Aortic aneurysms form when the aorta, the large artery that carries blood from the heart, weakens and expands. Smooth muscle cells (SMC) in the aortic wall regulate aortic structure and contractility and help preserve normal aortic diameter. Transforming growth factor beta (TGF-β) signaling is integral to SMC function and abnormal TGF-β signaling and is implicated in aneurysm formation. However, whether excessive or deficient SMC TGF-β signaling promotes aneurysms is controversial. To determine if increased SMC TGF-β signaling is sufficient to cause aneurysms, we expressed a Constitutively Active Type I TGF-β Receptor (TβR1-CA) transgene in SMC of male and female mice (both hemizygous). Because the transgene is integrated into the X chromosome, and half of female X chromosomes undergo inactivation, expression of TβR1-CA in males and females may differ. This difference could affect the interpretation of studies that investigate the role of SMC TGF-β signaling in aneurysmal disease. We hypothesize that hemizygous females will have lower levels of TβR1-CA expression than males due to random X-inactivation. To test our hypothesis, we will quantify the mRNA levels of TβR1-CA relative to that of Gapdh (a reference gene for normalization) in aortic SMC of male and female hemizygous transgenic mice using real time (RT)-qPCR. To confirm that we can detect X chromosome inactivation using RT-qPCR, we will also measure mRNA levels of a gene that is known to undergo X-chromosome inactivation (Pgk1) and a gene that is known to escape X-chromosome inactivation (Kdm5c). If our hypothesis is correct, we will find that the amount of aortic TβR1-CA mRNA is ~50% lower in aortas of female versus male transgenic mice. The results of this project will be essential in helping us to interpret results of parallel experiments that investigate whether TβR1-CA expression (and activated SMC TGF-β signaling) causes aneurysms in male and female mice.

The blood-brain barrier (BBB) is a highly specialized interface of brain microvascular endothelial cells (BECs). The main functions of BECs are to protect the brain against exposure to harmful substances in the blood, and to transport and secrete nutrients and other molecules that support normal brain functions. BECs can also alter their functions in response to signals from the brain or blood compartments. GLUT-1 is the predominant transporter at the BBB that regulates glucose entry into the brain, and does so by a mechanism of facilitated diffusion, which permits glucose transport in the blood-to brain or brain-to-blood direction. GLUT-1 dysfunction occurs in and may contribute to Alzheimer’s disease.The main hypothesis is that GLUT-1 malfunction in the BBB occurs as it deteriorates with age, which causes errors in other regulation methods of the BBB leading to the development of Alzheimer's. However, it is difficult to study mechanisms of GLUT-1 dysfunction at the BBB specifically because of the involvement of multiple cell types that regulate glucose uptake into the brain in vivo. In my research, I have utilized a model of iPSC-derived brain endothelial cells (iBECs) in order to study aspects of GLUT-1 regulation in an in vitro model of the BBB. Currently, my main findings have shown that the GLUT1 transporter is functional in our system, and that GLUT1 protein expression is increased and glycolytic enzymes are decreased when iBECs switch from a proliferative state to a quiescent state. Glucose transport in the blood-to-brain direction is decreased in the quiescent state.  Additionally, high glucose exposure causes downregulation of glucose transport.  My future studies aim to determine whether proliferating vs. quiescent iBECs respond differently to high or low concentrations of glucose. In summary, we have shown that iBECs are a translatable and effective model that allows us to further investigate the regulation of GLUT-1 at the BBB to further understand the physiological mechanisms involved in Alzheimer's Disease. 

Mycobacterium tuberculosis (Mtb) is a bacterium that kills nearly 2 million people annually and continues to be a large threat to the health of individuals around the world. T-cells play a key role in the immune response to Mtb infection, but there is still much we do not understand about their role in mediating protection. CD4 and CD8 are glycoproteins found on the surface of T cells. CD4 and CD8 T-cells recognize glycolipids associated with the bacterium, such as glucose monomycolate (GMM), that are presented by the cell-surface glycoprotein CD1b on a human antigen presenting cell. I am focused on identifying the T-cell receptor (TCR) of a T-cell line from the Rhesus Macaque species. Using newly validated CD1b tetramers, I sorted for T-cells that can bind to the GMM lipid antigen using flow cytometry. The tetramer is made up of four CD1b molecules which present the antigen and bind to T-cells that have a matching receptor. I used various molecular techniques such as RNA extraction, 5’ RACE PCR (a procedure for amplification of nucleic acid sequences using a messenger RNA template), and in fusion cloning to determine the TCR gene sequence of the alpha and beta chain regions. By identifying the sequence of a T-cell receptor in the Rhesus Macaque species we will be expanding our knowledge of how T cells recognize Mtb lipids in non-human primates. Since we have previously published that CD1d-restricted TCRs are very similar between NHP (non-human primates) and humans, we expect the same is true here. We can use tetramers and the NHP model to understand how lipid-specific T cells are important for controlling Mtb.

