Dissertations / Theses on the topic 'Molecular Physiology and Pharmacology'
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1
Bahnasi, Yahya Mohamed. "Molecular physiology and pharmacolgy of TRPC5 ion channels." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496554.
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Elnakish, Mohammad T. "Mechanisms and Functional Consequences of Cardiac Remodeling: Role of Myocardial Rac1 and Vascular Profilin1 Genes." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1363694358.
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Bonilla, Ingrid Marie. "Acquired Electrophysiological Remodeling and Cardiac Arrhythmias." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1396024058.
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Szabo, Elod Zala. "Molecular and cellular properties of the human brain Na+H+ exchanger isoform 5." Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38420.
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Na+/H+ exchangers (NHE) are transmembrane proteins that mediate the electroneutral exchange of Na+ and proton across the plasma membrane. There are seven mammalian isoforms of the gene family identified to date. The recently cloned isoform NHE5 seems to be the most highly cell type-specific being probably expressed only in neurons. The objective of my research was to describe the basic biochemical and some of the regulatory characteristics of NHE5 in an effort to understand its in vivo function. To this end, the full-length cDNA of NHE5 was reconstructed and transfected into the NHE-deficient Chinese hamster ovary cell line AP1.Pharmacological analyses demonstrated that H+ i-activated 22Na+ influx mediated by NHE5 was inhibited by several classes of drugs at half-maximal concentrations that were intermediate to those determined for the high-affinity NHE1 and the low-affinity NHE3 isoforms. Kinetic analyses showed that the extracellular Na+-dependence of NHE5 activity followed a simple hyperbolic relationship and, unlike other NHE isoforms, the intracellular H+-dependence also exhibited first-order kinetics. Extracellular monovalent cations, such as H+ and Li+, but not K+, acted as effective competitive inhibitors of 22Na+ influx by NHE5.
To find novel interacting proteins that are involved in NHE5 regulation, a yeast two-hybrid screen of human brain cDNA library was conducted using NHE5 as bait. A clone encoding the AMP-activated protein kinase (AMPK) alpha2 subunit was further analyzed. AMPK is a serine/threonine kinase that is activated by elevated ratios of [AMP]/[ATP], regulating various biological processes in response to hypoxia or exercise. AMPK alpha2 binds NHE5 in vitro and in vivo, and directly phosphorylates it in vitro. Activation of endogenous AMPK by AICAR, a membrane permeable AMP analogue, as well as heterologous expression of the full-length and constitutive active forms of alpha2 subunit increased the transporter activity measured by 22Na+ influx.
The regulatory protein arrestin3 was also found to interact with NHE5 in the yeast two-hybrid screen. Arrestins were previously shown to associate with and regulate transmembrane proteins of the G protein-coupled receptor family. We demonstrate that NHE5 binds arrestin3 both in vitro and in vivo; and the binding is phosphorylation-dependent. When co-expressed in CHO cells, arrestin3 and NHE5 co-localize, and arrestin3 expression seems to attenuate the basal activity of the transporter. The data presented in this thesis reveals new aspects of both NHE regulation, and AMPK and arrestin function.
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Zicha, Stephen. "Molecular basis for ion current heterogeneity in normal and diseased hearts." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85660.
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Cardiac action potential characteristics are known to vary in different species, but also in the different regions of the heart within a given species and in cardiovascular disease. The heterologous expression of voltage-gated ion currents is believed to underlie these differences. The purpose of this thesis is to elucidate the molecular mechanisms which may underlie some of the observed current changes in different species, as well as regions and diseases of the heart.Here, we describe the variable dependence on repolarizing K+ currents in different species as being the result of the lack of Ito subunits in guinea pig heart with a greater expression of IK subunits, while rabbits express all hypothesized Ito subunits, but express IK subunits at low levels. Humans are found to lie in between these two species in terms of the expression of these voltage-gated K+ channel subunits. The specialized function of certain regions of the heart, such as the ventricles and the SAN, have been attributed to the heterologous expression of Ito and the pacemaker current (I f) respectively. Here were demonstrate that both Kv4.3 and KchIP2 gradients underlie an observed Ito transmural gradient and contribute to the dispersion of repolarization, while a greater expression of HCN2 and HCN4 subunits in the SAN compared to the right atrium account for the larger I f current in this region. Cardiovascular diseases such as congestive heart failure (CHF) have been associated with ion channel remodelling. Here, we report the finding of changes in Nav1.5, Kv4.3, HCN2 and HCN4 expression which may underlie some of the electrophysiological changes associated with this disease. Furthermore, we characterise a genetic polymorphism which is associated with another disease, atrial fibrillation.
The heterologous expression of voltage-gated ion channel subunits may account for many of the species-, region- and disease-specific differences which have been observed in the heart. Such heterogeneity contributes to the proper functioning of the heart under normal conditions, but may also contribute to the pathogenesis of cardiovascular disease.
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Richman, Jeremy Golding 1970. "Characterization of α₂-adrenergic receptor localization and functional responses." Diss., The University of Arizona, 1998. http://hdl.handle.net/10150/282583.
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The three α₂-adrenergic receptors (α₂-ARs) were studied in both recombinant and endogenous systems. It was my general hypothesis that the three α₂-adrenergic receptor (AR) subtypes exhibit differential tissue and subcellular localization and couple to physiological pathways to directly elicit functional responses. It was our hope, that in identifying differences between the α₂-ARs we may elucidate their physiological roles and provide a potential means for the rational development of therapeutic drugs or models of such pathologies as atherosclerosis, hypertension and glaucoma. Subtype selective antibodies have been generated and these have provided a tool with which to study the receptors on a molecular and cellular level. Utilizing a number of biochemical, pharmacological, molecular and cellular techniques, I have identified differences between the three receptors with respect to the tissues in which they are expressed, the subcellular domains in which they localize and their patterns of sequestration and down-regulation. In addition, I have demonstrated the expression and co-localization of the α₂-ARs in a variety of native tissues, and a number of functional responses these receptors mediate. These functional responses may have ramifications relevant to tissue healing mechanisms as well as may play a role in a number of pathologies.
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Sinha, Sayantani. "Role of TRPA1 and TRPV1 in Propofol Induced Vasodilation." Thesis, Kent State University, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3618926.
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Aims: Propofol, clinically named as Diprivan is an intravenous anesthetic known to cause hypotension in patients presenting for surgery. We have investigated the vasodilatory signaling cascade by which propofol causes hypotension using both in vivo and in vitro experimental approaches.
