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1
Nasa, Zeyad, and nasa zeyad@med monash edu au. "Characterization of the Rat Relaxin-like Factor Gene." RMIT University. Medical Sciences, 2006. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20080514.100729.
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Relaxin-like factor (RLF), also known as Leydig insulin-like peptide (Ley-I-L) or Insulin 3 (INSL3), is a newly characterized member of the insulin peptide family. Amino acid sequence homology revealed that RLF is more closely related to relaxin than any other insulin-like hormones. The main aim of this thesis was to sequence the rat RLF (Relaxin-like factor) gene and determine the structure and organisation of the gene. Secondly to compare the structural organisation of the rat RLF/JAK3 genomic region with that of the mouse and human, using bioinformatic databases. Thirdly to further investigate the signalling pathways for the RLF receptor, in particular the NFÛB pathway. The homology between rat and mouse in the JAK3/RLF region revealed 84.4 % similarity over 1262 bp of DNA sequence, observing that unlike the mouse, the rat RLF promoter is separated from the JAK3 gene by around 700-1000 bp. Similarly in humans, the RLF gene is located around 4 kb downstre am from JAK3. Also Protein kinase A (PKA) was the only signalling pathway which dispalyed major induction and no inhibitory effects were observed through the NFÛB signalling pathway.
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Restrepo, Montoya Daniel. "Computational Identification, Phylogenetic and Synteny Analysis of Receptor-Like Kinases “RLK” and Receptor-Like Proteins “RLP” in Legumes." Diss., North Dakota State University, 2018. https://hdl.handle.net/10365/29870.
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Legumes are considered the second most important family of crop plants after the grass family based on economic relevance. In recent years, the field of legume genomics has expanded due to advancements in high-throughput sequencing and genotyping technologies. To date, no published comparative genomic analysis explores receptor-like kinases “RLK” and receptor-like proteins “RLP” among legume genomes. Evaluating these RLK and RLP should provide a source of new information because extensive genetic and phenotypic studies have already discovered the diverse roles of RLK and RLP in cell development, disease resistance, and stress responses among other functions. This study demonstrates that a computational logical approach for classifying the RLK/RLP in legumes/non-legumes is statistically well supported and can be used in other plant species. The analysis of RLK/RLP of 7 legumes and 3 non-legume species evaluated suggests that about 2% are RLK and less than 1% of the proteins are RLP. The results suggest a dynamic evolution of RLK and RLP in the legume family. In fact, between 66% to 85% of RLK and 83% to 88% of RLP belong to orthologous clusters among the species evaluated. The remaining RLK and RLP proteins are classified as singletons. The ratio of the pairwise synteny blocks of RLK/RLP among legumes shows a 1:1 relationship. The exception is G. max, which shows an approximately 2:1 ratio due to its recent whole genome duplication (G. max vs. the other six legumes). The other legumes show evidence of a similar proportion of plasma membrane proteins among the legume pairwise synteny blocks.Fulbright Scholarship
Francisco Jose de Caldas (Colciencias, Colombia) Scholarship
North Dakota State University. Dry Breeding Program
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Marcu, Jahan Phillip. "Novel Insights into CB1 Receptor Signaling and the Anabolic Role of Cannabinoid Receptors in Bone." Diss., Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/233543.
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Cell BiologyPh.D.
Activation of the CB1 receptor is modulated by aspartate residue D2.63176 in transmembrane helix (TMH) II. Interestingly, D2.63 does not affect the affinity for ligand binding at the CB1 receptor. Studies in class A GPCRs have suggested an ionic interaction between residues of TMHII and VII. In this report, modeling studies identified residue K373, in the extracellular (EC)-3 loop, in charged interactions with D2.63. We investigated this possibility by performing reciprocal mutations and biochemical studies. D2.63176A, K373A, D2.63176A-K373A, and the reciprocal mutant with the interacting residues juxtaposed, D2.63176K-K373D were characterized using radioligand binding and guanosine 5'-3-O-(thio)triphosphate functional assays. None of the mutations resulted in a significant change in the binding affinity of CP55,940 or SR141716A. Computational results indicate that the D2.63176-K373 ionic interaction strongly influences the conformation(s) of the EC-3 loop, providing a structure-based rationale for the importance of the EC-3 loop to signal transduction in CB1. Specifically, the putative ionic interaction results in the EC-3 loop pulling over the top (extracellular side) of the receptor; this EC-3 loop conformation may serve protective and mechanistic roles. These results suggest that the ionic interaction between D2.63176 and K373 is crucial for CB1 signal transduction. This work may help to aide drug design efforts for the effective treatment of different diseases. The cannabinoid receptors of osteoblasts may represent a target for the treatment of bone disorders such as osteoporosis. Our research demonstrates that cannabinoids can affect important signaling molecules in osteoblasts. In MC3T3-E1 osteoblastic cells, the CB1 antagonist, AM251, has been reported to induce increases in Runx2 mRNA, mineralized bone nodule formation, and activation of signaling molecules such as ERK and AKT (Wu et al., 2011). Studies from our lab characterizing mice in which both CB1 and CB2 receptors were inactivated by homologous recombination have demonstrated increased bone mass coupled with enhanced osteoblast differentiation of bone marrow stromal cells in culture (manuscript in preparation). We explored the effect of antagonizing CB1 and CB2 cannabinoid receptors in osteoblastic cells to gain insights into molecular pathways that may help to explain the effects of the endocannabinoid system (ECS) in bone development. Our data was generated by running time course experiments with MC3T3-E1 cells under the influence of SR141716A, SR144528 or both in combination. The cells were harvested with a lysis buffer at specific time points and analyzed by western blot analysis. Quantification of protein activation was calculated using LiCor imaging equipment and software. Within 15 minutes, treatment with the CB1 receptor antagonist SR141716A resulted in several fold increases in pERK, pSMAD158, and pAKT. SR144528, a CB2 receptor antagonist, caused increases in pERK and pSMAD158, but not pAKT. When both antagonists were applied together, pERK and pSMAD158 levels increased, while pAKT signaling was diminished compared to SR141716A alone. The finding that cannabinoid receptor antagonists alter the activity of the SMAD158 complex is a novel finding, which suggests that cannabinoids can influence bone morphogenic signaling pathways, and therefore play a significant role in osteoblast differentiation and function.
Temple University--Theses
4
Tumati, Suneeta. "Functional regulation of opioid receptor signaling." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/194989.
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Studies have shown that long-term opioid agonist (such as morphine) treatment produces antinociceptive tolerance and increased pain sensitivity (hyperalgesia and/or allodynia), limiting the clinical efficacy of morphine. Prolonged opiate administration also upregulates spinal pain neurotransmitter (such as calcitonin gene-related peptide (CGRP)) levels and enhances evoked CGRP release in the dorsal horn of rats. It was suggested that augmented spinal pain neurotransmission may contribute to paradoxical pain sensitization and antinociceptive tolerance. The cellular signal transduction pathways involved in sustained opioid mediated augmentation of spinal pain neurotransmitter are not fully clarified.Sustained morphine treatment was shown to augment the concentrations of inflammatory mediators, such as PGE2 in the spinal cord. Studies have shown that PGE2 stimulates cAMP formation and CGRP release by activation of Gs protein-coupled prostaglandin receptor types in primary sensory neurons. Interestingly, it was found earlier that sustained opioid agonist treatment leads to a Raf-1-dependent sensitization of adenylyl cyclase(s) (AC superactivation), augmenting forskolin-stimulated cAMP formation upon opioid withdrawal (cAMP overshoot). It is well demonstrated that cAMP activates cAMP-dependent protein kinase (PKA), which plays an important role in the modulation of presynaptic neurotransmitter release. Therefore, in this study, we investigate the physiological role of Raf-1 mediated AC superactivation and subsequent PKA activation in A. sustained morphine-mediated augmentation of basal or evoked pain neurotransmitter release in vitro, in cultured primary sensory neurons, and B. in vivo, in sustained morphine mediated paradoxical pain sensitization and antinociceptive tolerance in rats.Our data demonstrates that A. sustained morphine treatment augments both basal and capsaicin-evoked CGRP release from isolated primary sensory neurons in a PKA- and Raf-1- dependent manner. B. sustained morphine treatment- augments of PGE2-evoked CGRP release from these cells. C. selective knockdown of spinal PKA or Raf-1 protein levels by intrathecal PKA- or Raf-1-specific siRNA pretreatment completely attenuates sustained morphine-mediated thermal hyperalgesia, tactile allodynia and greatly reduces antinociceptive tolerance in rats.In conclusion, we suggest that Raf-1-mediated AC superactivation may have a crucial trigger role in sustained morphine-mediated compensatory adaptations in the nervous system. Thus, we expect that pharmacological attenuation of Raf-1-mediated AC superactivation may improve the clinical treatment of chronic and neuropathic pain.
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Chiu, Yi-Ting. "STUDIES ON NEURITE OUTGROWTH AND RECEPTOR PHOSPHORYLATION FOLLOWING KAPPA OPIOID RECEPTOR ACTIVATION." Diss., Temple University Libraries, 2016. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/384383.
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PharmacologyPh.D.
Kappa opioid receptor (KOPR) is involved in many physiological functions and pharmacological responses such as analgesia, anti-pruritic effect, sedation, motor incoordination and aversion (Simonin et al., 1998; Liu-Chen, 2004). The cellular mechanisms following activation of KOPR involve in part Gi/o protein-dependent pathways (Law et al., 2000). Following KOPR activation, the receptor is phosphorylated and arrestins are recruited. Arrestins mediate agonist-dependent KOPR desensitization, internalization and down-regulation (Liu-Chen, 2004). In recent years, arrestins were found to initiate arrestin-dependent downstream signaling. Thus, agonist-promoted KOPR phosphorylation plays a pivotal role in KOPR regulation and signaling. Previous studies from our lab showed that in Chinese hamster ovary (CHO) cells stably transfected with the human KOPR (hKOPR), U50,488H induced phosphorylation (Li et al., 2002a); however, sites of phosphorylation were not determined. Using LC-MS/MS, our lab recently identified four residues (S356, T357, T363 and S369) to be the sites of U50,488H-promoted phosphorylation in the mouse KOPR (mKOPR) stably expressed in N2A cells (Chen et al., 2016). Antibodies were generated against phosphopeptides and purified and three antibodies were found to have high specificity for the mKOPR phosphorylated at S356/T357, T363 and S369, respectively (Chen et al., 2016). Our lab previously showed that while U50,488H promoted robust hKOPR phosphorylation and internalization, etorphine induced little phosphorylation and internalization, although both were potent full agonists in enhancing [35S]GTPγS (Li et al., 2002a; Zhang et al., 2002; Li et al., 2003). Etorphine caused lower levels of KOPR phosphorylation at all the four residues than U50,488H by immunoblotting with the phospho-specific antibodies (Chen et al., 2016). Using the SILAC (stable isotope labeling by amino acids in cell culture) approach, we have found that compared to etorphine, U50,488H promoted higher levels of single phosphorylation at T363 and S369 and double phosphorylation at T363+S369 and T357+S369 as well as triple phosphorylation at S356+T357+S369 (Chen et al., 2016). These results indicate that an above-threshold phosphorylation is required for KOPR internalization. It has been reported that KOPR is involved in neuronal differentiation and neurogenesis. In the first chapter, I focused on whether there are differences in the mechanisms underlying neurite outgrowth induced by U50,488H and etorphine. In the chapter 2, mechanisms of KOPR phosphorylation were characterized in detail using phospho-specific KOPR antibodies. Protein kinase C was found, for the first time, to be involved in agonist-promoted KOPR phosphorylation. The roles of PKC in behavioral effects induced by KOPR agonists in mice were examined. For the chapter 1, in Neuro2a mouse neuroblastoma cells stably transfected with the hKOPR (N2A-3HA-hKOPR), U50,488H robustly induced neurite outgrowth, but etorphine caused outgrowth to a much lower extent. G protein-dependent pathway was found to be involved in the actions of both agonists, but β-arrestin-dependent pathway was not. Inhibition of ERK1/2 phosphorylation decreased neurite outgrowth promoted by both agonists, indicating the roles of MAP kinase cascades in KOPR agonist-induced neuritogenesis. In contrast, β-arrestin2, 14-3-3ζ, GEC1 and Rap1 are not involved in U50,488H- or etorphine-promoted neurite outgrowth. Thus, the two agonists appear to share the same signaling pathways and the difference between two agonists is likely due to the lower efficacy of etorphine. For the chapter 2, U50,488H caused phosphorylation of the mKOPR at S356, T357, T363 and S369 in N2A cells stably transfected with FmK6H (FmK6H-N2A cells). NorBNI abolished U50,488H-induced KOPR phosphorylation at all four residues. GRKs (GRKs2, 3, 5 and 6) and PKCs were involved in U50,488H-mediated KOPR phosphorylation. In addition, PKC also participated in agonist-independent KOPR phosphorylation. This is the first time that PKC was shown to be involved in agonist-induced KOPR phosphorylation. We found that U50,488H caused KOPR phosphorylation at T363 and S369 in the mouse brain and PKC participated in phosphorylation of S369, but not T363, by using the PKC inhibitor chelerythrine (CHL). Thus, we further characterized effects of PKC inhibition on KOPR-mediated behaviors in CD1 mice. PKC was involved in KOPR-mediated sedation, motor incoordination and conditioned place aversion, but not analgesia and anti-scratching effect in mice. Studies in this thesis revealed the mechanisms of KOPR-mediated neurite outgrowth and KOPR-mediated phosphorylation and the involvement of PKC in KOPR-mediated pharmacological effects in vivo. These studies push the frontier of molecular pharmacology of the KOPR, which may be useful for development of KOPR agonists for therapeutic use.
Temple University--Theses
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Dressano, Keini. "Interação do AtRALF1 com o receptor quinase1 associado ao BRI1 (BAK1)." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-26052015-164356/.
