Genomic engineering has a wealth of possible applications, from curing genetic disease to curbing the spread of malaria. Homing endonucleases (HEs), which cut DNA at long, specific DNA target sites, are useful for genomic engineering because of their high site specificity. However, to use HEs for engineering, we must be able to modify their target site. Though this can be done with current computational and experimental protein engineering techniques, some HEs are resistant to those approaches. Therefore, we are working to develop a general methodology to modify the site specificity of HEs, based on a new synthetic biology technique called phage-assisted continuous evolution (PACE). PACE is a continuous evolution system in which the evolution of a desired biomolecular activity is linked to the reproductive cycle of phage particles containing the evolving gene(s), and should allow us to develop proteins or nucleic acids with almost any desired activity or specificity. We have built an automated system to keep the growing host cells and phage at a constant cell density and temperature, which is required for PACE. We are also working to design a mechanism to link HE DNA binding activity to phage reproduction by engineering plasmids in the host cells and phage. In the future, we will be refining the hardware set-up and protocol even further, and looking for ways to modularize the PACE system.

The formation of amyloid ß (Aß) plaques, resulting from the inappropriate aggregation of the protein beta-amyloid, is a major pathology associated with Alzheimer's disease (AD). One potential therapy for Alzheimer's disease is to reduce cerebral Aß plaque deposition through stem cell therapies. We hypothesize that myeloablative therapy, using lethal irradiation, as well as non-myeloablative bone marrow transplantation, using non-lethal irradiation, will result in a reduction in Aß plaque accumulation in the cerebrum. To test the therapeutic potential of myeloablative and non-myeloablative bone marrow transplantation, we transplanted bone marrow into either lethally irradiated AD (APPswePSIdeltae9) mouse model recipients (myeloablative therapy) or non-lethally irradiated AD mouse model recipients (non-myeloablative therapy). The number of plaques present and the total plaque area are quantified using ImageJ software. Initial immunofluorescence quantification has shown that non-myeloablative bone marrow transplantation reduces total Aß plaque area in the cortex by 33% when compared to non-treatment controls. To further assess the therapeutic potential of both myeloablative and non-myeloablative bone marrow transplantation therapies, additional immunofluorescence, as well as immunohistochemistry, quantification of Aß plaque deposition will be performed. These preliminary results suggest non-myeloablative bone marrow transplantation is a potentially effective stem cell therapy in treating an Alzheimer's related pathology.

Aging is a process that causes decline in normal biological function, eventually leading to disease and death. Aging is influenced by a number of factors, including stress-response pathways such as the hypoxic response. Over-expression of the hypoxic response pathway regulator, Hypoxia-inducible factor (HIF-1), increases lifespan in the nematode Caenorhabditis Elegans. Under atmospheric oxygen, HIF-1 is targeted for degradation by the Von Hippel Lindau (VHL-1) tumor suppressor protein, whose deletion increases nematode lifespan through increased HIF-1 activity. Since knocking out VHL-1 in mammals is known to cause disease, it is important to better understand the hypoxic pathway in nematodes to develop targeted therapies for age-related diseases in humans. The goal of this research is to determine the tissues and genes downstream of HIF-1 that modulate aging in C. elegans. To determine tissue specificity, we have developed vectors designed to reintroduce VHL-1 into VHL-1 knockout worms under the control of tissue specific promoters. Lifespan and other age-related parameters will be measured in these transgenic animals and compared to that of control groups. To further test tissue specificity, we are also developing vectors to overexpress a mutated non-degradable form of HIF-1 in specific tissues in wild-type worms, essentially the reverse of the reintroduction of VHL-1. To test for genes downstream of HIF-1 important for longevity, we have performed a screen and identified several genes that are important for HIF-1-induced lifespan extension. Our experiments will examine whether C. elegans lifespan is increased by the over-expression of these proteins, which would then suggest further characterization and a search for conserved proteins. Together, the results from these experiments will provide important information on how we might utilize the hypoxic response pathway to improve human health.

