Osteocytes are the most abundant cell type in bone. Although our understanding of the function of osteocytes has progressed over the last decade, these cells remain the least understood cell in bone. Osteocytes are considered to be orchestrators of bone remodeling, mineral homeostasis, and hematopoiesis. The sympathetic nervous system (SNS) regulates almost every system in the body including bone. It is known that SNS suppresses bone formation but the mechanism is still not fully elucidated. Beta 2 adrenergic receptor (β2AR) is a G-protein coupled receptor that, upon binding to norepinephrine, activates the stimulatory subunit of the heterotrimeric G protein (Gαs) and the enzyme adenylate cyclase. The function of β2AR in osteocytes has not been studied. Therefore, the goal of this study is to delineate the role of β2AR signaling in osteocytes. To investigate that, an osteocyte-like cell line (Ocy454) was used. Ocy454 cells were treated with three different agents: norepinephrine (NE), isoproterenol (ISO), and butoxamine. Knock down of β2AR was achieved by using short hairpin RNA. Consequently, treatment with norepinephrine (NE) was done as well as fluid flow shear stress (FFSS) experiment. Taken together, this study shows that osteocytes do express β2AR and that β1AR and β2AR are almost equally expressed in osteocytes. Secondly, we have demonstrated that upon treatment of Ocy454 with NE and isoproterenol, there is a significant upregulation of Rankl, whereas Sost is not significantly regulated. Lastly, preliminary data suggest that β2AR might be involved in osteocyte’s mechanosensation.

The American Diabetes Association has determined that the number of people with diabetes in America was 30.3 million in 2015, and it is becoming increasingly more prevalent. Diabetes is a condition characterized by chronic hyperglycemia due to either insufficient insulin or reduced insulin sensitivity. Diabetes comes with a host of complications; one major complication is osteopenia, which increases fracture risks in both type 1 and type 2 diabetics14. The changes in diabetic bone may be due to reductions in lysyl oxidase (LOX) levels leading to decreased amounts of insoluble type 1 collagen fibers, which are necessary for bone strength28. LOX catalyzes oxidative deamination of the hydroxylysine and lysine side chains of the collagen molecules to create reactive aldehyde groups9,14,17. These aldehyde groups rapidly react with the lysine or hydroxylysine on the helical region of neighboring molecules of collagen fibrils, creating crosslinks between molecules leading to a mature cross-linked collagen matrix9,15,18. Our lab has now linked the LOX reductions in diabetes to incretins such as glucose-dependent insulinotropic peptide (GIP), which directly increases LOX, and anti-incretins such as dopamine, which reduces LOX by inhibiting GIP. Incretins and anti-incretins are gastric hormones released by the intestine in response to nutrient consumption4. Generally, incretins are hormones that stimulate insulin secretion from the pancreas where as anti-incretins inhibit that insulinotropic effect13,23. GIP is an incretin that also has an anabolic effect on bone in addition to its insulinotropic effect23. In diabetes there is an impaired cellular response to GIP in the pancreas, but its effects on the bone as related to diabetes are unknown. Additionally, it was observed in our lab that bone-derived LOX levels were decreased in diabetes while the serum level of anti-incretin gut-derived dopamine was vastly increased. Dopamine is known to inhibit GIP in the pancreas, but its effect on the bone was unknown. Prior to this experiment our lab had already started exploring a potential mechanism for diabetic osteopenia, where diabetes leads to an increase in gut-derived anti-incretin (dopamine), which interferes with GIP-stimulated LOX in osteoblasts. The decreased levels of LOX lead to lowered collagen integrity and a loss in trabecular bone structure. The current work is a sub-study within the larger aim to elucidate the mechanism for diabetic osteopenia. The aim of this study was to further verify the effects of dopamine by using amisulpride, a dopamine receptor antagonist, to determine if amisulpride could restore bone health in streptozotocin (STZ) induced diabetic mice. For this study C57BL/6 wild type mice were used. The mice were divided at random into three groups (N=10 per group) as follows: control (no STZ, no amisulpride), STZ diabetic (STZ, no amisulpride), and STZ + amisulpride. Twenty of the mice had low dose intraperitoneal injections of STZ for five days to induce diabetes and ten mice received vehicle injections for the control group. The mice were maintained diabetic or normal for 8 weeks, after which ten of the STZ mice received intraperitoneal injections of amisulpride daily for a month. It was found that STZ induced diabetic mice had a significant decrease in LOX levels and insulin levels. Amisulpride was able to rescue the decreases in LOX levels but did not alter insulin levels. Furthermore, micro-CT analysis and picrosirius red histology of long bones indicated that there is a decrease in bone volume, impaired trabecular structure, and disorganized collagen matrix in the STZ diabetic group. These impairments could be rescued by amisulpride administration, giving further evidence to the new mechanism for diabetic osteopenia. The increase in anti-incretin gut-derived dopamine signaling could be the cause of diabetic osteopenia. Inhibition of the anti-incretin gut-derived dopamine may possibly provide a therapeutic target for diabetic osteopenia.

