A sequential extraction technique was used to rapidly analyse AChE molecular form activity at the mouse neuromuscular junction and also in peripheral parts of muscle fibres. AChE in the synaptic cleft involved in the termination of cholinergic transmission was successfully assessed by the assay method and by an alternative method using a correlation equation which represented the relationship between synaptic AChE and the prolongation of extra-cellular miniature endplate potentials. It was found that inhibition after in vivo Carbamate (CB) dosing could not be maintained during tissue analysis because CB-inhibited enzyme complexes decarbamoylated vary rapidly and could not be prevented even when maintained on ice. The methods employed did not therefore give a measure of inhibition but presented a profile of metabolic responses to continual, low dose CB treatment. Repetitive and continual infusion with low doses of the CBs: pyridostigmine and physostigmine induced a variety of effects on mouse skeletal muscle. Both compounds induced a mild myopathy in the mouse diaphragm during continual infusion which was characterised by endplate deformation without necrosis; such deformation persisted on termination of treatment but had recovered slightly 14 days later. Endplate and non-endplate AChE molecular forms displayed selective responses to CB treatment. During treatment endplate AChE was reduced whereas non-endplate AChE was largely unaffected, and after treatment, endplate AChE recovered, whereas non-endplate AChE was up-regulated. The mechanisms by which these responses become manifest are unclear but may be due to CB-induced effects on nerve-mediated muscle activity, neurotrophic factors or morphological and physiological changes which arise at the neuromuscular junction. As well as inhibiting AChE, CBs also influence the metabolism and regulation of the enzyme and induce persistent endplate deformation.

The effect of L-dopa on rat brain protein synthesis was examined by studying the effect of the drug on the incorporation of radioactive amino acids into TCA-precipitable material. In the majority of experiments trace quantities of either L-[3,4(n)-H] valine or L-[4,5-3H] lysine were administered subcutaneously. Incorporation was expressed in terms of l) the relative incorporation (Irel) defined as the final TCA-insoluble radioactivity divided by the final TCA-soluble radioactivity, and 2) the apparent incorporation (I app) defined as the final TCA-insoluble radioactivity divided by the final specific activity of the soluble amino acid pool. It is shown that Irel is not, in general, a reliable index of rates of protein synthesis. L-dopa (500mg/kg,ip), administered 45 min previously, had no significant effect on either the apparent incorporation of L-[3H] valine, measured after a 15 min incorporation period, or that of L-[3H] lysine, measured after a 7.5 min incorporation period. The drug did, however, cause a significant 55-59% decrease in the apparent incorporation of L-[3H] lysine measured after a 15 min labelling period. Computer simulation was used to demonstrate that these results can be quantitatively accounted for if it is assumed that L-dopa has no effect on brain protein synthesis but does affect precursor lysine compartmentation. Temperature controlled experiments were performed in order to rule out the possibility that the difference between the effect of L-dopa on lysine and valine incorporation arose artifactually through the failure to control ambient temperature. Further, in order to obtain more reliable estimates of rates of brain protein synthesis, in two of these experiments, rats were given a quantity of L-[U-14C] valine sufficient to maintain a constant precursor specific activity throughout the incorporation period. The results confirmed those obtained at room temperature using trace quantities of labelled amino acids. While the possibility that L-dopa has a small inhibitory effect on brain protein synthesis could not be ruled out, it was concluded that a drug-induced effect on precursor lysine compartmentation was mainly responsible for the gross inhibition of lysine incorporation observed in some experiments. Preliminary experiments indicated that a similar effect may occur in rat liver.