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Os peptídeos hormonais vegetais, que vêm sendo caracterizados em plantas desde a década de 90, podem estar relacionados com defesa, reprodução, crescimento e desenvolvimento de plantas. O peptídeo RALF (Rapid Alkalinization Factor), ubíquo no reino vegetal, está envolvido com o desenvolvimento de plantas. Em arabidopsis há 37 genes que codificam peptídeos RALF (AtRALFs). A isoforma mais estudada é a AtRALF1, a qual regula de maneira negativa a expansão celular, inibindo o crescimento de raiz primária e o alongamento de hipocótilo quando aplicado exogenamente. Recentemente, demonstrou-se a existência de uma relação antagônica entre AtRALF1 e a via de brassinosteróides (BRs) no desenvolvimento de raízes. Quando mutantes da via de sinalização do BR foram avaliados quanto a sua resposta ao peptídeo AtRALF1, descobriu-se que mutantes para o receptor quinase1 associado ao BRI1 (bak1) são insensíveis a AtRALF1. Experimentos utilizando o sistema de duplo híbrido em levedura, a co-imunoprecipitação e a indução de genes marcadores em mutantes bak1 foram realizados e confirmaram o envolvimento da proteína BAK1 na percepção do peptídeo. Plantas transgênicas que superexpressam AtRALF1 apresentam um fenótipo semi-anão, no entanto, quando as mesmas foram cruzadas com o mutante bak1, suas progênies apresentaram um fenótipo similar ao de plantas selvagens. Ainda, quando plantas deste cruzamento foram novamente cruzadas com plantas selvagens, plantas com fenótipo semi-anão foram observadas na prole. Ensaios de ligação usando o peptídeo AtRALF1 marcado com éster de acridínio foram realizados e mostraram que em mutantes bak1, a ligação do AtRALF1 é menor em aproximadamente 30% quando comparada com plantas selvagens. Os dados obtidos mostram que a proteína BAK1 interage fisicamente com o AtRALF1, está envolvida na percepção do peptídeo, é essencial para a inibição do crescimento da raiz primária causada pelo AtRALF1 e é necessária para a indução dos genes responsivos ao AtRALF1.The plant peptides, which have been characterized in plants since the 90\'s, can be related to defense, reproduction, growth and development of plants. The RALF (Rapid Alkalinization Factor) peptide, ubiquitous in the plant kingdom, is related to the development of plants. In arabidopsis plants, there are 37 genes encoding RALF peptides (AtRALFs). AtRALF1 is the most studied isoform, which negatively regulate cell expansion, inhibiting primary root growth and hypocotyl elongation when it is applied exogenously. Recently, an antagonistic relationship between AtRALF1 and the brassinosteroids (BRs) to control root development had been demonstrated. When the response of mutants related to the BR signaling pathway to AtRALF1 peptide was investigated, it was found that mutants lacking the BRI1-associated receptor kinase1 (bak1) are insensitive to AtRALF1. Experiments using the two-hybrid system in yeast, co-immunoprecipitation, and the induction of marker genes in bak1 mutants were carried out, and confirmed the involvement of the BAK1 protein in the perception of AtRALF1 peptide. Transgenic plants overexpressing AtRALF1 are semi-dwarf. However, when those transgenic plants were crossed with bak1 mutant, their progeny showed a wild-type phenotype. Besides, when plants from this progeny were crossed again with wild-type plants, semi-dwarf phenotype plants were obtained in the offspring. Binding assays using AtRALF1 labeled with acridinium-ester were performed, and showed that in bak1 mutants, the AtRALF1 binding was reduced approximately 30% when compared to wild-type plants. All data indicate that BAK1 protein interacts physically with AtRALF1, it\'s involved with the peptide perception, essential for the primary root growth inhibition caused by AtRALF1, and required to the induction of genes responsive to AtRALF1.
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Fu, Hangfei. "Interleukin 35 inhibits ischemia-induced angiogenesis essentially through the key receptor subunit Interleukin 12 receptor beta 2." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/546609.
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Biomedical SciencesPh.D.
Peripheral arterial disease (PAD) is a worldwide disease caused by atherosclerosis. It is a circulatory condition where narrowed blood vessels reduce blood flow to the peripheral such as legs. Although current gold standard treatment for advanced PAD patients is still based on surgical revascularization, there is no effective therapy for many patients that are not suitable for surgery. In addition, better recovery from surgical revascularization largely relies on angiogenesis in the adjacent ischemic tissue. Thus, novel pro-angiogenic therapies to improve post-ischemic neovascularization are urgently desired. However, current poor understanding of the roles of anti-inflammatory cytokines in angiogenesis prevents the development of these new therapies. We and others have reported that IL-35 is a newly identified inducible immunosuppressive heterodimeric cytokine in the IL-12 family. IL-35 is composed of p35 (IL-12A) and EBI3, and its receptors are comprised of homodimers or heterodimer of IL-12Rb2 and gp130 (IL-6ST). We have shown that IL-35 inhibits endothelial cell (EC) activation induced by lipopolysaccharide (LPS) or atherogenic lysophosphatidylcholine (LPC). At least partially through these new EC-dependent mechanisms, IL-35 inhibits inflammation in autoimmune diseases, infectious diseases, atherosclerosis, and tumors. Recent studies have indicated the role of IL-35 in angiogenesis in rheumatoid arthritis and different tumors. However, whether and how IL-35 regulates post-ischemic angiogenesis in peripheral artery disease are unrevealed. In our study, we used hindlimb ischemia (HLI) and Matrigel plug assay as in vivo angiogenesis models and wound healing assay as in vitro angiogenesis model to study the role and underlying mechanisms of IL-35-mediated angiogenesis. We made the following findings: 1) muscle in human and mouse has high angiogenic potential in physiological conditions; 2) angiogenic cytokines and chemokines including anti-inflammatory cytokines are predominantly regulated by inflammatory transcription factors; 3) IL-35 signaling is induced in ischemic muscle; 4) IL-12Rb2, but not IL-6ST, is the key receptor component of IL-35 signaling in ischemic muscle and hypoxic human microvascular endothelial cells (HMVECs); 5) hyperlipidemia (atherogenic factor) impairs angiogenesis in vivo and in vitro, which partially acts through the induction of IL-35; 6) IL-12Rb2 deficiency improves HLI-induced angiogenesis in both WT or apolipoprotein E (ApoE) -/- mice (an atherosclerosis model); 7) IL-35 injection inhibits HLI-induced angiogenesis in WT mice but not that in the IL-12Rb2 deficient mice; 8) IL-35 injection enlarges the avascular area in gastrocnemius muscle after HLI; 9) IL-35 obstructs fibroblast growth factor-2 (FGF2)-induced angiogenesis in Matrigel plug assay in vivo; 10) CD45-CD31+ ECs from the IL-35-injected ischemic muscle at day 14 of HLI have an abnormal extracellular matrix organization, activated integrin pathways (cell-matrix adhesions), disrupted vascular endothelial (VE)-cadherin-plakoglobin complex (cell-cell adhesions), and increased infiltration and migration of bone marrow-derived leukocytes; 11) IL-35 inhibits HMVEC migration in wound healing assay in vitro presumably through upregulation of anti-angiogenic proteins including pigment epithelium-derived factor (PEDF), serpin family B member 5 (SERPINB5, Maspin), and thrombospondin (THBS)-1. These results suggest that anti-inflammatory cytokine IL-35, signaling through the key receptor subunit IL-12Rb2, inhibits HLI-induced angiogenesis and delays tissue repair by dysregulating cell-cell and cell-matrix adhesions, which leads to the impaired vascular adhesion junction and maturation of blood vessels.
Temple University--Theses
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Bryan, Anthony C. "Social Networks of Receptor-like Kinases Regulate Cell Identity in Arabidopsis thaliana." Diss., The University of Arizona, 2011. http://hdl.handle.net/10150/205180.
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Receptor-like kinases (RLKs) make up one of the largest gene families in Arabidopsis thaliana. These genes are required for various biological processes, including response to biotic stress, cell elongation, cell proliferation, and cell fate patterning. An emerging theme in Arabidopsis and other plants is that networks of RLKs are required to regulate a specific process throughout development involving spatial and temporal regulation of transcription factors. However, there are still many RLKs (>50%) with no known function.Several RLKs regulate epidermal development by contributing to early embryonic epidermal maintenance or to epidermal differentiation. In my first analysis, I characterize the role of two related RLKs GASSHO1 (GSO1) and GSO2 in epidermal differentiation. gso1 gso2 double mutants initially form an epidermis during embryogenesis, but analysis of post-embryonic root development indicates the mis-expression of epidermal-specific genes. Three previously characterized RLKs that are involved in epidermal development are also involved in meristem maintenance. In order to decipher the RLK gene networks controlling epidermal development and meristem maintenance, it is necessary to identify additional RLKs involved in both of these processes. I further identified roles for GSO1 and GSO2 in maintaining root growth and root apical meristem (RAM) activity. A future goal will be to elucidate the networks of RLKs, including GSO1 and GSO2 in regulating epidermal and RAM development.The development of the vasculature in plants is controlled by a vascular meristem, the procambium. Oriented cell divisions from the procambium produce phloem, to the periphery, and xylem, to the center of the plant. In a reverse genetic screen to determine to roles of the remaining RLKs with unknown function, we identified the RLK XYLEM INTERMIXED WITH PHLOEM1 (XIP1) that is required for vascular development. We show XIP1 is required for regulating the differentiation of the phloem and for the organization of xylem vessel elements. Our analysis indicates that XIP1 is part of a vascular meristem network, further emphasizing the importance of social networks of RLKs regulating a specific process in development.
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Geider, Kirsten [Verfasser], Bodo [Akademischer Betreuer] Laube, and Ralf [Akademischer Betreuer] Galuske. "Ionotropic glutamate receptor dysfunction in pediatric neurodevelopment / Kirsten Geider. Betreuer: Bodo Laube ; Ralf Galuske." Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2015. http://d-nb.info/1110981228/34.
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Inan, Saadet. "Pharmacological and Neuroanatomical Analysis of GNTI-Induced Repetitive Behavior in Mice." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/75332.
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PharmacologyPh.D.
This thesis is comprised of two parts. In the first part, we investigated a) the pharmacology of GNTI, a selective kappa opioid receptor antagonist, as a scratch-inducing compound in mice and b) possible mediators and receptors that may be involved in GNTI-induced scratching (itch). We studied if GNTI induces scratching through opioid, histamine, gastrin-releasing peptide (GRP) and/or muscarinic M1 receptors. In the second part, we established similarities and differences between pain and itch using GNTI-induced scratching and formalin-induced nociception models in mice. We found that GNTI (0.03-3 mg/kg, s.c., behind the neck) induces compulsive and vigorous scratching behavior in a dose-dependent manner. A standard submaximal dose (0.3 mg/kg) of GNTI caused animals to scratch 500-600 times in a 30 min observation period. Intrathecal (i.t.) or intraperitoneal (i.p.) administration of GNTI did not elicit scratching behavior. Duration of action of GNTI was 60-70 min and tolerance to the repetitive behavior did not develop. C-fos expressing neurons, in response to GNTI injection, were localized on the lateral side of the superficial layers of the dorsal horn of the cervical spinal cord. Compound 48/80, a chemically different pruritogen, evoked c-fos expression in neurons which are located on the lateral side of the superficial layer of the dorsal horn. These data suggest that both GNTI and compound 48/80 activate a group of sensory neurons located on the lateral side of lamina I and II. Pretreating (at -20 min) and posttreating (at +5 min) mice with the kappa opioid receptor agonist, nalfurafine (0.001-0.03 mg/kg, s.c.), significantly attenuated scratching induced by GNTI (0.3 mg/kg). These effects were not a consequence of behavioral depression. Tolerance did not develop to the anti-scratch activity of nalfurafine. Pretreating mice with nalfurafine (0.02 mg/kg) prevented both GNTI- and compound 48/80-provoked c-fos expression. Our c-fos results suggest that the preclinical antipruritic activity of nalfurafine occurs at the spinal level. Moreover, our results reinforce the need to evaluate nalfurafine as a potentially useful antipruritic in human conditions involving itch. GNTI still elicited excessive scratching in mice lacking mu, delta or kappa opioid receptors, respectively, as well as in mice pretreated with either naloxone or norbinaltorphimine. The H1 receptor antagonist, fexofenadine, or the H4 receptor antagonist, JNJ 10191584, did not attenuate GNTI-induced scratching. Also, pretreating mice with the peptide GRPR antagonist, [D-Phe6]bombesin(6-13) methyl ester, or the non-peptide GRPR antagonist, RC-3095, did not antagonize scratching induced by GNTI. Furthermore, GRPR mRNA levels did not change in response to GNTI injection. Telenzepine, a standard M1 receptor antagonist, had no marked effect against GNTI-elicited scratching, however (unexpectedly) McN-A-343, an M1 receptor agonist, attenuated this behavior in a dose-dependent manner. In the second part of our studies, we found that pretreating mice with lidocaine (i.d., behind the neck) inhibits GNTI-induced scratching and prevents GNTI-provoked c-fos expression in the dorsal horn of the spinal cord. Similarly, lidocaine (i.d., hind leg) inhibits formalin-induced nociception as well as formalin-provoked c-fos expression. While injection (s.c.) of formalin to the face of mice induced only wiping (indicating pain) by forepaws of the injection side, injection (s.c.) of GNTI to the face elicited grooming and scratching (indicating itch). In contrast to formalin, GNTI did not induce c-fos expression in the trigeminal nucleus suggesting that pain and itch sensations are projected differently along the sensory trigeminal pathway. In short, our main results indicate that a) the scratch-inducing activity of GNTI is not mediated by opioid, histamine or GRP receptors; b) kappa opioid receptors are involved, at least in part, in the inhibition of itch sensation and thus, on the basis of our results, nalfurafine holds promise as a potentially useful antipruritic in human conditions involving itch; and c) agonism at M1 receptors inhibits GNTI-induced scratching therefore the M1 receptor may be a key target for antipruritic drug development.
Temple University--Theses
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DiMattio, Kelly Marie. "Studies on Ligands of the Kappa Opioid Receptor." Diss., Temple University Libraries, 2016. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/334919.
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PharmacologyPh.D.
