The last decade for the Medical College of Georgia has yielded strategic, phenomenal growth in educational, research and clinical initiatives in diseases affecting every family in Georgia and the United States.

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Recent Submissions

  • Profiling the HCA Receptor Family through BRET Analysis of GPCR-G-Protein and GPCR-Arrestin Interactions

    Saj, Dalia; Department of Biological Sciences; Department of Pharmacology & Toxicology (Augusta University, 2020-05)
    Increasing obesity rates have put the American population at higher risk for developing obesity-related medical conditions such as hypertension, heart disease, and diabetes. The hydroxycarboxylic acid (HCA) receptor family is a family of G protein-coupled receptors (GPCRs) that are expressed in adipose tissue and function as metabolic sensors, making them potential pharmaceutical targets in the treatment of obesity and other metabolic disorders. The HCA receptor family consists of the HCA1, HCA2, and HCA3 receptors, which are activated by hydroxycarboxylic acids such as lactate and 3-hydroxybutyric acid. We utilized bioluminescence resonance energy transfer (BRET) to study agonist-induced coupling of luciferase-tagged HCA receptors to Venus fluorescent protein-tagged G protein heterotrimers or arrestins. Our results indicate that the three HCA receptors couple to the Gαi/o subfamily of G proteins. The data additionally confirms a lack of coupling to the other G protein subfamilies (Gαs, Gαq, and Gα12/13), and lacks evidence of arrestin recruitment to HCA receptors. Overall, our study highlights the use of BRET as a powerful tool for analysis of GPCR signaling and demonstrates its possible use for future studies to determine the potency of potential drugs targeting HCA receptors as a therapy for health-related problems such as obesity.
  • Hypothalamic AgRP and POMC neurons modulate stress-induced depression-related behaviors

    Fang, Xing; Department of Neuroscience and Regenerative Medicine
    Depression is a common and debilitating mental disease. Currently available antidepressants are not effective for many individuals with depression and our understanding of the underlying mechanisms remain limited. Evidence suggests that hypothalamic arcuate nucleus (ARC) is highly responsive to acute stress. The ARC contains two distinct subpopulations of neurons—expressing orexigenic agouti-related peptide (AgRP) and anorexigenic pro-opiomelanocortin (POMC). AgRP and POMC neurons regulate food intake and the food reward system. It is unknown whether AgRP and POMC neurons are recruited by chronic stress and if their dysfunction may contribute to the development of chronic stress-induced depression-related behaviors. To address this, we have developed a mouse model of chronic unpredictable stress (CUS), which can induce anhedonia and despair behavior that mimic symptoms in human depression. Using this animal model, I investigated the roles of AgRP and POMC neurons in stress responses and stress-induced depression-related behaviors. I demonstrated that CUS decreases activity of AgRP neurons but increases activity of POMC neurons. A chemogenetic approach was used to selectively manipulate the activity of POMC and AgRP neurons, leading to opposite effects of stress-induced depression-related behaviors. These results suggest that AgRP and POMC neurons are differentially involved in stress maladaptation and related behaviors. It provides insight into the mechanisms underlying the development of depression and novel strategies for the treatment of this mental illness.
  • OSTEOPONTIN AS A NOVEL IMMUNE CHECKPOINT

