Cardiac Purkinje fibers (PFs) play a very important role in cardiac electrophysiology. They are crucial for assuring appropriate timing and sequence of ventricle contraction and play an important role in cardiac arrhythmogenesis, largely via abnormalities in repolarization. Little work has been done to define the molecular electrophysiology of cardiac Purkinje cells (PCs). The primary hypothesis of this thesis was that PCs have unique molecular determinants of repolarization. The specific objectives were to characterize the repolarizing currents in isolated canine PCs, to clarify the molecular basis for these currents and to study role of PC current remodeling in a cardiac disease state. To achieve these goals, we used approaches at three different levels: the cellular level (with microelectrode techniques), the ionic level (with whole-cell patch clamp techniques) and molecular level (with competitive RT-PCR, Western blot analysis and immunocytochemistry). / We first optimized PC isolation techniques, which allowed us to characterize repolarizing currents and to visualize channel protein distribution by immunolocalization in cardiac PCs. We then characterized an important repolarizing current, the transient outward current (Ito) in canine PCs. We found that Purkinje Ito has some unique properties compared to those of atrial and ventricular Ito, suggesting a different molecular basis. We therefore characterized the expression of alpha-subunits encoding Ito-like currents and the K+-channel interacting protein 2 (KChIP2) beta-subunit. We demonstrated important differences in the expression of Kv3.4; encoding a TEA-sensitive Ito channel, and of KChIP2, that might play an important role in the specific molecular composition of Purkinje Ito. We also characterized another important repolarizing current, the delayed rectifier (IK) that had been reported to be absent or small in PCs. We found that I K· in PCs has properties typical of those observed in other regions of the heart; and IK channel subunits ERG, KvLQT1 and minK were more sparsely expressed in PCs than in ventricular muscle (VM), potentially explaining the tendency of PCs to generate arrhythmias due to abnormal repolarization. We also noted important differences in the expression of the Ca2+-channel subunits (Cav1.2, Cav3.1, 3.2 and 3.3), the Na+/Ca2+-exchanger subunit NCX1 and the hyperpolarization-activated channel subunits HCN1, 2 and 4. Studies in human PCs confirmed that some of the unique PC ionic properties observed in dogs are also present in man. Finally, we showed that an experimental cardiac disease paradigm (congestive heart failure) causes characteristic ionic remodeling in PCs that may explain their role in potentially lethal arrhythmias associated with heart failure. / Our findings support the hypothesis of a unique and important molecular basis for the control of repolarization in cardiac PCs.

The incidence of infections caused by antibiotic-resistant bacteria and fungi is rising rapidly. Once considered as little more than a nuisance, antibiotic resistance has become a serious threat. The mortality rate for some infections is approaching that of the pre-antibiotic era. New antimicrobials are needed urgently. Prior to the introduction of any new antimicrobial, comprehensive toxicity and efficacy profiles are assessed in preclinical studies. This thesis focuses on two key stages of preclinical antimicrobial drug development, specifically compound screening in vitro and animal efficacy testing in vivo. We developed a sensitive colorimetric platform with high-throughput capacity for the rapid screening of candidate antimicrobials. This platform could be adapted to assess compounds targeting a range of bacteria, fungi (such as Candida albicans), and protozoan parasites (such as Leishmania major). When this assay was modified to measure minimum inhibitory concentrations (MICs) for bacteria, 100% agreement within one dilution was achieved compared to the gold-standard method. A novel antifungal compound was taken forward to animal testing in an immunocompromised mouse model. We demonstrated herein that a histone deacetylase inhibitor in combination with an imidazole can synergise to produce a potent antifungal effect. A dose-dependent response, defined as a lower fungal burden and a higher survival rate, was achieved with increasing concentrations of the novel inhibitor.

