Heart failure (HF) is a constellation of symptoms caused by the heart's inability to pump blood to the rest of your body efficiently and keep up with its workload. As an increasing problem worldwide, there is a need to better understand the underlying mechanisms in HF to develop new therapeutics. HF activates many pathways that could potentially contribute to worsening conditions, such as deoxynucleotide biosynthesis which utilizes Ribonucleotide Reductase (RNR; rate-limiting step). RNR is a vital catalyst in converting nucleotide diphosphates (NDPs) to deoxynucleotide diphosphates (dNDPs) for further phosphorylation to deoxynucleotide triphosphates (dNTPs; utilized as building blocks of nuclear and mitochondrial DNA). RNR is made up of two subunits; Rrm1 which serves as a binding and catalysis domain, and Rrm2/2b which coordinates RNR activity. This project aims to understand how hearts respond with reduced RNR activity, which we investigate through a new mouse model that selectively removed Rrm2 from cardiac cells. We inject tamoxifen daily (25 mg/kg) for five consecutive days into two mouse lines we breed with LoxP sites in the Rrm2, one with and one without αMHC-MerCreMer (R2KO and control, respectively). Tamoxifen injections will conditionally knockout Rrm2 in adult mice with the αMHC-MerCreMer (R2KO). We used echocardiography to assess cardiac function which showed increased cardiac chamber dilation and reduced cardiac function in R2KO mice compared to control mice. We harvest the tissue for histological, DNA, and RNA analysis. Histological analysis suggests that loss of RNR activity does not have any significant effect on cell size or fibrosis in comparison to the control. We will isolate DNA from mice cardiac tissue and use quantitative PCR to assess mitochondrial DNA content, as well as, isolate RNA to perform quantitative Polymerase Chain Reaction (qPCR) to confirm Rrm2 deletion and assess expression of other genes involved in the pathway.

We are currently investigating how neonates establish a symbiotic relationship between their developing immune system and the microbes colonizing their gut immediately post birth, and what role factors (e.g. antibodies) from the mother play in establishing this relationship. Lactobacillus is a genus of commensal bacteria that is commonly found in the neonatal intestine of both mice and humans. To track immune responses in the gut, I will create a fluorescent Lactobacillus species by transforming (genetically altering a cell through uptake and incorporation of outside DNA) a plasmid encoding a fluorescent protein into the bacterial cell. By engineering a fluorescent commensal species, we can track a normal, healthy immune-commensal interaction in vivo (in an animal model). Using this novel tool, we will study and compare how neonatal mice that do and do not receive antibodies from the mother post-birth differ in their immune response against these commensal species.

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.

Advances in medicine have significantly increased life expectancy for much of the population. However, aging-associated diseases such as cancer, heart disease, and hypertension amongst others continue to significantly impact the aging population. Biological aging is defined by the gradual accumulation of cellular damage and development of physiological abnormalities from repeated acute insults or chronic disease states. A major driving force in biological aging is due to generation of reactive oxygen species (ROS), natural byproducts of cellular metabolism, possessing an unpaired electron that is highly reactive with essential biomolecules. ROS generation is increased by factors like lifestyle or exposure to toxic concentrations of drugs or environmental toxins.  One marker of oxidative stress (OS) is oxidative modification of nucleic acids, in particular the modification of guanine in RNA and DNA to 8-oxo-Gsn and 8-oxod-Gsn respectively. I hypothesize that 8-oxo-Gsn is a superior biomarker to 8-oxod-Gsn for assessing transient and acute incidences of elevated OS due to RNA having a higher turnover rate than DNA. Utilizing a recently published high pressure liquid chromatography- tandem mass spectrometry (HPLC-MS/MS) method, I assessed the production of 8-oxo-Gsn and 8-oxod-Gsn in human proximal tubule epithelial cell (PTECs) 2D culture and in 3D microphysiological systems (MPS) in response to induction of OS under experimental conditions. Accessible biomarkers can serve as early indicators of cellular stress, allowing for early intervention of aging-associated diseases.

Acute respiratory distress syndrome (ARDS) is a significant cause of morbidity and mortality in older people. ARDS is initially mediated by an acute lung injury (ALI) response. Studies are needed to investigate why aging increases the risk for more severe ALI and complications assoicated with ALI. Animal models are useful for these types of investigations but most studies use young animals and therefore do not replicate an aging environment and fail to provide valid translational information. The purpose of this pilot study was to determine the pulmonary response and serum cytokine levels in old mice exposed to lipopolysaccharide (LPS), designed to induce ALI. Twenty C57BL/6 mice, 22 months of age, were exposed to 800 ng LPS in 50 ul of saline or saline alone by endotracheal instillation. Two days later, mice were euthanized and serum, lung and other tissues were collected. Serum was tested by ELISA for inflammatory cytokines TNFalpha and IL-6. Lungs were formalin fixed for H and E staining, and slides were read by a veterinary pathologist. Serum TNFalpha and IL-6 were both significantly increased in LPS treated mice compared to baseline and saline controls. Lung pathology consisted of an acute response of proteinaceous exudate flooding alveolar spaces, inflammatory cells within interstitial and alveolar spaces, and inflammatory cells surrounding and within small blood vessels. Additional studies are needed to confirm the utility of the model but this preliminary observation suggests that LPS-induced ALI in old mice might enable more valid investigations into pathogenesis and interventions associated with ARDS and respiratory afflictions caused by SARS-Co-V2 and related virus infections in older people.

 The methionine salvage pathway (MSP) is a set of metabolic reactions that is highly conserved among species. The SPE3 gene of the budding yeast Saccharomyces cerevisiae, characterized as essential for growth, encodes spermidine synthase, which catalyzes the 3rd enzymatic step of the MSP and is involved in biosynthesis of spermidine. We investigated the effects of mutations in SPE3 and other genes of the MSP by comparing strains containing mutations in genes encoding for different MSP enzymes. Diploid heterozygotes were created via mating, and double mutants were created via knockout PCR, transformation, and recombination. Cell growth rate, viability, vacuolar morphology, and genetic relationships were analyzed via growth curves, viability tests, microscopy, and spot test assays. We found that SPE3 knockout haploid (spe3Δ) mutants are viable with a generation time unaffected by growth in minimal media. We also show that SPE3 mutations result in a hindered ability to respond to stressors. Characterization of these mutant strains and their responses to stressors will lead to better understanding of spermidine biosynthesis and the functions of the Spe3p enzyme and other MSP enzymes in S. cerevisiae.