    Klement, John; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
    The host adaptive immune system functions to discriminate self from non-self, eliminating threats from viral infection to tumors. Cytotoxic lymphocytes (CTLs) are the primary effector arm of adaptive immunity. To prevent aberrant activation and autoimmunity, immune checkpoints function physiologically to restrain the CTL response. Tumors pathologically express these checkpoints, preventing immune-driven tumor clearance. Accordingly, immune checkpoint inhibitors (ICIs) have shown remarkable clinical success. However, many types of malignancies, as well as many individual patients with responsive tumor types, fail to benefit from current ICI immunotherapies. This conundrum suggests that as-yet undiscovered immune checkpoints exist. We observed that mice deficient in the transcription factor interferon regulatory factor eight (IRF8) tolerated allogenic tumor grafts and demonstrated impaired CTL activation with an accumulation of CD44hi memory-like CTLs. We sought to investigate the mechanism of this immunosuppression. Conditional deletion of IRF8 in T cells, as well as a mixed chimera model, demonstrated that IRF8 did not directly control CTL activation or differentiation into a CD44hi population. Instead, global loss of IRF8 lead to an expansion of an immature myeloid CD11b+Ly6G+Ly6Clo population which highly expressed osteopontin (OPN), a physiological ligand for CD44. Elevated levels of OPN were shown to suppress murine CTL activation and proliferation. A similar IRF8-OPN-CD44 axis was observed in murine and human colorectal cancer, which is refractory to current ICI therapies. Malignant cells and human patients displayed enhanced OPN levels relative to healthy donor controls. This was shown to be mediated by loss of IRF8 expression, which directly bound to the OPN promoter to repress its transcription. Elevated levels of OPN similarly prevented human CTL activation, and higher levels of OPN were correlated with decreased survival in human patients. We have shown that the IRF8-OPN-CD44 axis functions as a novel immune checkpoint in both myeloid and tumor cells. Blockade of OPN may have potent anti-tumor activity, expanding the pool of patients responsive to ICI therapy.
  • ROLE OF CLASS III PHOSPHATIDYLINOSITOL 3-KINASE IN THE RENAL PROXIMAL TUBULE

    Liu, Ting; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
    Yeast only has a single phosphatidylinositol 3-kinase (PI3K) known as vacuolar protein sorting 34 (VPS34); however, mammals have evolved to express three structurally different classes of PI3Ks: class I, II, and III. Although the class III PI3K (Pik3c3) is the only PI3K evolutionarily conserved from yeast to man, its distribution in the mammalian kidney is unknown, and its role in the renal proximal tubule, especially under certain pathophysiological conditions such as nephron loss-induced Compensatory Nephron Hypertrophy (CNH), remains undefined. The goal of Aim 1 was to define the expression pattern and relevant biological function of Pik3c3 in the kidney. We found that the glomerular podocyte expresses the highest level of Pik3c3 in the kidney. Among all renal tubular cells, the specialized distal convoluted tubular epithelial cells called macula densa cells express the highest level of Pik3c3, and the renal proximal tubular cells (RPTC) express the second highest level of Pik3c3. This prompted us to perform additional experiments for Aim 1 that led to the demonstration of an essential function of Pik3c3 in regulating the degradation of epidermal growth factor (EGF) receptor (EGFR) and the termination of EGFR signaling in RPTC following EGF binding with EGFR. The goal of Aim 2 was to determine whether Pik3c3 is essential in mediating uninephrectomy (UNX)-induced compensatory nephron hypertrophy. We generated a global Pik3c3-hypomorphic mouse model and two slightly different proximal tubule-specific Pik3c3 knockout mouse models: Pik3c3Neo-ptKO and Pik3c3ptKO. Interestingly, CNH was markedly inhibited in the global Pik3c3-hypomorphic mouse model and proximal tubule-specific Pik3c3 knockout models. The goal of Aim 3 was to determine the effect and underlying mechanism of complete Pik3c3 deletion in renal proximal tubule cells. We found that complete Pik3c3 deletion in some renal proximal tubule cells resulting in marked cell death that subsequently progressed to tubulointerstitial fibrosis. My project has, for the first time, determined the expression pattern of Pik3c3 in the kidney and provided the first definitive evidence that Pik3c3 controls the degree of CNH and functions upstream of the mTORC1-S6K1-rpS6 pathway in the regulation of CNH. In addition, my project reveals an essential role of Pik3c3 in maintaining the homeostasis and survival of proximal tubule cells.
  • Role of Ufl1 in CD8+ Memory T Cell Survival and Function