This thesis is comprised of three parts. In the first part, we investigated zyklophin, a novel selective short-acting kappa opioid receptor (KOPR) antagonist, and its effects on scratching behaviors in Swiss-Webster mice. We investigated whether zyklophin was able to induce scratching in a dose-dependent fashion, and whether this scratching behavior could be blocked by pretreatment with nor-binaltorphimine (norBNI). We also used KOPR -/- mice to further clarify the role of the KOPR in this behavior. In the second part, we examined the role of the divergent amino acid at position 6.58 in the mu opioid receptor (MOPR) and the KOPR on the binding of beta-funaltrexamine ß-FNA). ß-FNA is an irreversible antagonist at the MOPR and a reversible agonist at the KOPR. Utilizing the recently published crystal structures of the MOPR and KOPR, we collaborated with Dr. Lei Shi, who employed molecular modeling to choose a residue in transmembrane helix 6 (TM6) to mutate at the same position in MOPR and KOPR. We then characterized the mutants by performing [3H]diprenorphine binding, competition binding by unlabeled β-FNA, irreversible ß-FNA binding and [35S]GTPγS binding. In the third part, we investigated the concept of functional selectivity, or ligand bias, at the KOPR. We studied 23 different KOPR agonists in vitro using [35S]GTPγS binding as a measure of G protein activation and the on-cell Western (OCW) as a measure of ß-arrestin-mediated receptor internalization at the human KOPR (hKOPR), and from the results, chose 13 ligands to study at the mouse KOPR (mKOPR). We then selected biased ligands from the in vitro mKOPR results and studied their effects on scratching behavior, inhibition of pain behaviors and dysphoria as measured by the conditioned place aversion (CPA) test. We predicted that the G biased ligand would produce analgesia and anti-scratching effects at lower doses than would produce aversion in the CPA test, since analgesia has been shown to be G protein mediated and CPA has been shown to be arrestin mediated. Our first set of studies revealed that zyklophin (0.1, 0.3 and 1 mg/kg, s.c., behind the neck), induced vigorous scratching in a dose-dependent manner. 0.3 mg/kg zyklophin induced 150 scratches over a 30 minute period. The scratching was not blocked by pretreatment with 20 mg/kg norBNI (i.p.) 18-20 hours before injection of 0.3 mg/kg zyklophin s.c. in the nape of the neck. The scratching also persisted in KOPR -/- mice, in which the absence of the KOPR was confirmed by [3H]U69,593 binding (2 nM). In our second set of studies, we mutated the lysine at position 303 in the MOPR to glutamic acid (K303E), and the glutamic acid at the equivalent position in the KOPR to lysine (E297K). We transfected these mutant receptors into mouse neuroblastoma (N2A) cells. We found that the mutations had no effect on [3H]diprenorphine binding affinity or competition binding with [3H]diprenorphine and β-FNA indicating a functional intact opioid receptor. The mutations also did not affect [35S]GTPγS binding EC50 or Emax values. The mutation K303E in the MOPR reduced irreversible binding by 2/3 compared to the wildtype MOPR. Finally, we found that there were several ligands that displayed bias at the hKOPR and the mKOPR. At the hKOPR, using dynorphin A as the reference ligand to calculate bias, ICI-199441 was the only G biased ligand, while enadoline, nalbuphine, pentazocine, salvinorin A, tifluadom and butorphanol were arrestin-biased. At the mKOPR, only salvinorin B methoxymethyl ether (MOM-SalB) was G-biased, and salvinorin B ethoxymethyl ether (EOM-SalB), ICI-199441, U50,488H, nalfurafine and 12-epi-salvinorin A (12epiSalA) were ß-arrestin-biased. Enadoline and salvinorin A were slightly arrestin biased with respect to dynorphin A. From the in vitro data at the mKOPR, we selected MOM-SalB as our G biased ligand, U50,488H as our arrestin biased ligand and additionally chose to investigate nalfurafine due to its use in clinical studies. We hypothesized that U50,488H and nalfurafine would produce aversion at lower doses than analgesia or anti-pruritic effects. We found that nalfurafine was the only ligand studied to have a separation between doses that produced analgesia and anti-scratching effects, with A50 values of 5.8 and 8 μg/kg, respectively, and only produced significant dysphoria at a dose of 20 μg/kg. U50,488H and MOM-SalB produced dysphoria at all doses tested (0.25-10 mg/kg and 0.01-0.3 mg/kg, respectively). U50,488H produced a dose-dependent analgesia and anti-scratching with A50 values of 0.58 mg/kg and 2.07 mg/kg, respectively. MOM-SalB was more potent than U50,488H in producing dose-dependent analgesia and anti-scratching, with A50 values of 0.017 mg/kg and 0.070 mg/kg, respectively. Therefore, we concluded that the in vitro bias is not able to accurately predict in vivo behaviors, and nalfurafine is the first selective full agonist at the KOPR to show ligand bias in vivo.
Temple University--Theses
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McDevitt, Jane K. "N-METHYL-D-ASPARTIC ACID RECEPTOR SUBUNIT NR2A REPEAT." Diss., Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/216597.
Full textAbstract:
KinesiologyPh.D.
During a concussion, mechanical forces cause neuron cell strain that initiates dysfunction through the indiscriminate movement of ions through protein channels. Extracellular glutamate binds with cell membrane proteins (e.g. NR2A), which exacerbates the Ca2+ ion influx and prolongs neuron dysfunction. Genetic variation may be a factor in regulating glutamate binding and therefore cell recovery time. The NR2A subunit of NMDA contains a variable (GT)n nucleotide tandem repeat (VNTR) within GRIN2A promoter region. This VNTR has been shown to regulate transcription levels in a length dependent manner, where longer repeat decreases transcription of the NR2A subunit. The purpose of this study was to determine the association of the GRIN2A VNTR and recovery (days) as well as concussion severity scores within concussed athletes. The independent variable was VNTR (long allele vs. short allele). The primary dependent variable, recovery time, was defined as injury date to return to play (RTP) clearance date as determined by the physician. Participant RTP time was categorized as normal ( 20 days). Secondary dependent variables were assessed at the initial evaluation and included vestibular ocular score, Balance Error Scoring System (BESS) score, and Immediate Post Concussion and Cognitive Testing (ImPACT) module scores. All 51 participants were athletes, comprised of 38 males and 13 females with a mean age of 18.69 6.65. Participants were evaluated at a university concussion center. The standardized concussion evaluation consisted of cranial nerve, vestibular ocular tests, balance (Balance Error Scoring System), signs and symptoms (s/s), and neurocognitive (ImPACT) testing. Each participant was genotyped via saliva sample for the GRIN2A (GT)n repeat polymorphism (rs3219790). Data analysis consisted of descriptive and inferential statistics. Chi-squares were used to assess the association between VNTR (long allele versus short allele) and concussion recovery (prolonged versus normal). Regression analyses were used to estimate extent to which non-genetic factors and genotype contributed to concussion recovery group assignment. One-way ANOVAs were used to identify any significant differences in vestibular ocular, BESS, and ImPACT module scores between long and short alleles. Primary potential non-genetic contributing factors were age, race, sex, concussion history, dizziness at time of injury, history of migraines, and history of ADHD. The alpha level was set at p ≤ .05. There were no differences between demographic or health history within the VNTR or recovery groups. There was a significant association (x2 = 4.01, p = 0.045) between the VNTR group (dominant model: LL versus SS + LS) and recovery, where the chance of prolonged recovery was 4.3 times greater for carriers of the homozygous long allele. There were no differences in concussion severity scores between VNTR group and concussion severity scores. This was the first study to investigate the association of the (GT)n VNTR within GRIN2A. We established a DNA collection, estimation, and genotyping protocol of the (GT)n VNTR for 96 samples and demonstrated accuracy of this genotyping method. Clinically, athletes carrying the long allele genotype may be predisposed to prolonged recovery following a concussive injury.
Temple University--Theses
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Huang, Xiaofang. "Functional study of amylin and regulation of amylin receptor." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/114036.
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PharmacologyPh.D.
Amylin, a 37 amino acid peptide secreted from pancreatic beta cells upon stimulation by meal/glucose, belongs to the family of the calcitonin or calcitonin gene-related peptide (CGRP) and shares up to 50% homology with CGRP, which is a well-documented pain-related peptide. The amylin receptor is composed of a calcitonin receptor (CTR) and receptor activity modifying proteins (RAMPs). Numerous studies have shown that amylin plays an important role in glucose homeostasis and food intake. Few studies have been conducted with respect to the effect of amylin in the central or peripheral neuraxis. In this thesis, immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the tail flick and acetic acid-induced writhing test. Amylin (1-10 µg, i.t.) reduced the number of writhing in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32) [sCT(8-32)]or AC187 by i.t. antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 µg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were up-regulated in the spinal cord of mice in acetic acid-induced visceral pain model and the increase was attenuated by pretreatment with amylin. Pretreatment of sCT[8-32] or AC187 significantly reversed the effect of amylin on c-fos expression in the spinal cord. As the neuronal response to amylin is closely dependent on the molecular property of amylin receptor, the localization, internalization and regulation of the calcitonin and amylin receptor were examined in the second part of the thesis. Immunofluorescence microscopy demonstrated the surface expression of CTRa, and intracellular distribution of RAMP1. Moreover, co-expression of CTRa translocated the RAMP1 to the cell surface and generated the amylin receptor phenotype. Both immunocytochemistry and on cell western analysis showed the internalization of CTRa and amylin receptor (CTRa/RAMP1) stimulated by different agonists, which was partially ß-arrestin dependent. Moreover, RAMP1 did not change the surface expression pattern of CTRa, but co-localized with the receptor with and without agonist treatment. sCT and amylin activated the ERK1/2 in HEK293 cells stably expressing amylin receptors, indicating the involvement of MAPK in amylin receptor signaling cascade. Collectively, these results led us to conclude that 1) irAMY is expressed in dorsal root ganglion neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the peptide by interacting with amylin receptors in the spinal cord may be antinociceptive; 2) RAMP1 does not change the pattern of CTR cell-surface localization and internalization, but receptor phenotype, presumably through a direct or indirect effect on the ligand-binding site; 3) amylin internalizes the amylin receptor (CTRa/RAMP1 complex); which is partially ß-arrestin dependent. Our studies extend the current knowledge of amylin on the spinal cord and new insight on the cellular and molecular mechanism underlying the antinociceptive effect of amylin. Also we demonstrate for the first time agonist induced-internalization of CTR/RAMP complex and its possible regulation pathway.
Temple University--Theses
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Deliu, Elena. "GPER/GPR30 Estrogen Receptor: A Target for Pain Modulation." Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/194862.
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PharmacologyPh.D.
The G protein-coupled estrogen receptor GPER/GPER1, also known as GPR30, was originally cloned as an orphan receptor and later shown to be specifically activated by 17-ß-estradiol. This has led to its classification as an estrogen receptor and expanded the perspective on the mechanisms underlying the rapid estrogenic effects reported over the years. GPER is strongly expressed in the central nervous system and peripheral tissues and appears to be involved in a wide variety of physiological and pathological processes. Estrogens are known to alter the processing of nociceptive sensory information and analgesic responses in the central nervous system. Both analgesic and pro-nociceptive effects of estrogens have been reported. Some pro-algesic estrogenic responses have a short latency, suggesting a non-genomic mechanism of action. Immunohistochemical studies in rodents prove the existence of GPER in pain-relevant areas of the nervous system such as dorsal root ganglia, superficial dorsal horn of the spinal cord, periaqueductal gray (PAG), amygdala, trigeminal sensory nucleus and thalamus. In the periphery, activation of GPER results in pro-nociceptive effects. However, GPER involvement in pain processing at central levels is largely unexplored. Thus, the work presented in this thesis was aimed at investigating whether GPER modulates nociception at spinal and supraspinal sites. The behavioral response to GPER activation in the spinal cord and PAG was evaluated in an acute grooming test (scratching, biting and licking behavior) and in the hot plate test, respectively. Intrathecal challenge of mice with the GPER agonist G-1 (0.1-1 nmol) induced a dose-dependent increase in pain-related behaviors, that was abolished by pre-treatment with the GPER antagonist G15 (1-10 nmol), confirming GPER specificity of the response. Likewise, intra-PAG microinjection of G-1 (10-100 pmol) to rats reduced the nociceptive threshold in the hot plate test, an effect that was G15 sensitive. To obtain further insight on the mechanisms involved in the behavioral effects observed in whole animals, we tested the effect of GPER ligands on neuronal membrane potential, intracellular calcium concentration ([Ca2+]i) and reactive oxygen species (ROS) accumulation. The membrane depolarization and the increases in [Ca2+]i and ROS levels are markers of neuronal activation, underlying pain sensitization in the spinal cord and pain facilitation in the PAG. Electrophysiological recordings from superficial dorsal horn and lateral PAG neurons indicate neuronal depolarization upon G-1 application, an effect that was fully prevented by G15 pre-treatment. Both cultured spinal neurons and cultured PAG neurons responded to G-1 administration by elevating [Ca2+]i and mitochondrial and cytosolic ROS levels. In the presence of G15, G-1 did not elicit the calcium and ROS responses. Collectively, these results demonstrate that GPER modulates both the ascending and descending pain pathways to increase nociception via cytosolic calcium elevation and ROS accumulation in spinal and PAG neurons, respectively. These findings broaden the current knowledge on GPER involvement in physiology and pathophysiology, providing the first evidence of its pro-nociceptive effects at central levels and characterizing some of the mechanisms involved. Moreover, we show for the first time ROS accumulation downstream of GPER activation, extending the current understanding of GPER signaling.
Temple University--Theses
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Khawaja, Rabia Raheel. "Role of calcium influx through glutamate receptors in white matter brain injury and oligodendrocyte regeneration." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/583654.
Full textAbstract:
Biomedical SciencesPh.D.
Calcium-influx through ionotropic glutamate receptors expressed on non-excitable cells, such as CNS glia, may regulate important cell events via intracellular signaling mechanisms. Oligodendrocytes and oligodendrocyte progenitors (OPCs), two glial populations supporting CNS myelination and myelin repair, express AMPA and NMDA receptors. Although calcium-influx through these receptors is thought to cause glutamate excitotoxicity to oligodendrocytes in CNS injuries, more recent studies suggest that AMPA or NMDA receptor-mediated synaptic transmission between neurons and OPCs plays a positive role in neuronal activity-dependent oligodendrocyte development and regeneration. Given the opposing roles of glutamate receptors in oligodendrocyte death and repair, the clinical relevance of these receptors in white matter injuries remain unclear. Another major challenge for exploring the role of these receptors in white matter injuries is that OPCs and neurons express a similar complement of AMPA and NMDA receptor subunits, which has complicated the interpretation of pharmacological manipulations and global genetic deletion approaches. To define the cell autonomous role of AMPA and NMDA receptor-mediated calcium signaling in oligodendroglia, I abolished the calcium influx through glutamate receptors using two different genetic approaches, and examined their impacts on oligodendrocyte development, injury-induced cell death, and regeneration. First, I employed a new mouse line which allows overexpression of GluA2, the calcium-impermeable AMPA receptor subunit, in a Cre activity-dependent manner. After crossing these mice with OPC- or oligodendrocyte-lineage-specific Cre mice, I applied hypoxic-ischemic injury to these multiple transgenic mice. Surprisingly, even though AMPA receptor-mediated calcium influx was blocked in OPCs, oligodendrogenesis or myelin integrity was not affected. However, GluA2 overexpression significantly promoted oligodendrocyte regeneration and OPC proliferation after injury, while the same manipulation in oligodendrocytes did not protect them from the initial cell loss. Moreover, GluA2 overexpression also stimulated transcriptional activities linked to myelinogenesis, even without injury. Second, I used conditional knockout mice for Grin1, the gene encoding an essential subunit of NMDA receptor complexes. As with GluA2 overexpressing mice, the removal of NMDA receptors from OPCs or all oligodendroglia did not significantly change normal oligodendrocyte development. However, the ablation of NMDA receptor in OPCs exacerbated oligodendrocyte loss by impairing new oligodendrogenesis in hypoxic-ischemic injury. These results suggest that neither AMPA receptors nor NMDA receptors mediate glutamate excitotoxicity in oligodendrocytes in neonatal hypoxic-ischemic injury. Instead, these receptors play distinct roles in post-injury oligodendrocyte development: AMPA receptor-mediated calcium suppresses oligodendrocyte regeneration, and NMDA receptor signaling supports oligodendrocyte regeneration after injury.
