Lipid peroxides (LOOHs) abound in processed food and have been implicated in the pathology of diverse diseases including gut, cardiovascular, and cancer diseases. However, the molecular mechanisms by which LOOHs contribute to disease have not been fully characterized. Caco-2 cells have been widely used to model human intestinal epithelium in metabolic studies. As differentiated (Diff) and poorly-differentiated (PDiff) Caco-2 cells represent good models of human enterocytes and intestinal tumor cells, respectively, we investigated the cellular response of Diff and PDiff Caco-2 cells to the most common dietary LOOH, 13-hydroperoxyoctadecadienoic acid (13-HPODE), in terms of differential gene expression, gene ontology and pathway analysis using transcriptomic profiling. This dissertation demonstrates the work conducted on Diff and PDiff Caco-2 cells in order to understand how LOOHs might contribute to disease in the intestinal epithelium. We also compare between the responses of Diff and PDiff cells to LOOHs as Diff cells resemble mature-like enterocytes (have brush borders) and PDiff cells model intestinal cancer cells or crypt cells (lack brush borders). To characterize gene expression and pathway dysregulation upon exposure to peroxidized linoleic acid, we incubated Diff and PDiff intestinal epithelial cells (Caco-2) with 100µM of 13-hydroperoxyoctadecadienoic acid (13-HPODE), linoleic acid (LA) or hydrogen peroxide (H2O2) for 24h. Total RNA was extracted for library preparation and Illumina HiSeq sequencing. This dissertation demonstrates that the Diff and PDiff Caco-2 cells, which differ in their phenotype, behavior and gene expression, show significant differences in their response to the most common dietary lipid peroxide, 13-HPODE, although some similarities in the enriched processes of both cell types were observed. Both cell types showed enrichment of PPAR signaling, cytochrome P450, oxidative phosphorylation and membrane transporters which support previous studies reported the effect of 13-HPODE on these processes. In addition, 13-HPODE treatment had a significant effect on steroid hormone biosynthesis, RNA processing and ribosome biogenesis in both types of cells. On the other hand, Diff cells showed enrichment of bile conjugation upon 13-HPODE treatment which could provide a link between 13-HPODE's detergent activity and cell phenotype. The significant impact of 13-HPODE on cell cycle and DNA replication/repair in Diff cells might indicate an effect on cellular differentiation and apoptosis. In PDiff cells, more defense mechanisms were triggered by 13-HPODE than in Diff cells. In addition to cytochrome P450, retinol metabolism and peroxisomal pathway were enriched in PDiff cells indicating a stronger defense mechanism was triggered in PDiff than Diff cells. Moreover, phospholipid biosynthesis, amino acid and glycogen metabolism enrichment in PDiff cells, but not in Diff cells, upon 13-HPODE treatment was an indication of tumorigenic environment and malignant transformation. This research using Caco-2 cells provides insights into the physiological changes that might occur in the intestinal epithelial cells upon exposure to 13-HPODE and the possible mechanisms by which it contributes to disease development or progression in intestinal epithelium. Our results also support that Diff and PDiff Caco-2 cells differ in their response to 13-HPODE.

The assembly, mechanics, and structure of actin cytoskeleton are critical for eukaryotic cellular processes such as structural support, cellular motility, and intracellular transport. Actin assembly occurs in crowded intracellular environments containing many ions and macromolecules. While the individual roles of cation and crowding on actin assembly and mechanics are well established, how the combined cation and crowding effects modulate the formation, mechanics and structural transitioning of actin filaments and bundles are not fully understood. Here, we investigate the impacts of various cation and crowded environments on the bending stiffness, length, organization, secondary structure, and kinetics of actin filaments and bundles. Fluorescence microscopy and biophysical analysis of filaments in crowding indicates an increase to filament bending stiffness and reductions in length, while molecular dynamics (MD) simulations demonstrate crowding alters filament conformations and inter-subunit contacts. Direct visualization and analysis of bundle stiffness and geometry by fluorescence imaging and atomic force microscopy (AFM) show changes to bundle longitudinal and transverse mechanics as well as bundle packing, displaying a dissimilar dependence on either cation or crowding conditions. Furthermore, spectroscopy results illustrate bundles experience changes to secondary structure and hydrogen bonding with shifts from a-helices to ß-sheets supporting observed bundle stiffness in cation and crowded environments. In addition, bundle assembly kinetics demonstrate that bundle formation is sensitive to variations in electrostatic and crowding conditions, potentially influencing bundle mechanics and structure. These investigations suggest that cation and crowding effects may work counteractively leading to alterations in bundle formation, mechanics, and structure.

