<p>  </p> <p>Calcium influx into pancreatic beta-cells is required for proper beta-cell growth and function. While the functional significance of calcium influx into the beta-cells is known, the significance of release of calcium from intracellular stores is less understood. Calcium-induced calcium release (CICR) is a process by which calcium influx into the cell through voltage-gated calcium channels activated release of calcium from intracellular stores. The functional significance of CICR is well understood in cardiac and vascular muscle cells in regard to excitation-contraction coupling. However, the functional significance of CICR in beta-cells in not well understood. </p> <p>To investigate the role of RyR2 in pancreatic beta-cell function, we utilized CRISPR-Cas9 gene editing to delete RyR2 from the rat insulinoma INS-1 cell line. we found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) which was sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. RyR2 KO and IRBIT KO cells had reduced glucose-stimulated insulin secretion and insulin content. RT-qPCR revealed that <em>INS2</em> transcript levels were reduced in both RyR2KO and IRBITKO. Nuclear localization of AHCY were increase in both the RyR2KO and IRBITKO cells, corresponding with increased levels of insulin gene methylation. Proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 and IRBIT activity regulate insulin content, insulin secretion, and regulate the proteome in INS-1 cells</p> <p>We next sought to assess the consequences on cellular Ca2+ handling in the absence of RyR2 and IRBIT in INS-1 cells. Store-operated Ca2+ entry (SOCE) stimulated with thapsigargin was reduced in RyR2KO cells compared to controls, but this was not different in IRBITKO cells. STIM1 protein levels were not different between the three cell lines. Basal and carbachol stimulated phospholipase C (PLC) activity was reduced specifically in RyR2KO cells and not IRBITKO cells. However, basal PIP2 levels were elevated in both RyR2KO and IRBITKO cells. Insulin secretion stimulated by tolbutamide was reduced in RyR2KO and IRBITKO cells compared to controls, but this was still potentiated by an EPAC-selective cAMP analog in all three cell lines. Cortical f-actin is known to regulate insulin secretion, and levels were markedly reduced in RyR2KO cells compared to control INS-1 cells. Whole-cell Cav channel current density was reduced in RyR2KO cells compared to controls, and Ba2+ current was significantly reduced by PIP2 depletion preferentially in RyR2KO cells over control INS-1 cells. Action potentials stimulated by 18 mM glucose were more frequent in RyR2KO cells compared to controls, and insensitive to the SK channel inhibitor apamin. Taken together, these results suggest that RyR2 plays a critical role in regulating PLC activity and PIP2 levels via regulation of SOCE. RyR2 also regulates beta-cell electrical activity by controlling Cav current density, via regulation of PIP2 levels, and SK channel activation.</p> <p>Lastly, we investigated the role of PDE subtypes cAMP in INS-1 cells and human islets. We utilized subtype selective inhibitors of PDE1, PDE3 and PDE8 to assess the potential of these PDEs as potential therapeutic targets. We found that PDE1 is the primary subtype in INS-1 cells, whereas PDE3 appears to be required in human pancreatic β-cells by cAMP measurements. PDE1 inhibition potentiated glucose-stimulated to the greatest extent in both INS-1 cells and human islets. PDE1 inhibition potentiated CREB phosphorylation to the greatest extent and was also capable of mitigating lipotoxicity in INS-1 cells. Collectivity, this work highlights the role of cAMP compartmentalized signaling in pancreatic β-cells, and this has drastic effects on pancreatic beta-cell function and survival.</p>

<p dir="ltr">In this dissertation, we utilized the zebrafish model and the human neutrophil model to investigate the novel mechanisms that regulate neutrophil motility and chemotaxis.</p>

<p>  </p> <p>The role of the endoplasmic reticulum (ER) Ca2+ release channels ryanodine receptor 2 (RyR2) and inositol 1,4,5-triphosphate receptor (IP3R) in pancreatic b-cell function are emerging, but are not well defined. It has been demonstrated that ER stress brought about by RyR2 dysfunction leads to impaired insulin secretion and contributes to the etiology of type 2 diabetes (T2D). Our work contributes to the understanding of the role of RyR2 in physiological pancreatic b-cell function and how loss of RyR2 contributes to the pathophysiology of T2D.