The longevity of an organism is influenced both by internal (genetic) and external (environmental) factors. With respect to internal factors, a significant effort is being made to identify novel pharmacological agents that extend lifespan by targeting genetic pathways with a defined role in the aging process. On the external side, the molecular mechanisms responsible for the positive influence of environmental interventions, such as dietary restriction and mild stress, are being widely explored. The environment experienced by humans in modern societies already contains countless compounds that may influence longevity. Understanding the role that the most prevalent of these compounds play in the aging process will be important to predicting and interpreting the outcome of introducing new interventions. Caffeine is the most widely used psychoactive drug worldwide. Prior studies indicate that caffeine may positively impact age-associated neurodegenerative diseases, such as Alzheimer’s. In the nematode, Caenorhabditis elegans, we observe that low concentrations of caffeine extend lifespan in worms in a temperature-dependent manner. Initial evidence from experiments combining caffeine with previously identified aging interventions suggests caffeine may increase life span by reducing signaling through the insulin/IGF pathway. We have also shown that caffeine delays pathology in a worm model of polyglutamine disease. Additionally, caffeine and dietary restriction by bacterial deprivation show non-additive life span extension, suggesting that they impact aging through a similar mechanism. Our initial effort is focused on characterizing the effect of caffeine on longevity and health span, and exploring the interplay between caffeine and other aging interventions.

Significantly reducing caloric intake without malnutrition (calorie restriction) has been shown to extend lifespan in many organisms including yeast, nematodes, and mammals. Since calorie restriction (CR) can also result in the extension of healthspan, CR or CR mimetics may be useful in the prevention of aging related disorders such as cancer, neurodegenerative disease, and cardiovascular disease. However, before long-term CR or CR mimetics can be considered safe for humans, more information is needed on the molecular mechanisms responsible for the effects of CR. In addition, since long-term voluntary CR may prove difficult to endure in humans, the consequences of reinstating a normal or ad libitum (AL) diet after prolonged CR must be investigated. We hypothesized that switching back to an AL diet after prolonged CR would reactivate pro-aging molecular pathways, such as the target of rapamycin (TOR) pathway. To test this, we obtained kidney tissue from aged F344 rats that had been subjected to long-term CR. Some of the long-term CR treated rats had been gradually switched to an AL diet and the rest had been maintained on the CR diet. Proteins were extracted from the kidney tissue and then analyzed by Western blot. Switching to an AL diet after long-term CR resulted in a significant increase in the phosphorylation of downstream signaling proteins in the TOR pathway in kidney tissue. In addition, after switching to an AL diet, there were significant changes in autophagy, which is regulated by the TOR pathway, as indicated by increases in the autophagy markers LC3 and p62. These results indicate that reinstating an AL diet after prolonged CR results in an increase in the activation of the pro-aging TOR pathway. This suggests that the TOR pathway may regulate some of the physiological effects of reinstating a normal or ad libitum (AL) diet after prolonged CR.

The mitochondrial unfolded protein response (mitoUPR) is a protective mechanism that is activated when the mitochondria experience stress. mitoUPR signaling has been linked to systemic inflammation and the innate immune response in multicellular organisms as well as being an important factor involved in the etiology and progression of many diseases, especially mitochondrial diseases such as Leigh Syndrome, and in aging. We are using HSP60 and HSP6 GFP reporter expressing Caenorhabditis elegans (C. elegans), which express green fluorescent protein when the mitoUPR is activated. By specifically inhibiting and altering mitochondrial function with different drugs, we hope to identify the signaling components of the mitochondria that induce (or inhibit) the protective mechanism. In addition, we are constructing HSP6 and HSP60 expressing plasmids to be taken up by the bacteria that the C. elegans eat. By feeding HSP6 and HSP60 to the worms we can determine if the mitoUPR can be induced through exogenous heat shock protein uptake in the gut. We are also feeding the C. elegans different strains of bacteria to observe the effect of how different bacterial proteins can affect the strength of mitoUPR induction and whether the innate immune response can induce the mitoUPR. From this data we hope to gain a better understanding of the mitoUPR, and configure a model on how this mechanism is expressed.