Transforming growth factor-$ beta$ (TGF-$ beta$) is a multifunctional polypeptide growth factor which is believed to play an important role in the growth and differentiation of uterine cells. Although the expression of TGF-$ beta$ in the uterus has been previously described, the receptors for TGF-$ beta$ in this tissue have not been characterized. In the present study, the cell surface receptors for TGF-$ beta$ were characterized on cultures of stromal cells prepared from human endometrial biopsies, and on a human endometrial epithelial cell line (RL95-2) using affinity labeling techniques. On stromal cells, five TGF-$ beta$ binding proteins were identified. Analysis of the sensitivity of these proteins to dithiothreitol and phosphatidylinositol-specific phospholipase C, together with results from immunoprecipitations with anti-TGF-$ beta$ receptor antibodies, confirmed that three of these binding proteins correspond to the cloned type I, II, and III TGF-$ beta$ receptors. The other two binding proteins exhibited characteristics of isoform-specific glycosyl-phosphatidylinositol-anchored TGF-$ beta$ binding proteins. On RL95-2 cells, three TGF-$ beta$ binding proteins, corresponding to the type I, II and III TGF-$ beta$ receptors, were identified. The number of receptors on endometrial cells and their relative affinity for TGF-$ beta$ was estimated by Scatchard analysis. These receptors are responsive to physiological concentrations of TGF-$ beta$ as demonstrated by the effect of TGF-$ beta$ on DNA synthesis in these cells. Accordingly, they have the potential to respond to TGF-$ beta$ expressed in the endometrium in an autocrine and/or paracrine manner.

The polyamines spermine and spermidine are found ubiquitously in all mammalian cells where they play a role in a variety of cellular processes. In recent years, the interaction of polyamines with a number of structurally and functionally distinct cation channels has been described. These studies have shown that intracellular polyamines cause a voltage-dependent block of currents from inwardly rectifying K+ channels, AMPA and kainate type glutamate receptors, and neuronal nicotinic acetylcholine receptors. As result of the voltage-dependent polyamine block, these channels conduct current at negative membrane potentials but do not conduct current at positive membrane potentials. This property is termed inward rectification and likely plays an important role in the function of these channels. / The focus of my thesis has been to study the polyamine mediated inward rectification of neuronal nicotinic acetylcholine receptors (nAChRs). / To determine whether spermine causes the rectification of nAChRs in native neurons, I used the Gyro mouse model that lacks spermine due to a deletion in the gene coding for spermine synthase, the enzyme that catalyzes the production of spermine. (Abstract shortened by UMI.)

Potassium currents play an essential role in integrating neuronal signals by modulating the excitability of neurons and determining the action potential waveforms and firing patterns. Previously, Cooper and colleagues characterized voltage-gated K currents in neonatal neurons from the rat superior cervical ganglia (SCG). They demonstrated a positive correlation between the appearance of fast-inactivated currents (IAf) and the mRNA levels for the Kv4.2 voltage-gated potassium channel gene. Particularly, in cultured postnatal day 1 (P1) SCG neurons, both IAf and Kv4.2 mRNA decrease to very low levels after 14 days in culture. However, mRNA levels for other Kv genes that could also be responsible for Af currents on SCG neurons remained to be determined. My hypothesis was that Kv4.2 encodes K channel subunits underlying IAf in sympathetic neurons. To test this hypothesis, I built an adenoviral construct with a Kv4.2 insert to manipulate Kv4.2 expression in these neurons and compare the electrophysiological and pharmacological profiles of native IAf and Kv4.2-induced currents. (Abstract shortened by UMI.)