Methods and Results: Using high-fidelity microtip transducer catheter, mean arterial blood pressure (MAP) was measured in control, transient receptor potential ankyrin subtype 1 knock-out (TRPA1-/-), transient receptor potential vanilloid 1 knock-out (TRPV1-/-) and TRPA1-TRPV1 double-knockout mice (TRPAV-/-) in the presence and absence of L-NAME (an endothelial nitric oxide synthase inhibitor) and penitrem A [a big-conductance calcium gated (BKCa) channel inhibitor]. To further support our in-vivo data, murine coronary microvessels were isolated and cannulated for vasoreactivity studies. Furthermore, NO production from endothelial cells isolated from mouse aorta was also measured and immunocytochemical (ICC) studies were performed to show the intracellular localization of TRPA1 and TRPV1. Our in-vivo data shows that the characteristic propofol-induced depressor response is dependent on TRPA1-NO-BKCa pathway. Interestingly, vasoreactivity studies in isolated murine left anterior ascending (LAD) arteries demonstrate that TRPA1 and TRPV1 communicate with each other and propofol-induced vasodilation is dependent on both TRPA1 and TRPV1. Moreover our data also suggest that NO production and BK channel activation are the downstream mediators in this pathway. Finally, we demonstrate that NO production is attenuated in primary endothelial cells isolated from TRPAV-/- mice. ICC data also shows the co-localization of these channels in mouse aortic endothelial cells.
Conclusions: This is the first study which has shown that propofol-induced vasodilation involves TRPA1 in-vivo and also there is an implication of cross-talk between TRPA1 and TRPV1 in the coronary bed. Furthermore by understanding the mechanisms by which this anesthetic causes hypotension and coronary dilation will help to mitigate the potential harmful side-effects of anesthesia in patients with little cardiovascular reserve. This will in turn ensure a better and faster post-operative recovery in patients, especially benefiting those suffering from diabetes and other cardiovascular disorders.
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Harris, Tanoya L. "Ouabain Regulates Caveolin-1 Vesicle Trafficking by a Src-Dependent Mechanism." University of Toledo Health Science Campus / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=mco1333732028.
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Barr, Larry A. "The Role of Calcium in the Regulation of Pathological Hypertrophy." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/254617.
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PhysiologyPh.D.
Pathological hypertrophy leads to cardiac dysfunction and heart failure. It is not clearly defined how this process occurs in the cardiomyocyte, or how the pathology can be effectively treated. There are numerous processes that lead to pathological hypertrophy. We developed two models to study pathological hypertrophy and the role that Ca2+ plays. In one model, we administered clinical doses of the leukemia therapeutic drug imatinib to neonatal ventricular cardiomyocytes. This drug has recently been found to be cardiotoxic, and we set out to understand if Ca2+ is involved. In the second model, we developed mice with overexpression of the Ca2+ entrance channel, the L-type calcium channel (LTCC), which leads to pathological hypertrophy over time. We instituted a chronic exercise regimen on these mice to learn if physiological hypertrophy can ameliorate detrimental aspects of pathological hypertrophy. After cardiomyocytes were treated with imatinib, they expressed enhanced Ca2+ activity. Levels of atrial natriuretic peptide (ANP) were up, signifying pathological hypertrophy. We determined that Ca2+ was activating Calcineurin, leading to translocation of nuclear factor of activated T-cells (NFAT) into the nucleus, resulting in hypertrophy. This activity was blocked by Ca2+ and Calcineurin inhibitors. We concluded that imatinib causes Ca2+ induced pathological hypertrophy. When mice with LTCC overexpression were exercised, they exhibited enhanced cardiac function. They also had thicker septal walls and increased chamber diameter, hallmarks of physiological hypertrophy. Heart weight to body weight ratio was significantly higher after exercise. When isolated hearts were administered ischemia/reperfusion injury, the exercised hearts showed a significant improvement in recovery compared to sedentary LTCC overexpressed hearts. Calcium activity was enhanced at the cardiomyocyte level in both mouse lines of exercised mice. In conclusion, hearts with a pathological hypertrophic phenotype can enhance function and achieve cardioprotection through chronic exercise.
Temple University--Theses
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Blatherwick, Eleanor Q. "Imaging mass spectrometry approaches for the detection and localisation of drug compounds and small molecules in tissue." Thesis, University of Warwick, 2013. http://wrap.warwick.ac.uk/57257/.
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A crucial and challenging aspect of the drug development process is the requirement to measure the distribution of a pharmaceutical compound and its metabolites in tissue. Industry-standard methods used to look at total localisation of drug-related material are limited due to their dependence on labels. These labelled techniques can have difficulty in distinguishing between the drug of interest and its metabolites. Imaging mass spectrometry is a technique that has the potential to spatially distinguish between drug and metabolites, due to its high chemical specificity and sensitivity. A number of imaging mass spectrometry approaches have been described for localisation of drug compounds in tissue, most notably matrix-assisted laser desorption/ionisation (MALDI) imaging, which can provide data complementary to existing imaging techniques. Two imaging mass spectrometry approaches have been evaluated and compared for use in the localisation of a range of drug compounds in target tissues. The techniques used were MALDI imaging and a recently described electrospray ionisation-based technique, liquid extraction surface analysis (LESA). Both techniques have been successfully used for the detection of drug compounds in dosed tissue sections. A major challenge associated with imaging techniques is the required selectivity of the experiment for the compound of interest, due to the complex nature of tissue sections. Combining the shape-selective method of ion mobility separation with MS/MS fragmentation has been shown to improve the selectivity of both imaging approaches for the compound of interest. Results obtained using LESA-MS have demonstrated the suitability of this technique as a rapid and sensitive profiling technique for the detection of drugs and metabolites in tissue, but with a lower achievable spatial resolution than MALDI imaging. Higher spatial resolution was achieved with MALDI imaging; however data acquisition times were longer and required higher dosing levels for successful detection of drug compounds in tissue. A biological application of MALDI imaging was also evaluated. Mobility-enabled MALDI imaging was used to assess differences in the localisation of important adenine nucleotides between control and metabolically stressed mouse brain sections. Tissue fixation methods were evaluated to overcome rapid post-mortem degradation of adenine nucleotides such that biologically relevant localisation images can be obtained. These studies highlight the crucial importance of appropriate biological sample preparation in MALDI imaging experiments.
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Shin, Jong M. "Role of C121A in mGluR2 homodimeric expression and function." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5576.
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The group II metabotropic glutamate receptors are known for their involvement in various psychiatric disorders. The mGluR2 in particular is linked with etiology of schizophrenia especially in the context of crosstalk with 5-HT2A. Thus, the mGluR2 has attracted attentions for its potential therapeutic applications. Despite numerous physiological evidences on the actions of mGluR2, its mechanism is still unclear to this day. It is partially due to the lack of understanding in characteristics of mGluR2 homodimer which is its functionally active form. Therefore, the characterization of dimeric interaction serves as a foundation to advanced understanding of the role of mGluR2. On that note, the role of the conserved cysteine residue (C121) in the ligand binding domain of mGluR2 has been evaluated in this study as they are known to play a critical part in homodimer formation. Collectively, C121 has been shown to affect the dimerization, subcellular localization, and pharmacokinetics of mGluR2. Lastly, the effect of mGluR2 on mouse behavior was examined in a partial effort to elucidate its role in crosstalk with 5-HT2A.
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Vohra, Hiba Z. "Molecular Targets of Psychedelics and Their Role in Behavioral Models of Hallucinogenic Action." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6012.
