Herpes Simplex Virus 1 (HSV1) is a prevalent lifelong virus transmitted through oral-to-oral contact resulting in painful oral sores. HSV1 may also give rise to encephalitis, or inflammation of the brain, and has even been linked to sporadic Alzheimer’s Disease (AD). Typically, HSV1 enters through the oral cavity, replicates, invades nearby clusters of sensory neurons that innervate mucosal membranes called the trigeminal ganglia, and can ultimately lead to infection of the brain. HSV1 can establish latency in these trigeminal ganglia and other neuronal nuclei, and can reactivate in the brain in response to external stresses at any point in time. Previous studies have even shown presence of HSV1 in the brain. However, this process of reactivation is still not understood. Our goal is to comprehend the mechanisms of HSV1 reactivation in the brain by generating two recombinant HSV1 through the CRISPR-Cas9 gene editing system. One strain will contain an insertion into the HSV1 genome of luciferase and mEmerald, a green fluorescent protein, for detection of cells with reactivating HSV1 through in vivo imaging of bioluminescence. The second strain will have an insertion of Cre recombinase and mEmerald. When this strain is injected into a mutant mice line with a Cre-dependent reporter gene, the Cre recombinase can lift genetic repression of a red fluorescence protein. This prompts all cells with an HSV1 infection, including those with past reactivation, to be detected. Through these novel recombinant HSV1 strains, we can monitor viral reactivation in brain cells in response to different external stresses in vivo, examine the time course of the infection, and distinguish areas of the brain with high viral load, especially cell types vulnerable to HSV1 infection. These experiments will be the stepping stone for improved investigation of viral latency and reactivation and understanding the connection between HSV1 and AD.

  My research focuses on ‘tauopathy’—the pathological effects of misfolding of the tau protein—using the fruit fly, Drosophila melanogaster, as a model system. In particular, I am interested in how the expression of tau affects locomotive function. Tau is a protein found in both humans and flies that is associated with stabilizing neuronal microtubules. However, under certain physiological conditions, human tau proteins form neurotoxic aggregates in the brain. This neuronal damage leads to dementia, a hallmark of Alzheimer’s Disease (AD). Aging comes with a multitude of age-related functional deficits, including decline in locomotor function. Some individuals with AD pathology are never diagnosed, however, because the cognitive impairment they experience is not sufficient for a clinical diagnosis of dementia, the most common symptom of AD. Consequently, it is imperative we understand AD as more than dementia. I study the climbing abilities of transgenic tau and control flies through negative geotaxis assays. Negative geotaxis refers to the tendency of flies to move vertically upward when startled. For my project, I am measuring the climbing performance of the flies to see the effects of tau on locomotor function as flies age. I hypothesize that the neurotoxic tau aggregates that form will interfere with neural activity involved in the flies’ motor function, resulting in decreased climbing performance. This research holds an abundance of biomedical implications and a capacity to better the lives of many. By using motor function to flag at-risk individuals early in their lives, they have the opportunity to participate in preclinical AD clinical trials that may prevent them from developing AD pathology later on. It is important that we, as a scientific community, strive to better understand the effects of aging and tauopathies, as it is through this understanding we can provide elderly individuals with care that transforms their quality of life.

Aging is the most prevalent risk factor behind many common diseases such as cancer, cardiovascular disease, and various neurodegenerative diseases. The changes that take place with age are complex and affect most of the human body; age-related changes in gait, in particular, have been found to be associated with the onset of disease. While it is generally understood that there is an effect of aging on walking speed in humans, the mechanisms underlying age-related locomotor impairment have not yet been fully characterized. The focus of my research is using Drosophila melanogaster as a model to investigate such mechanisms. My hypothesis is that D. melanogaster exhibit similar age-related changes in gait as those seen in humans, which includes a decrease in walking velocity and duration, and a significant change in limb coordination over their lifespan. I followed cohorts of D. melanogaster over their lifespans and recorded videos of their walking in an arena. I then used these videos to study factors such as walking velocity and duration by analyzing the trajectories of each fly. In addition, I investigated more in-depth limb coordination of individual flies by analyzing the movement of each individual leg. Previous studies using D. melanogaster have found a significant decrease in climbing behavior with age, but have not looked at gait on a finer scale. Therefore, upon completion of this study, I expect to see a decrease in limb coordination and population walking velocity as the flies age. With the findings from this study, I hope to establish a foundation for how gait changes with age in D. melanogaster and to further use gait analysis to help predict the onset of disease and to distinguish between diseased and healthy flies.

