Hyperkalemic periodic paralysis, HyperKPP, is an inherited progressive disorder of the muscles caused by mutations in the voltage gated sodium channel (NaV1.4). The objectives of this thesis were to develop a technique for measurement symptoms in vivo using electromyography (EMG) and to determine the mechanism by which Ca2+ alleviates HyperKPP symptoms, since this is unknown. Increasing extracellular [Ca2+] ([Ca2+]e) from 1.3 to 4 mM did not result in any increases in45Ca2+ influx suggesting no increase in intracellular [Ca2+] ([Ca2+]i) acting on an intracellular signaling pathway or on an ion channel such as the Ca2+sensitive K+ channels. HyperKPP muscles have larger TTX-sensitive22Na+ influx than wild type muscles because of the defective NaV1.4 channels. When [Ca2+] was increased from 1.3 to 4 mM, the abnormal 22Na+ influx was completely abolished. Thus, one mechanism by which Ca2+alleviates HyperKPP symptoms is by reducing the abnormal Na+ influx caused by the mutation in the NaV1.4 channel.

Activation of the intrinsic aqueous water-wire proton conductance (GAQ) in Hv1 channels is controlled by changes in membrane potential and the transmembrane pH gradient (ΔpH). The mechanism by which changes in ΔpH affect the apparent voltage dependence of GAQ activation is not understood. In order to measure voltage sensor (VS) activation in Hv1, we mutated a conserved Arg residue in the fourth helical segment (S4) to His and measured H+ currents under whole-cell voltage clamp in transfected HEK-293 cells. Consistent with previous studies in VS domain containing proteins, we find that Hv1 R205H mediates a robust resting-state H+ ‘shuttle’ conductance (GSH) at negative membrane potentials. Voltage-dependent GSH gating is measured at more negative voltages than the activation GAQ, indicating that VS activation is thermodynamically distinct from opening of the intrinsic H+ permeation pathway. A hallmark biophysical feature of Hv1 channels is a ~-40 mV/pH unit shift in the apparent voltage dependence of GAQ gating. We show here that changes pHO are sufficient to cause similar shifts in GSH gating, indicating that GAQ inherits its pH dependence from an early step in the Hv1 activation pathway. Furthermore, we show for the first time that Hv1 channels manifest a form of electromechanical coupling VS activation and GAQ pore opening. Second-site mutations of D185 markedly alter GAQ gating without affecting GSH gating, indicating that D185 is required for a late step in the activation pathway that controls opening of the aqueous H+ permeation pathway. In summary, this work demonstrates that the Hv1 activation pathway contains multiple transitions with distinct voltage and pH dependencies that have not been previously identified. The results reported here novel insight into the mechanism of VS activation in Hv1 and raise fundamental questions about the nature of pH-dependent gating and electromechanical coupling in related VS domain-containing ion channels and phosphatases.

Recent evidence has indicated that NSAIDs might have direct effects on CNS tissue in addition to their classical inhibitory action on COX enzymes. This thesis addresses this hypothesis using electrophysiological and behavioural techniques. The effects of fenamate and other NSAIDs on native neuronal GABA(_A), 5-HT(_3), nicotinic ACh, P2x and strychinine-sensitive glycine receptors, expressed on isolated vagus or optic nerves, was investigated using an extra-cellular recording technique. The fenamate NSAID, mefenamic acid (MFA) potentiated GABA (10µM)- evoked responses in the vagus nerve. Application of MFA also resulted in non-competitive inhibition of 5-HT-and a,βMeATP- evoked responses. Non-competitive like inhibition was also observed with flufenamic acid on DMPP- and a,βMeATP- evoked responses and with meclofenamic acid on GABA- evoked responses. Non-fenamate NSAIDs, including aspirin, did not significantly modulate the GABA(_A), 5-HT(_3), nicotinic ACh, P2x or glycine receptors. The cognitive and behavioural effects of fenamates and other NASIDs were then investigated. MFA (5-20mg/kg) caused a significant dose- and time-dependent enhancement in the non-spatial object discrimination working memory task when compared to saline controls. The enhancement observed with MFA was greater than that of the cognitive enhancer piracetam. This enhancement was not due to a change in non-mnemonic processes such as arousal, anxiety or locomotion. MFA also enhanced rats' performance in the spatial object location working memory task. The fenamate NSAID, meclofenamic acid (20mg/kg) mimicked the effect of MFA, but the non-fenamate NSAIDs aspirin and ibuprofen, did not enhance object discrimination indicating that these cognitive effects are not via inhibition of COX. The GABA(_A) receptor modulators diazepam, bicuculline and loreclezole, did not replicate the effect of MFA on object discrimination, suggesting that its effects do not depend entirely on the GABAa receptor. Scopolamine (0.25-lmg/kg) significantly impaired object discrimination in a dose-dependent manner. This action could be fully reversed by co-treatment with MFA (20mg/kg).In the T-maze task, MFA (20mg/kg) decreased the number of arm entry errors and days taken to reach criterion. The number of arm entry errors made when a 5-minute intra-trial interval was introduced was also significantly reduced by MFA compared with saline treated animals. In the radial maze, MFA (20mg/kg) did not decrease the number of never baited arm entries to reach criterion. However MFA did significantly reduce the number of re-entry errors to baited arms, compared to controls, when an intra-trial delay (10-30 sees) was introduced. These results support the hypothesis that MFA enhances spatial working memory and that these effects are not task-specific. Overall, the data in this thesis show that fenamate NSAIDs can directly modulate native neuronal ligand-gated ion channels and that MFA can enhance working memory in normal and scopolamine-impaired rats. These results suggest additional pharmacological potential for certain fenamate NSAIDs.