    Bhatt, Brinda Nikhil; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
    Immunity mediated by CD8+ T cells plays an integral part of the response to eradicate both infections and tumor cells. The recognition of antigen by the T cell receptor triggers a cascade of signaling events in naïve CD8+ T cells that leads to their proliferation and differentiation into memory and effector cells. Cytotoxic effector CD8+ T cells produce large amount of cytokines and effector molecules that play a critical role in the elimination of pathogens. Following pathogen clearance, a small population of memory T cells persists long-term, which has the ability to expand robustly upon re-exposure to antigen and provide the host with rapid and specific recall responses against the pathogen. Treatments boosting an individual’s own immune response have changed the landscape of cancer research. However there are still limitations with patient responsiveness, toxic side effects, and not achieving long-term remission. As advances in immunotherapy revolutionize cancer treatment, understanding the molecular networks governing CD8+ T cell function has become more important than ever. In our studies, we found that genetic knockout of UFM1 Specific Ligase 1 (Ufl1) in T cells leads to a CD8-specific loss of central memory cells. Flow cytometry staining revealed that the population of CD8+CD122+ T cells are significantly reduced in mice lacking Ufl1 in T cells. We observed that Ufl1 deficiency leads to apoptosis of these cells in vivo. Ufl1-deficient CD8+CD122+ T cells express higher amounts of Fas on the cell surface as well as activated cleaved caspase 3. RNA-sequencing analysis demonstrated that these cells also overexpress numerous genes associated with exhaustion, including PD-1, Lag3, Tim3, and 2B4. Interestingly, the Listeria monocytogenes disease model showed that Ufl1-deficient CD8+ T cells behave similarly to wild type cells during the acute effector response, but undergo a more dramatic contraction and subsequently launch an attenuated recall response. Consistent with this, a melanoma-specific vaccine failed to protect mice lacking Ufl1 in T cells. Our present study suggests that Ufl1 plays a critical role in the suppression of apoptosis and exhaustion of memory CD8+ T cells.
  • Gene regulation by the putative Campylobacter jejuni diguanylate cyclase CbrR

    Fulmer, Claudia; Department of Biochemistry and Cancer Biology (Augusta University, 2020-05)
    As a leading cause of bacterial gastroenteritis, Campylobacter jejuni incurs health care costs estimated at $290 million a year in the US and up to 40,000 deaths in children aged 5 and younger worldwide. As such, determining those proteins that regulate C. jejuni virulence factors are prime targets to possibly develop a prophylactic therapy, that as of yet does not exist. CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), the class of enzyme that catalyzes the synthesis of cyclic di-GMP, a universal bacterial second message molecule, from GTP. In C. jejuni strain DRH212, an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+, were constructed. Soft agar motility tests, biofilm formation assays, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) were performed. Site-directed mutagenesis was performed on cbrR to make alanine substitutions in both the autoinhibitory site (I-site) and active site and differential radial capillary of action ligand (DRaCALA) assays were performed to determine nucleotide binding by wild-type CbrR and the CbrR point mutants. Soft agar motility assays indicated a hyper-motile phenotype associated with the C. jejuni cbrR- mutant, whereas motility was all but negated in the cbrR+ complement. Biofilm assays and SEM demonstrated similar formation and robustness of biofilms between wild type and cbrR- mutant, however cbrR+ was unable to form significant biofilms in 72 hours. TEM images showed similar cellular morphology between cbrR-, wild type, and cbrR+, however cbrR+ cells had fewer flagella. DRaCALA assays showed wild-type CbrR and the active site mutant were both able to bind GTP and cyclic di-GMP, whereas the I-site mutant lost the ability to bind cyclic di-GMP, indicating the product binding site on CbrR. The highly conserved diguanylate cyclase CbrR is the only annotated DGC in the C. jejuni genome. Though the active site sequence is highly variant when compared to the consensus sequence, this protein is able to bind both substrate and product of the chemical synthesis of cyclic di-GMP and has now been shown to be a negative regulator of motility, a critical virulence factor in C. jejuni pathogenesis.
  • GLYCOSAMINOGLYCANS, CHONDROITINASE, AND MOLECULAR SUBTYPES IN BLADDER CANCER