<p> The regulation and mechanism of outer membrane (OM) lysis during spore wall assembly is not well understood. Although the timing of OM lysis has been established in previous studies, this event has never been assayed for. To this end, a new method was developed to assay the lysis of the OM during sporulation in <i>Saccharomyces cerevisiae</i>, the split GFP assay. By expressing complementary fragments of GFP in the ascal cytoplasm and the lumen between the OM and the spore plasma membrane (SPM), the lysis of the OM could be detected by the reconstitution of a functional GFP protein in the ascal cytoplasm as a result of these two compartments merging. A screen of several mutant strains with sporulation defects with published TEM images showing whether or not the lyse the OM then verified the split GFP assay for OM lysis as an effective method for detecting this event. This assay was then used to further investigate the sporulation defects associated with mutations in <i>FKS</i> genes, which all have 1,3-&beta;-glucan synthase homology. The results of this assay showed that mutation in <i> FKS2</i> or <i>FKS3</i> leads to a reduction on observed OM lysis. This provides further evidence for the current model that proper completion of the &beta;-glucan layer needs to occur before the OM will lyse. In addition, it was shown via dissection that <i>FKS3</i> is not able to substitute for <i>FKS1</i>, as <i>FKS2</i> has been shown to do. Although <i>FKS3</i> is very similar to the other 1,3-&beta;-glucan synthases homologically, the data suggest it has a function distinct from that of <i>FKS1</i> and <i>FKS2</i>.</p><p>

<p> Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42&bull;GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.</p><p>

<p>Many cell types are remarkably adept at tracking chemical gradients, but they use different mechanisms in order to properly migrate or grow up-gradient. Bacteria use a temporal sensing mechanism to determine if they are swimming up-gradient. In contrast, eukaryotes are thought to use spatial sensing mechanisms where they compare the chemical concentration on one side of the cell to the other. In the present study, we utilized budding yeast <italic>(Saccharomyces cerevisiae) </italic>mating as a model for gradient tracking. Yeast cells are thought to use a spatial gradient tracking mechanism to grow up the pheromone gradient created by their mating partners. However, yeast cells polarize their receptors towards the direction of growth thereby reducing the distance that they can use to compare pheromone concentrations. </p><p>Yeast cells grow towards their mating partners by establishing a polarity patch that concentrates the master regulatory GTPase Cdc42 and its associated polarity factors on the membrane. The Cdc42 polarity patch orients actin cables so vesicles trafficking along these cables fuse at the polarity patch. Therefore, the location of the polarity patch determines the direction of growth. During mating, the pheromone gradient is thought to bias the polarity patch to the up-gradient side of the cell, but especially in shallow gradients, sometimes yeast cells initially establish the polarity patch on the wrong side of the cell. Work from our lab has found that the polarity patch wanders along the cell cortex during pheromone gradient tracking, and suggests that wandering behavior could serve as a mechanism of reorientation for cells whose polarity patch is misaligned with the gradient. </p><p>In order for yeast cells to properly track the pheromone gradient, their polarity patch must spend more time on the up-gradient side of the cell. How does the pheromone gradient bias wandering of the polarity patch to achieve this? We suggest that by polarizing their receptors and G proteins, yeast cells create a sensitized zone of the plasma membrane that can locally influence wandering of the polarity patch. As the polarity patch wanders along the cell cortex, so too does this zone of polarized receptors. If the patch wanders to a side of the cell with higher pheromone concentration, then more active receptors near the polarity patch could slow its wandering and allow more growth to occur in that direction.</p> / Dissertation