Alzheimer’s Disease (AD) is a neurological disease that causes memory loss and neurodegeneration, and is one of the leading causes of death within the United States. Alzheimer’s is linked to the presence of neurofibrillary tangles within the brain, generated by hyperphosphorylation and aggregation of a protein called tau. However, the specific impact that tau has on biochemical pathways in neurons is largely unknown. My project attempts to fill this gap. I compared samples of wild type fruit flies, Drosophila melanogaster, with a strain which expresses the human tau gene in neurons. Despite the difference in human and fly brains, determining the affected biochemical pathway in flies could suggest similar effects in humans. I am using metabolomics, which quantifies the levels of approximately one hundred metabolites, to try to identify the biochemical pathways that are affected by expression of tau in the neurons. Head and body samples of these two fly strains were frozen at 12 days of age and assayed for a targeted set of metabolites. I analyzed these metabolome data using statistical methods in Python to search for potential differences between wild type and tau flies. Any metabolites found to be different will then be analyzed to see if they tend to represent similar biochemical pathways, allowing us to speculate about the effect of tau on those pathways, both in flies and humans. Future work aims to analyze fly brains and individual neurons, thus mapping the affected metabolic pathways more precisely. Importantly, metabolic pathways are often identical between organisms, allowing us to speculate on the effect tau has on similar pathways within humans, thus this work will inform our understanding of the mechanism of AD in humans.

The goal of the Kaeberlein lab is to understand the underlying molecular mechanisms of aging and how they can be perturbed in order to beneficially alter the aging process. To do so, we use the unicellular model S. cerevisiae or brewers yeast. In order to expedite the process of studying aging in large numbers of cells, the Kaeberlein lab has been using microfluidic devices that immobilize yeast cells throughout their lives so that visual data can be collected without manual interference of the cells. The question being investigated here is whether a neural network can be trained to automate the scoring tasks required for processing the image data generated by these microfluidic devices. Using python and the Yolov3 neural network architecture, I have created an AI-based screening tool to quickly analyze data generated with these microfluidic devices, such that the pipeline as a whole produces lifespan data for about 120 cells at a time, using stacks of image files as raw data. This work is significant as it provides insights into the issues of training a neural network on similar objects, and more importantly, insights into how these issues can be mitigated. In addition to increasing the speed with which data can be scored, this system will also increase the capacity for scoring experiments with large numbers of experimental groups. The system itself can also be used in order to expedite biology research.

Alzheimer’s Disease (AD) is a neurodegenerative disease that is the most common cause of dementia. One hallmark of AD pathology is hyperphosphorylation of Tau protein. Tau is a neuronal-specific protein that stabilizes microtubules. Hyperphosphorylation of Tau leads to loss of its normal function and promotes aggregation into neurotoxic fibrillary tangles. While Tau aggregation is well-documented, the exact role of Tau loss-of-function in AD pathogenesis remains uncharacterized. The goal of our project is to determine the mechanism by which Tau loss-of-function contributes to AD pathogenesis. We hypothesize that Tau loss-of-function contributes to AD pathogenesis by activating the cellular stress response in neurons, characterized by DNA damage, stress granule formation, and the upregulation of heat shock proteins, chaperones that stabilize and refold proteins damaged by cellular stress. To test this hypothesis, we cultured neural progenitor cells (NPCs) and neurons derived from human induced pluripotent stem cells (hiPSCs). We generated three cell lines: one in which the gene encoding Tau was knocked out (Tau KO), another in which Tau expression was knocked down (shTau), and a control line. To determine whether genes implicated in the cellular stress response are upregulated in Tau KO neurons, we used RNAseq and RT-PCR. Then, we used immunocytochemistry to detect protein markers of cellular stress in NPCs and neurons from all three lines. Preliminary results indicate that several genes encoding proteins involved in the cellular stress response are upregulated in Tau KO neurons, including the small heat shock protein HSPB8 and BAG3, a gene that is upregulated in the aging rodent brain and regulates the autophagic response. By immunostaining, we show that dsRNA aggregation, which may indicate stress granule formation, is more prevalent in Tau KO cell lines than control. By elucidating the role of Tau loss-of-function in AD pathogenesis, this research could inform therapeutic targets for AD.