    Morera, Daley S; Department of Biochemistry and Molecular Biology (Augusta University, 2020-05)
    There is a need for novel prognostic biomarkers and targeted treatments in bladder cancer (BC), even more so for muscle-invasive disease (MIBC). Discovery of molecular markers to predict outcome in BC patients may lead to identification of impactful therapeutic targets. The hyaluronic acid (HA) family of molecules and distinct molecular subtypes have both been investigated as potential prognostic markers. HA family and chondroitin sulfate proteoglycans, such as CD44, have been implicated in driving aggressiveness of disease; however, a chondroitinase enzyme that cleaves chondroitin sulfate proteoglycans has not been identified in any eukaryotic system. We evaluated molecular markers of BC and the first known eukaryotic/human chondroitinase, that we identified, for their ability to predict clinical outcome in patients, and for their roles as drivers of disease. We also investigated the anti-tumor effects of HA synthesis inhibitor 4-methylumbelliferone (4MU), a non-toxic orally bioavailable supplement. This study demonstrates that transcript levels of HA family members can predict metastasis and poor survival in BC patients. HA- family expression also correlated with epithelial mesenchymal transition (EMT) markers β -Catenin, Twist, Snail, and E-Cadherin. HA signaled through its receptors CD44/RHAMM and the PI3-K/AKT axis. 4MU targeted HA signaling, inhibiting proliferation and motility/invasion, inducing apoptosis in vitro, and preventing tumor growth in vivo. We discovered that a previously unidentified splice variant of HYAL-4 was elevated in bladder tumors. We named this variant "V1". Our studies showed that V1 had chondroitinase activity, cleaved chondroitin-6-sulfate from CD44, and consequently increased CD44 secretion. In vivo, V1-expressing urothelial cells formed muscle-invasive tumors and V1-expressing cancer cells developed metastatic tumors. Evaluation of the prognostic significance of the molecular subtypes of MIBC that were recently identified by other groups, showed the subtypes to have little to no predictive ability for clinical outcome in multivariate analyses that included standard clinical parameters. Consistently, clinical parameters such as histopathologic tumor grade, T-stage, and lymph node status, outperformed the molecular subtypes. Contrarily, V1 levels could independently predict metastasis and survival with high efficacy, suggesting that focusing on V1 as a functional biomarker may be a better strategy to improve clinical outcome of BC patients.
  • THE ROLE OF KYNURENINE, A TRYPTOPHAN METABOLITE THAT INCREASES WITH AGE, IN MUSCLE ATROPHY AND LIPID PEROXIDATION)

    Kaiser, Helen E.; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
    Loss of mobility and independence are risk factors for falls and mortality, and drastically reduce the quality of life among older adults. The cellular and molecular mechanisms underlying loss of muscle mass and strength with age (sarcopenia) are not well-understood; however, heterochronic parabiosis experiments show that circulating factors are likely to play a role. Kynurenine (KYN) is a circulating tryptophan metabolite that is known to increase with age and is implicated in several age-related pathologies. Here I tested the hypothesis that KYN contributes directly to muscle loss with aging. Results indicate that that KYN treatment of mouse and human myoblasts increased levels of reactive oxygen species (ROS) two-fold, and significantly increased lipid peroxidation enzymes. Small-molecule inhibition of the Aryl hydrocarbon receptor (Ahr), an endogenous KYN receptor, in vitro did not prevent KYN-induced increases in ROS, and homozygous Ahr knockout in vivo did not protect mice from KYN-induced stress, suggesting that KYN can directly increase ROS independent of Ahr activation. In vivo, wild-type mice treated with KYN had reduced skeletal muscle strength, size, and increased oxidative stress and lipid peroxidation. Old wild-type mice treated with 1MT, a small molecule that suppresses KYN production by IDO1, showed an increase in muscle fiber size, peak muscle strength, and oxidative stress. Protein analysis identified mitochondrial lipid peroxidation as a downstream mechanism that is increased upon KYN treatment. Lipid peroxidation enzymes increased with KYN have been shown to produce H2O2 outside of the electron transport chain. Our data suggest that IDO inhibition may represent a novel therapeutic approach for the attenuation of sarcopenia and possibly other age-associated conditions associated with KYN accumulation such as bone loss and neurodegeneration.
  • False coverage rate - adjusted smoothed bootstrap simultaneous confidence intervals for selected parameters