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Psychedelics are a subset of hallucinogenic drugs that exert their characteristic effects through agonist activity at the serotonin receptor 2A (5-HT2A). In this study, I aimed to characterize the modulatory role of the metabotropic glutamate subtype 2 receptor (mGluR2) in the 5-HT2A-specific rodent model of hallucinogenic action, head-twitch response (HTR). Secondly, I aimed to explore if 5-HT2A agonist-induced deficits in prepulse inhibition (PPI) of the startle response, an additional model of hallucinogenic action, could be produced in mice. Though 5-HT2A agonist-induced PPI deficits, which represent interruptions in normal sensorimotor gating, have been described in both rats and humans, attempts to translate this behavior to mice are rare. In contrast to prior gene knockout studies suggesting the mGluR2 is necessary for 5-HT2A agonist-induced HTR, mGluR2 knockout (Grm2-/-) mice still displayed HTR upon administration of the psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI). Additionally, DOI and lysergic acid diethylamide (LSD) produced unexpected improvements in PPI in male 126S6/Sv wild-type mice, depending on the experimental protocol used and the origin of the animals. Sex differences were observed as DOI-induced improvements in PPI were present in female 129S6/Sv mice of the same origin and tested with the same protocol as their male counterparts; this effect in females was absent in 5-HT2A knockout (Htr2a-/-) mice. The results of this study shed light on issues with replicability and reproducibility in science, the importance of highlighting the origin and background of animal subjects, and potential sex differences in hallucinogenic drug action.
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Pandey, Varunkumar Girijaprasad. "The Effect of Glucocorticoids on Regulation of the Human Angiotensinogen Gene and Blood Pressure." University of Toledo Health Science Campus / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1378647891.
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Muchhala, Karan Hitesh. "An investigation on the role of β-arrestin 2, protein kinase C and sex on the mechanism of morphine tolerance in the mouse ileum." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/6074.
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Opioids such as morphine are frequently used in the clinic to treat pain. However, the perennial bane of chronic opioid use is the rapid development of tolerance to the analgesic effects but delayed development of tolerance to the respiratory depressant and constipating effects. As constipation is one of the most common opioid-related adverse effects in humans, it is important to delineate mechanisms that drive opioid tolerance in the ileum and lack of it in the colon. The overarching goal of this thesis was to investigate mechanisms of morphine tolerance in the ileum by comparing the mechanism of morphine tolerance in ileum myenteric plexus neurons and dorsal root ganglia (DRG) neurons. Myenteric plexus neurons are integral to the motor function of the ileum, whereas DRG neurons are important components of peripheral nociceptive sensation. We also examined the mechanism of morphine tolerance development to small intestinal transit and to antinociception at the systemic level in male and female mice. Studies presented in this dissertation demonstrate that the mechanism of morphine tolerance in the mouse ileum is not contingent on b-arrestin 2. In fact, tolerance in the small intestine might be mediated by a b-arrestin 2-independent mechanism following protein kinase C-induced phosphorylation of the m-opioid receptor. We also demonstrate that morphine tolerance to antinociception is not solely dependent on b-arrestin 2, and is mediated by b-arrestin 2-dependent and-independent mechanisms. Lastly, we have shown how sex of the animal can influence mechanisms underlying the development of morphine tolerance. Collectively, the findings presented here increase our understanding of the mechanisms by which morphine tolerance develops in the ileum and to antinociception.
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Narayanaswami, Vidya. "DIET-INDUCED OBESITY: DOPAMINERGIC AND BEHAVIORAL MECHANISMS AS OUTCOMES AND PREDICTORS." UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/12.
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Obesity and drug abuse share common neural circuitries including the mesocoticolimbic and striatal dopamine reward system. In the current study, a rat model of diet-induced obesity (DIO) was used to determine striatal dopamine function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity. For the outcome study, rats were randomly assigned a high-fat (HF) or a low-fat (LF) diet for 8 wk. Following the 8-wk HF-diet exposure, rats were segregated into obesity-prone and obesity-resistant groups based on maximum and minimum body weight gain, respectively, and neurobehavioral outcomes were evaluated. For the predictor study, neurobehavioral antecedents were evaluated prior to an 8-wk high-fat diet exposure and were correlated with subsequent body weight gain. Striatal D2 receptor density was determined by in vitro kinetic analysis of [3H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [3H]dopamine uptake, methamphetamine-evoked [3H]dopamine overflow and no net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and Western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule for food-reinforcers, respectively. Relative to obesity-resistant, obesity-prone rats exhibited 18% greater body weight, 42% lower striatal D2 receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine concentration, and 2-fold greater methamphetamine-evoked [3H]dopamine overflow. Obesity-prone rats exhibited higher motivation for food, but were less impulsive relative to obesity-resistant rats. Neurobehavioral antecedents of DIO included greater motivation for high-fat reinforcers in rats subsequently shown to be obesity-prone relative to obesity-resistant. Impulsivity, DAT function and extracellular dopamine concentration did not predict the DIO-phenotype. Thus, motivation for food is linked to both initiation and maintenance of obesity. Importantly, obesity results in decreased striatal DAT function, which may underlie the maintenance of compulsive food intake in obesity.
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Ravilla, Nagendra Babu. "K-Cl Cotransport: Role of KCC3 in cellular Potassium (K) homeostasis in KCC3- transfected HEK-293 cells." Wright State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=wright1377519708.
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SINHA, SAYANTANI. "Role of TRPA1 and TRPV1 in Propofol Induced Vasodilation." Kent State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=kent1384901930.
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Abdul, Majid Aman Shah Bin. "The influence of selected sulphur containing compounds on retinal cell death : neuroprotective effects of hydrogen sulphide in a glaucoma model." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:bfb5ec82-9141-4784-94a8-0df3ce9d9471.
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Ganglion cell death in glaucoma is caused by a variety of insults that include ischemia, insufficient neurotrophic support and oxidative stress. Experimental studies were therefore conducted on cell cultures to determine how serum deprivation (to mimic insufficient neurotrophic support) or oxidative glutamate toxicity (GB), cause oxidative stress and induce retinal cell death. Moreover, studies were carried out to deduce whether selected sulphur containing compounds can blunt any negative influences to cells in culture and/or a defined ischaemic insult to the rat retina in situ, as this might suggest their use for the treatment of glaucoma. Serum deprivation and GB caused generation of reactive oxygen species (ROS) and apoptosis-like death to transformed retinal ganglion cells (RGC-5 cells). RGC-5 cells were more susceptible to the detrimental effects of GB than serum deprivation. RGC-5 cells subjected to serum deprivation appear to die by mechanisms that resemble classical apoptosis more closely than that caused by GB and the phase between the maximal generation of ROS and cell death were different. Cell death caused by serum deprivation was caspase-dependent but this was not the case for GB. Moreover, of the two sulphur compounds sulbutiamine and N-acetyl cysteine (NAC), sulbutiamine blunts the effect of serum deprivation more effectively. In addition, the pan caspase inhibitor z-VAD-fmk attenuated the negative effect of serum deprivation to RGC-5 cells while the necroptosis inhibitor (necrostsatin-1) counteracted solely the insult of GB. The sulphur containing compounds, ACS1 and ACS 67 which release hydrogen sulphide (H2S) slowly and NAC (a pro-cysteine GSH precursor) attenuated GB-induced cell death of RGC-5 cells. In contrast, sulbutiamine (a lipophilic thiolic thiamine derivative) was particularly effective in protecting RGC-5 cells from an insult of serum deprivation. Moreover, all of the sulphur compounds directly sequestered different types of ROS but with varying efficiency. Common features by which all tested sulphur containing agents seem to elicit a mode of action include the stimulation of GSH and the antioxidant enzyme glutathione-S-transferase (GST) as well as to scavenge excess free radicals. Moreover, the slow release of H2S from the ACS compounds appears to protect cells from oxidative stress through increasing the level of GSH, modulation of the cystine uptake transporter xCT, stimulation of the oxidative stress related transcription factor Nrf2 and the stimulation of pro-survival signalling pathways. The slow releasing H2S sulphur compound ACS67 also counteracts a number of detrimental influences to the rat retina in situ because of ischemia/reperfusion that includes damage to their ganglion cells. This suggests that such sulphur compounds might find a use in the treatment of glaucoma where ischemia probably plays a part in the disease process.