The complexity of the factors underlying the aging process and difficulty in categorizing definitive aging phenotypes complicates efforts to elucidate the pathology of aging. One approach toward understanding human biology is to look to model organisms as simpler starting points. Specifically, we can ask which aging genes have counterparts in more complex organisms and, of those, which also influence longevity. The Kaeberlein lab is in the process of completing a genome-wide screen of the budding yeast Saccharomyces cerevisiae for genes that alter its lifespan. The more difficult challenge is to piece together the pathways connecting the myriad products of these genes into a cohesive model for their effect on the aging process. A pathway of particular interest is the mitochondrial unfolded protein response (mtUPR). This stress response pathway is activated by accumulation of unfolded or misfolded proteins in the mitochondrion. After stress, signaling from the mitochondria to the nucleus can increase mitochondrial chaperone presence to promote proper protein folding. This pathway might play a role in some of the lifespan extending effects of the genes identified through the genomic screen, and there is evidence that some mechanisms of longevity in other organisms also involve the mtUPR. Our goal is to develop a GFP reporter system for the yeast mtUPR and investigate mtUPR activation in the lifespan mutants already identified by the lab. By creating a yeast GFP-promoter construct, we will be able to conduct experiments using fluorescence microscopy and flow cytometry to quantitatively determine mtUPR activation for a variety of genotypes and environmental conditions.

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

Glioblastoma Multiforme (GBM) is an aggressive type of primary brain tumor, which is treated by surgery followed by chemotherapy and radiation. A majority of cases prove to be highly resistant resulting in uniform fatality. At present, research has been moving towards developing a patient-centered model of treatment that is developed by finding the unique characteristics of a patient's tumor growth kinetics and response to therapy. GBM is a very heterogeneous disease despite having a generally poor prognosis. A promising method of investigating this heterogeneity has been through finding patterns in the molecular-genetic makeup of these tumors. Large-scale mathematical analysis on data from open-source repositories such as The Cancer Genome Atlas (TCGA) has already led to the discovery and validation of four GBM subclasses which point to characteristic areas of origination in the brain as well as differential growth behaviour. A limitation in this strictly data-oriented approach is the lack of meaningful clinical perspective applied which has resulted in the findings only being directed towards gauging patient survival. The focus of our research has been the discovery of information about individual characteristics of patients tumors that may have more clinical significance for each patient. Our hypothesis is that there is a correlation between the different biological subtypes classified by cluster analysis and the different kinetics of tumor growth measured by imaging. This work will be done by developing tools to investigate the genetic changes that may be driving different kinetics of disease progression (the proliferation and migration rates of the tumor cells) measured from actual patient imaging. This approach is guided by the belief that individual patient tumor biology can be used to inform the data analysis techniques and ultimately bring us closer to the goal of identifying the most discriminating treatment to effectively treat an individual's cancer.

Gliomas are invasive, fatal brain tumors that can be detected using magnetic resonance imaging (MRI). However, MRI does not reveal the entire size of tumor. By using a mathematical model developed by Kristin R. Swanson and colleagues, glioma growth can be quantified from the diffusion rate of the tumors cells, the net proliferation rate, and the carrying capacity of the tissue. These parameters are unique to each patient and can be determined by measuring the tumor volume from different MRI modalities as they change over time. Our lab collects data from patients consented to our study and records information about what treatments a given patient receives, since this can affect how the image appears. In my role as a member of the measurement team, I examine patient images to determine the extent of tumor burden on a given date. To obtain these measurements, I use Calcmri, a MATLAB program developed by our lab. Depending on the modality, enhancement on MRI slices can indicate tumor volume as well as other fluids. Calcmri can adjust the intensity threshold at which MRI enhancement will appear, which allows me to isolate the tumor and specify a region for the program to measure. I measure a series of two-dimensional cross section images in order to calculate a combined into 3-dimensional volume. These measurements are used to model the growth of the tumor, “in silico”. Our lab uses information from these models to discover clinical applications, such as to suggest more accurate treatment dosages and to predict life expectancy, which follows an overall goal to better capture the extent and invasion of tumor burden than what appears on MRI.