    Sun, Jing; Department of Biostatistics and Epidemiology (Augusta University, 2020-05)
    Many modern applications refer to a large number of populations with high dimensional parameters. Since there are so many parameters, researchers often draw inferences regarding the most significant parameters, which are called selected parameters. Benjamini and Yekutieli (2005) proposed the false coverage-statement rate (FCR) method for multiplicity correction when constructing confidence intervals for only selected parameters. FCR for the confidence interval method is parallel to the concept of the false discovery rate for multiple hypothesis testing. In practice, we typically construct FCR-adjusted approximate confidence intervals for selected parameters either using the bootstrap method or the normal approximation method. However, these approximated confidence intervals show higher FCR for small and moderate sample sizes. Therefore, we suggest a novel procedure to construct simultaneous confidence intervals for the selected parameters by using a smoothed bootstrap procedure. We consider a smoothed bootstrap procedure using a kernel density estimator. A pertinent problem associated with the smoothed bootstrap approach is how to choose the unknown bandwidth in some optimal sense. We derive an optimal choice for the bandwidth and the resulting smoothed bootstrap confidence intervals asymptotically to give better control of the FCR than its competitors. We further show that the suggested smoothed bootstrap simultaneous confidence intervals are FCR-consistent if the dimension of data grows no faster than N^3/2. Finite sample performances of our method are illustrated based on empirical studies. Through these empirical studies, it is shown that the proposed method can be successfully applied in practice.
  • DEFINING THE ROLE OF TROPOMYOSIN-1C IN CARGO TRANSPORT IN DROSOPHILA

    Boggupalli, Shankarappa Devi Prasad; Department of Cellular Biology and Anatomy (Augusta University, 2020-05)
    Cell polarity is the asymmetric organization of different organelles in a cell, including the plasma membrane and cytoskeleton. Such organization results from asymmetric sorting of proteins, either post-translationally or pre-translationally by messenger RNA localization. In Drosophila oocytes, posterior localization of oskar mRNA is required for germplasm assembly and establishing antero-posterior polarity. oskar mRNA is transported by Kinesin, however the adaptor that links Kinesin to oskar mRNA was not known. In Aim 1 of this thesis, we demonstrate that a novel isoform of Tropomyosin, namely Tm1C, binds directly to kinesin and functions as the adaptor in linking kinesin to oskar mRNA. Oskar expression is limited to female germline, however Tm1C is also expressed in male flies. This suggests that there might be additional cargoes for Tm1C. We attempted to identify novel cargoes of Tm1C by performing a proteomic assay in Drosophila S2 cells. Apart from Khc, we identified Supernumerary limbs (Slmb) as the main interacting partner. Our further investigation of Slmb suggests that it might not be a cargo. Instead, Slmb which is a component of E3 ubiquitin ligase, might regulate the expression of Tm1C. In Aim 2 of the thesis, we show that Slmb regulates the levels of Tm1C by ubiquitinating it and facilitating its degradation by the Proteasome.
  • Neuron-derived estrogen and neural function

    Lu, Yujiao; Department of Neuroscience and Regenerative Medicine (Augusta University, 2020-05)
    17β-estradiol (E2) is produced from androgens via the action of the enzyme aromatase. E2 is known to be made in neurons in the brain, but its precise functions in the brain are unclear. We created a forebrain neuron-specific aromatase knockout (FBN-ARO-KO) mouse model to deplete neuron-derived E2 in the forebrain of mice. Under normal conditions, FBN-ARO-KO mice showed a 70-80% decrease in aromatase and forebrain E2 levels. Male and female FBN-ARO-KO mice exhibited significant deficits in forebrain spine and synaptic density, as well as hippocampal-dependent cognitive functions. Reinstating forebrain E2 levels via exogenous in vivo E2 administration was able to rescue both the molecular and behavioral defects in FBN-ARO-KO mice. Furthermore, electrophysiological study suggested normal long-term potentiation (LTP) induction, but significantly decreased amplitude in FBN-ARO-KO mice which could be fully rescued by acute E2 treatment in vitro. Mechanistic studies revealed that FBN-ARO-KO mice had compromised rapid kinase (AKT, ERK) and CREB-BDNF signaling in the hippocampus and cerebral cortex. After global cerebral ischemia (GCI), ovariectomized female FBN-ARO-KO mice had significantly attenuated aromatase and hippocampal E2 levels. Intriguingly, FBN-ARO-KO mice exhibited a robust reduction in astrocyte activation, as well as exacerbated neuronal damage and worse cognitive dysfunction after GCI. Similar results were observed in intact male mice. RNA-seq analysis revealed alterations in pathways and genes associated with astrocyte activation, neuroinflammation and oxidative stress in FBN-ARO-KO mice. The compromised astrocyte activation in FBN-ARO-KO mice was associated with robust downregulation of the astrocyte-derived neurotrophic factors, BDNF and IGF-1, as well as the astrocytic glutamate transporter, GLT-1. In vivo E2 replacement rescued the compromised reactive astrogliosis and cognitive deficits. Moreover, neuronal FGF2, which acts in a paracrine manner to suppress astrocyte activation, was dramatically increased in FBN-ARO-KO neurons. Interestingly, blocking FGF2 signaling in astrocytes by central injection of an FGFR3 antibody was able to reverse the diminishment in neuroprotective astrocyte reactivity, and attenuate neuronal damage in FBN-ARO-KO mice. Collectively, our data provides novel genetic evidence for the roles of neuron-derived E2 in regulating synaptic plasticity, cognitive function in the non-injured brain, and astrocyte activation and neuroprotection in the injured brain.
  • Surgical Anatomy of the Nose and Ear

