1,721,022 research outputs found
An Automated System For High-Throughput Longevity and Healthspan Discovery in Caenorhabditis elegans
Full text linkThesis (Ph.D.)--University of Washington, 2022Over the last century, the study of aging biology has primarily been advanced through the development and use of animal models that share common molecular hallmarks with human aging. One major model system that has been widely used during the last several decades and through the present day is the roundworm Ceanorhabditis elegans. Its short lifespan, easy husbandry, and genetic tractability have allowed it to be easily adapted for studying the molecular biology of aging. For instance, research using C. elegans was used to discover the interplay between insulin signaling and rate of aging. However, while C. elegans research proved orders of magnitude more efficient (at the cost of a larger evolutionary gap) than using vertebrate models, such as mice or non-human primates, it’s use was still hamstrung by the necessity for manually collected lifespans. During the last decade, the lowering cost and increasing capabilities of digital microscopy and computer vision have led to researchers attempting to automate the most basic aging related experiment in C. elegans: the measurement of an individual’s lifespan relative to the population within which it resides. Some examples of this include the Automated Lifespan Machine, The WormMotel, as well as several different microfluidic platforms such as the NemaLife chip. These platforms sought to semi-automate or even fully-automate the collection and recording of lifespan measurements but often had large bottlenecks in their pipeline that precluded them from achieving the scale necessary to robustly advance the field of aging biology. This thesis will present the development and use of a novel robotics platform that, when paired with a suite of AI-powered computer vision, fully automates lifespan analysis of C. elegans
Analysis of lifespan extending compounds using growth kinetics and replicative lifespan in Saccharomyces cerevisiae
Full text linkThesis (Ph.D.)--University of Washington, 2023Advances in modern medicine have facilitated stunning rises in life expectancy over the last two centuries. This longer life, however, is concomitant with a gradual decline in health and the onset of multiple age-associated pathologies including heart disease, neurodegeneration and cancer. Risk of developing and dying from these age-associated diseases increases dramatically with age suggesting a common underlying cause. Targeting the aging process itself with clinical intervention may allow for amelioration of the onset and progression of multiple age-associated pathologies simultaneously. The mechanistic Target of Rapamycin (mTOR) signaling pathway has emerged as an important clinical target in this regard with its dysregulation being observed in multiple pathologies including many cancers, autoimmune disorders, heart failure, neurodegeneration and type 2 diabetes. Importantly, genetic or pharmacological inhibition of mTOR signaling results in increased life-span across evolutionary distant organisms suggesting a conserved role for this signaling pathway in longevity regulation. A novel yeast-based system to identify putative mTOR inhibitors is described. Utilizing differential growth kinetics of WT and mutant strains sensitized to mTOR perturbations, this system successfully discriminates between allosteric (i.e. rapamycin) and ATP competitive mTOR inhibitors. A number of nutraceutical compounds were screened and of these, caffeine was confirmed to be an mTOR inhibitor (Chapter two). Next a screen of natural products and natural product mixtures were screened for effects on growth rate, mTOR-mediated growth inhibition, and replicative lifespan (RLS). No mTOR inhibitors were identified but two treatments, berberine and green tea extract significantly reduced RLS. Pterocarpus marsupium extract (PME), a plant extract with a long history of use in Ayurvedic medicine, extended cellular lifespan (Chapter three). Constituents of this extract include several compounds with reported health span and life span extending properties: epicatechin, quercetin, berberine, and pterostilbene. Of these PME constituents, quercetin and epicatechin had no effect on cellular life span, while berberine decreased cellular life span. We describe the lifespan and outgrowth kinetics of pterostilbene-treated cells and report on dose-dependent effects on longevity, potent cytotoxicity, and a possible role for mitochondrial function in mediating these phenotypes (Chapter Four)
Cell aging preserves cellular immortality in the presence of lethal levels of damage