The study of chronological and replicative aging in Saccharomyces cerevisiae has provided both post-mitotic and mitotic aging models in this single-celled organism. In chronological aging, lifespan is determined by the length of time a yeast cell can survive in a non-dividing state, whereas in replicative aging, the lifespan is determined by the number of daughter cells produced by a mother cell prior to irreversible cell cycle arrest. The primary goals of this study were to observe how the chronological aging of yeast cells influences replicative lifespan (RLS) and to determine whether changes in the extracellular environment that influence chronological lifespan (CLS) would also influence subsequent RLS. By buffering the pH of the medium to 6.0 or reducing the concentration of available glucose (dietary restriction), we show that these manipulations were able to suppress the reduction in RLS associated with chronological aging, when compared with cells aged in synthetic complete medium with 2% glucose. The production of ethanol and acetic acid due to yeast fermentation may be responsible for cellular damage leading to reduced RLS, and it can be inferred that by buffering the pH to 6.0 or dietary restricting the cells, this damage is attenuated and replicative capacity is increased following chronological aging.

Hyperlipidema is associated with the formation of vascular plaque and development of atherosclerosis. Recently, hyperlipidemia was implicated in macrovasculature dysfunction including decreased nitric oxide production, angiogenesis, and pericyte density. Consequently, macrovascular dysfunction has been implicated in compounding the hemodynamics of atherosclerosis. However, the effects of hyperlipidemia on the microvasculature, an important component of vascular tone, has not been carefully examined. Apolipoprotein-E (ApoE) is a class of apolipoprotein found in chylomicrons and lipoproteins, which bind to specific receptors in both liver and peripheral cells. Previous literature has implicated the absence of ApoE in hyperlipidemia and atherosclerosis. Due to ApoE’s known effect on blood cholesterol levels, and the genetic homology of the ApoE locus between mammals, ApoE knock out (ApoE KO) mice are often utilized as a model organism for hyperlipidemia. My previous work with The Reyes Lab has demonstrated, through histological quantification, that the number of capillaries in the hearts of ApoE null mice are significantly reduced compared to wild type mice. In contrast to a decline in capillaries, the number of venuoles and coronary arteries, identified by the presence of a smooth muscle layer, remained unchanged between the two groups. In turn, our findings indicate that the decline of cardiac vasculature in ApoE animals occurs within the microvasculature. Herein I present our experiment aimed towards elucidating the mechanism of microvascular decline in ApoE animals. Specifically, I tested the hypothesis that microvascular decline is a result of endothelial cell dysfunction, attributed to elevated concentration of lipids and cholesterol in the circulation. In order to test this, wild type microvasuclar heart endothelial cells were exposed to hyperlipidemic serum and their response analyzed for nitric oxide synthase activity and the presence of reactive oxygen species. These studies will shed light into possible mechanisms of hyperlipidemia induced microvascular dysfunction.

Organisms have evolved specific mechanisms to respond to molecular and environmental stress. These stress-response pathways are evolutionarily conserved and can modify lifespan in model organisms, making them popular targets of aging research. Using the nematode Caenorhabditis elegans (C. elegans), we have found the loss of important genes in stress-response pathways can cause a phenomenon called a vulval integrity defect (Vid). Worms with Vid show a small protrusion near their vulva that can eventually lead to rupture of the gonad and premature death. While many labs have noted this phenotype, it is unknown if Vid is a natural part of C. elegans aging or if other genetic and environmental factors induce this phenotype, leading to confusion as to whether worms with Vid should be censored in aging experiments. The goal of our project is to determine the mechanism of Vid and whether it is part of normal worm aging. To further define the mechanism of Vid, we are using three experimental approaches: (1) using mutant worm strains, we are testing the genetic pathways important for causing and preventing Vid, (2) because Vid is temperature dependent, with frequency increasing inversely with temperature, we are testing at what point in their lifespan worms can still be rescued from Vid, (3) beginning with the gonad and vulval muscle, we are looking for cellular death and/or structural weaknesses that could be the direct mechanism behind Vid. Our initial work has identified several genes and genetic pathways important for Vid, including the hypoxic response pathway, the oxidative stress response pathway, and the protein assembly pathway. Our current work is close to identifying when worms are most susceptible to Vid and has begun to describe the physical mechanism of Vid. We intend to combine the results from these experiments to develop a model for Vid.