We can use human pluripotent stem cells to derive cardiomyocytes (hPSC-CMs) in vitro, with the goal of transplanting them into the hearts of individuals who have suffered from heart attacks and restore contractile function. After transplantation into animal models, however, hPSC-CMs produce arrhythmias (irregular heartbeats), likely caused by the immature state of hPSC-CMs. This immature state is associated with low expression of cardiac genes regulating heart muscle contraction and electrical properties. We aim to mature hPSC-CMs in vitro by controlling the expression of these genes, so we can engineer them to behave more like adult cardiomyocytes. To do this, I am looking at DNA methylation, a modification occurring at cytosine nucleotides that is associated with transcriptional repression or gene silencing. My project goal is to determine if DNA methylation plays a role in regulating gene expression patterns of cardiac genes in hPSC-CMs. To investigate this, I have treated hPSC-CM genomic DNA with bisulfite reagent, which converts unmethylated cytosine nucleotides to thymine nucleotides. This treatment will allow me to differentiate between unmethylated versus methylated DNA, and determine whether cardiac maturation genes are methylated at their promoters (where gene expression is typically regulated) by running PCR. Additionally, I have cultured hPSC-CMs with the DNA methylation inhibiting drug 5-azacytidine. By blocking DNA methylation, I will be able to determine if methylation has a direct effect on the expression of cardiac genes by measuring gene expression via quantitative real-time PCR. I hypothesize that DNA methylation regulates cardiac gene expression, and inhibiting methylation will cause expression to increase. Thus, if DNA methylation represses cardiac gene expression, we can mature hPSC-CMs by inhibiting methylation. Ultimately, we hope to prevent arrhythmias that occur after hPSC-CM engraftment and develop cell therapies using mature hPSC-CMs to restore heart function after a heart attack.

A survey in the existing literary sources says that there are three main diet-related patterns in Blue Zone communities that contribute to their longevity. After I cross-referenced the micronutrients in Blue Zone diets with the average American diet, I found that the Blue Zone diets are all enriched for some common vitamins and nutrients, specifically: retinol, thiamin, pyridoxine, ascorbic acid, vitamin E, vitamin K, magnesium, and potassium. All of these vitamins and minerals are known to affect cellular metabolism through a wide array of factors, potentially linking them to bettering health and increasing lifespan. I initialially will confirm that these compounds indeed play a vital role in extending the functionality of the biomechanical systems in C elegans. I will then further pursue whether combinations of the nutrients increase effectiveness, and which specific areas in the body they individually impact for promoting longevity. My first step in investigating the biomechanical effects of these micronutrients will be to incorporate each one individually into the C elegans’ diet. My long-term goal, once I test the wild type model, is to focus on how these micronutrients combat age related diseases. To do this I will use my results for optimal micronutrients to test the effectiveness of the specific micronutrients in the Alzheimer’s degenerative pathway. Specifically, I will use the GMC-101 transgenic worm, (genetically modified strain to exhibit the degenerating effects of Alzheimer's disease), which expresses human amyloid- beta - a protein implicated in the progression of human Alzheimer’s Disease - in the body wall muscle and becomes increasingly paralyzed with age. I will use a similar experimental set up, looking for a delayed onset of paralysis. I hope this research will be able to shed light on how micronutrients interact and affect degenerating biochemical pathways in aging C elegans; specifically, in a strain that models Alzheimer’s disease.