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Hirsch, Alexander M. "Embryonic Stem Cell-Derived Exosomes Increase the Antiproliferative Activity of Doxorubicin in Breast Cancer." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5981.
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The field of cancer research has grown immensely in recent decades and has led to a better understanding of the causes of the disease, as well as greatly improved treatment for various types of cancers, especially breast cancer. One of the most effective treatments involves the chemotherapeutic drug doxorubicin (DOX). DOX is an effective tool against all types of breast cancer, especially against triple negative breast cancer. However, DOX causes adverse side effects that include damage to the heart and skeletal muscle, particularly above specific cumulative doses. Recent evidence suggests that embryonic stem cell-derived (ES) exosomes, nanoscale extracellular vesicles that carry proteins, messenger RNA, and microRNAs, may be able to mitigate some of the cardio- and cytotoxic effects of DOX without reducing its efficacy.
The present study examined the effects of combined treatment with DOX (1 μM) and ES exosomes (10 μg/mL) on three cancer cell lines, MCF7, MDA-MB-231, and MDA-MB-468. The DOX/ES exosomes treatment increased cell death and increased apoptosis specifically compared to control, as measured via dye exclusion assay and flow cytometry. The treatment also decreased cell growth compared to control, as measured via MTS cell proliferation assay. In addition, DOX/ES exosomes treatment also increased expression of pro-apoptotic Bax while decreasing the expression of anti-apoptotic Bcl-2, as measured via Western blot. Finally, the DOX/ES exosomes treatment decreased expression of miR-200c, a microRNA associated with preventing epithelial-mesenchymal transition, a process that is integral to metastasis.
Although increased cell death and apoptosis and decreased cell proliferation implies that the DOX/exosomes treatment is effective against cancer, the decrease in miR-200c expression may suggest the opposite and will be investigated further in future studies. Even so, the results of this study suggest that exosomes may be an important component to reduce the harmful effects of cancer treatment in the future.
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Ahmad, Faizzan Syed. "A novel human stem cell platform for probing adrenoceptor signaling in iPSC derived cardiomyocytes including those with an adult atrial phenotype." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:17972018-6750-4e5c-8cc9-42e9c381f531.
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Scientific research is propelled by two objectives: Understanding and recognizing the essential biology of life, and deciphering this to uncover possible therapeutics in order to improve quality of life as well as relieve pain from disease. The aim of the work described in this thesis was to dissect the fundamental requirements of induced pluripotent stem cells both in pluripotency and differentiation with a particular focus on atrial specificity. Drug targeting of atrial-specific ion channels has been difficult because of lack of availability of appropriate cardiac cells, and preclinical testing studies have been carried out in non-cardiac cell lines, heterogeneous cardiac populations or animal models that have been unable to accurately represent human cardiomyocyte physiology. Therefore, we sought out to develop a preparation of cardiomyocytes showing an atrial phenotype with adult characteristics from human induced-pluripotent stem cells. A culture programme involving the use of Gremlin 2 allowed differentiation of cardiomyocytes with an atrial phenotype from human induced-pluripotent stem cells. When these differentiated cultures were dissociated into single myocytes a substantial fraction of cells showed a rod-shaped morphology with a single central nucleus that was broadly similar to that observed in cells isolated from atrial chambers of the heart. Immunolabelling of these myocytes for cardiac proteins (including RyR2 receptors, actinin-2, F-actin) showed striations with a sarcomere spacing of slightly less than 2um. The isolated rod-shaped cells were electrically quiescent unless stimulated to fire action potentials with an amplitude of 100 mV from a resting potential of approximately -70 mV. Proteins expressed included those for IK1, IKur channels. Ca2+ Transients recorded from spontaneously beating cultures showed increases in amplitude in response to stimulation of adrenoceptors (both alpha and beta). With the aim of identifying key signaling mechanisms in directing cell fate, our new protocol allowed differentiation of human myocytes with an atrial phenotype and adult characteristics that show functional adrenoceptor signaling pathways and are suitable for investigation of drug effects.
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Park, Sung H. "High Affinity Block of ICl,swell by Thiol-Reactive Small Molecules." VCU Scholars Compass, 2016. http://scholarscompass.vcu.edu/etd/4433.
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Ebselen (Ebs) is considered as a glutathione peroxidase (GPx) mimetic and primarily thought to function by scavenging intracellular reactive oxygen species (ROS). Previous to our work, Deng et al. (2010a) demonstrated complete block of ICl,swell with 15 microM Ebs following endothelin-1 (ET-1) induced activation of the current in cardiomyocytes. This block was presumed to take effect mainly via the quenching of ROS. Nonetheless, our work with DI TNC1 astrocytes strongly emphasizes that Ebs might function by an alternative mechanism based on its kinetic profile in blocking ICl,swell. Our experiments showed that 45 nM Ebs can fully block ICl,swell thus suggesting an apparent IC50 result, we predicted Ebs to possess a high kon with a low koff close to zero. As predicted, Ebs failed to washout in the timescale covered by our patch-clamp experiments. The block was also distal to H2O2, previously considered as the most proximate regulator of ICl,swell. And based on further evidence demonstrating irreversible block of ICl,swell distal to H2O2 with Ebs congeners, complete suppression of native ICl,swell with MTS reagents, and failure of Ebs to block ICl,swell from the cytosol, we concluded that Ebs and its congeners can covalently modify important –SH groups required for current activation while functioning as sulfhydryl reagents. Complete irreversible block of ICl,swell with 110 mM cell impermeant MTSES in native DI TNC1 astrocytes contrasts sharply to SWELL1 (Qiu et al., 2014) or LRRC8A (Voss et al., 2014), the latest molecular entity presumably responsible for ICl,swell, where 3.33 mM MTSES failed to demonstrate block of ICl,swell in the wild-type stably expressing SWELL1 (Qiu et al., 2014). Our data with Ebs, its congeners, and MTS reagents indicate the existence of a common extracellular binding site which involves a selenenylsulfide (Se-S) bond that critically modulates ICl,swell. We, therefore, synthesized a derivative of Ebs called ebselen-para-yne (Ebs-p-yne), which provided an even higher affinity for blocking ICl,swell with a presumed IC50 ~picomolar range. Ebs-p-yne is a promising novel molecule that may serve as a tag in identifying the molecular fingerprint ultimately responsible for ICl,swell. Furthermore, we can take advantage of click chemistry to ultimately pull out the channel or channel component which has remained elusive for greater than two decades.