Temple University--Theses
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Schmidt, Ralf [Verfasser], and Frank [Akademischer Betreuer] Würthner. "Hamilton-Receptor-Mediated Self-Assembly of Merocyanine Dyes into Supramolecular Polymers / Ralf Schmidt. Betreuer: Frank Würthner." Würzburg : Universitätsbibliothek der Universität Würzburg, 2012. http://d-nb.info/1021645737/34.
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McGlynn, Liane Marie. "The interaction between the type 1 receptor tyrosine kinases and the oestrogen receptor in human breast cancer : the role of the Ras/Raf-1/MAPK pathway." Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443199.
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Hendrikse, Megan. "Interactions of GPR54 and GPR147 receptors with RF-amide ligands." Doctoral thesis, University of Cape Town, 2014. http://hdl.handle.net/11427/8708.
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Includes bibliographical references.G protein-coupled receptors play a key role in cellular signaling by transducing extracellular signals via G proteins to elicit intracellular responses. Studies have provided evidence supporting the role of the GPCR GPR54 and its cognate peptide ligand, kisspeptin (an RFamide peptide), in the regulation of reproduction. Kisspeptin and GPR54 play a critical role in the control of the hypothalamic-pituitary-gonadal axis by regulating gonadotropin-releasing hormone secretion. Despite the physiological importance of GPR54/kisspeptin signalling, the GRP54 residues important for receptor activation and signalling have not been extensively investigated. Another hypothalamic peptide, gonadotropin inhibiting hormone (also known as RFamide-related peptide), which interacts with the GPCR GPR147, has been found to inhibit GnRH-induced gonadotropin release and is therefore also of importance in control of the HPG axis. As many of the RFamide and RFamide-related receptors and ligands can be promiscuous, there is the potential for crosstalk between the GPR54/kisspeptin and GRP147/RFRP systems (or other RFamides) which may be of importance in the regulation of reproduction. GPR54 chimeras and point mutants were constructed in order to investigate the residues important for kisspeptin binding and receptor activation. The data obtained indicate that the acidic residues within the extracellular loops of GPR54 contribute to cell surface receptor expression and play a role in receptor signalling. In order to investigate the interactions of kisspeptin/RFRP peptides at GPR147 and GPR54, binding and activation of these receptors was studied with a range of ligands and their analogs. In addition to RFRP and its analogs, kisspeptin and several kisspeptin analogs were found to act as agonists at GRP147. In contrast, of all the ligands tested, only kisspeptin was able to bind to GPR54 with high affinity and elicit a response, thus indicating that GPR54 has high specificity for kisspeptin in contrast to the more promiscuous GPR147. These data demonstrate the therapeutic potential of kisspeptin analogs, for the inhibition of gonadotropin secretion and treatment of sex steroid hormone disease. In addition, these data have identified ligand and receptor residues important for binding and activation of GRP54/GRP147 which may aid development of new analogs targeting these receptors and highlighted the importance of testing these analogs for receptor specificity.
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Finley, Matthew James. "Molecular Basis for Kappa-Opioid Regulation of Chemokine Receptor Function." Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/62878.
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Molecular Biology and GeneticsPh.D.
Opioid receptor-mediated regulation of chemokine receptors is vital for the host immune response, development, and neurological function. Previous studies have demonstrated that the kappa opioid receptor (KOR) activation results in decreased infectivity of human immunodeficiency virus 1 (HIV-1) in human peripheral blood mononuclear cells (PBMCs). We have found this effect is due to down-regulation of the major HIV-1 co-receptors, CCR5 and CXCR4. Using molecular techniques, CCR5 and CXCR4 mRNA levels drop dramatically following KOR activation. To dissect the mechanism involved, we used transcription factor binding arrays and compared control cell extracts to KOR activated cell extracts. We determined that the interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) could be involved in the KOR-mediated repression of CCR5 and CXCR4 transcription and protein expression. Using chemical inhibitors and small interfering RNA (siRNA) molecules, we determined that JAK2, STAT3, and IRF2 are critical members of this signal transduction pathway. The understanding of these particular mechanisms should prove to be beneficial for the development of potential pharmacological agents targeted at HIV-1 binding and infection since virus infection requires expression of the co-receptors CXCR4 and CCR5. Understanding the molecular basis for KOR-induced inhibition of co-receptor expression may provide a basis for the development of KOR agonist-based therapeutics to treat individuals infected with HIV.
Temple University--Theses
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PENG, LI. "Gene Expression Study and DNA Methylation Status of Aryl Hydrocarbon Receptor Gene in Rbf/f;Alb-Cre+ Mouse Liver Tumors." University of Cincinnati / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1186635329.
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Aramaki, Yo. "Lectin-like oxidized LDL receptor-1 (LOX-1) acts as a receptor for remnant-like lipoprotein particles (RLPs) and mediates RLP-induced migration of vascular smooth muscle cells." Kyoto University, 2008. http://hdl.handle.net/2433/124233.
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Gofman, Larisa. "Role of Purinergic Receptor (P2X4) in EtOH-Mediated Microglial Immune Responses." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/315641.
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PathologyPh.D.
Ethanol (EtOH) abuse is the third leading cause of preventable death in the United States. Mounting evidence indicates that EtOH-induced neuropathology may result from multicellular responses in which microglia cells play a prominent role. Purinergic receptor signaling plays a key role in regulating microglial function and, more importantly, mediates EtOH-induced effects. In our current study we sought to determine the effects of EtOH on microglial cell function, specifically the role of purinergic receptor X4 (P2X4) in EtOH-mediated microglial responses. Our results show a significant up-regulation of P2X4 gene expression as analyzed by real-time qPCR and protein expression as analyzed by flow cytometry in embryonic stem cell-derived microglial cells (ESdM) after 48 hours of EtOH treatment, as compared to untreated controls. Calcium mobilization in EtOH treated ESdM cells was found to be P2X4R- dependent using 5-BDBD, a selective P2X4R antagonist. Blocking P2X4R signaling with 5-BDBD decreased the level of calcium mobilization compared to EtOH treatment alone. EtOH decreased migration of microglia towards fractalkine (CX3CL1) by 75% following 48 hours of treatment compared to control. CX3CL1-dependent migration was confirmed to be P2X4 receptor-dependent using the antagonist 5-BDBD, which reversed the effects as compared to EtOH alone. Similarly, 48 hours of EtOH treatment significantly decreased phagocytosis of microglia by 15% compared to control. 5-BDBD pre-treatment prior to EtOH treatment significantly increased microglial phagocytosis. These findings demonstrate that P2X4 receptor may play a role in modulating important regulatory functions in microglia in the context of EtOH abuse. P2X4R plays an important regulatory function in microglia. P2X4 is involved in a myriad of molecular signaling such as proliferation, activation of transcription factors, specifically through the MAPK pathway, and ATP signaling. Here, we also investigated the intracellular signal transduction pathway that influences P2X4R expression in microglia in response to EtOH. We found EtOH (100 mM) decreased phosphorylated AKT and extracellular signal-regulated kinase (ERK) cascades in ESdM cells. EtOH effect on ERK phosphorylation was completely inhibited by U0126, an inhibitor of MEK 1 and 2. However, PD98095, a potent inhibitor of MEK1 but a weak inhibitor of MEK2 had modest effect on phosphorylated ERK1/2 suggesting a possible role of MEK2-dependent ERK signaling in modulating EtOH induced effects on microglia. Utilization of 5-BDBD, a selective P2X4R antagonist reversed the EtOH-induced effect on phosphorylated AKT and ERK. Next we wanted to examine the effects of EtOH on transcription factor activity to determine the signaling mediators, which may play a role in EtOH-induced increase in P2X4R in microglia. EtOH increased transcriptional activity of NFκB, NFAT, and CREB,, however 5-BDBD blocked the effect on CREB transcriptional activity alone, suggesting a specific role of CREB in EtOH-induced expression of P2X4R in microglia. In summary, EtOH affects the expression of P2X4R in microglial cells and contributes to aberrant microglial effector function including phagocytosis and migration as well as alterations in calcium mobilization. Furthermore, pharmacological blockade with a selective P2X4R antagonist reversed the action, suggesting that P2X4R may play a role in mediating EtOH-induced effects on microglia. EtOH decreased expression of ERK and AKT, which was blocked with the P2X4R antagonist, suggesting EtOH effect may contribute to irregular microglial signaling. Investigations regarding transcription factor NFκB, NFAT and CREB activity in response to EtOH, all showed an increase after EtOH treatment, however P2X4R antagonist only had an effect on CREB, blocking the effect of EtOH on its activity. Determining the mechanism underlying EtOH-induced increase in P2X4R expression still remains unclear. This research was conducted to investigate the importance of P2X4R signaling in EtOH-mediate microglial function. Although many more questions remain unanswered, these experiments have provided evidence to target purinergic receptor X4 as a potential mediator of EtOH-induced effects in microglia.
Temple University--Theses
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Brito, Filho Francisco de Assis. "VCO Banda Larga Integrado para Receptor a Cinco Portas." Universidade Federal do Rio Grande do Norte, 2009. http://repositorio.ufrn.br:8080/jspui/handle/123456789/15300.
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Previous issue date: 2009-09-03Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
This work presents an wideband ring VCO for cognitive radio five-port based receivers. A three-stage differential topology using transmission gate was adopted in order to maintain wide and linear tuning range and a low phase-noise. Monte-Carlo analysis were performed for phase-shift response of individual stages, which is an important figure of merit in five-port works. It was observed a fairly linear correlation between control voltage and oscillation frequency in the range between 200 MHz and 1800 MHz. The VCO was preliminarily designed for IBM 130nm CMOS technology
Este trabalho apresenta um VCO anel banda-larga para ser utilizado em receptores para R?dio Cognitivo baseados no correlator a cinco portas. Uma arquitetura diferencial de tr?s est?gios com porta de transmiss?o ? utilizada como forma de manter uma sintonia linear em larga faixa de frequ?ncias, bem como, um baixo ru?do de fase. An?lises de Monte-Carlo foram feita para avaliar as varia??es de fase em cada est?gio, o que constitui uma figura de m?rito importante em receptores baseados no correlator de cinco portas. Observou-se correspond?ncia razoavelmente linear entre tens?o de controle e freq??ncia de oscila??o na faixa compreendida entre 200 MHz e 1800 MHz. O VCO foi preliminarmente projetado para tecnologia CMOS IBM de 130 nan?metros
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Lemos, Sandra [Verfasser], Michael [Gutachter] Hollmann, and Heumann [Gutachter] Rolf. "Regulation of AMPA receptors by type II-TARPs / Sandra Lemos ; Gutachter: Michael Hollmann, Heumann Rolf." Bochum : Ruhr-Universität Bochum, 2017. http://d-nb.info/1129452433/34.
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Harb, Ali [Verfasser], and Ralf [Akademischer Betreuer] Mohrmann. "AMPA receptor trafficking and regulation by prominent auxiliary subunits in cultured hippocampal neurons / Ali Harb ; Betreuer: Ralf Mohrmann." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2019. http://d-nb.info/1197054693/34.
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Mao, Yingying. "ROLE OF PROTEASE-ACTIVATED RECEPTORS IN PLATELET ACTIVATION." Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/47279.
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PhysiologyPh.D.
Platelets act as a fundamental component of the hemostatic process and their activation leads to the formation of a stable clot at the injured endothelium surface. Thrombin, as the important physiological agonist, activates platelets through protease-activated receptors (PARs). Protease-activated receptors are one of the major receptors in platelets and belong to the seven-transmembrane G-protein couple receptor family. Four protease-activated receptors are found, named as PAR1, PAR2, PAR3 and PAR4. Human platelets express PAR1 and PAR4 and murine platelets express PAR4 and PAR3 instead of PAR1. Thrombin activates PARs through a unique mechanism, involving the cleavage of N-terminus of PAR receptors and the newly exposed N-terminus acts as its own tethered ligand to bind and activate the receptor. In this study, we characterized a new PAR1 specific activating peptide (TFRRRLSRATR), generated from the c-terminus of human platelet P2Y1 receptor, and evaluated its biological function. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Its activation is completely inhibited by using BMS200261, a PAR-1 specific antagonist. Its specificity to PAR1 receptor is further confirmed by using TFRRR-peptide-pretreated washed platelets and murine platelets. The shape change induced by 10 microM peptide was totally abolished by Y-27632, an inhibitor of p160ROCK which is the downstream signal of G12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increased concentrations. Similar to SFLLRN, the TFRRR-peptide caused phosphorylation of Akt and Erk in a P2Y12 receptor-dependent manner, and p-38 MAP kinase activation in a P2Y12-independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. Beside thrombin, PARs also can be activated by other proteases. Previous studies in our lab show that plasmin, a major extracellular protease, activates both human and murine platelets through prototypical cleavage of PAR4 (Quinton et al., 2004). In this study, we continue our study and investigate the molecular basis for the differential activation of murine and human platelets by plasmin. Plasmin-induced full aggregation is achieved at lower concentrations (0.1 U/mL) in murine platelets as compared to human platelets (1 U/mL). In COS7 cells expressing the murine PAR4 (mPAR4) receptor, 1 U/mL plasmin caused a higher intracellular calcium mobilization than in cells expressing the human PAR4 (hPAR4) receptor. This difference was reversed when the tethered ligand sequences of mPAR4 and hPAR4 were interchanged through site-directed mutagenesis. This difference between human and murine PAR4 is not because of the cofactor effect of PAR3 in murine platelets by showing that in both transfected cell lines and platelet system, PAR3 inhibits plasmin-induced PAR4 stimulation. All of the data suggest that murine platelets are more sensitive to activation by plasmin than human platelets due to differences in the primary sequence of PAR4. In contrast to thrombin-dependent activation of platelets, wherein PAR3 acts as a co-receptor, mPAR3 inhibits plasmin-induced PAR4 activation. Abnormal platelet activation causes thrombus formation and induces pathological conditions including stroke and atherosclerosis. Antithrombotic therapy is a widely used therapeutic method for stroke. However, currently used agents based on the irreversible inhibition of the platelet cyclooxygenases 1 and 2 or inhibition of P2Y12 receptors can cause unexpected bleeding or resistant side effects. Antithrombotic therapy targeting thrombin signaling is one of the new treatments under investigation and PAR1 antagonists are now in clinical trials. In this study, we investigate the effect of one of thrombin receptors, protease-activated receptor 4 (PAR4) in mice transient middle cerebral artery occlusion/ reperfusion (tMCAO/R) model. Our data show that PAR4 -/- mice have more than 80% reduction in infarct volume and significant improved neurological and motor function after 1 h MCAO followed by 23 h reperfusion. Examination of cellular responses to tMCAO/R indicates that PAR4-/- mice have less cellular death. Platelet/endothelial and leukocyte/endothelial interactions have been shown to play a critical role in the inflammatory responses during cerebral ischemic/reperfusion injury. Comparing wild-type with PAR4-/- mice platelets/endothelial and leukocyte/endothelial interactions, deficiency of PAR4 causes a significant decrease in both platelet/endothelial and leukocyte/endothelial interactions. In addition, PAR4-/- mice attenuate blood-brain barrier (BBB) disruption during tMCAO/R. All the data suggest that deficiency of PAR4 will protect against brain ischemic injury though attenuation of cerebral inflammatory responses including inflammatory cells extravasation and BBB disruption. Protease-activated receptor 4 (PAR4) is the only thrombin receptor existing in both human and murine platelets. The data we get in this study also have a beneficial effect for human study and inhibition of PAR4 may provide a novel potential therapeutic strategy for ischemic injury.