Caenorhabditis elegans is frequently used as a model organism for testing the effects of various compounds on longevity. A current limitation of running these experiments is the tremendous amount of work needed to collect large sample sizes of data when testing for several compounds in different genetic populations. Fortunately the Kaeberlein lab has developed the WormBot, an image capture robot used to take high resolution images of hundreds of experimental plates each containing ~30 worms. Researchers still have to rely on humans manually annotating tens of thousands of frames to extract valuable metrics for their analysis. I have developed the implementation of a neural network to automatically analyze these images so that the number of experiments and compounds that can be tested is increased exponentially. I utilize a network architecture known as Yolov3 to allow the computer to identify and track individual worms from the images. The results obtained from our new computational method extract data from the images that is equal to or even better than the human method while also requiring a fraction of the time. Using this novel platform, we are analyzing a broad spectrum of natural and synthetic compounds for their effects on longevity and health span in C. elegans.

We have established a head and neck squamous cell carcinoma (HNSCC) cell line resource to facilitate translational research on HNSCC in individuals with Fanconi Anemia (FA). FA is a rare genetic disorder characterized by bone marrow failure and predisposition to leukemia and solid tumors. FA individuals have an extraordinarily high lifetime risk of HNSCC compared to the general population and the pathogenesis of these cancers is not understood. Apart from surgery, effective treatment of these cancers is limited by patient hypersensitivity to standard-of-care therapies that include ionizing radiation and DNA cross-linking drugs. Insight into the mechanistic origins of these cancers and the identification of less toxic, more effective therapies are necessary to improve survival and quality of life. The FA Cancer Cell Line Resource was developed to provide well-characterized, experimentally tractable pre-clinical models to investigate the origins, pathogenesis, treatment and prevention of FA HNSCC. Isogenic cell line pairs or trios included in this Resource were generated from FA patient-derived or sporadic HNSCC cells using CRISPR/Cas9 technology. The biochemical and molecular characteristics of these models were confirmed to verify the presence or absence of FA-associated phenotypes following gene edits. This Resource addresses the critical need for well-characterized and tractable disease models that are FA patient-derived, and versatile enough to permit both in vitro and in vivo analyses. These cell lines are now available at no cost to foster research through the Fanconi Anemia Research Fund-sponsored “Fanconi Anemia Research Materials” repository at Oregon Health and Sciences University (https://apps.ohsu.edu/research/fanconi-anemia/celllines.cfm).

Cancer is an evolutionary process driven by mutagenesis and selection for malignant phenotypes. Correspondingly, mutations that elevate mutation rates, producing a “mutator phenotype,” accelerate tumor formation. Heterozygous mutator alleles affecting the catalytic subunit of DNA polymerase (Pol) epsilon (encoded by the POLE gene) or bi-allelic mutations affecting mismatch repair (MMR) components, such as MSH2, arise frequently in a subset of colorectal and endometrial cancers. In some tumors, mutator alleles from both classes occur together and synergistically cause tremendous genetic instability. A key unanswered question is whether the severe mutation accumulation compromises tumor cell fitness and imposes a selection for “antimutator” phenotypes that allow the tumor to escape extinction. Evidence supporting the selection for antimutator alleles in strong mutator cells have been obtained using budding yeast, whose DNA replication and repair machinery are highly conserved with their mammalian counterparts. Our initial studies in haploid yeast show that mutations in the pol2 mutator alleles represent a major class of antimutator alleles. However, mutator suppression in diploid cells remains understudied. The strongest mutator allele known in human disease is POLE-P286R, which corresponds to pol2-P301R in yeast. We previously evolved diploid pol2-P301R/POL2 msh2Δ/msh2Δ mutator yeast strains to identify candidate antimutators that may arise in diploid cells. We also identified putative human antimutator alleles from The Cancer Genome Atlas database. Here, we test whether these alleles do indeed exert antimutator phenotypes by re-engineering them into diploid yeast. Our findings will provide a direct test of the relevance of antimutators for tumor evolution and define likely antimutator candidates for further study.