The technique of Indirect Calorimetry (IC) allows for the non-invasive and continuous measurement of metabolically relevant functions of an animal. The Michael Schwartz lab uses IC techniques in the investigation of how brain regions, neuronal populations, and the larger neuronal circuitry that they connect with, defend either blood glucose or body weight. Taking IC measurements of treatment mice allows for quantification of glucose consumption and energy homeostasis. My project was to develop a programming pipeline for the analysis of circadian metrics from rodent populations placed on a 14-hour light and 10-hour dark cycle and placed on control or high-fat high-sucrose diets. The circadian cycling of oxygen consumption and respiratory energy ratio was quantified, daily maxima and minima were measured, and latency from dark cycle onset to peak was measured. Using C#, I generated a parsing software that could combine data from experiments, recalibrate time to the day-night cycle used, and pull out these parameters for future research.

MicroRNAs (miRNAs) are short 21-23 nucleotide (nt) nucleic acid species that have a seed-sequence complimentary to target RNAs which can then be negatively regulated via the RNA silencing pathway. miR-122 is the most highly expressed miRNA in the liver, accounting for 70% of the miRNA reads and is involved in hepatocyte differentiation and cholesterol/fatty-acid synthesis. Previous studies have indicated that total abrogation of miR-122 or competition of miR-122 by short hairpin RNAs (shRNAs) in mice caused an increased expression of the Dlk-Dio3 locus, which is highly expressed in development but downregulated in adults and ultimately leads to hepatocellular carcinoma (HCC). Furthermore, it is known that activation of the Dlk-Dio3 locus alone has been shown to lead to the development of HCC but the specifics of miR-122’s regulatory role have not been elucidated. To determine the effects of miR-122 on the Dlk-Dio3 locus, I am using human HCC cells that contain a 3-bp deletion down-stream from the miR-122 transcript. We predict that the 3-bp deletion prevents proper miR-122 transcription. By transfecting the cells with a miR-122 mimic transcript to introduce its expression, I can compare the levels of RNA expression in liver cells containing miR-122 to those without by using qPCR and small-RNA sequencing. Preliminary data indicates that cells transfected with the miR-122 mimic have decreased levels of expression of RNAs located in the Dlk-Dio3 locus such as Rtl1 and positive control targets such as Aldoa. To further confirm this relationship, I am planning on using CRISPR-mediated homology-directed repair to replace the 3-bp deletion in my cell line and endogenously re-express miR-122 in order to study its regulatory role at the Dlk-Dio3 locus.

5-hydroxymethylcytosine (5hmC) is a DNA modification studied in mammalian cells and tissues that has been implicated in embryonic stem cell differentiation, neuronal development, and formation of tumors. However, much is still unknown about the regulatory function of 5hmC and its genomic localization in diverse cell types. Recently, a novel, enzyme-based method was developed for 5hmC identification, APOBEC-Coupled Epigenetic Sequencing (ACE-seq), that is highly accurate, requires low input, and does not degrade DNA as commonly used bisulfite treatment methods do. ACE-seq is especially relevant to cellular environments that are epigenetically dynamic, including brain tissue and embryonic cell cultures. My project uses ACE-seq to generate high fidelity 5hmC characterizations in the Elf1 cell line across culture conditions that mimic early human embryonic development in which dramatic changes in DNA methylation occur. ACE-seq will be used on three human embryonic stem cell (hESC) conditions that mimic the transition from before embryonic implantation, naïve hESCs (two conditions), to near the time of embryonic implantation, primed hESCs. We hope to gain insight into 5hmC localization and regulatory function during this physiologically significant developmental event occurring during early embryogenesis. A more robust understanding of 5hmC regional abundance during this transition will help us elucidate the regulatory circuitry underlying early development. Knowledge gained in this project is especially relevant to the field of personalized medicine, as thorough understanding of pluripotency transitions will be significant to future applications of stem cell based therapies and precision healthcare. 