</p> <p>To investigate the role of RyR2 in pancreatic b-cell function, we utilized CRISPR-Cas9 to delete RyR2 from the rat insulinoma INS-1 cell line (RyR2KO). We found that RyR2KO cells displayed an enhanced glucose-stimulated Ca2+ integral (area under the curve; AUC) and were sensitive to inhibition by the IP3R antagonist, xestospongin C. Loss of RyR2 also resulted in a reduction in IRBIT protein levels. Therefore, we deleted IRBIT from INS-1 cells (IRBITKO) and found that IRBITKO cells also displayed an increased Ca2+ AUC in response to glucose stimulation. We discovered that total cellular insulin content and secretion were reduced in RyR2KO cells, but more modestly reduced in IRBITKO cells. We found that <em>INS2</em> mRNA levels were reduced in both RyR2KO and IRBITKO cells, but <em>INS1</em> mRNA levels were specifically decreased in RyR2KO cells. Additionally, nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in both RyR2KO and IRBITKO cells. DNA methylation of exon 2 of the <em>INS1</em> and <em>INS2</em> genes was more extensively methylated in RyR2KO and IRBITKO cells compared to controls. Proteomics analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. Our results suggest that RyR2 regulates IRBIT levels and activity, and together maintain insulin content and secretion, and regulate the INS-1 cell proteome, perhaps via DNA methylation.</p> <p>The role of interplay between RyR2 and IP3R in Ca2+ signaling and homeostasis in pancreatic b-cell function remains understudied. Stimulation with the sulfonylurea tolbutamide resulted in markedly delayed Ca2+ transients in both RyR2KO and IRBITKO cells. Xestospongin C significantly reduced the AUC of Ca2+ in RyR2KO and IRBITKO cells. Muscarinic receptor stimulation revealed a markedly increased AUC of Ca2+ in IRBITKO cells compared to both RyR2KO and control INS-1 cells. Assessment of PLC activity revealed that basal and stimulated PLC activity were reduced in the absence of RyR2 or IRBIT. Store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion revealed a decreased SOCE amplitude only in RyR2KO cells. Given evidence that phosphatidylinositol-4,5-bisphosphate (PIP2) depletion from the plasma membrane can regulate voltage-gated Ca2+ channel inhibition, we explored electrophysiological properties of all three cell lines. The frequency of glucose-stimulated action potentials was doubled in RyR2KO cells. Additionally, whole-cell voltage-gated Ca2+ current density was doubled in RyR2KO cells, and this current was more sensitive to hydrolysis of PIP2. These results evidence crosstalk between RyR2 and IP3R, and that RyR2 plays a critical role in maintaining proper Ca2+ homeostasis, PLC activity, and electrophysiological properties in pancreatic b-cells.</p>

<p dir="ltr">CaaX proteins, comprising approximately 300 members in the human protein database, represent a diverse group implicated in fundamental cellular processes, including proliferation, differentiation, trafficking, and gene expression. To carry out such vital cellular functions, CaaX proteins need to undergo three sequential post-translational modifications (PTM) through the CaaX pathway, which consists of isoprenylation (farnesylated or geranylgeranylated), endoproteolysis, and methylation. Among the CaaX family of protein, the Ras superfamily, plays a pivotal role in cell growth and survival. Mutations in <i>Ras proteins</i> are associated with a spectrum of cancers, presenting a significant challenge for therapeutic intervention. This thesis explores the intricate landscape of PTMs of CaaX proteins, with a focus on methylation, which is carried out by membrane protein isoprenylcysteine carboxyl methyltransferase (Icmt), and its potential as a therapeutic target, particularly for Ras-driven cancers.</p><p><br></p><p dir="ltr">Icmt is unique as it is the sole methyltransferase which carries out the third PTM of methyl esterification of CaaX proteins with the aid of co-substrate SAM, which serves as the methyl donor. Additionally, how Icmt, a membrane protein localized in the endoplasmic reticulum (ER), brings these two chemically diverse molecules in close enough proximity to promote catalysis, is very intriguing and is not yet fully understood. This thesis focuses on studying the structural and functional properties of Ste14, the yeast homolog of Icmt, in order to better understand the Icmt family of proteins. Ste14 is a functional homolog of human Icmt, sharing 41% sequence identity and 62% sequence similarity. Furthermore, Ste14 can be functionally purified unlike human Icmt. Together, these attributes make Ste14 an ideal system to study.</p><p dir="ltr"><br>The first project explores Ste14 and substrate binding, focusing on residues that determine farnesylated vs geranylgeranylated substrate specificity. It is essential to note that wild-type Ste14 recognizes farnesylated and geranylgeranylated substrate equally, with no preference to one over the other. Conserved residues found in Loop 2 and Transmembrane 3 of Ste14 were mutated to alanine and assessed for their activity with AGGC, the minimal geranylgeranylated CaaX substrate. Mutants which showed nearly zero percent activity with AGGC in comparison to wild type were further analyzed to understand if this loss of mutant activity with AGGC was potentially due to the mutant's inability to bind with AGGC. A photoreactive AGGC analog was used to carry out the photolabeling experiments and residues were analyzed for their binding ability with geranylgeranylated substrate. Mutants were further analyzed to understand the effect of mutation on structural integrity, to gauge which residues are essential for catalysis and for maintaining structural integrity of Ste14. Results demonstrated that residues F80 and E98 are essential for structural stability while L81 and L82 are essential for catalysis. This project would overall help better understand the lesser studied Ste14-substrate binding.