Full text linkCellular aging, a progressive functional decline driven by damage accumulation, often culminates in the mortality of a cell lineage. Certain lineages, however, are able to sustain long-lasting immortality, as prominently exemplified by stem cells. Here, we show that Escherichia coli cell lineages exhibit comparable patterns of mortality and immortality. Through single-cell microscopy and microfluidic techniques, we find that these patterns are explained by the dynamics of damage accumulation and asymmetric partitioning between daughter cells. At low damage accumulation rates, both aging and rejuvenating lineages retain immortality by reaching their respective states of physiological equilibrium. We show that both asymmetry and equilibrium are present in repair mutants lacking certain repair chaperones, suggesting that intact repair capacity is not essential for immortal proliferation. We show that this growth equilibrium, however, is displaced by extrinsic damage in a dosage-dependent response. Moreover, we demonstrate that aging lineages become mortal when damage accumulation rates surpass a threshold, whereas rejuvenating lineages within the same population remain immortal. Thus, the processes of damage accumulation and partitioning through asymmetric cell division are essential in the determination of proliferative mortality and immortality in bacterial populations. This study provides further evidence for the characterization of cellular aging as a general process, affecting prokaryotes and eukaryotes alike and according to similar evolutionary constraints.</div
Divergent Single-Cell Trajectories in Homeostatic Control and Genome Instability during Aging
No full textThesis (Ph.D.)--University of Washington, 2019The budding yeast has a long and storied history as a model organism for biological inquiry. Accordingly, experiments into the replicative aging of S. cerevisiae have yielded critical insights into evolutionarily conserved mechanisms of aging. Historically, replicative aging experiments have relied on labor-intensive techniques for lifespan measurement, and methods to observe physiological function across a cell’s lifespan were not available. Recently, novel microfluidic devices have been developed for the whole-lifespan monitoring of yeast cells during replicative aging. Using time-lapse microscopy, these devices allow researchers to measure replicative lifespan in much higher throughput and to quantitate various aspects of cell biology during aging, with single-cell resolution, across a cell’s entire lifetime. In this dissertation, I engineer a low-cost motorized light microscopy system with open-source software, aimed at increasing the accessibility this new technology. Recently, an early life decline in vacuolar/lysosomal acidity in the budding yeast has been found to underlie the aging process. I use our microfluidic device to investigate the consequences of lysosomal/vacuolar dysfunction during yeast replicative aging. I find that this loss acidity triggers an iron sulfur cluster deficiency and is associated with a age-associated genome instability. However, only a subset of cells mount the expected iron starvation gene expression program, leading to divergent single-cell trajectories of iron dyshomeostasis during aging. Cells which mount an iron starvation response during aging have limited iron sulfur cluster deficiency and extended survival and passage through periods of genome instability during aging
The regulation and function of C. elgans flavin-containing monooxygenase-2
No full textThesis (Ph.D.)--University of Washington, 2021In the last one hundred years, aging has become an increasingly tractable problem. The pioneering dietary restriction experiments of the 1920s and '30s, the robust evolutionary and molecular theories of the 1950s -'70s, and the identification of conserved longevity genes in the 1980s – 2000s all paved the way for the aging research field's current rapid expansion and mainstream traction. Along with metformin, senolytics, and numerous other promising avenues, the field is still characterizing the molecular mechanisms through which major interventions like dietary restriction and the inhibition of insulin and mTOR signaling promote longevity. In this dissertation, I use the nematode roundworm model species Caenorhabditis elegans to define the regulation and function of the conserved pro-longevity target gene flavin-containing monooxygenase-2 (fmo-2). I find that both the regulation and function of fmo-2 is dependent on endogenous sulfur amino acid metabolism, placing fmo-2 at a nexus of redox, cellular energetics, and other processes central to aging
Investigation into the effects and mechanisms of rapamycin treatment in two mouse models of Complex I-deficient neurological pathology
Full text linkThesis (Ph.D.)--University of Washington, 2014Treatments to stop or reverse the debilitating progression of early- and late-onset neurological diseases remain undiscovered. Collective evidence suggests that inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway is effective at reducing markers of pathology in experimental models of age-related and developmental neurological diseases. These include, but are not limited to, models for Alzheimer's disease, Parkinson's disease, Huntington's disease, Fragile X syndrome, Tuberous Sclerosis Complex and Leigh syndrome. How regulation of mTOR activity and its downstream effectors interact with underlying neural mechanisms of disease has been a topic of considerable debate. The studies presented here investigate the potential therapeutic effects of the mTOR inhibitor rapamycin in two different mouse models for neurological disease: the NDUFS4 knockout (NDUFS4 KO) mouse for Leigh Syndrome and the MPTP mouse model for Parkinson's disease. In these models, mitochondrial electron transport chain complex I activity is reduced but results in distinct patterns of neuronal pathology. Our major objectives were (1) to