Among the estimated 1.5 million adults and children living with HIV in Kenya, only 68% had suppressed viral loads in 2019. Improving adherence to antiretroviral therapy (ART) and minimizing ART failure is crucial in ensuring viral suppression. New types of viral load (VL) and drug resistance mutation (DRM) testing through point-of-care (POC) assays are potential solutions to optimize viral suppression rates. POC assays have faster turnaround times, can facilitate rapid clinical decision making, and are cost-effective, all of which can facilitate better patient outcomes. In the Opt4Kids and Opt4Mamas studies, children and pregnant/postpartum women either received the intervention, consisting of POC VL testing every three months with targeted DRM testing, or standard-of-care (SOC) testing according to national Kenyan guidelines. As undergraduate researchers, we are responsible for coding and analyzing 68 in-depth interviews collected from key informants and participants, including adolescents, children’s caregivers, and pregnant/postpartum women, to better understand how POC VL and targeted DRM testing influences viral suppression and may be utilized for use nation-wide. We used the socioecological model to identify the individual, interpersonal, organizational, societal/cultural, and structural/policy factors that influence viral suppression in children and pregnant/postpartum women. Through inductive coding and thematic analysis, we discovered six major domains of themes of interest amongst these populations: 1) HIV health literacy, 2) HIV testing and treatment experience, 3) differences between SOC VL testing and POC VL testing, and 4) DRM testing experience, 5) future improvements, and 6) impact of the COVID-19 pandemic on treatment and testing. Our findings will provide insight into ways of optimizing existing protocols and will determine whether our intervention is an improved alternative to current national standards. Ultimately, our research will shape national policies regarding HIV treatment and directly address ways to optimize HIV viral suppression.

Pseudomonas aeruginosa, an opportunistic pathogen that commonly infects cystic fibrosis patients, uses quorum sensing (QS), a form of cell-cell communication, to regulate the expression of virulence factors and public goods based on population density. P. aeruginosa QS consists in part of N-acyl homoserine lactone signal molecules that activate two separate regulatory proteins, LasR and RhlR, which in turn activates the transcription of other target genes in their respective regulons. The las and rhl regulons are hierarchical in lab strains, with LasR activating the transcription of rhlR; however, many pathogenic variants carry nonfunctional alleles of lasR and rely on rhlR as the dominant QS regulator. Two anti-activator proteins, QteE and QslA, restrict the expression of these two QS regulons; however, it is not clear how P. aeruginosa anti-activators function in many pathogenic strains. Identifying how anti-activators regulate QS in pathogenic variants could be crucial in developing therapies that do not rely on antibiotics. To investigate how QteE and QslA modulate QS, we overexpressed each anti-activator in P. aeruginosa and used transcriptional reporters to monitor the activity of rhlA, a RhlR regulated gene. Expression of both genes is reduced significantly in strains with over-expressed anti-activators. However, in a pathogenic variant, only over-expressing qteE delayed QS induction while over-expressing qslA had no effect. These results indicate that QteE can modulate QS by affecting LasR and RhlR levels, while QslA only modulates LasR levels. These experiments lay the foundation for therapeutic strategies centered on inhibiting QS rather than relying on conventional antibiotics.

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer with a recurrence risk of ~40%; however, prognosis for low-risk, stage I disease is excellent with primary surgical management. The role of post-operative radiation therapy (PORT) is controversial as it can cause significant and acute long-term side effects. Here, we assess the efficacy of PORT on local recurrence (LR) rates in patients with pathological stage I MCC with primary tumors on the head/neck (HN) vs. non-head and neck (Non-HN) sites. One hundred forty-seven MCC patients treated from 2006-2020 were identified from an IRB-approved prospective registry who had ‘low-risk’ disease: pathological T1 primary tumor resected with negative margins, negative pathologic node status, and no immunosuppression. LR was defined as tumor recurrence within 2 cm of the primary surgical bed. I led compilation of the cohort, and contributed to discussion of results, and development of the figures and manuscript. Seventy-nine patients received PORT (30 HN, 49 Non-HN), and 68 patients were treated with surgery alone (30 HN, 38 Non-HN). Addition of PORT was associated with a decreased risk of LR across the entire cohort (5-year rate: 9.5% vs. 0%, p=0.004), with 6 LRs in the surgery alone group. The addition of PORT significantly reduced LR rates among HN patients (21% vs. 0%, p=0.034). Conversely, no LRs were observed in Non-HN patients. No significant MCC-specific survival differences were observed. For low-risk MCC of the extremities and trunk, excellent outcomes were achieved with surgery alone. However, HN MCC was a risk factor for LR that was significantly reduced with PORT. Overall, this study demonstrates the importance of primary tumor site location for prognosis and treatment of MCC to determine patients that would benefit from PORT and those that can be spared the toxic side effects of radiotherapy.