Although aging has been studied extensively, the molecular mechanisms of longevity are unclear. Dietary restriction (DR), a reduction in calories without malnutrition, increases lifespan in most model organisms tested, including rats, flies, and the round worm, Caenorhabditis elegans. Although DR is the most proven longevity-promoting intervention, the mechanism by which it works has not been fully elucidated. A reduction of metabolic rate and reduction of insulin signaling have been implicated in longevity, though neither appear to be sufficient on their own. Another highly supported mechanism of longevity includes the link between TOR (Target of Rapamycin) signaling and DR. Recent data suggests that TOR and DR act along the same pathway in respect to longevity; however, it is widely accepted that DR influences other pathways in addition to TOR. There are many approaches to DR in C. elegans. Using a bacterium carrying an RNAi plasmid, we feed the worms from egg through the first four days of adulthood and are therefore able to knockout specific genes in temperature sterile nematodes. The worms are then transferred to a liquid medium in the absence of the bacterial food. This process allows us to test the effects of dietary restriction on specific genes and determine whether the mutants will respond synergistically or antagonistically to the intervention. These results will be followed up to determine common pathways relating to longevity. Caloric intake was believed to be the sole mechanism of lifespan extension in DR; however recent data supports the idea that different mechanisms appear to be involved. This screen will provide us with a basis of which knockouts extend or shorten lifespan allowing us to relate the biology of DR with the pathways recognized as determinants of aging. If these pathways are conserved, these research findings may be applicable to other organisms.

Aging in humans is often characterized by an accumulation of various multifocal degenerative pathologies whose distribution is relatively random within a particular anatomical site. The result is that no two people, not even identical twins, age in exactly the same way. Our hypothesis is that variegated gene expression may be an evolutionary adaptable mechanism which, via epigenetic drift, progresses to develop into age-related cellular degeneration. Our belief is that there exist genetic loci capable of diversifying the degree of gene expression over a range of different proteins. Using Ethyl Methane Sulfonate, we introduced a host of point mutations into cultured fibroblast clone cells. We followed this mutagenesis by a double-selection protocol with Cadmium and 6-Thioguanine. In order for the fibroblast cells to survive this selection, they must simultaneously down regulate the expression of a transferase enzyme while up regulating the expression of metallothionein proteins. We have successfully isolated an extreme class of mutant fibroblast cells which, compared to the parent cell line, show a high degree of variegation in protein expression. These preliminary studies suggest that individual cells can be induced to epigenetically regulate their gene expression. While this epigenetic gambling is adaptive under the selection conditions, once it is initiated, it can progress on its own towards deleterious consequences. It is possible that such epigenetic gambling can escape selection in aging humans and be responsible for a host of age-related diseases, including various neoplasms as well as Alzheimer's dementia.

Aging research seeks to determine the processes that cause degeneration and disease in aged organisms, and to discover ways to prevent and/or slow their progression. Previous studies have shown that aging can be slowed in several model organisms through various means; however much is still not understood. In the model organism Caenorhabditis elegans, previous work shows that decreasing oxygen levels (hypoxia) significantly increases lifespan. The hypoxia-induced lifespan increase is observed when worms are exposed to hypoxia either from a late larval stage for life or if they are deprived of oxygen for just one day at the same larval stage. The low oxygen environment activates the hypoxic response pathway, mediated by the hypoxia-inducible factor (HIF-1). Previous data show that an increase of the HIF-1 protein, via genetic mutation or alterations in atmospheric oxygen, is sufficient to extend lifespan. However, much about this pathway remains unknown, including the mechanism for increased longevity, the difference between genetic and environmental manipulations of HIF-1, and the downstream proteins involved. My project examines the genetic components responsible for extending lifespan in hypoxia. To test this, we measured whether hypoxia increases the lifespans of worm strains with deletions or transgenic overexpression of known aging genes. Our results suggest that disruption of most of the non-HIF-1-related pathways have no effect on hypoxia-induced lifespan extension. However, the data show that the forkhead transcription factor, DAF-16, is required for hypoxia to increase lifespan, and subsequent work has shown that hypoxia causes DAF-16 to enter the nucleus. Interestingly, results indicate that hypoxia decreases lifespan when the oxidative stress response factor, SKN-1, is overexpressed, suggesting that SKN-1 is somehow toxic to worms in low oxygen. Collectively, the results show that hypoxic treatment increases lifespan in worms, but that the hypoxic pathway is intertwined with other stress responsive pathways.