identify previously unreported effects of rapamycin treatment in these models; and (2) to identify the potential cellular mechanisms that mediate these effects. First, we demonstrate that daily treatment with high dose rapamycin effectively extends the short lifespan of NDUFS4 KO mice. Rapamycin treatment also resulted in prolonged healthspan in KO mice, as indicated by the offset of neurological damage, maintenance of weight and body fat, and the improvement of deleterious behavioral phenotypes. Systematic testing of potential mechanisms mediating these effects led us to favor a model in which rapamycin induces a metabolic shift in NDUFS4 KO brains toward amino acid catabolism and away from glycolysis, thus alleviating the buildup of glycolytic intermediates. Following this set of discoveries, we expanded our findings to test whether dietary rapamycin delivered at higher doses than previously used could improve lifespan and abnormal weight phenotypes in NDUFS4 KO mice, similar to what was found for high dose injections. Dietary rapamycin doses were tested at 42 ppm, 126 ppm and 378 ppm, corresponding to 3-fold, 9-fold and 27-fold increases from the standard 14 ppm dosage used by the NIH Interventions Testing Program, respectively. As a result of these treatments, NDUFS4 KO lifespan was significantly extended, with successively higher dosages correlating with increased survival. We also found that dietary rapamycin at 126 and 378 ppm had significant effects on body weight and fat mass in male and female wild-type mice. Finally, we conducted a pilot study investigating the effectiveness of high dose rapamycin treatment in treating the Parkinson's disease-like pathology of mice exposed to the toxic drug MPTP. Our results show that rapamycin treatment partially reduced neuron degeneration in the substantia nigra resulting from MPTP exposure, consistent with previous reports. In addition, MPTP mice showed evidence for hyperactive mTOR signaling compared to control mice, which could be potently reduced by rapamycin treatment. No significant changes in body weight or fast mass were found as a result of MPTP exposure, or as a result of an interaction between MPTP and rapamycin. When accounting for different age cohorts, middle aged mice that had been exposed to MPTP performed better on a rotarod task after receiving rapamycin treatment. Our young cohort, however, did not show any differences in performance between treatment groups. Thus, we believe that MPTP induces age-dependent phenotypes that may have been overlooked in previous studies utilizing young mice. Thus far, comparison of these studies suggests that rapamycin treatment has both overlapping and distinct effects that contribute to attenuation of neural pathology of NDUFS4 and MPTP mouse models
The consequences of mutator-driven mutagenesis and analysis of lifespan extending compounds using outgrowth analysis and replicative lifespan in Saccharomyces cerevisiae
No full textThesis (Ph.D.)--University of Washington, 2018The hope of dramatically extending our lifespan has captivated humanity for millennia. Over the last two decades, the biology of aging has matured as a field of study and led to greater engagement and investment in aging as a biological problem that can be understood at the molecular level and treated. Translational Geroscience is an interdisciplinary field descended from basic gerontology that seeks to identify, validate, and clinically apply interventions to maximize healthy, disease-free lifespan. To identify and validate interventions, a translational geroscience research pipeline is proposed that begins with identifying and characterizing interventions in wild type model systems (either invertebrate or vertebrate), validating these interventions using models of genetic diversity and disease models, and finally, testing validated interventions in companion animals and humans (Chapter one). A model of mutator-driven mutagenesis, a potent mechanism to induce genetic diversity in cancer and other microbial populations, using budding yeast is then described. Defects in DNA polymerase δ or ε proofreading alone or combined with defects in mismatch repair are used to model mutator phenotypes and mutator-driven mutation burden in haploid and diploid yeast. This model is used to understand the effects of active mutagenesis and accrued mutation burden on cellular aging (Chapter two). Next, a new system to identify chemical inhibitors of mechanistic Target Of Rapamycin (mTOR) is described. This yeast outgrowth based system measures sensitivity of WT and mutant strains sensitized to mTOR inhibition. A set of nutraceutical compounds were screened using this assay. Of these compounds, caffeine was confirmed to be an mTOR inhibitor (Chapter three). Lastly, this set of nutraceuticals were screened for changes in yeast replicative lifespan (RLS). Two treatments, berberine and green tea extract, reduced RLS. Only one treatment, Pterocarpus marsupium extract (PME), extended cellular lifespan. Within this extract are two molecules with reported healthspan and lifespan properties: pterostilbene and (-)-epicatechin. We tested concentrations of these compounds comparable to those found in PME but did not recapitulate extended lifespan (Chapter four)
The Regulation of Mitochondrial Stress Responses in Caenorhabditis elegans