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Yan, Dejun. "THE EFFECT OF CURCUMIN ON LEWIS LUNG CARCINOMA." Bowling Green State University / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1308588440.
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Vallaster, Markus Parzival. "Intergenerational Effects of Nicotine in an Animal Model of Paternal Nicotine Exposure." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/913.
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Environmental conditions imposed onto organisms during certain phases of their life cycles such as embryogenesis or puberty can not only impact the organisms’ own health, but also affect subsequent generations. The underlying mechanisms causing intergenerational phenotypes are not encoded in the genome, but the result of reversible epigenetic modifications. This work investigates in a mouse model the impact of paternal nicotine exposure on the next generation regarding addictive behavior modulation, metabolic changes, and molecular mechanisms. It provides evidence that male offspring from nicotine-exposed fathers (NIC offspring) is more resistant to lethal doses of nicotine. This phenotype is gender-specific and depends on short-term environmental challenges with low doses of nicotine prior to the LD50 application. The observed survival phenotype is not restricted to nicotine as drug of abuse, but also presents itself, when NIC offspring is challenged with a cocaine LD50 after acclimatization to low doses of either nicotine or cocaine. Functionally, NIC offspring metabolizes nicotine faster than control. Mechanistically, NIC offspring livers show global up-regulation of xenobiotic processing genes (XPG), an effect that is even more pronounced in primary hepatocyte cultures. Being known targets of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR), these XPGs show higher baseline expression in naïve NIC offspring livers. Nicotine’s action on the brain’s reward circuitry does not appear to be of biological significance in our model system. Taken together, paternal nicotine exposure leads to a non-specific and conditional phenotype in male NIC offspring that may provide a general survival advantage against xenobiotic challenges.
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Vallaster, Markus Parzival. "Intergenerational Effects of Nicotine in an Animal Model of Paternal Nicotine Exposure." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/913.
Full textAbstract:
Environmental conditions imposed onto organisms during certain phases of their life cycles such as embryogenesis or puberty can not only impact the organisms’ own health, but also affect subsequent generations. The underlying mechanisms causing intergenerational phenotypes are not encoded in the genome, but the result of reversible epigenetic modifications. This work investigates in a mouse model the impact of paternal nicotine exposure on the next generation regarding addictive behavior modulation, metabolic changes, and molecular mechanisms. It provides evidence that male offspring from nicotine-exposed fathers (NIC offspring) is more resistant to lethal doses of nicotine. This phenotype is gender-specific and depends on short-term environmental challenges with low doses of nicotine prior to the LD50 application. The observed survival phenotype is not restricted to nicotine as drug of abuse, but also presents itself, when NIC offspring is challenged with a cocaine LD50 after acclimatization to low doses of either nicotine or cocaine. Functionally, NIC offspring metabolizes nicotine faster than control. Mechanistically, NIC offspring livers show global up-regulation of xenobiotic processing genes (XPG), an effect that is even more pronounced in primary hepatocyte cultures. Being known targets of Constitutive Androstane Receptor (CAR) and Pregnane X Receptor (PXR), these XPGs show higher baseline expression in naïve NIC offspring livers. Nicotine’s action on the brain’s reward circuitry does not appear to be of biological significance in our model system. Taken together, paternal nicotine exposure leads to a non-specific and conditional phenotype in male NIC offspring that may provide a general survival advantage against xenobiotic challenges.
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Ragas, Moner A. "Refining a Post-Stroke Pharmacological and Physical Treatment to Reduce Infarct Volume or Improve Functional Recovery, Using Gene Expression Changes in the Peri-Infarct Region to Examine Potential Mechanisms in Male and Female Rats." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1470395029.
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Appleby, Hollie Leanne. "Orai channel physiology and pharmacology." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/15950/.
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Background: Cardiovascular disease is often characterised by functional and structural changes in the blood vessel wall, usually associated with endothelial dysfunction. Therefore, interest in targeting the dysfunctional endothelial cell has heightened. Ca2+ signalling is crucial to endothelial cell physiology as it drives a number of intracellular signalling pathways. Store-operated Ca2+ entry (SOCE) mediated by Orai1 channels is of particular interest as it provides a major Ca2+ influx pathway in endothelial cells, driving cell migration and proliferation, ultimately contributing to endothelial repair and integrity, angiogenesis and wound healing events. The novel hypothesis is that modulation of Orai1 channels will have important physiological effects on endothelial cell function. Methods and Results: A novel series of Orai1 inhibitors has been identified in this thesis with improved pharmacological and physicochemical properties compared to existing SOCE inhibitors. Optimisation of compounds’ structure-activity relationships (SAR) via Ca2+ measurement assays using human umbilical vein endothelial cells (HUVECs) has revealed important insights into functionally important features of SOCE blockers. In conjunction with in silico modelling, a novel binding site in the Orai1 channel located in a small extracellular pocket has been proposed. The generation and subsequent testing of a quaternary ammonium analogue of the parent compound, JPIII supports the hypothesis. The novel SOCE inhibitors suppressed endothelial cell migration and proliferation without affecting cell viability. Reassuringly, the small molecules were well tolerated in vivo, supporting further development and testing of such blockers in animal models of cardiovascular disease. Here, a novel transgenic murine model with the potential for temporal and conditional disruption of Ca2+ permeation in Orai1 channels has been generated and characterised, offering new possibilities for better understanding of the physiological and pathological roles of Orai1 and the therapeutic potential of targeting this channel. Conclusion: Novel SOCE inhibitors have been characterised in vitro, their mechanism of action interrogated and their properties optimised for the next stages of in vivo testing in an animal model of cardiovascular disease.
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Prachanronarong, Kristina L. "Understanding Drug Resistance and Antibody Neutralization Escape in Antivirals: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/840.
Full textAbstract:
Antiviral drug resistance is a major problem in the treatment of viral infections, including influenza and hepatitis C virus (HCV). Influenza neuraminidase (NA) is a viral sialidase on the surface of the influenza virion and a primary antiviral target in influenza. Two subtypes of NA predominate in humans, N1 and N2, but different patterns of drug resistance have emerged in each subtype. To provide a framework for understanding the structural basis of subtype specific drug resistance mutations in NA, we used molecular dynamics simulations to define dynamic substrate envelopes for NA to determine how different patterns of drug resistance have emerged in N1 and N2 NA. Furthermore, we used the substrate envelope to analyze HCV NS3/4A protease inhibitors in clinical development. In addition, influenza hemagglutinin (HA) is a primary target of neutralizing antibodies against influenza. Novel broadly neutralizing antibodies (BnAbs) against the stem region of HA have been described and inhibit several influenza viral subtypes, but antibody neutralization escape mutations have emerged. We identified potential escape mutations in broadly neutralizing antibody F10 that may impact protein dynamics in HA that are critical for function. We also solved crystal structures of antibody fragments that are important for understanding the structural basis of antibody binding for influenza BnAbs. These studies can inform the design of improved therapeutic strategies against viruses by incorporating an understanding of structural elements that are critical for function, such as substrate processing and protein dynamics, into the development of novel therapeutics that are robust against resistance.