Metabolic syndrome, first described as Syndrome X, includes a cluster of metabolic abnormalities including visceral obesity, insulin resistance, dyslipidaemia and hypertension. The pathophysiology of metabolic syndrome is complex, however, visceral adiposity and insulin resistance are thought to play a central role and both are implicated in the development of type 2 diabetes. Both metabolic syndrome and type 2 diabetes increase the risk of cardiovascular disease, by initiating and accelerating the progression of atherosclerosis, the hallmark of cardiovascular disease. Chronic low-grade inflammation of adipose tissue and vascular endothelium is well documented in type 2 diabetes and associated cardiovascular disease. In addition, stress has also been identified as one of the factors that increases the risk of cardiovascular disease. The endocannabinoid system is a physiological system that includes cannabinoid (CB1/CB2) receptors, their endogenous ligands and the enzymes responsible for their biosynthesis and degradation. Activation of the endocannabinoid system in the central nervous system leads to increased appetite and food intake while in the periphery it causes lipogenesis. Chronic over activation of the endocannabinoid system has been seen in both obesity and type 2 diabetes. Attempts to modulate the endocannabinoid system, by using CB1 antagonist/inverse agonist, rimonabant, led to positive metabolic and cardiovascular effects, but were associated with adverse psychiatric events. Cannabidiol (CBD) and delta 9 tetrahydrocannabivarin (Δ9 THCV or THCV), are two phytocannabinoids, obtained from Cannabis Sativa L. Both show a distinct pharmacological profile, separate from rimonabant and delta 9 tetrahydrocannabinol (Δ9 THC or THC), the parent compound obtained from Cannabis Sativa L. Both have potent anti-inflammatory and anti-oxidant properties and show multiple desirable cardiovascular and metabolic effects in preclinical studies. The aim of this project was to investigate the metabolic and cardiovascular effects of these two phytocannabinoids, by employing in vivo and in vitro techniques. Chapter 2 describes in detail the clinical study in type 2 diabetic subjects, where we used CBD and THCV, alone and in combination and studied their effects on various metabolic, cardiovascular and inflammatory parameters. Chapter 3 presents acute study of the cardiovascular effects of CBD in young healthy volunteers. Chapters 4 and 5 deal with the effects of CBD and THCV on mature human adipocytes and human aortic endothelial cells respectively. Last chapter includes work done on plasma samples and homogenised femoral arteries from Zucker Diabetic Fatty rats and their lean counterparts, treated with CBD. We found that THCV improves glycaemic parameters in type 2 diabetes, while CBD affects resting cardiovascular parameters and cardiovascular response to stress. CBD affects the release of leptin and IL-6 from adipocytes, while both CBD and THCV affect the release of endothelin-1 and vascular cell adhesion molecule-1 from aortic endothelial cells. CBD also shows positive impact on cytokines in diabetic rats.

The application of fluorogenic and chromogenic enzymatic substrates in culture media continues to offer huge potential for accessible bacterial detection and identification. However, their clinical use poses two major disadvantages: the presence of commensal and/or coliform bacteria camouflages the detection of bacteria of interest, and false results may arise due to secretion of a similar enzyme by a different species (or strain of) that acts upon the same enzymatic substrates. In the current work, selective antimicrobial agents or suicide substrates were developed and investigated in order to prevent the growth of these interfering bacteria, thus improving the integrity of the detection and diagnosis of common clinical infections. This project has been carried out under collaboration between University of Sunderland (UK), bioMérieux (France) and Freeman Hospital, Newcastle-upon-Tyne (UK). The L-alanine analogues D/L-fosfalin and -chloro-L-alanine were synthesised, both of which target alanine racemase, and different peptide derivatives based on these moieties were prepared using the well-established IBCF/NMM coupling approach with a series of protection/deprotection methods. Two dipeptide derivatives based on para-aminobenzoic acid (PABA) were also investigated. A series of synthetic derivatives were subjected to microbiology evaluation using 20 different clinical strains of clinical bacteria at the Freeman Hospital. The stability of -Cl-L-Ala-D/L-Fos was investigated at pH 6.1 – 9.1; hydrolysis products were formed above pH 6.1. The minimum inhibitory concentration (MIC) of L-Ala-L-Ala-D/L-Fos was double the concentration of L-Ala-L-Ala-L-Fos, showing that only the diastereoisomer with L-fosfalin exhibited significant inhibitory activity against the bacteria. The introduction of -chloro-L-alanine into the phosphonopeptide derivatives enhanced the antibacterial activity; for example the growth of commensal bacterium, Escherichia coli, was inhibited at a low MIC (0.125 – 0.5 mg/L) by most X--Cl-L-Ala-D/L-Fos tripeptide analogues, except when X: sarcosine (MIC: 4 mg/L). However, no significant antibacterial activity was found in the PABA derivatives. These di- and tri-peptide fosfalin-containing derivatives, especially with the inclusion of -Cl-L-alanine, offer improved selectivity for the detection and identification of pathogenic bacteria in clinical samples by restricting the over-growth of commensal bacteria.

The aim of this thesis is to investigate possible mechanisms that may contribute to neutrophil hyperactivity and hyper-reactivity. One possibility is the presence of a neutrophil priming factors within the peripheral circulation of periodontitis patients. To examine this possibility differentiated HL-60 cells and primary neutrophils were studied in the presence and absence of plasma from periodontitis patients. In independent experiments, plasma was depleted of IL-8, GM-CSF, interferon-a, immunoglobulins and albumin. This work demonstrated that plasma factors such as IL-8, GM-CSF, and interferon-a present during periodontitis may contribute towards the reported hyperactive neutrophil phenotype. Furthermore, this work demonstrated that products from Pg may regulate neutrophil accumulation at infected periodontal sites by promoting gingipain-dependent modification of IL-8-77 into a more biologically active chemokine. To elucidate whether the oxidatively stressed environment that neutrophils are exposed to in periodontitis could influence hyperactivity and hyper-reactivity, neutrophils were depleted of glutathione. This work showed that during oxidative stress, where cellular redox-levels have been altered, neutrophils exhibit an increased respiratory burst. In conclusion, this work highlights the multiple mechanisms that may contribute to neutrophil hyperactivity and hyperreactivity including gingipain-modulated activity of IL-8 variants, the effect of host factors such as IL-8, GM-CSF, interferon-a on neutrophils priming and activation, and the shift of neutrophil GSH:GSSG ratio in favour of a more oxidised environment as observed in periodontitis.