    Potter, Kathryn Anne; Medical College of Georgia (Augusta University, 2020-03-23)
  • Families and Addictions: Forgiveness as a Powerful Clinical Tool

    Camino-Gaztambide, Richard F.; Malavé de León, Eunice; Department of Psychiatry and Health Behavior
    Short Description: Addictions are complex behaviors that have a profound impact on the individual, family, and society. Forgiveness can transform negative emotions for oneself or others to achieve or sustain recovery. The purpose of the workshop is to offer the clinical underpinnings that can facilitate the implementation of forgiveness in practice. Abstract: Addictions are complex behaviors that have a profound impact on the individual, family, and at a societal level. Although many see addictions as fundamentally a disease of the brain and clearly brain structures and functions are significantly involved, nevertheless, brain function alone does not address the consequences and profound effects that addictions have on the patient's ecosystem. Family, friends, co-workers, and neighborhood, all are altered with frequent feelings of anger, shame, guilt, and rejection present in all parties. Usually, these feelings are in response to real or perceived transgressions by one or more persons, and it is not uncommon that trauma is present, producing persistent stress which can interfere with recovery. The concept of forgiveness can be a powerful tool to help patients address the injury and trauma that they have done or received by others. Shame, defined as a “flawed self, often accompanied by feelings of worthlessness and powerlessness” is associated with negative feelings and poorer recovery. In contrast, guilt, that focuses more on the behavior not necessarily reflected as the total self, is more amenable to forgiveness. Forgiveness as a disposition to where the use of negative emotions for oneself or others can be transformed to achieve or sustain recovery. The Twelve-step facilitation model can be integrated, especially focusing on steps four through nine, as other models like Narrative, ACT, and CBT are also able to use the concept of forgiveness in effective ways. The purpose of the workshop is to provide basic theoretical and clinical underpinnings, use case presentations, interactive discussions, to provide skills that can facilitate the implementation of forgiveness in clinical practice. “Addiction is more than a disease and involves more than the brain: it is a systemic behavioral disorder.”
  • Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neurovascular Abnormalities

    Gutsaeva, Diana; Lamiaa, Shalaby; Wetzstein, Sara; Thounaojam, Menaka; Jadeja, Ravirajsinh; Powell, Folami; Martin, Pamela; Kwok, Hang Fai; Bartoli, Manuela; Department of Ophthalmology
    Retinal ischemia contributes to visual impairment in ischemic retinopathies such as diabetic retinopathy and other potentially blinding conditions. A member of the ADAM family of a disintegrin and metalloproteinases, ADAM17, contributes to multiple vascular pathologies through its ability to regulate inflammatory signals via ectodomain shedding. Here we investigated the specific contribution of endothelial ADAM17 to neurovascular damage associated with retinal ischemia using a mouse model of ischemiareperfusion (IR) injury.
  • SELECTIVITY AND PRODUCTIVITY OF GPCR-G PROTEIN INTERACTIONS