Temple University--Theses
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Limaye, Arati. "RECEPTOR MEDIATED ORAL DELIVERY OF BIOENCAPSULATED GREEN FLUORESCENT PROTEIN EXPRESSED IN TRANSGENIC CHLOROPLASTS." Master's thesis, University of Central Florida, 2005. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4295.
Full textAbstract:
The skyrocketing costs of prescription medicine in developed countries and their lack of availability in developing countries are the most challenging problems of human health. Primary reasons for such high cost are fermentation-based production, expensive purification methods, the need for low temperature storage and transportation and the delivery through sterile injections. Most of these expenses could be minimized or eliminated when therapeutic proteins are expressed and orally delivered via plant cells. Chloroplasts have the machinery to fold complex and biologically active eukaryotic proteins in the soluble chloroplast stromal compartment. Protein expression through chloroplast transformation system offers a number of advantages over nuclear transformation such as a high level of transgene expression (up to 47% of the total soluble protein), due to the presence of 10,000 copies of the transgene per cell, which is uniquely advantageous for oral delivery of adequate amounts of the therapeutic protein or vaccine antigen. It is also an environmentally friendly approach due to effective gene containment and lack of transgene expression in pollen since the chloroplast genome is maternally inherited. To study receptor-mediated oral delivery of therapeutic proteins using the transmucosal carrier cholera toxin B subunit (CTB), a CTB-GFP fusion protein separated by a furin cleavage site was expressed via the tobacco chloroplast genome and used as a visible marker. Site specific integration of the transgene was confirmed by PCR analysis. Southern blot analysis confirmed homoplasmy. Immunoblot analysis confirmed the expression of both the monomeric as well as the pentameric forms of CTB-GFP in transgenic plants. Expression levels of upto 21.3% were obtained and the functionality of the CTB-GFP pentamers was confirmed by an in vitro GM1 binding assay. GFP was seen in the intestinal mucosa, liver and spleen of mice orally fed with CTB-GFP expressing leaves, while CTB was detected only in the intestinal cells. Intestinal macrophages and dendritic cells stained positive for both the CTB as well as GFP. These results suggest successful cleavage of the foreign protein from the transmucosal carrier and its delivery to various organs. These investigations should facilitate the development of a novel cost-effective oral delivery system for plant-derived therapeutic proteins.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
28
Robinson, Rebecca Hartzell. "Cannabinoid Receptor 2-Selective Ligands as Immunosuppressive Compounds: Utility in Graft Rejection." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/246094.
Full textAbstract:
Microbiology and ImmunologyPh.D.
Cannabinoids are known to have anti-inflammatory and immunomodulatory properties. Cannabinoid receptor 2 (CB2) is expressed mainly on leukocytes and is the receptor implicated in mediating many of the effects of cannabinoids on immune processes. The capacity of delta-9-tetrahydrocannabinol (delta-9-THC) and of two CB2-selective agonists to inhibit the murine Mixed Lymphocyte Reaction (MLR), an in vitro correlate of graft rejection following skin and organ transplantation was tested. Both CB2-selective agonists and delta-9-THC significantly suppressed the MLR in a dose dependent fashion. The inhibition was via CB2, as suppression could be blocked by pretreatment with a CB2- selective antagonist, but not by a CB1 antagonist, and none of the compounds suppressed the MLR when splenocytes from CB2 deficient mice were used. The CB2 agonists were shown to act directly on T-cells, as exposure of CD3+ cells to these compounds completely inhibited their action in a reconstituted MLR and proliferation of purified T-cells by anti-CD3 and anti-CD28 antibodies was inhibited. Treatment of both CD4+ and CD8+ T-cells with a CB2-selective agonist inhibited the MLR, though significantly less than when both cell types were treated. T-cell function was decreased by CB2 agonists, as an ELISA of MLR culture supernatants revealed IL-2 release was significantly reduced in the cannabinoid treated cells. Further, treatment with O-1966 dose- dependently decreased levels of the active nuclear forms of the transcription factors NF- kappa-B and NFAT in wild-type T-cells, but not T-cells from CB2 knockout (CB2R k/o) mice. Additionally, a gene expression profile of purified T-cells from MLR cultures, generated using a PCR T-cell activation array, showed that O-1966 decreased mRNA expression of CD40 ligand and CyclinD3, and increased mRNA expression of Src-like-adaptor 2 (SLA2), Suppressor of Cytokine Signaling 5 (SOCS5), and IL-10. The increase in IL-10 was confirmed by measuring IL-10 protein levels in MLR culture supernatants. An increase in the percentage of regulatory T-cells (Tregs) was observed in MLR cultures and pretreatment with anti-IL-10 resulted in a partial reversal of the inhibition of proliferation and blocked the increase of Tregs. Additionally, O-1966 treatment caused a dose-dependent decrease in the expression of CD4 in MLR cultures from wild-type, but not CB2R k/o, mice. The ability of O-1966 treatment to block rejection of skin grafts in vivo was also tested. Mice received skin grafts from a histoincompatible donor, and the time to graft rejection was analyzed. Compared to mice that received the vehicle, mice that received O-1966 treatment had significantly prolonged graft survival and increased Tregs in the spleen. The spleen cells from O-1966-treated mice had reduced proliferation in an MLR and an increased percentage of Tregs. Together, these data support the potential of this class of compounds as useful therapies to prolong graft survival in transplant patients and possibly as a new class of immunosuppressive drugs.
Temple University--Theses
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Regan, Patrick M. "Regulation and Functional Impact of Opioid Receptor Splicing in Response to Morphine." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/344730.
Full textAbstract:
Biomedical NeurosciencePh.D.
Multiple classes of pharmaceuticals, including acetaminophen, aspirin, and other nonsteroidal anti-inflammatory drugs (NSAIDs), are used to relieve mild to moderate pain; however, one of the oldest classes of pharmaceuticals, opioids, remains the primary class of drugs used in the management of severe pain. For decades, the unique pharmacological profiles of opioid compounds have suggested the existence of multiple opioid receptor subtypes and, accordingly, four opioid receptors have been cloned to date; the mu (μ)-opioid receptor, the kappa (κ)-opioid receptor, the delta (δ)-opioid receptor, and the nociceptin/orphanin FQ receptor. Additionally, each receptor is encoded by its own distinct gene; the OPRM1, OPRK1, OPRD1, and OPRL1, respectively. Despite the identification and characterization of these four opioid receptor subtypes, pharmacological data, particularly from opioid receptor knockout mice, does not conform to the predications of a four opioid receptor model and instead suggests the existence of additional receptor subtypes. Additional opioid receptors have since been proposed but corresponding genes have either been unidentified or found to be genetically unrelated. Interestingly, this problem is not unique to opioid receptors, as there is a large discrepancy between the number of protein encoding genes and the repertoire of mRNA transcripts and encoded proteins they produce, with gene products far more numerous than estimates would predict. It is now understood that this discrepancy is due to the generation of multiple RNA transcripts from a single gene. Several mechanisms are utilized in order to generate mRNA transcript variants, or isoforms, from a single gene; however, the primary mechanism, known as alternative splicing, involves a complex macromolecular machine, referred to as the spliceosome, through which specific portions of the precursor mRNA (pre-mRNA) sequence are selectively removed and the remaining nucleotide sequences are ligated to form a unique mRNA transcript. Recently, multiple opioid receptor isoforms, particularly for the μ-opioid receptor, have been identified; however, both their regulation and their functional significance are poorly characterized. As such, multiple studies are needed to more precisely describe alternatively spliced μ-opioid receptor isoforms, particularly the regulation of spliceosome components that determine the splicing specificity of particular isoforms as well as the distinct signaling pathways utilized by particular isoforms both constitutively and following agonist binding. Using a model of dopaminergic neurons, this study sought to examine these questions and found that expression of a particular splice variant, MOR-1X, was up-regulated by morphine through a mechanism involving the essential splicing factor ASF/SF2. Structural comparison of this isoform to the prototypical variant MOR-1 found that the unique distal portion of C-terminal domain contains two additional PKA phosphorylation sites as well as a second agonist-induced phosphorylation motif highly conserved among opioid receptors. Functional comparison of MOR-1 and MOR-1X found distinct signaling differences, both constitutively and following morphine treatment, in MAPK signaling cascades, particularly ERK1/2. While the pharmacological significance of MOR-1X expression and signaling remains unclear, the clinical importance of this finding extends beyond a mechanism of opioid analgesic variability, as the physiological roles of opioids also include immunomodulation and have been implicated specifically in the exacerbation of HIV viral replication and pathology, particularly neurocognitive dysfunction. Accordingly, the HIV viral protein Tat was found to block morphine-mediated increases in MOR-1X expression by similarly blocking morphine-mediated increases in ASF/SF2 expression. Consequently, MOR-1X and HIV viral proteins were found to have a unique and synergistic role in the regulation of intrinsic apoptotic signaling cascades, specifically Bax expression, and in cell proliferation. Therefore, the regulation of alternative splicing events by both opioids and HIV viral proteins involves, in part, the inverse regulation of ASF/SF2 protein expression, through which the expression of the MOR-1X isoform is subsequently and significantly altered. This, in turn, may lead to functional consequences in opioid pharmacokinetics as well as in opioid-related pathology, such as the exacerbation of HIV associated neurocognitive dysfunction, as MOR-1X contains unique functional regions which may be responsible for the observed differences in MAPK and intrinsic apoptotic signaling and cellular proliferation. Collectively, these findings support previous studies that suggest alternative splicing of the MOR is altered by exogenous factors, such as morphine and HIV, identify unique signaling pathways for various opioid receptor isoforms, and are the first to suggest a potential mechanism through which pharmacological interventions could be utilized to alter opioid receptor isoform expression, thereby altering the pharmacological and physiological effects of opioids.
Temple University--Theses
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Bettembourg, Mathilde. "Caractérisation de récepteurs à activité kinase impliqués dans la mise en place de l'architecture racinaire chez le riz." Thesis, Montpellier, SupAgro, 2016. http://www.theses.fr/2016NSAM0026.
Full textAbstract:
Les racines ont deux grands rôles. Le premier est le prélèvement de l’eau et des éléments nutritifs et le second est l’ancrage dans le sol. Identifier les gènes responsables de la mise en place des tissus et de l'architecture du système racinaire est donc essentiel pour pouvoir améliorer les variétés de riz soumises à des stress abiotiques de plus en plus fréquents et nombreux du fait du changement climatique. Au cours de cette thèse, j'ai réalisé une analyse fonctionnelle du gène DEFECTIVE IN OUTER CELL LAYER SPECIFICATION (DOCS1) qui appartient à la famille des récepteurs kinases à répétitions riches en leucine (LRR-RLK). Ces protéines sont composées de deux domaines principaux: un domaine extra-cytoplasmique composé de répétitions LRR et un domaine kinase intra-cytoplasmique. Un mutant de ce gène, nommé c68, possède une mutation non-sens dans le domaine kinase. Les plantes mutantes c68 présentent plusieurs phénotypes: une sensibilité accrue à l'aluminium, une réduction du nombre et de la taille des poils absorbants dans les racines, et des couches d’exoderme/épiderme d’identité mêlée. Le premier chapitre de la thèse porte sur l’étude conjointe de lignées knock-out CRISPRs du gène DOCS1 et de c68. Nos résultats ont montré que les mutants c68 et CRISPRs présentaient les mêmes phénotypes : sensibilité à l’aluminium, défauts des poils absorbants et tissus externes d’identité mixte. Ces résultats suggéraient que chez le mutant c68, soit la protéine DOCS1 n'était pas fonctionnelle, soit elle n'était pas traduite. Nos analyses phénotypiques ont aussi révélé que tous les mutants présentaient des défauts de réponse à la gravité à différents stades de développement. A 3 jours, un retard de réponse à la gravité était observé pendant la première heure après gravistimulation. Les plantules mutantes présentaient aussi des défauts de localisation d’un transporteur d’auxine. A 40 jours, nous avons observé que l'angle du cône racinaire des plantes mutantes était plus ouvert que celui des plantes sauvages. Deux gènes liés à l’auxine et plusieurs QTLs ont déjà été identifiés comme participant à ce phénotype chez le riz. Dans la suite de notre étude, nous avons donc cherché à identifier de nouveaux QTLs et gènes impliqués dans ce phénotype morphologique par étude d'association pan-génomique dans deux panels Indica et Japonica. Toutes les accessions de l'écotype bulu d'Indonésie et trois japonicas tempérés d'Asie du Sud présentaient un angle du cône racinaire très ouvert. En utilisant un modèle mixte associé à une technique de ré-échantillonnage, 55 QTLs ont été détectés. L'analyse des gènes sous-jacents ou voisin (+/- 50kb) a identifié 539 gènes, dont 6 LRR-RLK, 5 gènes liés à l’auxine et 5 gènes avec une fonction validée dans le développement ou l'architecture racinaire. Une approche complémentaire par cartographie génétique classique est proposée pour identifier les gènes en cause dans la ou les mutations à angle du cône racinaire très ouvert. Des perspectives de poursuite du travail effectué sont aussi présentées afin de déterminer si le phénotype affectant l'angle du cône racinaire induit par les mutations du gène DOCS1 ou des nouveaux gènes identifiés est lié à des perturbations des flux d’auxineRoots have two major roles. The first one is to uptake water and nutrients and the second one is to anchor plants into the ground. Identifying the genes responsible for the establishment of tissues and architecture of the root system is essential to improve rice varieties subject to increasingly frequent and numerous abiotic stresses due to climate change. During my PhD, I undertook a functional analysis of the DEFECTIVE IN OUTER CELL LAYER SPECIFICATION (DOCS1) gene which belongs to the Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) family. These proteins are composed of two main domains: an extra-cytoplasmic domain containing LRR repeats and a cytoplasmic kinase domain. A mutant of this gene, named c68, carries a nonsense mutation in the kinase domain. The c68 mutant plants show several phenotypes: increased sensitivity to aluminum, reduced number and size of root hairs, and layers of external tissues with exodermis/epidermis mixed identity. The first chapter of the thesis focuses on the joint study of knockout CRISPRs lines of the DOCS1 gene and c68. Our results showed that the c68 and CRISPRs mutants displayed the same phenotypes: sensitivity to aluminum, defects in root hairs and mixed identity of external tissues. These results suggested that in the c68 mutant, either the DOCS1 protein was not functional, or the protein was not translated. Our phenotypic analyses also showed that all mutants exhibited impaired gravity responses at different development stages. At 3 days, a delay of response to gravity was observed during the first hour after gravistimulation. Mutant seedlings also had defects in an auxin transporter localization. At 40 days, we observed that the root cone angle of mutant plants was more open than that of wild-type plants. Two genes associated with auxin and several QTLs have been identified as contributing to this phenotype in rice. In the rest of our study, we therefore tried to identify new QTLs and genes involved in this morphological phenotype by a genome-wide association study in two Indica and Japonica panels. All accessions of the bulu ecotype from Indonesia and three South Asian temperate japonica had a very open root cone angle. Using a mixed model associated with a resampling technique, 55 QTLs were detected. The analysis of the underlying or neighbor (+/- 50kb) genes identified 539 genes, including 6 LRR-RLK, 5 genes related to auxin and 5 genes with a function validated in root development or architecture. A complementary approach by classical genetic mapping is proposed to identify genes involved in the mutation(s) involved in very open root cone angle. Prospective research lines are also presented to determine if the root cone angle phenotype , induced by DOCS1 or by newly identified genes, is linked with disruption of auxin fluxes
31
Salvadeo, Danielle Marie. "TO PEE OR NOT TO PEE: A CHARACTERIZATION OF CANINE BLADDER PHYSIOLOGY FOLLOWING LONG-TERM LOWER SPINAL ROOT TRANSECTION AND SURGICAL REINNERVATION." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/561128.
