces cerevisiae as a model organism in the study of aging. S. cerevisiae is a serviceable model organism for this task because its lifespan is relatively short and simple to assess. Furthermore, many genetic tools are available to study the effect of individual genes on lifespan. Two experimental paradigms have been extensively used to quantify lifespan in yeast: chronological and replicative. In the chronological lifespan model, cells are maintained in a non-dividing state and viability is determined over time by assessing the ability of cells to re-enter the cell cycle upon exposure to growth promoting conditions. In the replicative lifespan (RLS) model, mitotically active cells are incubated until they divide and produce newly budded daughter cells, which are then micro-dissected away from their mother cells and counted. This process is repeated until daughter cells are no longer produced by the mother cell, and the total number of daughters produced prior to irreversible cell cycle arrest is quantified. After analyzing numerous strains of S. cerevisiae, many different genes and environmental conditions that modulate replicative lifespan have been identified. Importantly, many longevity modifiers identified in yeast have been found to be conserved in higher eukaryotes.

One goal of aging research is to identify genes that modulate aging, in order to eventually develop therapies that delay the onset of age-associated diseases. In particular, genes that control mitochondrial function appear to play an important role in aging. Although severe mitochondrial dysfunction causes childhood diseases such as Leigh syndrome, there is growing evidence that moderate mitochondrial stress can extend lifespan in yeast, worms, fruit flies, and even mice. In the nematode Caenorhabditis elegans, a long-lived mitochondrial mutant called isp-1(qm150) has a defect in the Rieske iron-sulfur protein. The Rieske iron–sulfur protein is a primary electron acceptor located in complex III of the mitochondrial electron transportation chain. Complete RNAi knockdown of isp-1 results in larval arrest, while partial knockdown extends lifespan, further supporting the idea that milder forms of mitochondrial stress can increase lifespan while severe mitochondrial stress is detrimental. We are conducting a mutagenesis screen to identify genetic suppressors of the larval developmental arrest induced by isp-1(RNAi). Through exposure to EMS/ENU mutagen, wild-type worms will have random mutations generated throughout their genomes. These mutants will then be screened for the ability to develop on isp-1(RNAi) . We speculate the novel suppressor mutations from the screen may affect proteins that interact with ISP-1 or cause mutations that bypass the need for ISP-1 in the mitochondria. Identification of such suppressors will help to clarify the mechanisms linking mutation of isp-1 to development and aging. The isp-1 gene and its function in the electron transport chain are conserved in humans. Therefore these studies may be relevant for human aging and disease.