    Okashah, Najeah; Department of Pharmacology and Toxicology (Augusta University, 2020-03)
    Hundreds of human G protein-coupled receptors (GPCRs) converge on activation of four families of heterotrimeric G proteins. Individual receptors select a subset of G proteins in order to produce appropriate cellular responses. While the precise mechanisms of coupling selectivity are uncertain, the G alpha subunit carboxy (C) terminus is believed to be the primary region recognized by GPCRs. We directly assessed coupling between 14 representative GPCRs and 16 G alpha subunits, including one wild-type G alpha subunit from each of the four families and 12 chimeras with exchanged C termini. We found that Gi-coupled receptors were relatively selective for Gi1 heterotrimers, while Gs-, Gq-, and G12- coupled receptors were more promiscuous and always coupled in some measure to Gi1 heterotrimers. Our tests with G alpha subunit chimeras show that the G alpha subunit core and C terminus both play important roles in selectivity. This suggests that the key G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein. While promiscuous GPCR-G protein coupling is often observed. These interactions behave as expected with receptor-G protein coupling and activation being almost synonymous. Agonist bound GPCRs activate the G protein heterotrimers they interact with, while ignoring G protein subtypes that they cannot activate. However, we have shown that GPCRs can form unproductive complexes with G12 heterotrimers. Vasopressin 2 receptor (V2R) forms agonist-dependent complexes with G12 heterotrimers. Unlike V2R complexes with cognate Gs heterotrimers, V2R-G12 complexes do not dissociate when GDP or GTP is present. Stimulating V2R with arginine vasopressin (AVP) does not activate signaling responses downstream of G12 activation. Evaluation of several G12-coupled receptors demonstrated that agonist induced GPCR-G12 complexes have a wide range resistance to GDP. Like V2R receptors, formyl peptide 2 receptors (FPR2) and smoothened receptors (SMOR) formed complexes with G12 heterotrimers that were relatively resistant to GDP. Our results indicate that several GPCRs can form agonist-dependent unproductive complexes with G12 heterotrimers that are relatively resistant to GDP. Suggesting that for some GPCRs agonist-dependent association with G12 heterotrimers is weakly coupled to nucleotide exchange
  • Roles of Astrocyte-Derived Estrogen in the Brain

    Meyre, Pornjittra (Ja); Department of Neuroscience and Regenerative Medicine (Augusta University, 2019-12)
    The steroid hormone, 17β-estradiol (E2) is an important hormone that regulates many functions in the body. Traditionally, E2 was believed to be produced primarily by the ovaries in females, but a number of studies have shown that brain cells such as neurons and astrocytes can also make significant quantities of E2. The study presented in this thesis examined the role of astrocyte-derived E2 in exerting neuroprotection in the CA1 region of the hippocampus, as well as its ability to regulate two specific pathways implicated in neuroprotection - the LIF and STAT3 pathways. Since the hippocampal CA1 region is known to be highly vulnerable to global cerebral ischemia (GCI), such as occurs after cardiac arrest, we used a mouse GCI model to examine the neuroprotective role of astrocyte-derived E2 in the hippocampal CA1 region. The results of the study indicate that mice that lack the enzyme aromatase in astrocytes and were unable to produce astrocyte-derived E2, have decreased reactive astrocyte activation after GCI, greater neuronal deficits after GCI in both genders, and they have significantly decreased LIFSTAT3 signaling in the hippocampus.
  • Influence of Porphyromonas gingivalis on Anti-Apoptotic/Autophagic Signaling Pathways in Human Dendritic Cells

    Meghil, Mohamed; Tawfik, Omnia; Elashirty, Mahmoud; Rajendran, Mythilypriya; Arce, Roger; Schoenlein, Patricia V.; Cutler, Christopher; Department of Oral Biology & Diagnostic Sciences, Department of Periodontics, Department of Cellular Biology and Anatomy (Augusta University, 2019)
    The purpose of this study was to investigate the molecular mechanisims of P. gingivalis-mediated disruption of homeostatic apoptosis and autophagy in DCs.
  • Maxillary growth in patients with complete unilateral cleft lip and palate treated with Nasoalveolar molding