Full textAbstract:
Biomedical SciencesPh.D.
Bladder incontinence in patients who suffer from sacral spinal cord injury can wreak havoc on one's quality of life. A 2012 survey suggests that patients who sustain spinal cord injury prioritize the recovery of bladder function over other faculties. With about 12,000 new spinal cord injury cases reported in the United States each year, finding ways to combat the disabilities that result from lower spinal cord dysfunction should be of utmost importance to the scientific research community. Prior to studying the effects of surgical reinnervation on the bladder after long-term decentralization, it was critical to understand the effects that decentralization had on the integrity of both smooth muscle and intramural nerves of the bladder, the function of which could determine the success of surgical reinnervation. Chapter 2 describes in vivo stimulation, ex vivo smooth muscle contractility studies, and immunohistochemical techniques that were used to assess the condition of the functional components of the bladder. Collective results showed that although pelvic plexus-induced stimulation decreased when decentralization included the bilateral transection of the L7 dorsal root, smooth muscle cells and intramural nerves maintained their function after long-term bladder decentralization. Thus, preservation of at least some nerve activity may allow for successful surgical reinnervation after long-term injury. Following confirmation of smooth muscle and intramural nerve viability after decentralization, we sought to determine if nerve transfer after long-term decentralization restores bladder function in canines. In Chapter 3, we detail both decentralization and surgical reinnervation procedures used in our model. Briefly, decentralization of the bladder included bilateral transection of hypogastric nerves, as well as all spinal roots caudal to L7, with a subset of animals undergoing additional transection of the dorsal root of L7. One year after decentralization, animals that showed consistent loss of sensory and motor function underwent surgical reinnervation, which included the bilateral transfer of part of the obturator nerve to the anterior vesical branch of the pelvic nerve and the semimembranosus branch of the sciatic nerve to the pudendal nerve. Behavioral observations, in vivo stimulation of transferred nerves, and retrograde tracing studies were used to explore the efficacy of reinnervation on both sensory and motor components of bladder function. Ultimately, results showed that the new neuronal pathways created by nerve transfer can restore bladder sensation and possibly motor function in lower motor neuron-lesioned canines. Beyond the effects of surgical reinnervation on bladder function, we were interested in taking a closer look at the mechanisms that dictate function after decentralization and reinnervation (Chapter 3). Based on our previous work that found that transfer of somatic nerves resulted in bladder smooth muscle expression of a nicotinic receptor subunit thought to be expressed primarily in striated muscles, we were interested in assessing changes in the profile of nicotinic receptors responsible for bladder function. Ex vivo smooth muscle contractility studies showed that response to nicotinic receptor agonists were not altered after decentralization or reinnervation. Furthermore, the α1 nicotinic receptor subunit was expressed in bladder smooth muscle across all surgical groups. Future studies are necessary to better elicit the physiological relevance of these nicotinic receptors in the bladder. Additionally, due to the complexity of surgical reinnervation, it was important to understand all contributions to bladder innervation (Chapter 4). We previously identified that cells in the ventral horns of spinal cord levels rostral to the sacral cord can directly innervate the bladder via retrograde tracing. Because these direct inputs were not in proximity of the spinal root transections made during decentralization, we wanted to know how decentralization and reinnervation impacted their effects on the bladder when stimulated. L2-mediated detrusor contractions were significantly decreased by transection of the hypogastric nerves, suggesting that many of the nerves originating from the L2 cord are sympathetic in nature; however, treatment with phentolamine did not completely eliminate the increase in pressure in response to L2 stimulation. Therefore, the remaining inputs likely act upon the bladder through a yet undefined pathway. The quantity of positively labelled cells did not change in sections of the L2 ventral horn across all surgical groups, suggesting no change in the contribution of direct inputs to bladder innervation. Finally, anatomical feasibility of the obturator and semimembranosus branch of the sciatic nerve transfers has been assessed in an unembalmed cadaver, the results of which have not yet been published. Overall, this research gives us reason to believe that surgical reinnervation is a viable option for patients who develop lower neurogenic bladder after injury to the sacral cord, cauda equina, or peripheral nerves mediating bladder function.
Temple University--Theses
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Fan, Yanfeng. "THE APPLICATION OF SPIROLIGOMERS TOWARDS MOLECULAR RECOGNITION AND ORGANOCATALYSIS." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/576554.
Full textAbstract:
ChemistryPh.D.
This thesis presents the development of bis-amino acid-based spiroligomer applications in the areas of molecular recognition and organocatalysis. By taking advantage of the high degree of functionality and chirality of the unique bis-amino acid building blocks, spiroligomer backbones can be synthesized with predefined shapes, functioning as molecular hosts or as enzyme active-site-like pockets. Firstly, we demonstrated that spiroligomers can be designed to act as anion receptors. We designed a collection of spiroligomers that each display two urea groups. The spiroligomer that displayed the two urea groups in a way that they pointed at each other acts as an anion receptor and binds hydrogen pyrophosphate H2PPi anion (H2P2O72−), as demonstrated by an NMR titration experiment. Other spiroligomers that displayed the two ureas demonstrated a variety of behaviors including self-association and gel formation. In later work we explored the use of spiroligomers to develop catalysts. We attempted to design bipyridine/TEMPO-based bifunctional catalysts but they failed to achieve a faster alcohol oxidation rate than the background reaction. We then demonstrated the successful incorporation of metal-salen functional groups into spiroligomers in Chapter 4. Several bis-amino acid-based metal-salen complexes were synthesized and examined as asymmetric catalysts. Although only moderate enantio-selectivity was detected from synthesized Mn-salen catalyzed epoxidation reactions, it provides the first direct evidence that chiral bis-amino acid backbone can act as a chiral pocket that influence substrate selection and the stereochemical outcome of reactions.
Temple University--Theses
33
Venkataraman, Rajarajeshwari. "ROLE OF TRANSIENT RECEPTOR POTENTIAL CANONICAL-6 (TRPC6) CHANNEL IN METASTASIS OF GLIOBLASTOMA MULTIFORME." Master's thesis, University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4353.
Full textAbstract:
Glioblastoma multiforme (GBM) is one of the extremely fatal brain tumors. The main reason that makes it so lethal is its capability to invade and spread to other parts of CNS producing secondary tumors. Among other factors hypoxia, reduced oxygen availability, is linked to higher metastatic potential of cancers. Hypoxia causes numerous changes in genome and proteome of the cell. These changes help a normal cell to adapt to nutritional deficiency, but the same changes can increase the malignancy and metastasis in tumor cells. Extensive research by a number of curious scientists reveal that various pathways involving numerous proteins cross-talk and interact with each other and execute a response to hypoxia. We are trying to establish the link between two such pathways HIF1-alpha pathway and Notch pathway. Both, HIF1-alpha, which is a transcription factor that becomes active in hypoxic conditions and Notch, which is an evolutionarily conserved cell-fate determinant, are implicated in hypoxia-induced metastasis of cancer. In this given project, we confirm the cross talk between Notch and HIF1-alpha pathway and further continue our study to show that TrpC6 is the downstream mediator of this pathway, leading to metastasis of GBM. Expression analysis of hypoxia-induced U373 cells (Grade 3 glioblastoma cells), using Real-time PCR, western blot and immunocytochemistry, revealed elevated levels of Notch, Hif1 and TrpC6 indicating that these proteins might be important for the cellular response to hypoxia. Blocking Notch and/or HIF1-alpha, either by DAPT or HIF1-inhibitor, confirmed the communication between these two pathways. Role of TrpC6 in metastasis was demonstrated by knocking down this gene using siRNA against TrpC6. Inhibition of TrpC6 markedly decreased cell proliferation, migration, angiogenesis and tumorigenesis in these hypoxia-induced Glioblastoma cells. In summary, all these results reveal that TrpC6 is indeed an important member of the Notch-mediated metastasis of Glioblastoma under hypoxic conditions. This role of TrpC6 can therefore be utilized for pharmacological intervention to prevent hypoxia-induced metastasis in GBM.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular and Microbiology MS
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Chang, Jen-Kuan. "The Biochemical Basis of The miR-21 Expression by The Mu-Opioid Receptor." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/352105.
Full textAbstract:
Molecular Biology and GeneticsPh.D.
Opioid receptors are members of the superfamily of seven transmembrane G protein-coupled receptors (GPCRs) which share several structural and functional characteristics. There are 3 subtypes of opioid receptors, designated μ (MOR), δ (DOR), and κ (KOR) opioid receptors, have been found in the immune, nervous, gastrointestinal and other tissues. We have attempted to clarify the nature of MOR-induced signal transduction pathways in leukocytes. We found that the activation of MOR leads to a significant induction of ERK phosphorylation in peripheral blood mononuclear cells from normal donors using the MOR-selective agonist DAMGO. We are also interested in determining the role of this signaling pathway in the regulation of the immune response. Recent experiments using selective inhibitors suggests that the activation of ERK involves a pathway composed of Raf, Ras, and MEK1/2 kinases, but is independent of PI3 kinase. After treatment of multiple protein kinase inhibitors we found the PKC inhibitor Go-6983 and PLC inhibitor U73122 could also inhibit ERK phosphorylation in MOR stable line (HEK-MOR). According to the results from the Go-6983 and H-89 inhibitor treatment experiments, we found PKCμ/PKD1, a family member of Protein Kinase D, may be involved in MOR-induced ERK phosphorylation. We also found PKCμ/PKD1 S916 phosphorylation after MOR activation and the PKCμ specific inhibitor CID755673 inhibited the MOR-mediated ERK activation. ERK phosphorylation activated several transcription factors in human monocytes, the activation of transcription factors has been proved to induce miRNA expression. We have initiated a series of experiments to study the regulation of miRNA expression by MOR in human monocytes. We found miR-21, miR-155, miR-29a, miR-20b expression were significantly up-regulated following morphine treatment, and morphine-induced miR-21 expression is down-regulated following pretreatment with the ERK inhibitor U0126 and PKD inhibitor CID755673 in human primary monocytes. The results suggest that morphine-induced MOR activation results in up-regulation of miRNA expression human monocytes and this may regulate monocyte and/or macrophage function thought PKCμ/Ras/Raf/ERK signaling pathway.
Temple University--Theses
35
Happel, Christine. "Molecular Basis for Mu-Opioid Regulation of Chemokine Gene Expression." Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/28438.
Full textAbstract:
Molecular Biology and GeneticsPh.D.
Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines.
Temple University--Theses
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Lorch, Robert A. "MicroRNA regulation of prostate cancer desensitization to androgen receptor antagonist drugs during androgen deprivation therapy." Honors in the Major Thesis, University of Central Florida, 2011. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/462.
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The current standard treatment of prostate cancer by androgen deprivation therapy involves using drugs such as bicalutamide (Casodex) to antagonistically block androgen receptors that are normally present within prostate cells. Usually, the therapy is successful in the short run at limiting the growth of prostate cancer. However, in virtually all cases tumors begin to grow aggressively again after several months of treatment and new therapies must be started. The mechanism by which these prostate cells transform from androgen sensitive to androgen independent and anti-androgen resistant is unclear. In this study, we investigated the role of microRNAs, small 15 to 18 nucleotide regulatory RNAs, in regulating the desensitization of prostate cancer cells to the androgen receptor antagonist drug bicalutamide. In order to identify significant microRNAs, quantitative PCR was used to obtain genome-wide microRNA expression levels of 885 human microRNAs at different timepoints for androgen sensitive LNCaP cancer cells treated with bicalutamide and for untreated control cells in tissue culture. Analysis of microRNA expression by clustering analysis and by statistical comparisons of treatment groups resulted in identification of 28 microRNAs that have altered expression in the progression process. In silico target prediction analysis was performed with the microRNAs shown to have altered expression, and a group of genes predicted to be under microRNA regulatory control during cancer progression to resistance was identified. A microRNA expression profile can be useful in developing more effective prognostic and therapeutic tools for prostate cancer.B.S.
Bachelors
Medicine
Molecular Biology and Microbiology
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Bright, Nieka L. "Glutamate Receptor, Ionotropic N-methyl-D-aspartate 2B Polymorphisms and Concussive Recovery in Athletes." Diss., Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/216565.
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KinesiologyPh.D.