Oxidative stress and aggregation of misfolded proteins have been linked to aging in a range of model organisms, including the nematode Caenorhabditis elegans. Reduced expression of genes involved in mitochondrial respiration, such as those coding for electron transport chain (ETC) components, have been found to ameliorate these decreases in lifespan. This rescue is thought to act through upregulation of the mitochondrial unfolded protein response (UPRmt) in a cell-non-autonomous manner. Induction of the UPRmt is essential for degradation of misfolded mitochondrial proteins, and has been correlated with longevity in ETC mutant worms. While the mechanism underlying the UPRmt and its effect on gene expression knockdown is not well understood, there is evidence that knockdown induces the UPRmt. Previous studies demonstrate that knockdown of ETC components, as well as knockdown of a subset of mitochondrial proteins both induce the GFP reporters for the mitochondrial chaperone, hsp-6. To identify novel genes involved in the UPRmt, we are performing a forward genome-wide RNAi screen using the hsp-6::GFP reporter and measuring the UPRmt activity by fluorescent microscopy. We expect genes induced by the UPRmt to cluster into groups with common functionality, or along the same metabolic pathways. These hits may include genes coding for the ETC or other mitochondrial proteins, as well as genes outside of the mitochondrion. We will follow up on our screen data results by sequence verifying RNAi mutants and completing full lifespans to elucidate the interaction between the UPRmt and gene knockdown.

Gliomas are a uniformly fatal type of primary brain cancer characterized by their diffuse and invasive nature. Dr. Kristin Swanson and her team have developed a mathematical model of glioma growth that uses an individual’s unique tumor characteristics to derive metrics that enable the prediction of patient specific growth patterns and survival times. To date, relatively few studies have looked at predicting clinical progression of disease, which may be a function of disease localization. Clinical progression of disease incorporates variables such as mental cognition and impairment and is typically quantified using the widely used Karnofsky Performance Score (KPS). KPS is an accepted prognostic marker of a patient’s likelihood of tolerating chemotherapy and radiotherapy. In one study accessing functional status after gross total resection (GTR) of a patient’s enhancing tumor region, only 28% of patients had higher KPS post-operatively. 49% had stable scores, while 23% had declines in KPS of 10 to 30 points (Barker 1998). The variability in post GTR KPS scores suggests that there are patient-specific drivers of KPS. We will do a multivariate analysis to investigate which functional brain regions influence the mental and physical impairments that determine KPS scoring. We will identify brain regions, “functional drivers of KPS”, where tumor presence, as calculated by Dr. Swanson’s mathematical model, is well associated with a particular range of KPS scores. For each functional driver, we will use the region’s tumor cell concentration at time of tumor diagnosis, to quantify the future KPS. We will compare the predicted KPS to the actual KPS. If the initial and next time points are located pre and post- radiotherapy, chemotherapy, or resection, this will allow us another way of assessing the efficacy of therapies in palliating symptoms.

 The research conducted by the Kaeberlein Lab is focused on identifying conserved longevity genes in evolutionarily divergent model organisms. The research methods discussed here concentrate particularly on the mechanism of aging in budding yeast. Aging in the budding yeast Saccharomyces cerevisiae was described more than fifty years ago with the observation that mother cells produce a finite number of daughter cells and then cease replication. Our poster describes the method utilized in our lab for quantifying replicative lifespan. In short, individual yeast cells are grown on solid media and monitored from their initial undivided state until they undergo senescence. Mother cells that produce more daughter cells are defined as having a longer replicative lifespan. Over the past several years, we have measured replicative lifespan on several hundred thousand mother cells, resulting in the identification of dozens of genes that modulate aging in yeast.  By identifying these genes and determining their mechanism of action it may be possible to one day develop therapies that delay the onset of aging and age-related disorders in people. 