As scientists collect ever larger volumes of data, methods to deal with these data have evolved as well. One of the fields that has thus emerged is the field of network science. Network science has many applications in biological fields as it allows scientists to connect variables of any type in a quantitative way. This versatility makes network science ideal for studies on comorbidity, or the consistent cooccurrence of various diseases in individuals. By analyzing comorbidities, we gain greater insight into interactions between diseases and systems of the body. This helps us understand how potential risk factors such as age, sex, and genotype affect various disease risks as well as the risk of comorbidity. We applied these methods to data on age of diagnosis for over 300 diseases collected from more than 28,000 owner reported surveys through the Dog Aging Project. We constructed comorbidity networks from these data and analyzed these networks using quantitative network statistics which allowed us to compare nodes both in and between networks. We first constructed undirected networks with the nodes representing various diseases to establish and identify pairs of diseases with significantly higher rates of cooccurrence than expected by chance. Using this network, we assigned directions to edges based on temporal data on the relative ages of diagnoses, which allowed us to identify which diseases are precursors to others. We observed how these networks changed through stratifications based on age, sex, and size to identify disease progression through age. In doing so, we hope that we can identify how age affects comorbidity in dogs, which can help researchers identify and develop therapies for lengthening dog lifespans. This knowledge will also provide insight into the mechanisms behind certain disease connections that were previously unknown.

Periodontal disease stands to be the leading cause of tooth loss in older adults and is characterized by inflammation of tissues supporting the teeth and periodontal bone loss. This oral disease also occurs during normative aging in mice models. Thus, targeting the biological aging process may provide an innovative approach to reduce the impact of this disease in the elderly population. Rapamycin is a specific mTOR inhibitor shown to delay age-related decline and significantly extend the lifespan of mice models. Our lab has demonstrated that an 8 week treatment with rapamycin in aged animals reversed age-related periodontal disease. However, whether this improvement persists even after the treatment ends is still unknown. To determine whether reversal of periodontal disease persists even after stopping rapamycin treatment, cohorts of aged mice were either started with 8 week treatment with control (eudragit) food or rapamycin (42ppm) food, and then switched to either rapamycin (42ppm) food or control (eudragit) food for another 8 weeks, respectively. High resolution microCT imaging was completed to measure the amount of periodontal bone, and western blot was performed on total alveolar bone extracts and probed for the osteoclast marker RANKL. Our microCT analysis showed there was less periodontal bone loss in aged mice treated with rapamycin, and this result persisted even after 8 weeks on the control diet. Additionally, the age-related increase in RANKL expression was decreased in both treatment groups. In conclusion, the regain of lost periodontal bone in aged mice persisted even after cessation of rapamycin treatment, and the age-related increase in the osteoclast marker RANKL was decreased after rapamycin treatment and such decrease persisted. Overall, our studies indicate that short term treatment with rapamycin is sufficient to rejuvenate oral health and the benefits are maintained even after stopping the treatment.

Bile acids are intestinal metabolites that are biotransformed into diverse secondary bile acids to aid with digestion and absorption. However, once modified by the gut microbiome, they can produce serious health implications including colorectal cancer risk. We hypothesized that this bile acid metabolism is reflected in bacterial cell morphologic changes. To test this hypothesis, we anaerobically cultured and generated light microscopy images of Clostridium scindens in media containing 100 μM cholic acid (a known substrate in the production of the carcinogenic bile acid deoxycholic acid), C. scindens in media containing NaCl (a positive control; 1% NaCl is shown to cause shrinkage through osmosis in bacterial cells), and C. scindens in media alone (negative control) (8 images per group; 500 bacterial cells per image). We developed an image-based model using MicrobeJ (an ImageJ plug-in developed for analysis of bacterial images) by using smoothed particle contours and a skeletonization algorithm adjusting parameters to accurately detect cells. We observed a significant difference in shape descriptor analysis between curvature of the end points and center of the medial axes, width of the medial axes, ratio between the major and minor axes of the cells, ratio between area and convex area, angularity, roundness, length of the medial axes, circularity, and perimeter of the outside boundary between C. scindens with and without the presence of cholic acid (p < 10^-5 for all comparisons; Welch’s two-tailed t-test). Of note, this represented a larger difference than the delta between the two controls. Our data demonstrate that image-based analysis can enable detection of cellular morphologic differences of C. scindens based on metabolic profile with respect to bile acids. In principle, this approach could be expanded to other bile acids and/or beyond bile acid metabolism to identify bacterial metabolic behaviors of interest (e.g. assessing or predicting effects of clinically relevant compounds targeting the microbiome).