The thesis focuses on gated communities (GCs) as an example of residential separation in today's Prague. The GCs are a new specific phenomenon characterising the society in present post-industrial cities. It is a type of residence where high social status population concentrates behind walls and fences. The number of GCs grows more or less globally which results in increased experts' interest in GCs. This type of residence occurs also in Czechia, primarily in Prague. Experts' interest in the subject in our environment is, however, not as high as in other post-communist countries. This thesis follows up with previous studies and assesses in detail if - in context of the post-industrial transformation - the high social status population is separated in Prague GCs and why such type of residence appears. The document also partly focuses on the development and situation of residential separation and the consequences of GCs formation. It is based on several quantitative (data analysis, questionnaire survey) and qualitative (interviews) research methods. It turns up that on one hand the level of residential segregation and separation in Prague decreases, on the other hand we can see that the number of specific separated locations such as the GCs where the high social status population concentrates grows....

Student Number : 9709049J - MSc research report - School of Construction Economics and Management - Faculty of Engineering and the Built Environment / Spiralling violent crime in South Africa has led to the increased use of access control to restrict access to suburbs and thus make it more difficult to perpetrate violent crime against residents. This neighbourhood enclosure normally takes the form of booms, palisade fences and security guards to close off entire suburbs that were once or still are public property. This study examines the effect of the implementation of neighbourhood enclosure on property values. The primary methodology used to research this concentrated on the analysis of residential property values, bond approvals and relative amounts of property transferred within the period of late 2002 and mid 2004. Analysis was based on information available at the deed’s office. Limitations included the small sample size and the availability of information. Applicants for suburban closures can use the results of the study to verify or discredit claims of property value increases. Municipalities can match property values to services and rates. Results showed that property values that were found to be either similar in adjacent open and enclosed areas or more expensive in enclosed areas. The latter trend was evident in higher value properties. This illustrates a higher demand for properties in these enclosed areas than in the adjacent open areas. Further analysis of prices showed that, in all cases over the same period, prices increased by more in enclosed areas than in open areas. This statistic proves the hypothesis, that neighbourhood enclosure increases the value of properties within the enclosure when compared to similar properties in adjacent open areas. Analysis of the value of bonds granted in an area showed that banks appear to ignore the enclosure status of the immediate area.

It is clear that the voltage-gated proton channel HVCN1 plays an essential role in a range of cell types, in particular immune cells. Previous published work has confirmed the existence of proton channels in both murine and human macrophages. However, the role of HVCN1 in macrophages has not been investigated. Given that the current literature on voltage-gated proton channels in immune cells has found HVCN1 to be involved in several cellular processes (such as the respiratory burst and signalling events) it is important to establish its functional role in macrophages, which are a crucial constituent of the immune system. The aim of my thesis was to investigate the function of voltage-gated proton channels in macrophages with the use of mice with a disrupting mutation within the Hvcn1 gene, which results in HVCN1 loss. In particular, I wanted to address how Hvcn1-/- macrophages responded to LPS activation. I hypothesised that HVCN1 regulates the respiratory burst of macrophages and that it potentially modulates mitochondrial ROS production, and in doing so, may affect several functional aspects of macrophage biology.