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Novozhilova, Ekaterina B. "Physiology and pharmacology of flatworm muscle." [Ames, Iowa : Iowa State University], 2008.
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Spratt, James Christopher Samuel. "Endothelin : cardiovascular pharmacology, physiology & pathophysiology." Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/23202.
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Stechschulte, Lance A. "The Co-chaperones FKBP51 and PP5 Control Nuclear Receptor Phosphorylation and Adipogenesis." University of Toledo Health Science Campus / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1370871316.
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Ordway, Gregory A. "Molecular Pharmacology of Antidepressants." Digital Commons @ East Tennessee State University, 2006. https://dc.etsu.edu/etsu-works/8657.
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Maharjan, Chandra Kumar. "Interaction of Na+/K+ ATPase with Bcl-2 Proteins: Isolated Enzyme vs Epithelial Cell Extracts." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1464692938.
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Cumming, Paul Kenneth. "Pharmacology of cerebral histamine." Thesis, University of British Columbia, 1990. http://hdl.handle.net/2429/30687.
Full textAbstract:
Four aspects of the function of histaminergic systems were studied in the rat brain: toxicology, catabolism, release in vivo, and high affinity binding of histamine. Preparations of histamine-N-methyltransferase (HNMT) derived from kidney and brain were employed in the radioenzymatic quantification of histamine in biological samples. Tritiated S-adenosyl-L-methionine ([³H]-SAM) served as the co-substrate.
A toxicological study was conducted to determine the sensitivity of the HA innervation to prenatal treatment with methylazoxymethanol (MAM), an inhibitor of mitosis. In adult rats, the MAM treatment was without effect on cerebral histamine content, although forebrain HNMT activity was 50% reduced. In another study, C-57 mice were treated with the selective dopamine neurotoxin l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Substantial dopamine depletions were not associated with alterations in the cerebral histamine content.
In a study of the structural requirements for HNMT inhibition, 9-amino-1,2,3,4-tetrahydroacridine (THA), was found to be one of the most potent inhibitors yet described. The β-carboline alkaloids, of which harmaline is the prototype, were also found to be moderately potent HNMT inhibitors. Because of the lack of high-affinity re-uptake and the absence of alternate catabolic pathways, blockade of HNMT can potentially alter central histaminergic tone. Peripheral administration of THA was able to produce dose-dependent increases in cerebral histamine content, as was the more potent HNMT inhibitor, metoprine. The issue of structural requirements for HNMT inhibition are discussed in the light of these results.
The in vivo release of histamine was studied by the cerebral microdialysis technique. After chronic implantation of horizontal probes, TTX-insensitive and partially calcium-sensitive efflux of histamine was detected in the dorsal striatum and the bed nucleus of the stria terminalis. In striatum, histamine efflux was elevated 50% after peripheral histidine loading (500 mg/kg, i.p.). After synthesis blockade with α-fluoromethylhistidine (100 mg/kg,i.p.), extracellular histamine levels in striatum disappeared in a bi-exponential manner. The half-lives of this disappearance, 32 minutes and 7 hours, indicate the presence of at least two histamine pools. Striatal histamine efflux was elevated by yohimbine treatment (10 mg/kg, i.p.), suggesting the presence of a tonic α₂-adrenergic inhibition of histamine release in vivo.
In addition to the classical H₁ and H₂ receptors, histamine is able to bind to a pharmacologically distinct site, H₃, recently characterized as an autoreceptor regulating the synthesis and release of histamine. The binding properties of the H₃ ligand [³H]-N[symbol omitted]-methylhistamine ([³H]-N-MeHA) were studied in forebrain cryostat sections by
autoradiography. Determination of Bmax (25 fmole/section) and displacement studies indicated that [³H]-N-MeHA bound to the same site as [³H]-histamine: the high affinity
histamine binding site. Binding was greatest in the basal ganglia and had a complex
distribution within the cerebral cortex. Quinolinic acid lesion studies indicated that the
majority of the binding in the basal ganglia was on striato-nigral projection neurons. Cortical
binding was also sensitive to local excitotoxic lesions. Therefore, the majority of H₃ binding
is located on postsynaptic structures intrinsic to these brain regions, rather than on presynaptic
autoreceptors on terminals of histamine neurons.Medicine, Faculty of
Graduate
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Bolton, John Francis. "Urinary tract smooth muscle physiology and pharmacology." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.432685.
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Pearson, Hugh Anthony. "Physiology and pharmacology of insect calcium channels." Thesis, University College London (University of London), 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.308295.
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Farquhar, Michelle Jane. "Molecular pharmacology of chimeric peptides." Thesis, University of Wolverhampton, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.247780.
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Barkan, Kerry. "An investigation into glucagon receptor pharmacology." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/98781/.
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The glucagon receptor (GCGR), a family B G protein-coupled receptor (GPCR), plays an important role in regulating blood glucose levels through its ability to bind the 29 amino acid peptide hormone, glucagon (GCG). Antagonising GCG action is a potential therapeutic option for reducing hepatic glucose production. However, GCG-based therapy is currently limited to acute emergency treatment of hypoglycemia in patients with type 1 diabetes (T1D) (Habegger et al., 2010). Further insight into GCGR-mediated signalling pathways and mechanism of activation may provide the basis for therapeutic development. We investigated the ligand stimulated GCGR signalling using multiple assays including those measuring cAMP accumulation, ERK1/2 phosphorylation and intracellular Ca2+ (Ca2+ i) mobilisation. Through site directed mutagenesis and FACS analysis for the investigation of cell-surface expression, we identified several important residues. They included K16812.49, L16912.50 , H17012.51 and T1722.45 of the ICL1 region (G1651.63 -T1722.45 ) as potential determinant in signalling bias at the GCGR through Gq/11-coupling, C1712.44 as a critical determinant of GCGR expression and R1732.46 as an essential residue for G protein-coupling. Helix 8 residue E4068.49 was implicated in maintaining GCGR in an inactive state. Finally, TM4 residues G2714.49 , L2774.55 and V2804.58 were found to plays an important role in successful translation and/or trafficking of GCGR and (Ca2+ i) mobilisation. The GCGR-mediated pERK1/2 response way found to be both Gq/11 and β-arrestin1/2 mediated, whereas it was independent of PKA or Gβγ-subunits. The GCG analogue TH-GCG, contrary to previous reports (Wakelam et al., 1986, Lenzen et al., 1990), was characterised as a partial agonist at the GCGR, inducing a robust cAMP response but fails to induce a detectable Ca2+ i or IP1 response. We also identified a RAMP2-dependent potentiation of the GCG stimulated cAMP response at the GCGR. The research described in this thesis has produced novel data that contributes to a clearer understanding of GCGR pharmacology.
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Baker, Jillian G. "Molecular pharmacology of β-adrenoceptor ligands." Thesis, University of Nottingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401579.