Fatty acids are vital for proper cell function, serving as a source of energy, a structural component of the plasma membrane, and a signaling molecule. Imbalance of these fatty acids can change the homeostasis of the cell and can lead to major consequences, including cancer. We propose that lipid levels could be analyzed and serve as a diagnostic and preventative measure for human hepatocellular carcinoma (HCC). In our study, we use a hepatic-specific PTEN knockout mouse which recapitulates the progression of HCC. Phosphatase-and-tensin homologue (PTEN) is a tumor suppressor gene involved in cell cycle regulation and mutations have been observed in many types of cancers, including HCC. We collected plasma and liver tissue from 12 month old PTEN KO mice and proper controls to undergo quantitative lipid analysis. Human serum samples from cirrhotic patients with and without HCC underwent similar lipid analysis. This analysis was done for 4 lipid fractions (FFA (free fatty acids), PL (Phospholipids), TG (Triglycerides) and CE (Cholesterol esters) and determined the relative abundance of nearly 30 lipids. In addition, we quantified the abundance of several genes important in lipid metabolism using q-RT PCR. We found several lipids that showed strong correlation to tumor development. Further, the abundance of the genes involved in metabolism of these specific lipids was altered in the PTEN KO mice and showed significant correlation to both lipid levels and tumor development. Similar results were found through analysis of human samples of HCC compared to the cirrhotic controls. This infers that analyzing lipid levels and related gene expression may serve as a potential method for diagnosis and, eventually, prevention of human HCC.

Type 1 Diabetes Mellitus (T1D) is caused by a permanent destruction of insulin-producing β-cells of the pancreas by autoreactive T cells. Despite advances in insulin therapy, patients often face serious cardiovascular and neurological complications. Since most patients are children and young adults it is imperative to identify therapeutic targets to prevent the disease without causing generalized immunosuppression. Using autoimmune prone non-obese diabetic (NOD) mice (the widely used model of T1D) with spontaneous gene mutations in either Fas (lpr) or FasL (gld), we hypothesize the involvement of a specific population of B cells in the disease process. These B cells express Fas ligand (FasL), which is part of the pathway involved with T cell homeostasis. FasL expressed in these cells break immune tolerance in autoimmune prone NOD mice. Using flow cytometry B cell populations were characterized in spleen, pancreatic and inguinal lymph nodes, and in peritoneal cavity of NOD mice as well as in C57BL/6 and Balb/c mice. We detected FasL expressing B cells in spleen of all three mouse strains tested (NOD, C57BL/6, and Balb/c), but higher levels were detected in NOD mice. In addition, gld/gld mice B cells did not up-regulate FasL, CD23, or CD86 like the NOD wild-type mice which was illustrated by activation with LPS and anti-IgM. We looked for apoptosis among FasL+ B cells in the spleen of NOD, B6 and Balb/c and found higher annexin V levels in B6 and Balb/c than NOD, supporting the hypothesis that B cells in NOD mice, are pathogenic.

Research studies suggest that up to 40% of hepatocellular carcinomas may be caused by liver stem cell carcinogenesis, in which stem cells, due to their high proliferative abilities, become neoplastic and maintain cancer growth and metastasis. MicroRNA are post-transcriptional gene regulators that have been found to be involved in many cellular processes such as differentiation of stem cells. This project focused on the role microRNA play specifically in liver stem cell differentiation. Finding which microRNA are important in liver cell differentiation could provide new methods for diagnosis and treatment of liver cancer originating from stem cells. I used a stem-cell-like liver progenitor cell line capable of differentiating into mature liver tissue from a proliferative state, providing an ideal in vitro model for liver stem cell differentiation. Relative quantification of microRNA by real-time PCR and microarray was performed on samples taken from various time points during differentiation leading to the characterization of two microRNAs strongly upregulated in differentiation. In order to validate the importance of these microRNAs, I will use a viral vector to induce their overexpression and monitor any subsequent changes in the differentiation process. In the end, this project may provide additional insights into neoplastic liver stem cells.