Cette thèse étudie l'utilisation de la microscopie FRET en décalage temporelle pour la détection de co-localisation des deux protéines membranaires E- et N-cadhérine. Ces protéines sont importantes pour les contacts cellule-cellule et jouent un rôle important dans la transition épithélio-mésenchymateuse (EMT), un processus clé dans la métastase du cancer. Dans EMT cellules perdent leurs marqueurs épithéliaux (par exemple E-cadhérine) et acquièrent des marqueurs mésenchymateuses (par exemple N-cadhérine), ce qui augmente leur motilité et le caractère invasif pour échapper à la tumeur primaire dans la circulation sanguine en tant que ce qu'on appelle des cellules tumorales circulantes (CTC). Ce manuscrit porte sur la détection des CTC qui ont subi une EMT partielle, montrant un phénotype hybride (épithélio-mésenchymateuse) et co-expriment E et N-cadhérine, par des études de co-localisation en utilisant le FRET sur une lignée modèle de cellules. FRET (Transfer d’énergie par résonance de type Förster) est un transfert d'énergie non-radiatif entre deux molécules qui sont en résonance et à proximité (environ 1-20 nm). Une co-localisation d’E- et N-cadhérine en clusters serait donc détectable par FRET. La coloration des cadhérines qui a été fait en utilisant des anticorps spécifiques marqués avec une donneur qui a un longue durée de vie de fluorescence, le complexe de terbium Lumi4-Tb (TbL4) de Lumiphore, Inc., et diverses accepteurs. La longue durée de vie du donneur et la longue durée de vie d’accepteur sensibilisé (FRET) pourraient être imagés dans une configuration de microscopie en décalage temporelle. L’imagerie en décalage temporelle présente plusieurs avantages par rapport à l'imagerie stationnaire en termes de suppression efficace du bruit de fond dans des échantillons biologiques. L'installation décrite dans ce manuscrit est basée sur l'utilisation d'une caméra CCD intensifiée, une source d'excitation laser pulsé en UV et un décalage temporel défini de quelques microsecondes entre l'excitation et l'acquisition d'image. L’imagerie de FRET en décalage temporelle a été utilisée pour étudier des différents échantillons biologiques (intracellulaire et située à la membrane). Bien que les deux marqueurs protéiques pourraient imager correctement sur les mêmes cellules, FRET entre E- et N-cadhérine ou E- et E-cadhérine ne pouvaient pas être détectés. Des expériences de contrôle avec des anticorps contre le même anticorps primaire ont révélé des signaux de FRET forts à cause de la reconnaissance des anticorps donneurs et accepteurs aux mêmes anticorps primaires. Ces résultats de FRET entre deux anticorps différents séparés par quelques nanomètres démontrent la faisabilité de mesurer des interactions protéine-protéine et la co-localisation sur des membranes à l'aide de l’imagerie de FRET en décalage temporelle en utilisant TbL4. Imagerie en décalage temporelle de quelques microsecondes est particulièrement intéressante pour l'enquête des interactions protéine-protéine dans des échantillons biologiques hautement autofluorescentes, tels que les tissus cancéreux. / This thesis investigates the use of time-gated FRET microscopy for detection of colocalization of two membrane proteins, E- and N-cadherin. These proteins are important for cell-cell contacts and have an important role in the epithelial to mesenchymal transition (EMT), a key process in cancer metastasis. In EMT cells lose their epithelial markers (such as E-cadherin) and gain mesenchymal markers (such as N-cadherin), increasing their motility and invasiveness, enabling escape from the primary tumor into the bloodstream as so called circulating tumor cells (CTCs). This manuscript focuses on the detection of CTCs that have undergone partial EMT, displaying a hybrid phenotype (epithelial-mesenchymal) and co-express E- and N-cadherin, by FRET co-localization studies on a model cell line. FRET (Förster resonance energy transfer) is a non-radiative energy transfer between two molecules that are in resonance and in close proximity (ca. 1-20 nm). A co-localization of E- and N-cadherin in clusters would therefore be detectable by FRET. The staining of the cadherins was done by using specific antibodies labelled with a long lifetime donor, the terbium complex Lumi4-Tb (TbL4) from Lumiphore, Inc., and various acceptors. The long lifetime donor and long lifetime sensitized acceptor emission (FRET) could be imaged in a time-gated microscopy setup. Time -gated imaging has several advantages compared to steady state imaging in terms of efficient background suppression in biological samples. The setup described in this manuscript is based on the use of an intensified CCD camera, a pulsed UV-laser excitation source, and a defined (µs) delay between excitation and image acquisition. In addition to the E- and N-cadherin FRET experiments the time-gated FRET imaging microscopy was used to investigate different biological samples (intracellular and membrane located). Although both protein markers could be successfully imaged on the same cells, FRET between E- and N-cadherin or E- and E-cadherin could not be detected. Control experiments with antibodies against the same primary antibody revealed strong time-gated FRET signals due to binding of both donor and acceptor antibodies to the same primary antibodies. The successful time-gated imaging of two different antibodies separated by a few nanometers demonstrates the feasibility of probing protein-protein interaction and co-localization at membranes using TbL4 based time-gated FRET imaging. Microsecond time-gated imaging is especially interesting for the investigation of protein-protein interactions in highly autofluorescent biological samples such as cancer tissues.