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Slusarczyk, Adrian L. (Adrian Lukas). "Molecular imaging with engineered physiology." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/104229.
Full textAbstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2016.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 125-133).
Using molecular imaging in vivo, biomolecular and cellular phenomena can be investigated within their relevant physiological context, addressing a central challenge for 21st century biomedicine and basic research. To advance neuroscience in particular, molecular-level measurements across the brain inside the intact organism are required. However, existing imaging strategies and available probes have been limited by serious constraints. Magnetic resonance imaging (MRI) provides deeper tissue penetration depth than optical imaging and better spatial resolution and greater versatility in sensor design than radioactive probes. The most important drawback for MRI probes has been the need for high concentrations in the micromolar to millimolar range, leading to analyte sequestration, complications for noninvasive brain delivery, and toxicity. Efforts to address the sensitivity problem, such as nuclear hyperpolarization, introduce their own technical constraints and so far lack generality. Here, we introduce a conceptually novel molecular imaging technique based on artificially induced physiological perturbations, enabling molecular MRI with nanomolar sensitivity. In this imaging strategy, we take advantage of blood as an abundant endogenous source of contrast compatible with multiple imaging modalities including MRI and optical imaging to decouple the concentration requirement for molecular sensing from the concentration requirement for imaging contrast. Highly potent vasoactive peptides are engineered to respond to specific biomolecular phenomena of interest at nanomolar concentrations by inducing dilation of the microvasculature, increased local bloodflow, and consequently, large changes in T₂*-weighted MRI contrast. This principle is exploited to design activatable probes for protease activity based on the calcitonin gene-related peptide (CGRP) and validate them for brain imaging in live rats; to use CGRP as a genetic reporter for cell tracking; and to create fusions of a vasoactive peptide from flies to previously characterized antibodies capable of crossing the blood-brain barrier (BBB), suggesting the possibility of minimally invasive brain delivery of such probes. We demonstrate the feasibility of highly sensitive molecular MRI with vasoactive probes at concentrations compatible with in situ expression of probes and delivery across the BBB, and show that vasoactive peptides are a versatile platform for MRI probe design which promises unprecedented in vivo molecular insights for biomedicine and neuroscience.
by Adrian L. Slusarczyk.
Ph. D.
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Thornton, S. "The physiology and pharmacology of oxytocin during human pregnancy." Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234659.
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Sam, Cynthia Laura Sandra. "Atrial myocyte physiology and pharmacology in health and disease." Thesis, Kingston University, 2013. http://eprints.kingston.ac.uk/27782/.
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Atrial fibrillation is the most common form of cardiac dysrhythmia in the world. According to the British Heart Foundation, about 1 million people in the UK are currently living with this condition and they are up to five times more likely to suffer a stroke than the rest of the population. Atrial fibrillation is the result of irregular spontaneous contractions of the atrial chambers of the heart separate to the contractions generated by the sino-atrial node activity. At a cellular level, pro-arrhythmogenic calcium handling has been observed upon the stimulation of atrial cells with the partial agonist CGP12177 at a novel low affinity beta1-AR (beta1L-AR). The aim of this thesis is to characterise the relationship between the morphology of the left and right atrial myocytes and the initial calcium release sites, and alterations in calcium handling protein phosphorylation state during cardiomyocyte contraction. We study how this relates to atrial arrhythmias via spontaneous calcium release activity in quiescent rat atrial myocytes by stimulating the propranolol-insensitive beta1-ARs using CGP12177. We aim to contribute towards understanding specific alterations at the cellular level in atrial myocytes during arrhythmias and heart failure (HF). Quiescent WKY rat left and right atrial cells were obtained from Langendorff perfusion of the whole heart and subsequent atrial cell isolation. Left atrial cells (13.6 [plus or minus] 0.3 urn) were wider on average compared to right atrial cells (9.9 [plus or minus] 0.25 um). Di-8ANEPPS stained cells confirmed networks of t-tubules in left atrial myocytes that facilitate t-tubular transportation of calcium during excitation contraction coupling. However no t-tubules were reported for right atrial myocytes presumably due to their narrower widths which are sufficient for calcium diffusion. Cardiac dysfunction results in cardiac proteins (sarco(endo)plasmic calcium ATP-ase (SERCA2a) protein, phospholamban (PLB) and ryanodine receptors (RyR2) having abnormal expressions. Western blotting studies have demonstrated high abundance of PLB and a tendency for lower abundance of SERCA2a in hypertensive and volume overload HF tissues as a result of the increased the workload on the heart and prolonged calcium release from the calcium store. Phosphorylated proteins of PLB in hypertensive tissues have lower population of beta-ARs and hence there is a reduction in the stimulation of these receptors in the heart. On the other hand, a deterioration of RyR2 of the sarcoplasmic reticulum leads to a reduced amount of calcium being stored in the calcium store and consequently contributes to a contractile deficit. This may eventually result in arrhythmias, commonly observed in people with HF. A pharmacological study of spontaneous calcium release (calcium sparks, wavelets and waves) using a series of cardioactive agents suggests pathways of pro-arrhythmogenic calcium release via the low affinity beta1-ARs. In particular, CGP12177 stimulating the ß1L-ARs of quiescent rat atrial myocytes has revealed that there may be a pro-arrhythmogenic pool of calcium in these atrial cells and a beta-blocker such as bupranolol may be efficient in supressing such stimulation as a therapeutic solution.
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Ferro, Charles Joseph. "Endothelin in man : studies in pharmacology, physiology and pathophysiology." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/21238.
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In this thesis I review the biology of endothelin as well as discuss the evidence supporting a pathophysiological role for endothelin in a number of cardiovascular and renal diseases. In the studies described, I have further investigated the physiology of the endothelin system as well as examined the pharmacology of endothelin receptor antagonists in man. Finally, I have also examined the role of endothelin in the pathophysiology of chronic renal failure and essential hypertension. Study 1: Inhibitors of the enzyme, neutral endopeptidase cause forearm vasoconstriction when infused into the brachial artery. This enzyme appears to significantly contribute to the breakdown of endothelin in vivo. Study 2: Big ET-3 is converted to the mature peptide ET-3 in the forearm circulation, but not in capacitance vessels. This study suggests the existence of a third endothelin-converting enzyme capable of converting big ET-3. Study 3: Systemic doses of TAK-044, a non-selective endothelin receptor antagonist, lowers vascular resistance and blood pressure in man, demonstrating that endothelin contributes to the maintenance of blood pressure in health. Study 4: Systemic TAK-044 completely blocks the local vasoconstriction produced by intrabrachial artery infusion of ET-1 for up to 3 hours. This finding has implications for the treatment of acute vasospastic conditions. Study 5: Systemic doses of TAK-044 only partially block ET-1 mediated vasoconstriction for 12 hours. This has important implications for dosing schedules in potential therapeutic interventions. Study 6: TAK-044 causes renal vasodilatation and lowers effective filtration fraction by a relative decrease in glomerular filtration rate and increase in effective plasma flow in healthy volunteers. This study demonstrates that endothelin has a role in controlling the renal circulation. Study 7: TAK-044 lowers systemic vascular resistance and blood pressure in patients with chronic renal failure, with a reduction in effective filtration fraction.