No description available.

No description available.

Despite the extensive research on Notch signaling involvement in inflammation, its role in macrophage response in autoimmune diseases and defense mechanisms against bacterial infection such as Mycobacterium avium paratuberculosis (MAP) remains unknown. In this study, we investigated the molecular role of Notch-1 signaling and its downstream modulation in the macrophage response during infection and disease treatment. We demonstrated that MAP infection significantly upregulated Notch-1 and IL-6, which hijacked myeloid cell leukemia sequence-1 (MCL)-1 dependent inhibition of apoptosis and caused chronic MAP persistence and significantly led to successive inflammation. Blocking Notch signaling with gamma-secretase inhibitor (DAPT) decreased MAP survival and burden, increased apoptosis, and diminished the pro-inflammatory response. We have also unmasked the mechanism involved in Notch-1 signaling in response to anti-TNF-Alpha therapeutics. Anti-TNF-Alpha induced Notch-1 signaling in macrophages and consequently upregulated IL-6 and MCL-1, increased intracellular MAP Infection, and further inflammation. This may explain the short-term benefits and poor response to anti-TNF-Alpha therapy in most patients. Furthermore, we reported that polymorphisms in microRNA (miR)-146a rs2910164 GC occurs significantly in Crohn's disease leading to loss of miR-146a function and appears to upregulate Notch-1/IL-6 signaling and further worsens MAP infection and inflammation in patients. We were intrigued by the fact that the majority of fatalities of Coronavirus disease 2019 (COVID-19) were among patients with underlying conditions, including those with autoimmune diseases, many of whom were on anti-TNF-a therapy. In this study, we discovered that anti-TNF-Alpha therapy is involved in acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry and COVID-19 severe complications. Specifically, we found that anti-TNF-a monoclonal antibodies (Humira) via Notch-1/IL-6 signaling modulate TNF-a converting enzyme (TACE) signaling and angiotensin-converting enzyme II (ACE2) expression and its extracellular shedding by increasing type II transmembrane serine proteases (TMPRSS2)/TACE ratio; a high circulating ACE2 level is protective against viral infection. Clinically, ACE2 level was significantly lower in the plasma from RA patients on anti-TNF-Alpha treatment compared to healthy controls in our study. More follow up studies are needed to validate the safety of anti-TNF-Alpha therapy in high risk populations during the COVID-19 Pandemic. Overall, our data clearly demonstrate the significant importance of Notch-1/IL-6 signaling in macrophage response to infection and therapeutics especially in patients with underlying conditions.

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When cells undergo mitosis, they must replicate all six billion base pairs of DNA within the nucleus. With the sheer volume of information, it is impossible to replicate with 100% accuracy each time. Homologous recombination (HR) is one of many mechanisms the body has developed to correct and repair replication errors to DNA. HR is specific to double-stranded breaks to DNA, and it requires a sister chromatid to preserve the genetic code. BRCA1 and Rbbp8 interact to form a complex that is heavily involved in this process. Although there is a strong consensus about the involvement of these proteins in a cell cycle-dependent manner, there are discrepancies in the current literature regarding when homologous recombination repair occurs. The goal of this thesis is to elucidatethe true activity of BRCA1-Rbbp8 complex in the HR process. First, a meta-analysis was performed to review current research to understand the various experimental protocols that led to the conflicting conclusions about cell-cycle activity of HR. Then, we measured the mRNA levels of BRCA1 and Rbbp8 during different phases of the cell cycle.In order to accomplish this, the cell cycle of the L cells was synchronized using thymidine and RO-3306 for the S and G2phases, respectively. The RNA was collected and converted to cDNA via reverse transcriptase. Next, RT-qPCR was performed to measure the expression of BRCA1 and Rbbp8. The levels of Rbbp8 are not significantly different between these phases, though there was a clear downward trend worth noting. Furthermore, a statistically significant increase in BRCA1 as the cells moved from S phase to G2phase was observed. When normalized tothe housekeeping gene, GAPDH, the levels of mRNA for BRCA1 during the S phase were significant lower than the control group. These results suggest that HR occurs during both S and G2, but BRCA1 and Rbbp8 interact only during G2.

No description available.

No description available.