No full textThesis (Ph.D.)--University of Washington, 2016-12Organismal aging has been proposed to result, at least in part, from mitochondrial dysfunction and oxidative stress. Mitochondria play an important role in energy metabolism, molecular biosynthesis, apoptosis, and cellular signaling and are therefore complexly tied to cellular and organismal health. Paradoxically, there are numerous cases in Caenorhabditis elegans, in addition to other organisms, where inhibition of mitochondrial respiration is sufficient to extend lifespan. One proposed mechanism for this pro-longevity effect is the induction of the mitochondrial unfolded protein response (UPRmt), which upregulates expression of mitochondrial-specific chaperones and proteases to re-establish protein homeostasis in the mitochondria. This thesis describes the use of C. elegans to understand the genetic regulation of the UPRmt and importantly, the role of this response in stress resistance and aging. I first describe a genome-wide RNAi screen for negative regulators of the UPRmt that takes advantage of a highly sensitive UPRmt fluorescent reporter and RNAi feeding in C. elegans. I identify 95 inducers of the UPRmt (RNAi gene knockdowns that increase reporter expression), which are enriched for mitochondrial genes that affect respiratory chain function. A subset of these positive hits differentially affect lifespan and for those that increase lifespan, do so independently of the UPRmt transcription factor ATFS-1. I also find that constitutive activation of the UPRmt is not sufficient for lifespan extension in C. elegans, and in fact, seems to harm animals. In the second part of this thesis, I follow-up on a specific gene, the cytosolic pentose phosphate pathway enzyme transaldolase, whose connection to mitochondrial proteostasis is not well understood. I find that transaldolase deficiency alters multiple parameters of mitochondrial function including respiration and mitochondrial dynamics, and promotes longevity through activation of redox-sensitive MAPK pathways and the autophagy regulator TFEB/HLH-30. I also discover that ETC RNAi extends lifespan through identical JNK MAPKs, implicating adaptive responses aside from the UPRmt in longevity control from mitochondrial stress. Finally, I describe another genome-wide RNAi screen to identify positive regulators of UPRmt signaling, to elucidate the complex network of regulatory factors that control this response. Further understanding of the mechanistic details of UPRmt regulation will provide us with insights into the evolution of mitochondrial-nuclear communication and the growing list of human diseases associated with mitochondrial dysfunction
An Exploration of the Genetics and Molecular Mechanisms Underlying Conserved Longevity Interventions
No full textThesis (Ph.D.)--University of Washington, 2012Aging is a degenerative process that causes a time-dependent deterioration of virtually every biological system in the majority of species. Age is the primary risk factor for many human diseases, including the top causes of death modern societies. Developing treatments to slow the aging process has the potential increase human life span and simultaneously prevent or improve outcomes in countless diseases. Studying aging in mammals is challenging due to the relatively high longevity of most mammalian species and the costs associated with maintaining populations of mammals in the laboratory for their entire life span. The invertebrate organisms Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster have emerged as central models in aging due to relatively short life spans, ease of maintenance in the laboratory, well characterized genetics, and the availability of a wide range of genetic and biochemical tools. By focusing on genetic pathways and interventions that influence longevity in a similar manner across these evolutionarily divergent species, we can gain insight into the biology of aging in mammals. The application of genome-scale techniques in aging research has started to define the range of genetic and environmental factors involved in longevity determination, and the high degree of intercommunication between these factors. This dissertation reviews current progress toward identifying and understanding conserved longevity interventions and presents several current lines of investigation aimed both at developing tools for analyzing the complex interactions between aging factors and at probing the mechanism of action of specific longevity interventions
Elucidation of the molecular pathways of lifespan extension by dietary restriction in yeast
No full textThesis (Ph.D.)--University of Washington, 2012In this dissertation, we demonstrate strong support for the antagonistic pleiotropy view of aging, reveal the illusion of sirtuin mediated longevity in 32 long lived alleles (hopefully steering the field farther from sirtuin research and toward more interesting things), show that there is indeed a conserved aging factor between CLS and RLS, indicate that aging in yeast is complex like any other organism and use phenotype clustering to show afg3∆ was within the Gcn4 pathway of lifespan extension, and finally bring nuclear tRNA and Gln3 into the light- showing that these could positively affect lifespan without reducing translation
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