There is a complex relationship between genetic mutations and their phenotypic expression. Two patients who carry the exactly same disease-causing mutation can have drastically different severity in disease symptoms. Such phenotypic variations may be due to environmental factors, such as diet, stress and temperature, as well as genetic variations, including single nucleotide polymorphisms and copy number variations in other loci. From a clinical point of view, the environmental and genetic factors that modify phenotypic severity are important because, even when we cannot correct the original mutations, it may be able to reduce the burden of genetically inherited disorders by manipulating these modifiers. To study environmental and genetic modifiers, we used Shudderer (Shu), a Drosophila mutant for the voltage-gated sodium (Nav) channel gene, as an experimental model. Nav channels are essential for generation and propagation of action potentials in neurons. Reflecting their functional importance in neural function, mutations in the Nav channel genes are associated with a variety of human neurological disorders. Shu mutants display severe behavioral defects (e.g., spontaneous jerking and heat-induced seizures) and morphological abnormalities (e.g., indented thorax and down-turned wings). The goal of this study was to identify and characterize the environmental and genetic factors that modify behavioral and morphological phenotypes of Shu mutants. For environmental modifiers, we serendipitously discovered that a diet supplemented with milk dramatically reduces the Shu phenotypes. To identify genetic modifiers, we took two independent approaches, microarray analysis and unbiased forward genetic modifier screening. We found that reduction of Gadd45 or GstS1 activity leads to suppression of the Shu phenotypes to different degrees. Intriguingly, the effects of these genetic and environmental modifiers apparently converge into enhancement of the GABAergic inhibitory system. Because the molecular and cellular mechanisms underlying the basic neurobiological processes are highly conserved between flies and humans, our findings are expected to provide fundamental insights into genetic and environmental modifiers for human Nav channel gene mutations, leading to the future development of novel strategies for preventing and treating disorders caused by dysfunctional Navchannels.

Neurological disorders, such as epilepsy, often result from inherited or newly acquired genetic mutations. However, individuals possessing the exact same disease-causing mutations can exhibit dramatic differences in the severity of their symptoms. These differences can be explained in part by environmental factors, such as the microbes in our gut, that play an important role in the manifestation of disease symptoms. Within the last decade, microbes living in the gut have established themselves as an environmental factor with profound effects on our health and well-being. Of special interest is the relationship between the gut microbiota and neurological disease. The goal of my thesis was to: 1) characterize the gut microbiota composition and 2) understand how the gut microbiota modulates seizure-like behavior using Shudderer, a fruit fly (Drosophila melanogaster) model of epilepsy. Shudderer flies possess a mutation in the voltage-gated sodium channel gene and display seizure-like behavioral abnormalities including spontaneous tremors and heat-induced seizures. We identified differences in the microbial composition of the gut microbiota between Shudderer and control (healthy) flies. We also found that by removing the gut microbiota we could improve seizure-like behavior of Shudderer flies as well as another Drosophila mutant harboring a similar genetic mutation. Together, these findings provide evidence that a bidirectional interaction exists between the gut microbiota and neurological function. Since the molecular and cellular mechanisms controlling basic biological processes are highly conserved between fruit flies and humans, these findings are expected to be applicable to mammalian systems, including humans, and may lead to the future development of novel therapeutics to treat epilepsy and other neurological disorders.

Neuronal nicotinic acetylcholine receptors can have their function modulated by zinc. Depending on concentration and subunit composition, zinc either inhibits or potentiates receptor function. The zinc ion potentiates the alpha4beta4 receptor at non-agonist binding interfaces or "pseudo sites." Zinc potentiation is reduced if certain residues are mutated or spatially interfered with. The residue contributing most to this potentiation reduction effect is histidine 162 on the alpha4 subunit. The anthelmintic compound levamisole potentiates acetylcholine response of certain neuronal nicotinic receptors. Levamisole and its functional analogues morantel, oxantel, and pyrantel all were found to potentiate alpha4beta4 receptors at low (µM) concentrations and inhibit them at high (mM) concentrations. Oxantel showed the greatest degree of potentiation, about a third of the maximal zinc potentiation measured. Oxantel was screened using the substituted cysteine accessibility method (SCAM) against the residue histidine 162 as well as nearby alpha4 residues histidine 61 and glutamate 59 and the beta4 residue aspartate 195. Screening was carried out by mutating said residues into cysteine, followed by covalent linkage with a disulfide bridge of that residue with a methanethiosulfonate compound. SCAM experiments allowed testing of the effects of a single residue and the area immediately adjacent to it. Receptors that lost zinc potentiation capacity from site-directed mutagenesis at the his 162 residue and subsequent methanethiosulfonate reaction still showed regular potentiation following oxantel treatment. Although these compounds exhibit similar biphasic potentiation dose-response curves as zinc, their mechanism for potentiation is not through the same mechanism.