Cachexia is a debilitating condition characterized by the loss of muscle strength and mass. Affecting 50-80% of advanced cancer patients, this condition is associated with weakness, fatigue, poor tolerance to chemotherapy, and decreased quality of life. The mortality rate of patients with cancer-induced cachexia can be as high as 80%, and there is no current effective treatment. Ghrelin has recently been proposed as a therapeutic option for cancer cachexia due to its effects on preventing appetite, muscle, and fat loss. Its orexigenic effects are mediated by the growth hormone secretagogue receptor GHSR-1a, but the extent to which GHSR-1a mediates ghrelin’s effects on preventing muscle mass and function loss is unknown. This study characterizes the pathways involved in muscle mass loss and weakness in the Lewis lung carcinoma (LLC)-induced cachexia model and the effects of ghrelin in wildtype GHSR+/+ and knockout GHSR-/- mice. 5-6 month old male C57GL/6J mice were injected with LLC cells. When the tumor was palpable, tumor-bearing mice were injected with vehicle (saline solution) or ghrelin (0.8 mg/kg). We quantified body mass over 3 weeks and dissected and weighed hindlimb muscles. We analyzed mitochondrial protein concentrations via BCA and measured oxidative phosphorylation markers through Western blotting. Ghrelin attenuated LLC-induced muscle wasting in both genotypes but only prevented the decrease in grip strength in GHSR+/+. There was no significant difference in muscle mass between the two genotypes. The ubiquitin-proteasome system (UPS) and autophagy-lysosome pathways were activated by LLC in both genotypes, and these changes were more pronounced in GHSR -/-. In tumor-bearing mice, ghrelin mitigated the increased UPS markers (atrogin-1, MuRF1) independently of GHSR-1a. Ghrelin only prevented tumor-induced increases in mitophagy markers in GHSR+/+ (p62, Bnip3), and the levels of these mitophagy markers were negatively correlated with muscle strength. In conclusion, GHSR-1a is required for ghrelin’s effects on attenuating LLC-induced loss of muscle strength but not muscle mass, and this is likely due to the alterations in the autophagy-lysosome pathway and impaired mitophagy. By characterizing the preventative role of GHSR-1a in muscle wasting, we hope to improve hormonal therapy options for tumor-induced cachexia patients.

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).

Skin cancer is the most prevalent cancer in the United States, and its annual incidence exceeds all other cancers combined. There is thus a pressing need to develop novel approaches to inhibit skin cancer. Inactivation of tumor suppressor genes is a frequent event in carcinogenesis. In skin cancer, the CDKN2A gene, which encodes the p16^INK4A tumor suppressor protein, is often silenced by epigenetic abnormalities such as promoter DNA methylation and histone deacetylation at this genomic locus. The p16^INK4A tumor suppressor functions as a cell cycle regulator and plays an important role in tumor growth and metastasis. Unlike gene loss, gene silencing by aberrant epigenetic modifications can be reversed by small-molecule compounds such as DNA methyltransferase inhibitors and histone deacetylase inhibitors. However, these small-molecule inhibitors are nonspecific, affecting epigenetic modifications globally. Here we use CRISPR-Cas9-based epigenome editing tools for targeted histone acetylation at specific genomic loci. To induce histone acetylation at the CDKN2A promoter, nuclease-deactivated Cas9 (dCas9) is fused to histone acetyltransferase p300, and the dCas9-p300 fusion protein can be recruited to the target site by using guide RNA (gRNA) that binds to the CDKN2A promoter. After dCas9-p300 with gRNA was introduced into the A431 skin cancer cell line to target the CDKN2A promoter, RT-qPCR revealed that p16^INK4A mRNA expression levels increased by approximately 30-fold. Cell proliferation assays further revealed that proliferation of A431 cells decreased by approximately 20%. To fully inhibit proliferation of skin cancer cells, further investigations are needed to evaluate the combined effects of targeted histone acetylation and DNA demethylation at the CDKN2A promoter. This study provides insight into how epigenetic abnormalities can be targeted by novel epigenome editing tools in order to reactivate dormant tumor suppressors and inhibit cancer phenotypes.