</p><p><br></p><p dir="ltr">In the second project, the focus shifts to study Ste14 and co-substrate SAM binding by using electron paramagnetic resonance spectroscopy (EPR) and site directed spin labeling (SDSL). The biophysical technique of EPR requires much less protein and serves as great tool to study conformational change Ste14 undergoes on SAM binding, 3 non conserved residues found in the SAM binding region of Ste14, were individually mutated to cysteine, and had a spin label MTSL attached to their purified active mutant forms. Through EPR the conformational changes of Ste14 during methylation specifically during SAM binding was analyzed by visualizing the movement of MTSL attached residue. Results showed of the three non-conserved residues, A223 and E227 were immobile during SAM binding while T164 residue displayed flexibility during SAM binding during SAM binding and release process. This study would help understand the protein dynamics that Icmt undergoes upon SAM binding.</p><p><br></p><p dir="ltr">The final section centers on inhibiting the third step of the CaaX pathway, which is methyl esterification, by targeting Icmt. The project involved testing a library of Icmt inhibitors and evaluating their ability to inhibit Icmt activity. Of this library of bi-substrate analog inhibitors, compounds YD 1-66, YD 1-67 and YD 1-77 emerge as promising inhibitors against human Icmt, laying the foundation for further studies to develop more potent inhibitors. This section accentuates the strategies employed to target Icmt and the potential of these inhibitors in combating Ras-driven cancers.</p><p><br></p><p dir="ltr">This thesis provides an extensive analysis of the structure and function of Ste14. The varied studies and their insights contribute to a comprehensive understanding of Icmt and pave the way for the rational design of potent chemotherapeutic inhibitors for Ras-driven cancers. The multifaceted research presented in this thesis reveals several new possibilities for targeted therapies in the field of oncology.</p>

<p>Two species of <i>Neisseria</i>, <i>N. meningitidis</i> and <i>N. gonorrhoeae</i>, are obligate human pathogens that cause meningitis and gonorrhea, respectively. Although generally asymptomatic, <i>N. meningitidis</i> can cause invasive meningococcal disease with high mortality rate. Due to emerging antibiotic resistance strains of <i>N. gonorrhoeae</i>, the Centers for Disease Control and Prevention (CDC) have designated it as an urgent threat to public health. Therefore, immediate interventions are required for fight against these Neisserial pathogens. Iron is an essential nutrient for all bacteria, including <i>Neisseria</i>. However, free iron is scarce in human, therefore, <i>Neisseria</i> have evolved to acquire iron from host proteins. These iron acquisition systems are immunogenic and important for infection and are promising therapeutic targets.</p> <p> In the host, lactoferrin sequesters free iron and limits iron availability to pathogens. However, <i>Neisseria</i> have evolved machinery to hijack iron directly from lactoferrin itself. Lactoferrin binding proteins, LbpA and LbpB, are outer membrane proteins that together orchestrate the acquisition of iron from lactoferrin. Additionally, LbpB serves an additional role in providing protection against host cationic antimicrobial peptides and innate immune response. Despite studies aimed at deciphering the roles of LbpA and LbpB, the molecular mechanisms underpinning iron acquisition and immune protection remain unknown. Here, we investigated the role of the lactoferrin binding proteins in iron acquisition and protection against cationic antimicrobial peptides. We obtained three-dimensional structures of <i>Neisseria</i> LbpA and LbpB in complex with lactoferrin using cryo-electron microscopy and X-ray crystallography. These structures show that both LbpA and LbpB bind to C-lobe of lactoferrin, albeit at distinct sites. Structural analyses show that while lactoferrin maintains its iron-bound closed conformation in the LbpB-lactoferrin complex, it undergoes a large conformational change from an iron-bound closed to an iron-free open conformation upon binding to LbpA. This observation suggest that LbpA alone can trigger the extraction of iron from lactoferrin. Our studies also provide an explanation for LbpB’s preference towards holo-lactoferrin over apo-lactoferrin and LbpA’s inability to distinguish between holo- and apo-lactoferrin. Furthermore, using mutagenesis and binding studies, we show that anionic loops in the C-lobe of LbpB contribute to binding the cationic antimicrobial peptide lactoferricin. Solution scattering studies of the LbpB-lactoferricin complex showed that LbpB undergoes a small conformational change upon peptide binding.</p> Together, our studies provide structural insights into the role of the lactoferrin binding proteins in iron acquisition and evasion of the host immune defenses. Moreover, this work lays the foundation for structure-based design of therapeutics against <i>Neisseria</i> targeting the lactoferrin binding proteins.