Ischemic injury is a common and debilitating outcome of natural illness and as a complication of commonly performed medical procedures. Whereas naturally occurring ischemic insults are often the result of unpredictable events, such as in the case of stroke or heart attack, the risk of operative and perioperative ischemia is somewhat better characterized in the clinical setting. Given the prevalence and severity of outcomes in ischemic injury, there is significant interest in developing better pharmacological and procedural approaches to improve patient outcomes. One approach that has shown significant promise in the laboratory setting, particularly in the context of planned medical procedures, is the use of delayed anesthetic preconditioning. Delayed anesthetic preconditioning is a phenomenon whereby a prior exposure to clinical concentrations of commonly used inhaled anesthetics, including isoflurane, induces the production of endogenous protective proteins that are able to provide robust protection against subsequent, potentially toxic, ischemic insults. Although many aspects of delayed anesthetic preconditioning have been previously described, a complete understanding of preconditioning mechanism has yet to emerge. The studies described in this dissertation aim to further our understanding of molecular mechanisms involved in delayed anesthetic preconditioning. In the first project, I used DNA microarray to identify genes that were differentially expressed in adult rat liver, kidney and heart following a clinically relevant exposure to the inhaled anesthetic isoflurane. By selecting those genes that were differentially expressed in multiple tissues, I was able to identify a small group of interesting genes for further study. In my second study, I chose from our list two related genes, metallothioneins I + II, to analyze for a role in anesthetic-mediated protection. Using a combination of approaches, I was able to establish that metallotioneins I + II play an essential role in delayed anesthetic preconditioning. In the final study of this dissertation I explore a possible role for metallothioneins I + II as sensor molecules, involved in detecting cellular oxidative stress. Taken together, these three studies represent an important contribution to our understanding of the mechanisms of delayed anesthetic preconditioning and how they might contribute to protecting against ischemic stroke.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.

The Lyme disease spirochete Borrelia burgdorferi is the only known human pathogen that directly activates invariant natural killer T (iNKT) cells. The number and activation kinetics of iNKT cells vary greatly among different strains of mice. Here, we report the role of the iNKT cell response in the pathogenesis of Lyme disease using C57BL/6 (B6) mice, a strain with optimal iNKT cell activation that is resistant to the development of spirochetal-induced inflammation. During experimental infection of B6 mice with B. burgdorferi, iNKT cells localize to the inflamed heart where they are activated by CD1d-expressing macrophages. Activation of iNKT cells in vivo results in the production of IFNγ, which we demonstrate controls the severity of murine Lyme carditis by at least two mechanisms. First, IFNγ greatly enhances the recognition of B. burgdorferi by macrophages, leading to increased phagocytosis of the spirochete. Secondly, IFNγ activation of macrophages increases the surface expression of CD1d, thereby facilitating further iNKT activation. Collectively, our data demonstrate that in the resistant background, B6, iNKT cells modulate acute murine Lyme carditis through the action of IFNγ, which appears to self-renew through a positive feedback loop during infection. Inflammation during infection with B. burgdorferi is dependent on the ability of the spirochete to evade local mechanisms of clearance. Even though macrophages are the main infiltrating cell during Lyme carditis, the identification of a receptor capable of mediating phagocytosis of B. burgdorferi has been elusive. Here, we demonstrate that the integrin CR3 is able to mediate binding to the spirochete and facilitate phagocytosis in a complement-dependent and independent manner. Expression of CR3, but not CR4, in CHO cells markedly enhanced their capacity to interact with B. burgdorferi, in the absence and presence of complement opsonization. Furthermore, the interaction between CR3 and B. burgdorferi is dependent on the metal-ion-dependent adhesion site (MIDAS) and could be blocked with EDTA. Inhibition of CR3 with blocking antibody was able to completely abrogate phagocytosis of B. burgdorferi by the macrophage-like RAW264.7 cells and partially block uptake by bone marrow-derived macrophages (BMMs), a finding that was recapitulated with CD11b-deficient BMMs. We further show that activation with recombinant IFNγ increases the transcription of CD11b and CD18, which correlates with increased surface expression of CR3, and that the effect of IFNγ on the phagocytosis of B. burgdorferi is circumscribed to CR3 activity, because inhibition of CR3 is able to completely diminish the effect of IFNγ on the phagocytosis of the B. burgdorferi. Lastly, our results demonstrate that CR3 is a negative regulator of proinflammatory cytokine induction in macrophages responding to B. burgdorferi. Overall, our data demonstrate roles for CR3 in the binding, phagocytosis and proinflammatory cytokine elicited by B. burgdorferi and shed light on the role of IFNγ in mediating the clearance of the spirochete during Lyme disease.