Athletes vary in their ability to recover from concussions. Following a concussion, a pathophysiological cascade of events transpires, rendering symptoms. One such event, the indiscriminate release of the excitatory neurotransmitter glutamate, may result in hyperactivation of glutamate receptors (e.g., N-methyl-D-aspartate receptors [NMDARs]) and self-propagate a state of neurotoxicity that may be enhanced via the concomitant release of Ca2+, particularly through NMDARs containing the NR2B subunit. Genetic variation in regulatory regions of the glutamate receptor, ionotropic N-methyl-D-aspartate 2B (GRIN2B) gene, which codes for the NR2B subunit, may play a role in varied recovery among concussed athletes. Indeed, the rs1019385 promoter single nucleotide polymorphism (SNP) has been shown to alter transcription in dominant versus recessive allele carriers such that expression of the T allele results in increased upregulation of the GRIN2B gene. Therefore, the primary purpose of this study was to determine the association of this GRIN2B SNP and concussive recovery; a second GRIN2B SNP (rs890), in the 3'untranslated region, was also explored. A secondary purpose was to examine SNP associations with initial evaluation concussion severity scores. A triple-blind, between-subjects, genetic association design was utilized. The independent variable was genotype for both GRIN2B SNPs (rs1019385, rs890). The primary dependent variable, concussive recovery, was defined as the number of days from the time of injury until full return-to-play (RTP) clearance was granted by a university concussion center's physician; recovery was categorized as either normal (≤ 20 days) or prolonged (> 20 days). The secondary dependent variables were initial evaluation concussion severity scores and consisted of: (a) vestibulo-ocular reflex (VOR) result, (b) Balance Error Scoring System (BESS) sum, and (c) Immediate Postconcussion Assessment and Cognitive Testing (ImPACT) composite scores. Fifty-three, mostly White (69.7%), male (75.0%) concussed athletes (18.96 ± 6.31 years of age) participated in the study; two participants were excluded due to inconclusive genetic results. Participants were evaluated at a university concussion center per standardized concussion assessment battery, using the aforementioned severity indicators, and provided saliva samples for genotyping experiments. Follow-up visits were performed, as needed, until participants were asymptomatic and cleared for full RTP. No significant associations were demonstrated for the codominant (p = .35, p = .70), dominant (p = .39, p = 1.00) or recessive (p = .72, p = .51) genetic models for the rs1019385 and rs890 SNPs (respectively). Similarly, there were no significant differences in any initial evaluation severity scores between genotype for any genetic model. This exploratory study investigated the association between two GRIN2B SNPs and varied concussive recovery among athletes. Although no statistical and minimal clinical significance was demonstrated, future investigations should incorporate a larger sample and next-generation sequencing to investigate the 21,000 to 25,000 genes and their variations across the human genome as complex disorders (e.g., concussions) likely involve a multitude of genetic variations (and their interactions), many with small effects. Further elucidation of genetic factors involved in concussive recovery could equip clinicians with superior counseling methods and treatment options for athletes at-risk for prolonged recovery.
Temple University--Theses
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Guo, Shuchi. "EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) – AN ESSENTIAL MEDIATOR OF CARDIAC CONTRACTILE FUNCTION AND REMODELING." Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/555214.
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Biomedical SciencesPh.D.
Epidermal Growth Factor Receptor (EGFR), a member of the Erbb receptor tyrosine kinase family, is essential to the development of multiple tissue and organs. Due to the embryonic lethality of global EGFR deletion, the impact of EGFR signaling in the adult heart, normally or in response to pathological stimuli, has not been well-explored. Using recently attained mice with floxed EGFR alleles crossed with αMHC-Cre mice, we have generated a cardiomyocyte-specific constitutive EGFR knockout mouse model (CM-EGFR KO) to address its role in the heart. Compared to their wild-type (WT) littermate controls, CM-EGFR-KO mice displayed age-related development of cardiac dysfunction and remodeling, occurring between 7 and 9 weeks of age, as monitored via echocardiography and immunohistochemistry analyses. Although contractile responsiveness to β-adrenergic receptor stimulation was unaffected by EGFR deletion, RNASeq analysis of CM-EGFR-KO hearts within this timeframe revealed alterations in myofilam
Temple University--Theses
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Lougnon, Géraldine. "Analyse fonctionnelle des gènes précoces impliqués dans la symbiose fixatrice d'azote entre Medicago truncatula et Sinorhizobium meliloti." Paris 11, 2007. http://www.theses.fr/2007PA112103.
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Les légumineuses utilisent une voie de signalisation commune pour établir l’association symbiotique avec les rhizobiums et les champignons endomycorhiziens. La perception du microorganisme a lieu à la membrane plasmique et active une cascade de transduction du signal jusqu’au noyau pour la transcription de gènes spécifiques. Le gène NORK chez les espèces de Medicago code pour un récepteur kinase-like, composant de la voie de signalisation des facteurs Nod et essentiel pour le développement de la nodosité. Pour comprendre le rôle de NORK, nous avons cherché des protéines partenaires agissant en aval par double hybride et nous avons identifié une 3-hydroxy-3-méthylglutaryl Coenzyme A réductase (MtHMGR1). MtHMGR1 fait partie d’une famille multigénique chez M. Truncatula contrôlant la biosynthèse de mévalonate et en testant d’autres membres nous avons trouvé une spécificité pour cette unique protéine. La répression spécifique de l’expression de MtHMGR1 entraîne une inhibition de la nodulation. Ainsi, MtHMGR1 est un nouveau composant de la voie de signalisation symbiotique qui pourrait contrôler la production de cytokine et la biosynthèse de stérols. Des études surd’autres mutants, présentant le même phénotype, ont révélé l’existence du gène DM11 codant un canal ionique potentiel. Alors que ses homologues chez L. Japonicus, CASTOR et POLLUX, sont localisés dans les plastres, nous avons localisé la protéine de fusion DM11 :GFP, fonctionnelle, à la membrane nucléaire des cellules de racines de M. Truncatula lorsque elle est exprimée sous promoteur 35S ou natif. Il se peut que la région N-terminal de DM11 soit en partie responsable de sa localisation. Ces nouvelles informations placent DM11 dans l’enveloppe nucléaire, proche du lieu d’origine des oscillations de la concentration en calcium cytoplasmique induits par les facteurs NodLegumes utilize a common signaling pathway to form symbiotic associations both with rhizobial bacteria and arbuscular mycorrhizal fungi. The microbe perception is believe to take place at the plasma membrane, activating a cascade that converges on the nucleus where transcriptional reprogramming takes place. The NORK gene in Medicago species encodes a receptor-like kinase, component of the Nod signal transduction pathway and essential for root nodule development. NORK is required for the generation of nucleus associated calcium spikes in response to Nod factors as well as for infection of plant cells with rhizobia. To understand the mode of NORK action, we searched for its downstream targets by yeast two-hybrid screens. We identified 3-hydroxy-3-methylglutaryl Coenzyme A reductase (MtHMGR1) as a interacting partner of NORK. MtHMGR1 belong to a multigene family in M. Truncatula whose members are key component of the mevalonate pathway. Testing other members, the interaction specificity was revealed for MtHMGR1. Downregulation of MtHMGR1 expression affect dramatically the nodulation process. Thus, MtHMGR1 is a novel component of the symbiotic signaling pathway that could control biochemical pathway such as cytokinin and sterol synthesis for protein isoprenylation. Genetics studies on other mutants with same phenotypes have identified in M truncatula DM11 gene that encodes a putative ion channel. While the DM11 homologs from Lotus japonicus, XASTOR and POLLUX, were recently reported to localize in plastids, we report here that a functional DM11:GFP fusion localizes to the nuclear envelope in M. Truncatula roots when espressed both a constitutive 35S promoter and from a native DM11 promoter. Localization may be mediated in part by sequences located within the amino-terminus of DM11. These new data place DM11 in the nuclear envelope in close proximity to the origin of Nod factor induced calcium spiking
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Albano, Jennifer Nicole. "Localization of Human Prostaglandin E2 Receptors in Polarized Epithelial Cells." Diss., Temple University Libraries, 2008. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/17898.
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PharmacologyPh.D.
The underlying mechanisms of protein sorting in polarized epithelial cells are poorly understood. Several studies have determined membrane targeting of G protein-coupled receptors (GPCRs) using epithelial cells such as Madin-Darby canine kidney (MDCK) cells. Polarized epithelial cells are composed of apical and basolateral plasma membrane domains with specific protein compositions separated by tight junctions. Purinergic, muscarinic, and adrenergic receptors are a few examples of GPCRs that have been shown to localize to specific membranes in MDCK cells. The current work seeks to determine the differences in subcellular localization of the human prostaglandin E2 receptors. The EP receptors are all GPCRs, which differ in their second messenger pathways. The EP3 receptor is unique in that it has eight different isoforms that differ in the lengths of the carboxyl tail. The EP3 isoforms, as well as the EP2 and EP4 receptors, have distinct properties, including different agonist-induced internalization patterns. We have also shown the EP3 isoforms have tissue-specific distribution patterns. To further study the differences among the PGE2 receptors, we examined their subcellular localization patterns in polarized epithelial cells. We have determined the unique subcellular localization patterns for the receptors, as well as three mutants in MDCK cells. The localization patterns for these receptors in human bronchial epithelial (BEAS-2B) cells were quite similar to the MDCK cells, suggesting that receptor localization is not dependent on cell type. Additionally, in an attempt to locate structural motifs responsible for apical or basolateral localization, receptor chimeras between the purinergic P2Y2/P2Y4 receptors and EP3.VI isoform were constructed. Overall, the aim of our work was to define the subcellular localization patterns of various human prostaglandin E2 receptors and our results suggest the differences among them may correlate to the diverse physiological actions of PGE2 throughout the body.
Temple University--Theses
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Nattkemper, Leigh. "MEDIATORS AND RECEPTORS OF CHRONIC ITCH IN PRIMATES AND HUMANS." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/360207.
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Biomedical SciencesPh.D.
Chronic itch has a significant impact on quality of life for millions of patients worldwide, on a level comparable to that of chronic pain. Yet, although there are a host of effective drugs available for pain, there are no therapies that specifically target chronic itch. Current experimental approaches to investigate the pathogenesis of chronic pruritus and to test novel therapeutic agents are largely limited to rodent models. However, rodent models display significant dermatological, neurophysiological, and immunological differences from humans with chronic itch. The disadvantages of the current rodent paradigms call for the design of a valid primate model of chronic itch. For four years, we have monitored scratching behavior in a primate colony (n=35) of Cynomolgus macaques (Macaca fascicularis) suffering from idiopathic chronic itch. By comparing molecular and genetic analyses of the primates’ skin to their quantified scratching behavior, we attempted to characterize the underlying mechanisms of chronic itch in this model. Furthermore, the expression of itch-related proteins was examined in both the primate model and in humans with pruritic diseases. The first aim of the study was to characterize the underlying molecular and genetic basis of chronic itch in the primate model. We were able to distinguish specific peripheral targets related to pruritus by correlating the genetic and protein expression results to the primates’ scratching severity. In Aim 1a, RNA-sequencing was performed on skin biopsies from the primates to identify differentially expressed genes in pruritic, lichenified versus non-pruritic, non-lichenified skin. These results were then correlated to the quantified primate scratching behavior. This led to the identification of over 400 genes that were differentially expressed in the skin based on scratching intensity. Many of these differentially expressed transcripts were associated with sensory nerve fibers, keratinocytes, mast cells, or lymphocytes. Selected genes that were overexpressed and correlated to itch intensity were then targeted for immunohistochemical and proteomic analysis in Aim 1b. Immunohistochemical examination of the primate skin biopsies revealed that histamine levels were not elevated in primates that exhibited increased scratching behavior. However, mast cells containing tryptase were significantly increased in the skin of primates with severe scratching as compared to primates with mild scratching. The increased levels of gastrin-releasing peptide and substance P in lichenified skin were also found to be correlated to the primates’ scratching behavior. Of note, transient receptor potential channels V1, V3, and A1 were increased in the epidermis of primate skin, but the numbers of TRPV1+ and TRPA1+ nerve fibers were not significantly different between lichenified and non-lichenified skin. Transcriptome analysis of the opioid receptors and their ligands showed that primates with severe scratching behavior had a significant imbalance between the µ- and κ-opioid receptors and ligands. The µ-opioids had upregulated gene expression, while the κ-opioids were downregulated. In Aim 2, to further characterize this primate model of chronic itch, we compared immunohistochemical results from the primate studies to human findings. Lesional and non-lesional skin biopsies from patients with atopic dermatitis, psoriasis, and cutaneous T-cell lymphoma underwent immunohistochemical analysis in order to reveal the similarities and differences between the primate model and different types of chronic itch in humans. As in the primate model, substance P was found to be increased in the skin of lesional atopic and psoriasis skin. Additionally, similar to primate skin, human atopic and psoriatic skin had high levels of tryptase and its receptor in the epidermis. While IL-31 was only slightly elevated in primates, patients with cutaneous T-cell lymphoma or atopic dermatitis showed a significant correlation between itch severity and IL-31 levels. In conclusion, our primate model displayed expression patterns of many endogenous pruritogens and receptors that were similar to those of humans with atopic dermatitis or psoriasis. While the primate model did not completely mimic these specific pruritic diseases, the overlap of pruritic components suggests a commonality of signaling pathways across several different chronic itch states. The similarity of this primate model to human disease offers the combined advantages of experimental modeling and long-term behavioral follow-up.
Temple University--Theses
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Nguyen, The Duy [Verfasser], Rolf [Gutachter] Marschalek, and Christian [Gutachter] Brandts. "The role of the selective autophagy receptor p62 in acute myeloid leukemia / The Duy Nguyen ; Gutachter: Rolf Marschalek, Christian Brandts." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2018. http://d-nb.info/1157097979/34.
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Reichenbach, Zachary Wilmer. "Modulation of the Endogenous Cannabinoid System to Attenuate Inflammation in Central Nervous System Injury." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/253514.
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PhysiologyPh.D.
In non-pathological states the central nervous system maintains a degree of immunological privilege. When illness or injury occur, this privilege can be lost and the immune system drives pathology in the brain and spinal cord. More so, resident immune cells, the microglial, act as major effectors of this response. Cerebral ischemia, or stroke, is the fourth leading cause of death in developed nations. After the initial ischemia, the inflammatory response propagates further injury and cell death. Another affliction of the central nervous system, chronic pain and persistent use of the opioid analgesic, morphine, leads to tolerance and ineffectiveness of the drug. Currently, only one in three patients receive adequate pain relief from their pharmacological regiment. This loss of efficacy in morphine is also driven by an inflammatory response. Thus, a way to quell inflammation in both disease states could lead to better treatments for both disorders. The endogenous cannabinoid system has two known receptors, CB1 and CB2. Both of these receptors have been intimately linked to inflammation and the activation or antagonism of the receptors can impart desired outcomes in modulating the immune response. Primarily the CB1 receptor expression is on presynaptic terminals of neurons to modulate neuronal firing. The CB2 receptor's expression predominates on immunological cells including microglial. However, some degree of expression exists with reports of neuronal CB2 receptors and immunological CB1 receptors. This makes pharmacological therapies targeted at both receptors ideal candidates in treating not only stroke and but also preventing the induction of morphine tolerance. In the studies described here, we sought to investigate the role of the endogenous cannabinoid system in both stroke and as a way to prevent the induction of morphine tolerance. The results showed that CB1 -/- CB2 -/- receptor mice were able to maintain greater blood flow during cerebral ischemia. More so, CB1 antagonism in a permanent occlusion of cerebral vessels showed a protective effect independent of the serotonin receptor. Lastly, a CB2 agonist was able to limit the degree of tolerance that developed from chronic morphine therapy and also prevent hyperalgesia in addition to showing a reduction in pro-inflammatory cytokines. Acutely, this same agonist was found to antagonize the morphine receptor but this could be avoided if morphine was administered before the CB2 agonist. In brief, the studies at hand show that the endogenous cannabinoid system can attenuate inflammation in central nervous system injury and shows great promise as a future therapeutic for clinical use.