The overall aim of this thesis was to analyze the expression of key vasoregulatory genes in the vascular endothelium and smooth muscle following hypoxia. Hypoxia caused a reversal in endothelial cell function such that the vascular endothelium enhanced alpha-agonist induced contractions. Downregulation of endothelial nitric oxide synthase (eNOS) and impaired endothelium-dependent vasodilation were observed in the aortas of rats exposed to hypoxia for 12 and 48 hours. Following stimulation of aortic rings with acetylcholine, cyclic GMP and nitrite levels were also reduced. Decreased endothelial NO release may impair the vascular responses which defend vital organ oxygenation. This decrease in the capacity for vasodilator synthesis following hypoxia cannot by itself, however, mediate the observed enhancement of contractility. An increase in aortic endothelin-1 (ET-1) mRNA and protein was localized to the vascular endothelium. Treatment with the ET-1 receptor antagonist, BQ123, mimicked endothelial denudation and confirmed that locally secreted ET-1 accounts for endothelium-dependent enhancement of contractility. Concurrently, hypoxia caused an endothelium-independent impairment of smooth muscle reactivity to alpha-adrenoreceptor activation. Despite a decrease in eNOS in the aorta of rats exposed to hypoxia, Ca2+-dependent NOS activity remained unchanged suggesting the upregulation of neuronal NOS (nNOS). In endothelium-denuded aortic rings from hypoxic rats, L-NAME enhanced the contractile response to phenylephrine. In addition, it normalized the hypoxia-induced hypocontractility in intact aortic rings. The presence of nNOS was confirmed by Western blot analysis, immunohistochemistry, in situ hybridization and RT-PCR. Rat nNOS mRNA transcript bearing the alternately spliced exon 1b was shown to be expressed in the medial layer of aortas from hypoxic rats and in vascular smooth muscle cells cultured under hypoxic conditions. Cloning and analysis of the 5' regulatory

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na$ sp+$/H$ sp+$ exchanger (NHE) activity in various types of cells such as osteoblastic cells and renal proximal tubule OK cells. Na$ sp+$/H$ sp+$ exchangers are plasma membrane transporters catalyzing the electroneutral exchange of 1 H$ sp+$ for 1 Na$ sp+.$ Several mammalian isoforms of NHE have been so far identified with each mediating a variety of specific functions. Parathyroid hormone, playing an essential role in the physiology of blood Ca$ sp{2+}$ and phosphate homeostasis, inhibits renal proximal tubular bicarbonate reabsorption by inhibiting the apically located Na$ sp+$/H$ sp+$ exchanger. However, which specific isoform of NHE mediated this effect and the specific signaling components involved were unknown. In our studies we determined that Na$ sp+$/H$ sp+$ exchanger NHE-3 isoform is expressed in the renal proximal tubule OK cells and that N-terminal PTH and PTHRP analogues upon binding to their receptor stimulate both the PKA and the PKC pathways, each of which can independently lead to inhibition of this exchanger activity. NHEs also play an important role in the regulation of intracellular pH which is subject to fluctuation occurring during the process of hormone stimulated bone formation and bone remodeling. Again the specific NHE isoform(s) mediating this effect and the signaling pathways involved were unidentified. It is determined by our studies that NHE type 1 is expressed in osteoblastic cell line, UMR-106 cells, and that PTH and PTHRP stimulate this exchanger via a cAMP-dependent pathway exclusively. It was believed that motivation of NHE-1 in the UMR-106 cells and inhibition of NHE-3 in the OK cells by N-terminal analogues of PTH and PTHRP involves binding of these analogues to a common G protein-coupled receptor called the "classical" PTH/PTHRP receptor. However, with the recent discovery of other PTH and/or PTHRP receptor, this hypothesis is no longer clear

Human seminal plasma contains a protein factor that can inhibit the motility of both demembranated-reactivated and intact spermatozoa. This factor, named seminal plasma motility inhibitor (SPMI), was orignally isolated from seminal plasma and shown to orginate exclusively from the seminal vesicles, where its specific activity is 5- to 10-fold higher than it is in seminal plasma. The present study aimed at investigating the mechanism responsible for this difference in activity. Analysis of semen after ejaculation allowed to demonstrate that this difference in SPMI specific activity is due to the presence of a predominant 52 kDa SPMI precursor form in seminal vesicle fluid which is rapidly degraded after ejaculation by prostatic proteases. In addition, SPMI precursor was found to be associated with semen coagulum proteins and abnormal processing of the precursor in semen was associated with poor sperm motility. / A novel method was developed to purify SPMI precursor from seminal vesicle fluid and semen coagulum. Prostate-specific antigen (PSA) hydrolyzed SPMI precursor in a manner reminiscent of its processing in whole semen. Biochemical analysis of the precursor protein and its hydrolysis products provided evidence that SPMI precursor is identical to semenogelin, the main structural protein of semen coagulum. The purified precursor inhibited the motility of intact spermatozoa in a reversible and dose-dependent manner. / Finally, the characterization of SPMI molecular processing by PSA was addressed. The results directly demonstrate for the first time the restricted chymotrypsin-like specificity of PSA on its major physiological substrate. The sites of hydrolysis by PSA were identified along the precursor molecule and specific domains within the SPMI precursor responsible for biological activity and reactivity with various antibodies were mapped. / Overall, these results shed light on the photeolytic precessing of SPMI precursor occurring after ejaculation, and the associated change in SPMI activity on spermatozoa. The present findings provide evidence, for the first time, that a specific protein appears responsible for the observed low sperm motility in freshly ejaculated semen, and that its processing by PSA parallels the progressive increase in sperm motility observed during semen liquefaction.