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Liu, Tongyu. "Ethanol Effect on Three Distinct Types of Ependymal Cells and the Intracellular Calcium Oscillation Property." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1396351727.
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McGonigle, Ian Vincent. "Molecular pharmacology of an insect GABA receptor." Thesis, University of Cambridge, 2010. https://www.repository.cam.ac.uk/handle/1810/226857.
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Cys-loop receptors are ligand-gated ion channels that are involved in fast synaptic neurotransmission in the central and peripheral nervous system. The Cys-loop receptor RDL ('resistant to dieldrin') is a GABA-gated chloride channel from Drosophila melanogaster and is a major target site for insecticides. The aim of this dissertation was to characterise RDL receptors with particular focus on the agonist binding site. To assess the potency of a range of GABA analogues on RDL receptors, I expressed receptors in Xenopus oocytes and used voltage-clamp electrophysiology to detect receptor responses. I carried out computational modelling of these analogues to determine the dipole separation distances and atomic charges. Computational calculations and functional experiments revealed that agonists require a charged ammonium and an anionic centre, with the most potent agonists having a dipole separation distance of ~5 Å. I made a homology model of the extracellular domain of RDL and docked the active analogues into the putative binding site. I then conducted mutagenesis studies to test the accuracy of this model. Functional data from mutagenesis studies broadly support the location of GABA within this model. This model may be useful for further structure-activity studies and rational drug design. Natural compounds from the traditional Chinese medicine 'Ginkgo biloba' (ginkgolide A, ginkgolide B and bilobalide) have potent insecticidal properties and are similar in structure to picrotoxin. I tested the effect of these compounds on RDL receptor function using voltage-clamp electrophysiology. All compounds were found to inhibit RDL receptor function. I probed the binding site of these compounds using site-directed mutagenesis and electrophysiology. Mutations to the 2'A and 6'T channel-lining (M2) residues greatly reduced the potency of these compounds. I then made a homology model of the transmembrane domain of RDL and docked these compounds into the channel. Compounds docked into the channel pore close to the 2' and 6' channel-lining residues and H-bonding interactions were detected at these locations. Ginkgolides are therefore antagonists of RDL receptors, binding in the channel close to the 2' and 6' residues and this may be the mechanism underlying their potent insecticidal properties. The 5-HT3 receptor is a member of the Cys-loop receptor family and shows homology to RDL receptors. To explore different techniques for studying Cys-loop receptor function I assessed the functionality of two brain derived transcripts of the 5-HT3B subunit (Br1 and Br2) using single-channel electrophysiology and a fluorometric assay. Receptors containing Br1 were found to have a conductance identical to the 5-HT3B subunit whilst Br2 receptors were found not to be expressed. This finding has implications for 5-HT3 brain signalling, in which Br1 may play an important role. In conclusion, work here has described how agonists bind to and activate RDL GABA receptors and I have identified a candidate mechanism for the potent insecticidal properties of Ginkgo biloba extracts. I have also confirmed that 5-HT3 receptor brain transcript Br1 forms functional channels with similar properties to the 5-HT3B subunit.
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Whittaker, Steven Robert. "The molecular pharmacology of purine CDK inhibitors." Thesis, Institute of Cancer Research (University Of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.404908.
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Kasorn, Anongnard. "The molecular pharmacology of lysophosphatidic acid receptors." Thesis, University of Nottingham, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.441003.
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Lee, Ka Cheong. "Molecular pharmacology of DNA topoisomerase II drugs." Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3780.
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Topoisomerase II (TOP2) is an important anti-cancer drug target. This study demonstrates that proteasomal inhibition by MG132 or PS341 potentiates the effect of TOP2 poisons on cell growth inhibition. Mitoxantrone was potentiated the most. The presence of the proteasome inhibitor MG132 prolonged the half-life of drug-induced DNA-TOP2 complexes stabilised by mitoxantrone or etoposide. Genotoxicity was measured in K562 cells using in vitro micronucleus assays for combinations of a proteasome inhibitor (MG132 or PS341) and mitoxantrone and for each agent alone. Combinations that potentiated the cytotoxicity reduced the genotoxicity. This suggests that combining a proteasome inhibitor with a TOP2 drug has the potential to reduce late toxicities such as therapy related leukaemia. The genotoxicity of six TOP2 poisons was determined by high throughput in vitro micronucleus assays in three Nalm-6 cell lines with differing TOP2 levels. Lower genotoxicity was observed in TOP2B knock-out and TOP2A knock-down cells, suggesting both TOP2A and TOP2B have a role in genotoxicity triggered by TOP2 poisons.
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Lin, Kuan-Hung. "Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/841.
Full textAbstract:
Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.
First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.
Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.
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Lin, Kuan-Hung. "Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation." eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/841.
Full textAbstract:
Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.
First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.
Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.
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Knight, Anthony. "A systems pharmacology approach to the adenosine A1 receptor." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/75201/.
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The majority of drugs are prescribed on the premise that their desired and undesired effects are well characterised. However, the mechanisms underlying these effects can be elusive and are of interest to the pharmaceutical industry in terms of rational drug design. G protein-coupled receptors are a significant class of drug target that are capable of influencing multiple signalling processes, and downstream effects, simultaneously through a variety of effectors, such as G proteins or –β-arrestins. The effector activated by a given receptor is often a function of the ligand. This is termed functional selectivity and can contribute to adverse drug effects. Understanding functional selectivity in a mammalian setting is hindered by cross-talk between many competing signalling components. The Sc. cerevisiae pheromone response can be modified to isolate individual mammalian receptor- G protein interactions. Therefore, this simple organism represents an excellent tool to study functional selectivity. Further, the simplicity of this organism allows this pathway to be mathematically modelled. By applying mathematical models to mammalian GPCR signalling in yeast it is possible to extract experimentally inaccessible quantitative parameters underlying functional selectivity. This interdisciplinary approach to pharmacological mechanisms is an example of systems pharmacology. Here a systems pharmacology approach is applied to adenosine receptor signalling in yeast with a view to understanding the contribution of the ligand, receptor and G protein to functional selectivity. The first stage of this process was expression and characterisation of adenosine A1R, A2AR, A2BR and A3R subtypes in yeast. Here, the A1R and A2R subtypes were shown to be functional in yeast, but the A3R response was limited. The A1R signals through G proteins representing the inhibitory G αi family in yeast, while the A2AR and A2BR signal through both inhibitory and stimulatory G protein equivalents. Here ligand bias is quantified but further extended to describe adenosine receptor selectivity. Further, the yeast system was used to inform novel fluorescent compound development. Fluorescent ligand-binding rates would ultimately inform modelling studies. A minimal mathematical framework was developed to described A1R signalling in yeast. Ordinary differential equation models recreate dynamic cellular processes. Here an ODE model was applied to experimental time course data to predict rate constants throughout the yeast G protein cycle in the presence of the mammalian A1R. This model predicts that G protein subtype influences the ligand-receptor-G protein interactions of the A1R in yeast. Further modification of the system and fluorescent technologies may help validate these predictions.