 Chronic Mucocutaneous Candidiasis (CMC) is a recurring Candida infection that occurs in patients with T-cell deficiencies, and specifically infects skin, nails, and mucous membranes (Okada, Satoshi et al, Clinical & translational immunology, 2016). Signal Transducer and Activator of Transcription 1 (or STAT1) is a gene known for its role in the IL-17 signaling pathway, which functions in immune response; this specific pathway is critical to fighting off infections such as Candidiasis . Previous studies have shown that autosomal dominant mutations in STAT1 on exon 11 can lead to chronic Candida infections . We have identified a case of CMC which we suspect are caused by gain-of-function mutations in the STAT1 gene. To confirm this suspicion, we performed genetic testing of archival tissue samples. We designed primers targeting exon 11 of the STAT1 gene, and conducted polymerase chain reactions followed by Sanger sequencing. I will be working with the team to anayze the samples and determine whether or not STAT1 mutations are present in these patients, and if these are novel mutations. Identification of STAT1 mutations will be paramount to further understanding the genetic background of patients with CMC, and informing future studies to potentially target the IL-17 pathway in treating CMC.

Many advancements have been made in differentiating human pluripotent stem cells to cardiomyocytes (hPSC-CMs), with respect to obtaining large numbers with high purity. However, a limitation of hPSC-CMs is that they have an immature phenotype and behave like fetal cells. This immaturity limits the application of cardiomyocytes for cell transplantation that would help repair the heart after myocardial infarction. Our lab has identified candidate master transcriptional regulators of cardiac maturation. These regulators have low expression in immature hPSC-CMs and high expression in mature cardiomyocytes. My project goal is to test the role of PPARG coactivator 1 beta (PGC1B), a candidate transcriptional regulator, in regulating hPSC-CM maturation. PGC1B is involved in mitochondrial biogenesis and increases number of mitochondria, which are highly abundant in adult cardiomyocytes. I hypothesize that activating transcription for PGC1B will enhance maturation of hPSC-CMs through mitochondrial biogenesis. To upregulate gene expression, we use a CRISPR activation (CRISPRa) system with a modified version of Cas9 fused to a transcriptional activator VPR (dCas9-VPR) to upregulate transcription of target genes upon introduction of a specific guide RNA (gRNA). I have differentiated WTC11 stem cells into cardiomyocytes, introduced dCas9-VPR and gRNAs for PGC1B via lentivirus, and performed measurements after 2 weeks. I validated the increased expression of PGC1B at the RNA (using quantitative reverse transcriptase PCR) and protein levels (western blot). To assess relative abundance of mitochondria in PGC1B-expressing versus control hPSC-CMs, I will label mitochondria with MitoTracker and quantify using flow cytometry and microscopy. From these experiments, I expect that PGC1B-overexpressed hPSC-CMs would have a higher relative abundance of mitochondria and increased expression of metabolic and maturation genes compared to control hPSC-CMs. My findings will provide insight on the role of PGC1B in mitochondrial biogenesis and stem cell-derived cardiomyocyte maturation.

