Arboleda-Tham Syndrome (ARTHS) is a rare genetic neurodevelopmental disorder caused by mutations on the KAT6A gene and characterized by global developmental and speech delays. The genotype-phenotype correlation studies on the KAT6A patient population has shown that patients with late-truncating mutations have more severe phenotypes than patients with early-truncating mutations. There is currently no treatment for patients with ARTHS. Understanding the differing mechanisms driving the early- and late-truncating KAT6A mutations is essential to identifying potential therapeutic targets. We hypothesize that early-truncating phenotypes occur as a result of a loss of function mechanism, whereas late-truncating mutations lead to a gain of function resulting in elevated levels of acetylation. To understand the mechanistic differences between early and late truncating mutations, we developed mouse models harboring patient specific mutations and cultured mouse embryonic fibroblasts (mEFs). Western blot analysis from these mEFs has shown an increased level of H3k23ac in late-truncating samples. We also observe the late-truncating copy of the protein, suggesting a negative dominant effect. The absence of any early-truncating copy points towards non-sense mediated decay of the truncated copy. We also performed ChIP-Seq and RNA-Seq on control, early, and late-truncating mEFs. Together, this information provides insight on the different mechanisms that drive the early- and late-truncating mutations, allowing for proper drug development.

Melanoma is an aggressive and immunogenic cancer for which current immunotherapies remain insufficient. Although immune checkpoint inhibitors and tumor-infiltrating lymphocyte (TIL) therapy have improved outcomes, they do not directly target innate immune regulators in the tumor microenvironment (TME). Tumor-associated macrophages (TAMs) are abundant in melanoma and can adopt pro-inflammatory (M1-like) or anti-inflammatory (M2-like) states that shape tumor progression. Autophagy regulates inflammatory signaling and cellular stress responses, yet its role in macrophage polarization remains unclear. This thesis examines how canonical (Atg7-dependent) and noncanonical (Atg16l1-dependent) autophagy pathways influence macrophage activation. Bone marrow–derived macrophages (BMDMs) from autophagy-proficient and -deficient mice were stimulated with LPS to induce an M1-like state. Polarization was measured by iNOS expression using flow cytometry. LPS increased iNOS in both genotypes, with variability across experiments. Preliminary Atg16l1-deficient samples showed high dispersion, indicating the need for additional replicates. Autophagy may regulate polarization through NF-κB signaling, mitochondrial quality control, and reactive oxygen species (ROS), influencing metabolic rewiring. Overall, these findings support robust M1 induction and motivate further investigation into whether autophagy deficiency stabilizes pro-inflammatory states and resists tumor-driven reprogramming.

Rationale: Disruptive exogenous events during neonatal cardiac maturation predispose individuals to cardiac disease later in life. While it has been speculated that this window of time coincides with the regulation of sarcomeric assembly in cardiomyocytes, it remains unclear how its components may contribute to functional remodeling or how their functions may be altered upon disruptive cues during this time. Hypothesis: We hypothesize that sarcomeric proteins TCAP and Myl2 are regulated by clock and exogenous cues, which help drive cardiac maturation. Methods: NRVM culture, 21% vs. 0% O₂ exposure, catecholamine (phenylephrine, isoproterenol) treatments, 2x cell density, HW/BW, immunoblot, RT-qPCR, ICF. Results: TCAP and Myl2 expression are spatiotemporally regulated and influenced by the environment. Phenylephrine treatments induce oscillatory expression of Myl2 and temporally regulate accumulation of TCAP. Hypoxia induces de novo TCAP transcription and other clock-targets, and synchronized oscillatory transcription of TCAP is induced by increased cell density. TCAP and Myl2 also underwent sarcomeric organization and localized to the nucleus on postnatal day 9 in rats. Conclusions: TCAP and Myl2 are spatiotemporally controlled through a clock-environment relationship and serve non-sarcomeric functions correlated to the neonatal window of maturation. These findings suggest that early-life environmental factors influence cardiac tissue during maturation, which may impact cardiac development.

Cellular apparatuses like cilia and flagella contain long arms called axonemes composed of a central pair of microtubules connected to outer microtubules via radial spokes. The head regions of each radial spoke is a quaternary protein structure of two identical polypeptides containing radial spoke head protein 9 (rsph9) along with several other structural proteins. Despite its recognition as a ciliary motility regulator in humans, rsph9’s functional role in sea urchin development has yet to be understood, and cloning of the gene is the first step to investigate this purpose. After rsph9 was identified as the unknown sequencing using BLAST and was confirmed via InterPro, Echinobase, and phylogenetic analysis, PCR was to be used to amplify the gene from S. purpuratus genomic DNA. This PCR product was then purified and ligated to a vector and transformed into competent E. coli cells. The transformed cells had their plasmids isolated for restriction digestion to confirm cloning success. Results found several discrepancies in the restriction digest where there were partial digestions or multiple segments of the wrong size. This could indicate that plasmids transformed into the bacterial cells never contained correctly-ligated vectors and the results were overall unsuccessful. Better understanding of the rsph9 gene could lead to advancements in treatment of certain human genetic diseases in the respiratory tract and infertility.

Systemic sclerosis (SSc) is an incurable multisystem autoimmune disorder characterized by fibrosis of the skin and internal organs. Cutaneous fibrosis is caused by the presence of myofibroblasts which excrete excessive amounts of fibrillar collagens and extracellular matrix proteins in the dermis. Previous studies have identified the adipocyte-myofibroblast transition (AMT) as a key event in onset of cutaneous fibrosis pathogenesis and highlighted the unknown role of Vitamin C in fibrosis progression. This study investigates the effects of varying levels of Vitamin C on the AMT in vitro in human adipose derived stem cells (hADSC) and in vivo via a mouse model. While experimentation is still in progress, in vitro AMT progression was assessed through changes in cellular morphology, the transcriptome, and protein expression. The mouse model explores the effect of Vitamin C on fibrosis progression within the context of whole organism physiology assessed through changes in histology and the metabolome. Results indicate that hADSC were successfully differentiated into adipocytes. Though further investigation is needed to confirm initial findings, adipocytes appeared to have differentiated into myofibroblasts. The in vivo results suggest that higher Vitamin C levels may potentially accelerate the loss of adipose tissue in the dermis in a mouse model of systemic sclerosis. Continued and future experimentation on this project can potentially aid in developing new therapeutic and dietary approaches for SSc patients.