Previous studies indicate that CNS administration of oxytocin (OT) reduces body weight in male high fat diet-induced obese (DIO) rodents by reducing food intake and increasing energy expenditure (EE). We recently demonstrated that hindbrain [fourth ventricular (4V)] administration of OT elicits weight loss and elevates interscapular brown adipose tissue temperature (TIBAT; surrogate marker of increased EE) in male DIO rats. What remains unclear is whether chronic CNS OT can impact body weight in female high fat diet-fed (HFD) rats and whether this involves activation of hindbrain OT receptors. We hypothesized that OT-induced stimulation of hindbrain OT receptors reduces weight gain and adiposity, in part, by reducing energy intake and increasing BAT thermogenesis in female HFD-fed rats. To test this hypothesis, we measured the effects of chronic 4V OT (≈16.1 ug/day) or vehicle infusions over 28 days on body weight, adiposity and energy intake in female HFD-fed (60% kcal from fat) rats (N=7-8/group). We found that chronic 4V OT reduced weight gain (P<0.05) and relative fat mass (P<0.05) in randomly cycling female HFD-fed rats. These effects were attributed, in part, to reduced energy intake evident during weeks 2 (P<0.05), 3 (P<0.05) and 4 (P<0.05). To assess if hindbrain OT administration also elevates BAT thermogenesis, we examined the effects of acute 4V OT (1, 5 ug) or vehicle on TIBAT in a separate group of female HFD-fed rats (N=8/group). We found that the low dose (1 ug) elevated TIBAT at 0.75, 1, 1.25, 1.5 and 2-h post-injection (P<0.05); the higher dose (5 ug) elevated TIBAT at 0.75, 1, 1.25, 1.5, 1.75 and 2-h post-injection (P<0.05). Together, these findings support the hypothesis that oxytocin action in the hindbrain reduces body weight gain and adiposity by reducing energy intake and increasing BAT thermogenesis in female HFD-fed rats.

Arrhythmogenic cardiomyopathy (AC) is a devastating inheritable heart disease characterized by lethal heart rhythms and abnormal contractile function that can lead to sudden cardiac death or congestive heart failure. More than 70% of AC cases are due to mutations in desmosomal proteins, which are essential for maintaining structural integrity and intercellular junctions in the heart. Frameshift mutations in desmoplakin (DSP), a desmosomal protein, are a common cause of AC, therefore my project aims to use human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to model AC due to premature truncating DSP variants and to determine if the mechanism results from haploinsufficiency of DSP protein. From two unrelated patients with AC carrying different pathogenic DSP variants, p.Leu463Serfs*22 and DSP p.Arg941*, we generated patient-specific iPSC-CMs from each patient. With these cells, we created engineered heart tissues (EHTs) and found preliminary data that suggests DSP L463Sfs*22 EHTs generate less active twitch force compared to wild type, indicating that this human iPSC model can recapitulate the salient clinical phenotype. Protein analysis of these cells revealed that patients with premature truncating mutations in desmoplakin have lower desmoplakin levels when compared to wild type cells (DSP L463Sfs*22 iPSC-CMs have 38% less DSP protein levels compared to wild type iPSC-CMs). There was no evidence of the predicted truncated protein to suggest a dominant negative mechanism. Furthermore, we found a significant reduction in DSP transcript in the DSP L463Sfs*22 iPSC-CMs that was partially rescued by knocking down UPF1, a key regulator of the nonsense-mediated decay (NMD) pathway, suggesting a NMD-mediated clearance of DSP transcripts that results in haploinsufficiency. Should we also find that increasing desmoplakin protein rescues the phenotypes observed in patients, there is potential to find a novel treatment option for patients with desmoplakin-associated cardiomyopathy.

Human Immunodeficiency Virus type 1 (HIV-1) is currently a major cause of morbidity and mortality worldwide. HIV-1 isolates are classified into genetic groups M, N, O, and P. Group M is the most widespread and is further divided into at least nine genetic subtypes and over 100 circulating recombinant forms, the prevalence of which vary by geographic locations. Subtype B is the most common form in Europe and North America and as a result, receives the majority of funding and research. Because of this, most of what we know about antiretroviral drug resistance in HIV-1 is based on studies of subtype B. However, the majority of HIV-1 infections worldwide involve other “non-B” subtypes, and much less is known about drug resistance in these subtypes. Knowledge of resistance to integrase inhibitors (INIs) is particularly important, since INI-based regimens are now recommended as first-line treatment for HIV-1–infected individuals globally. My objective is to genetically alter a full-length HIV-1-encoding plasmid (HIV-1 pNL4-3) that can used as a “cassette” for cloning non-B, patient-derived integrase sequences. To do this, I have identified restriction sites flanking the integrase region that can be used to remove and replace the integrase region with a patient-derived sequence, but site-directed mutagenesis must be used to knock out additional restriction sites for the same enzymes within the plasmid to avoid undesired enzymatic activity. This will result in only the desired sites, without changing the amino acid sequence. This vector will enable us to generate recombinant viruses from patients who are failing INI-based treatment, which can be tested for resistance to INIs in cell culture. The resulting data will be used to improve prediction of drug resistance in non-B HIV-1 based on sequence information alone. These studies will help guide treatment recommendations for both individuals and populations infected with non-B-HIV-1.

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.