Spasticity is a common presentation in a wide variety of neurological disorders like cerebral palsy (CP), multiple sclerosis, and spinal cord injury. Management of spasticity involves multiple modalities such as physical and occupational therapy, oral medicines, Botulinum toxin injection, and orthopaedic and neurosurgical intervention. Intrathecal Baclofen (ITB) therapy is one neurosurgical intervention to control spasticity in CP patients. The ITB pump is implanted subcutaneously or sub-facially in the abdomen which delivers the baclofen directly to the intrathecal space via a catheter. As a result of by-passing the blood-brain-barrier, intrathecal administration of a hundredth part of the oral baclofen dose is sufficient to relieve spasticity and therefore preventing the peripheral side-effects seen with oral administration. Although the ITB delivery systems demonstrate significant effectiveness in improving spasticity, the ITB delivery system is associated with a high complication rate which could interfere with the desired effect of ITB therapy. Therefore, in a retrospective observational study we aimed to analyse the ITB complications in a large (n=222) consecutive series of patients. The complication rate in relation to the long period of follow-up (939 pump years),was found to be similar or less than those reported in literature. Dystonia, young age and presence of gastrostomy tube were significantly associated with infective complications. Catheter complications were influenced by presence of dystonia and the surgical technique, whether it was a subfascial or subcutaneous implantation. The pump is refilled by baclofen solution. The frequency of refilling is dependent on the daily dosing regime and the concentration of the aqueous solution of baclofen in the pump. Baclofen solutions are available as commercial and compounded products. An experimental controlled study was conducted to evaluate the safety of using higher concentrations of compounded baclofen in comparison to commercial baclofen. Baclofen concentration of the compounded solution was found to be less accurate than the commercial product although it was within an acceptable range from the expected value. The number of invisible particulates was significantly higher in the compounded solution than in commercial baclofen. However, no clinical complications were reported in the compounded or in the commercial baclofen groups. As patients receiving ITB therapy may have clinical benefits of reduction of the severity of spasticity, they could also have improvements in their functional status and quality of life. A survey study to evaluate changes in functional status and heath related quality of life, showed improvement in both aspects, moreover, the improvement in the health related quality of life was more significant than the change in functional status.

No description available.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Acetylcholine (Ach) has been shown to have excitatory effects on the mammalian heart, and it is possible that these actions may be related to certain arrhythmias. To test this hypothesis experiments were designed to study in vivo and in vitro these effects of Ach on atrial refractoriness, excitability, and rate and tension [TRUNCATED] / 2031-01-01