In response to various forms of intrinsic and extrinsic stresses such as heat shock, electrical stimulation, and viral infection, cells produce non-membrane-bound aggregates of mRNA and proteins called stress granules. These granules sequester mRNA and ribosomal subunits to halt the production of proteins unnecessary for the immediate survival of the cell, thus allowing more energy to be used in combatting the stress. Stress granules are beneficial in the short term, but the chronic presence of stress granules can be cytotoxic. If stress granules are not cleared, hyperaggregation of misfolded proteins, which is thought to play a role in neurological diseases, can occur. After myocardial infarction (heart attack), the heart experiences a lack of oxygen which is known to create free radicals and metabolic stress. Whether the stress response is involved in this process is unknown, as most research on stress granules, especially their role in disease, comes from work in neuronal and cancer cells. To test whether the stress granules response is conserved across cell types and how cardiomyocytes (heart muscle cells) specifically respond to stress, I cultured cancer cells, embryonic stem cells, and embryonic stem cell-derived cardiomyocytes and subjected these cells to various forms of stress, including sodium arsenate poisoning and heat shock. Using fixed immunofluorescence and spinning disk microscopy, I imaged each treatment and quantified the number of stress granules per cell. The sodium arsenate treatment induced stress granule formation in all three cell types, but surprisingly, the heat shock treatment only induced stress granule formation in the stem cells. It is widely believed the stress response is conserved across a wide range of cell types, but these results indicate some stress pathways differ between cardiomyocytes, cancer cells, and stem cells. Future experiments will test additional types of stress and how stress granules contribute to cardiomyocyte function.

COVID-19, the viral disease caused by the novel coronavirus SARS-CoV-2, is associated with cardiovascular complications such as arrhythmias, myocarditis, and even cardiac arrest. There are two possible mechanisms of SARS-CoV-2 entry into human cells; the endosomal-mediated pathway which requires intracellular processing by intracellular proteases, and the membrane fusion pathway mediated by secreted proteases. Importantly, SARS-CoV-2 entry relies on the expression of the transmembrane receptor ACE2, which interacts with the viral spike protein. It’s still unclear if ACE2 is required for both viral entry pathways. Cardiomyocytes express ACE2, thus SARS-CoV-2 can enter heart tissue; however, the mechanism by which this occurs and how it may lead to cardiac dysfunction is unknown. We previously demonstrated that SARS-CoV-2 significantly impairs mechanical and electrical function of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our goal is to understand if ACE2 is required for viral entry into the heart, using hiPSC-CMs as a model, in order to better understand COVID-19 pathology affecting the heart. Using a CRISPR/Cas9 system, we targeted the ACE2 gene at three loci to effectively knockout (KO) gene expression from WTC11 iPSCs. Two KO clones were selected and isolated after sequencing. Wild type (WT) and KO iPSCs were directly differentiated into CMs over a 17-day period. Preliminary results confirmed the absence of ACE2 protein expression in both KO clones by western blot. Fluorescent imaging of CMs infected with GFP-tagged SARS-CoV-2 showed severe infection and cell death at varied time points and multiplicities of infection (MOI) in WT WTC-CMs, while ACE2 KO-CMs showed absence of prominent infection and cell death. These data indicate that the lack of ACE2 markedly prevents SARS-CoV-2 entry into CMs, and understanding if blocking viral entry is sufficient to prevent functional impairment will provide key insights into the development of cardiomyopathies in COVID-19 patients.

