Diabetes results in hyperglycemia and elevated lipid levels (diabetic dyslipidemia), both of which contribute to complications such as atherosclerotic cardiovascular disease. Preliminary data from our laboratory suggest that monocytes are lipid-loaded in diabetes, and the Cluster of Differentiation 36 (CD36) receptor mediates monocyte lipid-uptake. My preliminary data indicate that Cd36 mRNA expression increases in monocytes treated with high glucose. Thus, I hypothesize that hyperglycemia augments uptake of the Very-low density lipoprotein (VLDL), a triglyceride-rich lipoprotein (TRL) elevated by diabetes. To address this, bone marrow monocytes will be cultured in low and high glucose, ex vivo. An osmotic control will be included. Following the glucose stimulation, monocytes will be challenged with fluorescently labeled VLDL (Dil-VLDL), and the uptake will be measured by fluorescent microscopy. Furthermore, to verify that CD36 is critical for monocyte VLDL-uptake, bone marrow monocytes from control and CD36-deficient mice will be used. This study will help us clarify the relationship between lipid metabolism and hyperglycemia in diabetes-induced monocyte lipid loading.

Over 38 million Americans have diabetes, and over 90% of people with diabetes have Type 2 Diabetes. Diabetes increases the risk for complications, including Diabetic Kidney Disease (DKD), a disease which impacts filtration of the kidneys. This filtration occurs in the glomerulus, a specialized capillary network lined with a single layer of endothelial cells. The glycocalyx, an extracellular matrix (ECM), produced by the endothelial cells, plays a crucial role in regulating filtration. Injury, reduced function, or changes in the ECM of endothelial cells cause abnormal filtration and kidney disease. Previously generated single-cell RNA sequencing data from our lab and the Kidney Precision Medicine Project indicated an upregulation of genes involved in ECM remodeling, such as Adamts6, in diabetes, both in mice and humans. My hypothesis is diabetes induces degradation of the endothelial cell glycocalyx through increased expression of ECM degrading enzymes, contributing to glomerular endothelial cell dysfunction. To test if diabetes induces ECM degradation, I examined the abundance of glomerular glycocalyx in a mouse model of DKD in Type 2 Diabetes and in nondiabetic mice. Glycocalyx levels were assessed using wheat germ agglutinin staining and quantified through immunohistochemistry and flow cytometry. Diabetes significantly reduced glomerular glycocalyx in diabetic versus nondiabetic mice. Additionally, I investigated the role of hyperglycemia and dyslipidemia individually on ECM degradation in cultured glomerular endothelial cells. Further investigation will focus on the role of ADAMTS6 on ECM remodeling in diabetes by using a siRNA to block ADAMTS6 expression in cultured endothelial cells. Additionally, to further test the role of hyperglycemia in glycocalyx degradation, an SGLT2 inhibitor was given to diabetic mice to reduce blood glucose levels and examine the impact on endothelial cell glycocalyx. ECM remodeling may be induced by increased expression of ECM-degrading enzymes in diabetes, contributing to the glomerular filtration barrier breakdown seen in DKD.

Type 1 Diabetes is characterized by a dysfunctional response of the immune system, with our project focusing on CD8+ T-cells. Studying epigenomics provides us with information about differential gene expression, as well as distal enhancers and their targets. Understanding this genetic background enables more efficient means of treatment. In this paper, we look at three different kinds of sequencing: ATACseq, RNAseq, and Hi-C. Our focus up until this point has primarily rested upon the first, as we have used it to analyze chromatin accessibility across the genome in patients with T1D and healthy controls. To do so, we have found peaks within the reads for both demographics, which we then used to examine the individual peaks for each subject and the consensus peaks between each condition to see which are especially prominent. These peaks are then the focus of our differential expression analysis, which will allow us to understand the areas of significance and perform further exploration: variance calling and footprinting. As we continue with this project, we hope that RNAseq and Hi-C will provide us with information on gene expression levels and the physical structure of chromatin, respectively. The former was run and sequenced within our lab, but the latter is pre-existing data we will be drawing from for this analysis. Understanding the regulatory landscape allows for better informed treatments, not just for T1D but for autoimmune diseases as a whole.