The first part of my work evaluates bilateral supramaximal transcutaneous phrenic nerve stimulation as a diagnostic test for respiratory muscle fatigue. I found that twitch transdiaphragmatic pressure (Pdi,T) was inversely and linearly related to lung volume (V$ sb{ rm L}$) both before and after fatigue. Although fatigue caused significant decrease in Pdi,T amplitude at all V$ sb{ rm L}$, the fractional decrease in Pdi,T was greater at high V$ sb{ rm L}$, indicating the importance of V$ sb{ rm L}$ as an independent variable that needs to be controlled whenever Pdi,T is determined. Twitch mouth pressure (Pm,T) was found to be linearly related to twitch esophageal pressure (Pes,T), to Pdi,T, and to V$ sb{ rm L}$. All these relationships were reproducible. Diaphragmatic fatigue resulted in significant decrease in Pm,T proportional to the decrease in Pdi,T for a given V$ sb{ rm L}$ so that Pm,T-Pes,T and Pm,T-Pdi,T relationships were unchanged. Thus the Pm,T-V$ sb{ rm L}$ relationship can be used to assess diaphragmatic fatigue non-invasively. Paired phrenic nerve shocks which were well tolerated by normal subjects can be used to obtain a measure of the pressure-frequency curves of the diaphragm, which were reproducible. In particular, I showed that the pressure ratio of diaphragmatic twitch elicited by the second shock at 10Hz over that at 100Hz (T2$ sb{10/100}$) is a valuable index of low frequency fatigue. / In the second part of my work I studied the effect of respiratory muscle fatigue on ventilatory response to CO$ sb2$ and respiratory muscle recruitment. The data showed that ventilatory response and respiratory muscle recruitment patterns were different in a number of aspects between diaphragmatic fatigue and global inspiratory muscle fatigue. After diaphragmatic fatigue, the only change was an increase in the recruitment of rib cage muscles, which fully compensated for decreased diaphragmatic contractility because all the ventilatory parameters were constant. After global fatigue, both the diaphragm and rib cage muscles contributed less to breathing but expiratory muscles were recruited resulting in a decrease in end-expiratory P$ sb{ rm L}$ and an increased contribution of elastic energy stored within the respiratory system to inspiratory tidal volume generation. In spite of this, rapid shallow breathing developed while minute ventilation remained constant. These data suggest that the ventilatory control system can detect fatigue and has sufficient plasticity to alter inspiratory drive appropriately. The overall ventilation level can thus be maintained.

The actions of internal choline on single "maxi" Ca$ sp{2+}$-activated K$ sp{+}$ channels were studied in excised patches of chromaffin cell membrane. The channels had a unit conductance of approximately 200 pS in a physiological K$ sp{+}$ gradient. / Choline (20-70 mM) applied to the intracellular membrane surface dose dependently reduced outward current flow through the channel. The reduction in single channel current amplitude increased with depolarization. / These data suggest that choline is a fast blocker that binds within the channel pore. The K$ sb{ rm d}$(0) for the reaction is 90 mM while $ delta$ is 0.27, suggesting that the choline binding site senses 27% of the transmembrane electric field. / The average open state probability appeared not to be affected by choline at any of the membrane potentials that were studied.