The IKs potassium channel, critical to control of heart electrical activity, requires assembly of pore-forming alpha subunits (KCNQ1) and accessory beta (KCNE1) subunits. IKs is the slowly activating component of delayed rectifier K+ current in the heart and is a major contributor to the timing of repolarization of the cardiomyocyte membrane potential. Inherited mutations in either IKs channel subunit are associated with cardiac arrhythmia syndromes, including long QT syndrome (LQTS), short QT syndrome (SQTS) and familial atrial fibrillation (FAF). The biophysical properties of IKs channel current are dramatically altered when KCNE1 associates with the KCNQ1 channel. Functional tetrameric channels can be formed by KCNQ1 alone, but co-assembly with KCNE1 is required for the unique kinetics necessary to regulate human cardiac electrical activity as well as for the channel's functional response to the sympathetic nervous system. Specifically, KCNE1 co-assembly results in a depolarizing shift in the voltage dependence of activation, an increase in the single channel conductance, and an increase in current density. IKs channel current is also characterized by slow activation and deactivation kinetics, with little or no inactivation, in contrast to the KCNQ1 homomeric channel, which is characterized by fast activation and deactivation kinetics and clear inactivation. We wanted to understand how KCNE1 modulates the KCNQ1 channel functionally and investigate the structural determinants of this modulation. In Chapter II, we explore the role(s) of KCNE1 in the context of two KCNQ1 atrial fibrillation associated mutations, S140G and V141M. In contrast to published results, we find distinct dependence on the KCNE1 subunit for V141M, but not for S140G. Having determined the importance of KCNE1 for V141M functionally, we continued to explore the role of KCNE1 structurally for this mutation. Using cysteine substitution in both KCNQ1 and KCNE1 subunits, we monitored spontaneous disulfide bond formation and find that V141C crosslinks to KCNE1, while S140C does not. Having established the functional and structural importance of KCNE1 for V141M, we proposed that there could be mutations in KCNE1 that could reverse the consequences of slow deactivation in the V141M mutation. In Chapter III, we engineer amino terminal KCNE1 mutations and demonstrate that this domain is important for controlling deactivation, but not activation, kinetics of the KCNQ1 channel. We find two KCNE1 mutations, L45F and Y46W, which when co-expressed with either V141M or S140G mutations in KCNQ1, help restore the mutant channel back towards a wild-type IKs channel. From these results, we propose that the amino-terminal domain could play an important role in mediating the rate of deactivation in KCNQ1/KCNE1 channels. After testing mutations on KCNE1 that could affect normal channel function, we continued with a project to study mutations on KCNQ1 that would have similar dramatic effects on the channel. In Chapter IV, we mutated KCNQ1 residue S140 to Threonine and found that S140T co-assembled with KCNE1 produced a channel having functional characteristics opposite to that of S140G/KCNE1 channels. In contrast to S140G/KCNE1 channels, where channels tend to stay open due to very slow deactivation kinetics, S140T/KCNE1 channels tend to be stabilized in the closed state and require more depolarized pulses to open channels. In addition, we find that a mutation at position Y46 in KCNE1, when co-expressed with the S140T mutation in KCNQ1, helps restore the mutant channel back towards a wild-type channel. Again, here we provide evidence that the amino terminal end of KCNE1 could play a role in controlling deactivation. In Chapter V, we investigated the importance of where KCNE1 is located in the channel and also how KCNQ1/KCNE1 subunits assemble using a tandem construct, with 1 KCNE1 subunit tethered to 2 KCNQ1 subunits (EQQ). To investigate the significance of KCNE1 location, we explored the functional consequences of having the S140G or V141M mutations in the proximal (closest to KCNE1) or distal (farthest from KCNE1) KCNQ1 subunit. We find that having a mutation in the proximal subunit is subject to modulation by KCNE1, but not the distal subunit. Using crosslinking, we want to confirm proper assembly of the heterotetrameric channel to verify that KCNE1 assembles between S1 from one KCNQ1 subunit and the S6 domain of an opposing KCNQ1 subunit. Taken together, we demonstrate that the proximity between the N-terminus of KCNE1 and the S1 domain of KCNQ1 could play a role in modulating deactivation kinetics of KCNQ1. These findings will be of great importance in understanding normal IKs channel function, which will be essential for maintaining proper heart function.

Dopamine receptors mediate various important neurophysiological functions. At a molecular level, G protein coupling is considered the main activation mechanism for most of the receptor-mediated cellular processes. A number of studies using native tissue have supported the idea that receptors can interact to form dimers or higher order oligomers. Particularly in medium spiny neurons of the striatum, dopamine receptor subtypes are reported to form dimers with themselves or other receptors (e.g. adenosine receptor A2A). Although a functional relevance for these dimers has been proposed, current assay systems are not capable of teasing out dimer-specific signaling events from those from other receptor populations. We have developed an assay that allows investigation of receptor-effector coupling specifically with defined dimer pairs. Using this assay, we investigated putative dopamine D1-D2 and A2A-D2 receptor dimer functions and studied the issue of a purported G protein coupling switch in the D1-D2 receptor dimer in which the heteromer was proposed to activate Gq, unlike D1 or D2 receptor when expressed alone. We were unable, however, to find evidence for Gq activation by the D1-D2 heteromer, as the protomers in the heteromer maintained fidelity of signaling to their cognate G proteins. We also developed and optimized a series of novel Gs biosensors to elucidate differences in heterotrimeric G protein conformational changes triggered by dopamine D1 and A2A receptors, two of the prominent pharmacological targets in the striatum. In addition to G protein signaling, intracellular calcium is also involved in many important cellular functions in all cell types. In neurons, intracellular calcium is implicated in learning and memory (synaptic plasticity) as well as neurodegeneration (apoptosis). In medium spiny neurons, dopamine-mediated calcium release from internal stores has been reported to result from activation of phospholipase C (PLC). However, different subtypes of dopamine receptors and intermediary proteins have been proposed to play a role in this dopamine-mediated PLC activation, and the underlying mechanisms are unclear. We found that activation of D1 and D2 receptors expressed individually can mobilize calcium in a PLC-dependent manner. In parallel, we also examined D1 and D2 receptor colocalization in striatal brain slices as well as in cultured medium spiny neurons. Although we found evidence using bacterial artificial chromosome-D1 and D2 reporter mice that D1 and D2 receptors are co-expressed in a small number of brain regions, we failed to observe D1-D2 receptor colocalization, suggesting the possibility that in neurons the receptors are somehow segregated.