Temple University--Theses
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Oppong, Gertrude Odamea. "The Role of Bacterial Amyloids In Regulating Gastrointestinal Homeostasis." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/323094.
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Microbiology and ImmunologyPh.D.
Many bacterial species exist in nature as part of highly structured multicellular communities known as biofilms. Amyloids, proteins with a conserved β-sheet quarternary structure, show high resistance to many chemical and enzymatic processes including proteinase K and SDS treatments and are produced as essential adhesins during biofilm formation. Curli fibers expressed by Enterobacteriaceae family members including E. coli and S. Typhimurium are the most studied amyloids to date. Curli-like fibers are also produced by members of the predominant phyla found in the host gastrointestinal microbiota in environmental biofilms. Curli fibers are the predominant microbial-associated molecular pattern (MAMP) on enteric bacteria recognized by the Toll-like receptor (TLR) 2/1-heterodimer complex. Interestingly, the TLR2/1 complex has been implicated as a key player in modulating gastrointestinal homeostasis. The focus of the current studies centered on the innate immune recognition of curli fibers by cells of the gastrointestinal tract and how that contributes to gastrointestinal homeostasis. In the first phase of our studies, utilizing intestinal epithelial cells polarized on semi-permeable tissue culture inserts (Transwells®), we observed that the recognition of curli fibers on Salmonella enterica serovar Typhimurium by intestinal epithelial cells led to the augmentation of the intestinal epithelial barrier in a PI3K-dependent manner. We also observed that bacterial translocation of S. Typhimurium from the apical side to the basolateral side of the Transwell system was limited when curli fibers were present. Furthermore, infection of mice with S. Typhimurium showed that translocation of bacteria from the intestinal lumen into the cecal tissue and mesenteric lymph nodes was limited in C57BL/6 mice as compared to TLR2 knockout mice. In the second phase of our studies, we sought to further investigate the effect that curli fibers exert on gastrointestinal homeostasis through the induction of immunomodulatory cytokines such as Interleukin 10 (IL10) from subepithelial lamina propria cells. IL10 has been shown to contribute to the maintenance of the intestinal epithelial barrier and IL10-deficient mice develop lethal colitis within the first 2-3 months of life. 6-8 week-old female C57BL/6 and TLR2-/- mice were given 5mg/kg of curli fibers via intraperitoneal injection. Subsequent RT-PCR analysis of the small intestine showed a significant expression of Il10 in C57BL/6 that was absent in TLR2-/- mice. Interestingly, no changes in Ifnγ or Tgfβ mRNA were observed in these mice. This response was gut-specific, as Il10 was not detected at all in the spleen. Furthermore, in a chemically-induced colitis model, we observed that the administration of curli fibers to 8-week old Balb/c mice ameliorated disease severity as compared to colitic mice that received mock treatments. Interestingly, Il10 was also induced in the colons of colitic mice that received curli and which were euthanized 6 days after colitis was induced. Our results suggest that curli fibers induce IL10 production via a TLR2-dependent manner to dampen inflammation in the gastrointestinal tract. Overall, our results partially describe a novel role for curli amyloid fibers produced by commensal bacteria in modulating gastrointestinal health and homeostasis. We propose that the induction of immunomodulatory cytokine such as IL10 by amyloid fibers is an important mechanism utilized by commensal bacterial to confer beneficial effects that benefit both the host and microbe. We also propose curli fibers as a potential alternative in the treatment of inflammatory bowel disease.
Temple University--Theses
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Dunn, Stanley Ingrid P. "The neurofibromatosis type 2 gene product, merlin, binds, directly to the epidermal growth factor receptor, ErbB2." Honors in the Major Thesis, University of Central Florida, 2000. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/188.
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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.Bachelors
Health and Public Affairs
Molecular and Microbiology
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Liang, Chengya. "Extracullular ATP Regulates IL-1Beta Release from Microglial Cells Via Purinergic Receptor After In Vitro Trauma." Master's thesis, University of Central Florida, 2004. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4420.
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Traumatic brain injury (TBI) induces a state of microglialactivation, which includes upregulation of macrophage functions and release inflammatory mediators such as certain inflammatory cytokines. Current literature suggests that interleukin-1Beta is an important cytokine mediator, which is dramatically increased after brain injury. Previous studies indicate that ATP is released by traumatically injured astrocytes and serves as a cell-to-cell mediator through purinergic receptors after in vitro injury. However, the mechanism of interleukin-1Beta release after traumatic brain injury remains poorly defined and is difficult to study using in vivo models. Using an in vitro model for traumatic brain injury (cell strain or stretch), we investigated the role of the extracellular nucleotides (ATP) in regulation of interleukin-1Beta release in rat cortical brain cells. We now report that activated microglia constitute the major source of interleukin-1Beta release after in vitro trauma. ATP is a powerful stimulus for interleukin-1Beta release from microglial cultures. Glutamate inhibits interleukin-1Beta release. ATP-induced interleukin-1Beta release was blocked completely by the P2X7 receptor antagonist, oxidized ATP, and partially by the P2X7 receptor antagonist suramin, suggesting that ATP stimulates interleukin-1Beta release from microglia via purinergic receptor and the P2X7 receptor is responsible for the interleukin-1Beta release. Blockage of interleukin-1Beta release by the purinergic receptor antagonists oATP and suramin decreased cell damage in uninjured mixed organotypic brain cell culture exposed to activated microglia. Taken together, these results suggest that interleukin-1Beta mediated inflammatory events are regulated in activated microglia by extracellular nucleotides (ATP) via purinergic receptors in central nervous system after in vitro trauma.M.S.
Department of Molecular Biology and Microbiology
Burnett College of Biomedical Sciences
Molecular Biology and Microbiology
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Bhandare, Richie R. "Investigation into the bioisosteric approach in the design, synthesis and evaluation of muscarinic receptor ligands." Diss., Temple University Libraries, 2013. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/214985.
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Pharmaceutical SciencesPh.D.
The acetylcholine (ACh) receptor system belongs to rhodopsin GPCR family and is an integral membrane protein divided into two types: muscarinic and nicotinic. The naturally occurring neurotransmitter acetylcholine binds to these two receptor systems non- selectively. The regulatory effects of the neurotransmitter acetylcholine are diverse ranging from autonomic nervous system and the central nervous system through different types of neurons innervated by cholinergic inputs. Muscarinic acetylcholine receptors (mAChRs) are divided into five receptor subtypes (M1-M5). In general, M1, M3 and M5 receptor subtypes are coupled via Gq like proteins; while M2 and M4 subtypes are coupled to Gi-proteins. Muscarinic receptors are widely distributed in the body where they mediate a variety of important physiological effects. mAChRs have been the target of drug development efforts for the treatment of various disorders including overactive bladder, Alzheimer's disease, pain, cognitive impairment, drug addiction, schizophrenia and Parkinson's disease. The development subtype selective ligands possess a challenge due to a high degree of homology among mAChR subtypes, however the recent availability of the X-ray crystal structure for the M2 and M3 receptor can be utilized for the design of new ligands. The pharmacophoric requirements for cholinergic ligands have been reported by numerous investigators based on structure-activity relationship (SAR) and/or molecular modeling data of known muscarinic ligands. These fundamental requirements are useful when designing muscarinic ligands but have provided little guidance in the design of subtype selective compounds. Our interest in developing novel muscarinic receptor ligands led to the design of lactone-based ligands using an approach similar to that reported by Kaiser et al. Preliminary binding studies of our previously synthesized lactone based compounds indicated that several were nonselective, low affinity (IC50 = µM range) muscarinic agonists (based on preliminary in vivo data). Hence based on the background information, we decided to utilize the previously synthesized lactone parent compound as lead molecule set out to investigate a new series of lactone based compounds in order improve the affinity and later the selectivity of ligands. Bioisosteric approach has been investigated for the metabolic lability of the lactone ring. Four probable bioisosteres have been evaluated: tetrahydrofuran, 1,3-benzodioxole, oxazolidinone and chromone. Thermal/microwave assisted synthesis has been utilized in the generation of intermediates as well as final compounds. Preliminary screening and further evaluation (IC50/ subtype selectivity) has resulted in the identification of promising fragments as bioisosteres for the lactone ring.
Temple University--Theses
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Nwaneshiudu, Chinwe A. "Characterization of a functional role of the neurokinin-3 receptor in behavioral effects of cocaine." Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/62614.
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PharmacologyPh.D.
The tachykinin NK-3 receptor is a G-protein coupled receptor activated by mammalian tachykinin neuropeptides, which can modulate dopaminergic neurotransmission, and alter dopamine-mediated behaviors. The NK-3 receptor is currently under investigation as a novel therapeutic target for cocaine addiction. Our studies, as outlined in this dissertation, sought to determine if NK-3 receptors have a functional role in the acute as well as long-term behavioral effects of cocaine. Administration of NK-3 receptor agonists or antagonists potentiates or attenuates dopamine-mediated behaviors, respectively. Based on these findings, we hypothesized that blockade of neurokinin-3 receptors would alter acute and long-term behavioral responses to cocaine. We investigated whether acute and repeated administration of the NK-3 receptor antagonist SB 222200 altered hyperactivity induced by cocaine, and determined a possible mechanism involving dopamine D1 receptors in the striatum. We also determined whether NK-3 receptor blockade altered the development and expression of behavioral sensitization after repeated cocaine administration. Lastly, we investigated whether modulation of behavioral effects of acute and repeated cocaine by NK-3 receptors involved GSK3 phosphorylation in the nucleus accumbens. As described in this dissertation, we show that acute administration of the NK-3 receptor antagonist SB 222200 before a cocaine injection attenuated stereotypic responses produced by cocaine. Repeated administration of SB 222200 enhanced stereotypic activity produced by either cocaine or a low dose of SKF 82958 (0.125 mg/kg, i.p.) when administered seven days later. Dopamine receptor binding studies were performed to determine the mechanism of enhanced stereotypic responses. Binding studies showed a 19.7% increase in dopamine D1 receptor density in the striatum seven days later after repeated SB 222200 administration. These findings demonstrate that acute blockade of NK-3 receptors attenuated cocaine-induced behaviors in agreement with previous studies. Furthermore, these studies also show novel effects of repeated blockade of NK-3 receptors, which causes subsequent enhancement of cocaine and dopamine D1 receptor-mediated behaviors, possibly resulting from dopamine D1 receptor up-regulation in the striatum. In order to determine a role of NK-3 receptors in the development of cocaine-induced behavioral sensitization, the NK-3 receptor antagonist SB 222200 (2.5 or 5 mg/kg, s.c.) was administered prior to daily cocaine injections for 5 days. After a 7-day drug-free period, behavioral responses to a cocaine challenge were measured. Repeated administration of cocaine for 5 days induced a sensitized response upon a cocaine challenge 7 days later. Administration of SB 222200 prior to daily cocaine attenuated the development of behavioral sensitization. Moreover, administration of SB 222200 prior to the cocaine challenge blocked the expression of behavioral sensitization. These findings demonstrate that NK-3 receptor activity is involved in the development and expression of behavioral sensitization to cocaine. Lastly, we examined GSK3 phosphorylation in the nucleus accumbens induced by acute and repeated cocaine administration and determined if phosphorylation was altered by NK-3 receptor blockade. Similar to the drug administration regimens used in the behavioral studies, the NK-3 receptor antagonist SB 222200 was administered 30 mins prior to an acute cocaine injection. The nucleus accumbens was examined for changes in GSK3 phosphorylation by Western blot analysis. Increases in phosphorylation of the isoforms, GSK3α and GSK3β in the nucleus accumbens were detected 20 mins after an acute injection of cocaine. NK-3 receptor blockade prior to cocaine administration did not alter the cocaine-induced increase in GSK3 phosphorylation. Similar to the behavioral sensitization studies, SB 222200 was administered prior to repeated cocaine for 5 days, and 7 days later GSK3 phosphorylation was measured after a subsequent cocaine challenge. In contrast to the increases in GSK3α and GSK3β in the nucleus accumbens after an acute cocaine injection, no regulation of GSK3 phosphorylation was found after prior repeated cocaine administration and cocaine challenge. Administration of SB 222200 prior to repeated cocaine produced an increase in GSK3α and GSK3β phosphorylation after a cocaine challenge. Collectively, these data point to involvement of NK-3 receptor activity in changes in the phosphorylation of GSK3 in the nucleus accumbens produced by cocaine. In summary, functional involvement of NK-3 receptors in acute and long-term behavioral effects of cocaine was investigated. In agreement with previous findings, studies in this dissertation demonstrate that acute blockade of NK-3 receptors attenuates cocaine-induced behaviors. In addition, we found novel effects of repeated blockade of NK-3 receptors on cocaine-induced hyperactivity. There is enhancement of subsequent cocaine and dopamine D1 receptor-mediated behaviors possibly due to dopamine D1 receptor up-regulation in the striatum. NK-3 receptor activity was shown to be involved in long-term behavioral effects of cocaine and molecular changes in GSK3 phosphorylation in the nucleus accumbens. Blockade of NK-3 receptors prevented the development and expression of behavioral sensitization to cocaine and also blocked the changes in the phosphorylation of GSK3 in the nucleus accumbens. This dissertation has demonstrated a role of NK-3 receptors in modulating acute as well long-term cocaine-induced behavioral hyperactivity. Therefore, there is potential clinical relevance of NK-3 receptors in cocaine abuse and dependence as a therapeutic target for treatment, which warrants further characterization in future preclinical and clinical investigations.
Temple University--Theses
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König, Christopher Verfasser], Sylvia [Gutachter] Anton, Rolf G. [Gutachter] [Beutel, and Bill S. [Gutachter] Hansson. "Chemosensory receptors in the tobacco hawkmoth Manduca sexta / Christopher König ; Gutachter: Sylvia Anton, Rolf Beutel, Bill S. Hansson." Jena : Friedrich-Schiller-Universität Jena, 2016. http://d-nb.info/1177612232/34.
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König, Christopher [Verfasser], Sylvia Gutachter] Anton, Rolf G. [Gutachter] [Beutel, and Bill S. [Gutachter] Hansson. "Chemosensory receptors in the tobacco hawkmoth Manduca sexta / Christopher König ; Gutachter: Sylvia Anton, Rolf Beutel, Bill S. Hansson." Jena : Friedrich-Schiller-Universität Jena, 2016. http://d-nb.info/1177612232/34.
Full text