Various fatal and debilitating genetic diseases are caused by mitochondrial dysfunction. To study mitochondrial diseases in humans, the Kaeberlein Lab uses NDUFS4-KO mice as a model of the human mitochondrial disease Leigh Syndrome. These mice have a non-functioning protein in their energy-producing oxidative phosphorylation process. We previously observed an increase in total iron in livers of NDUFS4-KO mice, which can damage cells by producing reactive oxygen species. Thus, we hypothesized a low-iron diet may alleviate the effects of the mitochondrial disease. My project aimed to study the molecular consequences of a low-iron diet in NDUFS4-KO mice by quantifying mRNA transcript levels and protein expression of genes involved in iron uptake, storage, or efflux, and comparing these levels between wild-type (WT) and NDUFS4-KO mice fed a normal or low-iron diet. First, I purified mRNA through cell lysis from 35-day old mice liver samples and used the mRNA to perform a one-step Quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Next, to quantify protein levels, I performed western blot analysis in collected protein extracts. I observed an increase protein expression and mRNA transcript levels of iron-uptake proteins such as TfR1 in both WT and NDUFS4-KO low-iron diet mice compared both control groups. This suggests iron levels were reduced to safer levels, supporting my hypothesis. Furthermore, when analyzing the expression of genes which respond to iron overload, such as ferritin, I observed a decrease in the low-iron diet NDUFS4-KO mice compared to control diet NDUFS4-KO mice. This observation shows that low iron diet contributes to a reduction in excess iron. My data may illuminate the involvement of iron in mitochondrial diseases as well as support further research into the consumption of low-iron containing foods, and eventually lead to the development of therapeutic drugs to treat humans who suffer from such ailments.

Friedreich's ataxia (FRDA) is an extremely destructive neurodegenerative mitochondrial disease with no cure to date. The disease is characterized by mutations in the FXN gene, resulting in a deficiency of functional frataxin protein. These reduced levels of frataxin protein cause multitudes of metabolic problems, including oxidative stress, disruption in iron-sulfur cluster synthesis, and iron overload in mitochondria. Recently, it has been discovered that hypoxia can rescue frataxin deficiency in various types of model organisms, including yeast, cultured human cells, nematodes, and mice. However, despite frataxin and oxygen’s integral relationship in mitochondrial function, the exact genetic pathways by which they interact remain elusive. I aim to bridge this gap by using yeast homolog and studying its oxygen dependence. YFH1 is the yeast frataxin homolog that can mimic FRDA pathology. I first created a yeast model of ∆yfh1. I am currently in the process of creating synthetic lethals and rescues of ∆yfh1 by mating it with previously identified yeast knockout strains that are known to show hypoxic resistance. After I obtain these double mutants, I plan to screen them for oxygen dependence by subjecting them under hypoxia, normoxia, and hyperoxia. Finally, I will replica plate my experiments in order to confirm the double mutants. Because ∆yfh1 is known to grow better in hypoxia, genes that create synthetic lethals in hypoxia with ∆yfh1 will most likely be the genes involved in genetic pathways of hypoxic rescue of frataxin deficiency. This will not only streamline the process of searching for the genetic basis of the disease but also serve as a platform for novel therapy to treat FRDA at its biochemical basis.

Alzheimer’s disease, the most common human neurodegenerative disorder, is characterized by hyperphosphorylation of the protein tau, leading to the protein’s aggregation and the formation of neurofibrillary tangles. The subsequent neurodegenerative consequences of these tangles may influence the biological response and sensitivity to neurological stressors, such as a traumatic brain injury (TBI). A TBI usually results from a strong blow to the head that leads to damaged brain cells and neurodegeneration. In the Promislow Lab, we are currently examining how neuronal tau affects the mortality response to a TBI-like trauma in the fruit fly, Drosophila melanogaster. Using the UAS-GAL4 gene expression system, we are able to induce the expression of the tau gene in fly neurons from eclosion. From both an experimental fly genotype (tau expression) and a control fly genotype (no tau expression), we are currently sampling flies at various time points during their lifespan and administering a TBI-like trauma on the flies using a high-impact trauma device. To quantify the impact of tau on the flies’ response to the TBI-like trauma, we are recording the percentage of flies dead 24 hours later (24 hour mortality index). We hypothesized that inflicting a TBI-like trauma would lead to a significantly increased 24-hour mortality index in the experimental genotype compared to the control genotype due to increased sensitivity in the former. Thus far, we have observed significantly increased mortality in response to a TBI-like trauma in the control genotype compared to the experimental genotype. The decreased mortality in the experimental genotype suggests a novel positive role of tau in the response to a TBI, which holds significant implications for targeting clinical TBI treatments and therapies. Further analysis and follow-up experiments will provide useful insight into understanding the mechanisms of tau’s role and the pathways of both Alzheimer’s and TBIs.