    Manente, M; Levy-Bercowski, D; Abreu, A; Fortson, W; Deleon, E; Yu, J; Looney, S; Department of Orthodontics; Department of Plastic Surgery; Department of Biostatistics and Data Science (Augusta University Libraries, 2019)
    In patients with cleft lip and/or palate (CLP), the nasolabial defect has a significant esthetic impact on the face and may impair psychosocial development. Nasoalveolar molding (NAM) is a pre-surgical orthopedic technique aimed to improve the alveolar and nasolabial morphology of patients with cleft lip and palate. This technique is used to facilitate and improve the future surgical correction in cleft lip and palate patients. Influences such as differences in patient age and gingivoperiosteoplasty procedures are among many that have made it difficult for conclusive results to be found and published on the impact of the NAM technique on maxillary growth in patients with complete unilateral cleft lip and palate (CUCLP).
  • HLA-G DIMER PROLONGS KIDNEY ALLOGRAFT SURVIVAL BY INHIBITING CD8+T CELL ACTIVATION AND GRANZYME B EXPRESSION

    Ajith, Ashwin; Biomedical Sciences (Augusta University, 2019-12)
    Solid organ transplantation is the preferred therapy for many patients diagnosed with end stage organ failure, however allograft rejection is a significant barrier for graft survival. Patient care involves heavy immunosuppressive drug treatment leading to elevated risk for cancer and other opportunistic infections. Hence there is a need to develop effective alternative approaches to minimize graft rejection. We focused on Human leukocyte antigen G (HLA-G), a nonclassical HLA class Ib molecule critically involved in the maintenance of maternal tolerance to semi-allogeneic fetal tissues during pregnancy and has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics, molecular and cellular analyses of cells from T-cell–mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the downregulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.
  • Investigating the Role of the Hdac3 Co-Repressor Complex in Glucocorticoid Signaling-Mediated Bone Marrow Lipid Storage with Age

    Pierce, Jessica Liane; Biomedical Sciences (Augusta University, 2019-12)
    Aging bone is characterized by loss of tissue density, marrow fat accumulation, and dysregulated bone marrow stromal cell (BMSC) differentiation. The contribution of the epigenetic regulator histone deactylase 3 (Hdac3) is of increasing interest in bone biology. Hdac3 expression decreases with aging, and the current model for conditional deletion of Hdac3 in Osterix-expressing osteoprogenitor cells (Hdac3-CKOOsx) exhibited an aged bone phenotype in young mice along with the novel finding of osteoblastic (Runx2+ osteogenic cells) lipid droplet storage. In addition, bone-specific loss of Hdac3 activity increases expression of the glucocorticoid (GC)-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (Hsd11b1), suggesting a mechanism for the increased lipid accumulation in aged and Hdac3-deficient BMSC-derived osteoblasts. The cofactor nuclear receptor corepressor 1 (NCoR1), which mediates Hdac3 enzymatic activity in a co-repressor complex (CRC), was proposed as a regulator of Hdac3 activity in bone. Both Hdac3 and NCoR1 expression decreased in aged osteoblasts, and the two factors exhibited synergy in downregulating the promoter activity of glucocorticoid-responsive elements. Because of the relationship between increased GC signaling and osteoporosis, the glucocorticoid receptor (GR) was investigated as a mediator of the marrow fat phenotype, with the hypothesis that loss of GR function in bone would be protective against common forms of osteoporosis. Chronic caloric restriction in WT and GR-deficient (GR-CKOOsx) mice was used as a short-term stressor to induce an osteoporotic phenotype, while aging of GR-deficient mice (where Hdac3 CRC expression naturally decreases) was used as a biologically-relevant model for dual loss of Hdac3 and the GR. Surprisingly, the loss of GR function in osteoprogenitors exacerbated bone loss and marrow fat accumulation in both models—and induced a chronic stress phenotype by increasing cellular bioenergetics and whole-body metabolic rate—providing evidence of a role for the GR in facilitating healthy bone maintenance as well as evidence for compensatory mechanisms that regulate bone biology through GC signaling. GR-deficient bone also induced changes to whole-body physiology (e.g., sarcopenia, decreased physical activity, metabolic dysfunction) that further demonstrate the intricacies of bone as an endocrine organ. The current study provides new avenues to investigate cell signaling, bioenergetics, and tissue crosstalk in osteoporotic bone.

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