<p>Plant height is an important agronomic trait and a major target for crop improvement. Owing to the ease of detection and measurement of plant stature, as well as its high heritability, several height-related mutants have been reported in maize. The genes underlying a few of those mutants have also been identified, with a majority of them related to the biosynthesis or signaling of two key phytohormones - gibberellins (GAs) and brassinosteroids (BRs). However, most other maize dwarfing mutants, and especially those that result from gain-of-function mutations, remain uncharacterized. The present study was undertaken to characterize a novel dominant dwarfing mutant, named <i>D13</i>. This mutant appeared in the M1 population of the inbred B73 that was generated by mutagenesis with ethyl methanesulfonate (EMS). Like most other maize dwarfing mutants, the reduction in <i>D13</i> height was largely due to the compression of the internodes. However, unlike the GA or BR mutants, <i>D13</i> had no defects in the female or male inflorescences. Further, in contrast to the GA and BR mutants, the mesocotyl elongation during etiolation was not impacted in <i>D13</i>. <i>D13</i> seedlings developed red coloration in two to three lowermost leaves. In addition, <i>D13</i> also showed enhanced tillering when the phenotype was very severe. The size of the shoot apical meristem of <i>D13</i> was reduced slightly, and significant aberrations in the structure of vascular bundles in the mutant were observed. All anatomical and phenotypic features of <i>D13</i> were highly exaggerated in homozygous state, indicating the partially dominant nature of the <i>D13</i> mutation. Interestingly, the heterozygous mutants showed remarkable variation in their phenotype, which was maintained across generations. Moreover, the <i>D13</i> phenotype was found to be sensitive to the genetic background, being completely suppressed in Mo17, Oh7B, enhanced in CML322, P39 and changed to different degrees in others. To identify the genetic defect responsible for the <i>D13</i> mutant phenotype, a map-based cloning approach was used, which identified a single base-pair change from G to A (G2976A) in the coding region of a glutamate receptor gene (Zm00001d015007). The G2976A missense mutation resulted in the replacement of alanine with threonine at the location 670. The replaced alanine is highly conserved in glutamate receptors across all domains of life from cyanobacteria to plants to mammals, suggesting a causal relationship between the G2976A substitution and the <i>D13</i> phenotype. To validate this relationship, a targeted EMS-based mutagenesis approach was used to knock-out (inactivate) the <i>D13</i> mutant allele. A suppressor mutant was found in which the <i>D13</i> mutant phenotype reverted to the normal tall phenotype. The sequence of the revertant allele, designated <i>D13</i>*, revealed that the original <i>D13</i> mutant allele underwent a second G to A mutation (G1520A) to change glycine into aspartic acid at position 473. This intragenic second-site mutation in the <i>D13</i> allele suppressed the function of the <i>D13</i> allele, thereby preventing it from interfering with the function of the wild type allele. To further unveil the genes and underlying mechanisms that enable the <i>D13</i> mutant to confer a dwarf phenotype, transcriptomic and metabolomic analyses of <i>D13</i> mutants were conducted and compared to the wild type sibs. While the omics analysis confirmed that stress responses were upregulated and genes related to shoot system development were downregulated in the mutant, the data did not allow us to pinpoint the underlying mechanisms that connect the <i>D13</i> mutation with its dwarfing phenotype. Furthermore, it remains unclear whether these stress and shoot system-related changes result in the manifestation of <i>D13</i> phenotype, or the dwarf phenotype due to <i>D13</i> mutation activates the stress-related mechanisms. This is the first study that signifies the importance of a glutamate receptor gene in controlling plant height.</p>

<p>Peptides, specifically cell-penetrating peptides (CPP), have become wonderful research tools due to their enhanced stability, solubility, and ease of synthesis. They have been used for a wide range of biomedical applications, from insecticides to biosensors and drug-delivery scaffolds. The work presented in this dissertation characterizes the biophysical properties of two different CPPs. The first is the cationic amphiphilic polyproline helix (CAPH) peptide, P14LRR. In addition to cell penetration, this CPP has demonstrated broad spectrum antibacterial properties. Fluorescence polarization (FP) and SEC-MALS were conducted to understand the dissociation constant (KD) and oligomerization effects of P14LRR with respect to its putative molecular target in Staphylococcus aureus (S. aureus). A biotinylated derivative of this peptide was also used as a drug-delivery scaffold to transport fluorescently conjugated streptavidin into mammalian cells. A second CPP, DAN13, was also developed as a biosensor for phosphoinositide lipids, specifically PI(4,5)P2. This was effected through careful calibration using stacked supported lipid bilayers (SSLB) in combination with total internal reflection fluorescence (TIRF) microscopy. This was then used to determine the absolute densities and spatial distribution of PIP2 in live KRas mutant cells.</p>