Pseudomonas aeruginosa (Pa) is an opportunistic pathogen that infects the airways of people with cystic fibrosis, a genetic disease that increases susceptibility to lung infections. Pa uses an intercellular communication system called quorum sensing (QS) that allows bacteria to sense cell density and coordinate behaviors among the population, including regulation of virulence. In the laboratory strain PAO1, there are three complete QS systems in Pa that are regulated by the transcription factors LasR, RhlR, and PqsR. PAO1 QS is organized hierarchically with LasR regulating RhlR, and the hierarchy is influenced by the transcription factor MexT that delays RhlR activity. However, it is unknown if QS hierarchy is found widely in Pa strains. My project tested whether the QS hierarchy exists in clinical isolates of Pa. We obtained 3 clinical isolates with intact lasR, rhlR, and mexT genes and created lasR and mexT knockout mutants for each strain to test the effects on RhlR activity compared to wild-type. To measure RhlR activity, we transformed each strain with a RhlR reporter plasmid. We found that a PAO1 mexT mutant shows greater RhlR activity compared to wild-type, while each clinical isolate showed similar RhlR activity between wild-type and the mexT mutant. We observed lower RhlR activity in clinical-isolate lasR mutants compared to wild-type, demonstrating LasR-dependent QS like PAO1. In PAO1, a ∆lasR∆mexT double knockout mutant restored RhlR activity. Interestingly, in clinical isolates, we observed no change in RhlR activity in these ∆lasR∆mexT double knockout mutants as compared to the lasR mutant, indicating MexT is not regulating QS hierarchy in these clinical isolates. Altogether, the clinical isolates displayed a LasR-dependent QS architecture similar to PAO1, but this was not dependent on MexT. Thus, my work points to undiscovered factors that influence QS architecture and highlight the diversity of QS regulation in strains of Pa.

Pseudomonas aeruginosa, a rod-shaped, Gram-negative bacteria, can cause various opportunistic infections, and it is a common pathogen in hospitals because of its antibiotic resistance and virulence. In P. aeruginosa, virulence is primarily regulated by cyclic adenosine monophosphate(cAMP), which binds to two effector proteins: virulence factor regulator(Vfr) and cAMP-binding protein A(CbpA). As cAMP binds to Vfr, this secondary signal promotes transcription of genes involved in virulence, such as the type IV pili system, which mediates twitching motility, and the Type III secretion system, which releases toxins into the host cell cytoplasm. However, regarding CbpA, all that is known so far is that its expression is strongly regulated by cAMP-Vfr signaling, and cAMP-CbpA binding localizes this protein to the P.aeruginosa cell pole. My project aims to determine the function of CbpA and how this effector protein regulates the cAMP-related processes of P.aeruginosa. To meet these goals, I have generated a construct that overproduces CbpA and am making mutant strains lacking cbpA. Using these constructs, I will evaluate how CbpA influences cAMP levels using a fluorescence reporter and assess its function in twitching and swimming motility using macroscopic assays.  Given that cbpA is regulated by cAMP-Vfr signaling, I will perform these experiments in strains of the wild-type (normal cAMP levels), ∆cyaAB(lacking cAMP synthesis, low cAMP levels), and ctx::araBAD-cyaB(inducible cAMP synthesis, high cAMP levels).  These experiments will provide insights into the roles of CbpA in P.aeruginosa virulence and motility. A deeper understanding of cAMP signaling and its effectors will enhance our understanding of the pathogenesis of P. aeruginosa, facilitating the development of therapeutic strategies against its infections.

In Escherichia coli, the Cpx system is understood to be a two-component cell envelope stress response system. In Pseudomonas aeruginosa however, the Cpx system is largely unstudied. Based on predictive modeling, the Cpx two-component system in P. aeruginosa is thought to involve interactions with two novel accessory proteins, PA3203 and PA3207. Previous genetic analysis in our lab has indicated that PA3207 acts as a negative regulator of Cpx signaling, while PA3203 promotes activity of the system. I evaluated biochemical interactions between these two proteins using the Bacterial Two-Hybrid assay. I generated N- and C-terminal fusions to two functional domains (T18 and T25) of an adenylate cyclase enzymatic reporter. Adenylate cyclase activity, occurring when T18 and T25 were brought into proximity by fusion protein interactions, was measured by a qualitative color assay on MacConkey agar. By this method, I confirmed functional interactions between PA3207 and cytoplasmic signaling domains of both CpxS and CpxR. Interactions between PA3203 and CpxSR were also detected, but were more dependent on the orientation of protein fusions. These findings indicate that CpxSR signaling is regulated through protein-protein interactions with multiple accessory proteins, a unique mechanism among bacterial two-component systems.