Mouse metaphase II (MII)-stage eggs exhibit oscillatory Ca2+ responses ([Ca2+]i oscillations) following fertilization. The wealth of information regarding Ca2+ signaling pathways in eggs has allowed these cells to become an ideal model system for the study of general Ca2+ signaling pathways. This dissertation provides data that contribute to the elucidation of the mechanism that culminates in Ca2+ release at fertilization, and to our understanding of the functional regulation of the inositol 1,4,5-trisphosphate receptor (IP 3R) using the mouse egg as a model system. We first present data indicating that injection of mouse eggs with porcine sperm factor (SF) induces [Ca2+]i oscillations through activation of a phospholipase C (PLC). U73122, a PLC inhibitor, prevented SF-induced [Ca2+]i oscillations whether SF or eggs were treated with the inhibitor. We also show that SF injection elicits inositol 1,4,5-trisphosphate (IP3) production and Ca2+ release in single Xenopus oocytes. Thus, SF induces [Ca2+]i release by stimulating the phosphoinositide pathway. We next show that KN-93, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor, antagonizes IP3R function independently of effects on CaMKII in mouse eggs and permeabilized A7r5 cells. This inhibition is not due to a block of IP3 production, Ca2+ store filling, or IP3 binding to the IP3R. KN-93 interferes with Ca2+-induced Ca2+ release by the IP 3R, indicating that KN-93 may prevent the ability of IP3 and/or Ca2+ to induce activatory conformational changes to the IP3R. KN-93 directly interacts with and alters the conformation of the IP3R, based on in vitro and in vivo proteolysis experiments. Finally, KN-93 does not interact with the IP3R via a calmodulin binding site, as hypothesized based on its mechanism of CaMKII inhibition. Lastly, we present preliminary data toward the development of an IP 3R overexpression system in mouse eggs. We successfully generated enhanced yellow fluorescent protein (eYFP)-tagged murine IP3R-1 mRNA in vitro. Injection of eYFP-IP3R-1 mRNA into mouse eggs resulted in expression of exogenous IP3R-1 protein. This system will provide new opportunities for the use of the mouse egg as a model system for the study of IP3R signaling, and may help facilitate the elucidation of the mechanism by which KN-93 inhibits the IP3R.

Cell adhesion to an extracellular matrix plays a critical role in many aspects of normal cell function. Cells display various modes of interaction with the extracellular matrix; they may attach and spread, become immobilized, or become motile. These cellular responses are regulated by intracellular signals, which modify the organization of the cytoskeleton. One common characteristic of malignantly transformed cells is alteration in one or more aspects of adhesion. Most notably, cancer cells often display enhanced motility and there is a positive correlation between cell mobility and metastatic potential in situ. HeLa cells, a cell line derived from a cervical carcinoma, were used as a model system for this investigation. It has been shown, in HeLa cells, that cell attachment to a gelatin-coated substrate results in the release of arachidonic acid, which is metabolized by lipoxygenase. A subsequent cascade of lipid second messengers activates protein kinase C, which triggers actin polymerization leading to cell spreading. This work employed inhibitor studies, and biochemical analysis to elucidate a parallel branch of arachidonic acid signaling that reorganizes the actin cytoskeleton into small bundles. This branch of the pathway is initiated by cyclooxygenase, which generates prostaglandins and causes the downstream activation of cyclic AMP-dependent protein kinase. The results suggest that arachidonic acid functions at a branch point in signaling to the cytoskeleton. The lipoxygenase branch provides polymerized actin; the actin filaments then act as a substrate for the cylooxygenase branch to generate actin bundles. These actin bundles were shown to associate with myosin and small adhesion complexes. Activation of cyclooxygenase signaling and the subsequent cytoskeletal organization were found to increase cell motility. Overexpression of the small GTPases rho and cdc42, also induces cell crawling, and these signaling molecules seem to interact with cyclooxygenase in directing organization of the cytoskeleton. In sum these results suggest that faulty regulation of arachidonic acid signaling can result in the pathological cell motility that characterizes the most aggressive cancers.