The voltage gated potassium channel Kv1.2 plays a key role in the central nervous system and mutations in Kv1.2 cause neurological disorders such as epilepsies and ataxias. In the cerebellum, regulation of Kv1.2 is coupled to learning and memory. We have previously shown that blocking Kv1.2 by infusing its specific inhibitor tityustoxin-kα (TsTX) into the lobulus simplex of the cerebellum facilitates eyeblink conditioning (EBC) and that EBC itself modulates Kv1.2 surface expression in cerebellar interneurons. The metabotropic glutamate receptor mGluR1 is required for EBC although the molecular mechanisms are not fully understood. Here we show that infusion of the mGluR1 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) into the lobulus simplex of the cerebellum mimics the facilitating effect of TsTX on EBC. We therefore hypothesize that mGluR1 could act, in part, through suppression of Kv1.2. Earlier studies have shown that Kv1.2 suppression involves channel tyrosine phosphorylation and endocytocytic removal from the cell surface. In this study we report that an excitatory chemical stimulus (50mM K+-100µM glutamate) applied to cerebellar slices enhanced Kv1.2 tyrosine phosphorylation and that this increase was lessened in the presence of the mGluR1 inhibitor YM298198. More direct evidence for mGluR1 modulation of Kv1.2 comes from our finding that selective activation of mGluR1 with DHPG reduced the amount of surface Kv1.2 detected by cell surface biotinylation in cerebellar slices. To determine the molecular pathways involved we used an unbiased mass spectrometry-based proteomics approach to identify Kv1.2-protein interactions that are modulated by mGluR1. Among the interactions enhanced by DHPG were those with PKC-γ, CaMKII, and Gq/G11, each of which had been shown in other studies to co-immunoprecipitate with mGluR1 and contribute to its signaling. Of particular note was the interaction between Kv1.2 and PKC-γ since in HEK cells and hippocampal neurons Kv1.2 endocytosis is elicited by PKC activation. We found that activation of PKCs with PMA reduced surface Kv1.2, while the PKC inhibitor Go6983 attenuated the reduction in surface Kv1.2 levels elicited by DHPG and PMA, suggesting that the mechanism by which mGluR1 modulates cerebellar Kv1.2 likely involves PKC. mGluR1 has been shown to signal independently of the agonist through a constitutively active, protein kinase A-dependent pathway in the cerebellum. Using HEK293 cells we show that co-expression of mGluR1 increases the surface expression levels of Kv1.2. This effect occurs in absence of mGluR1 agonists and in the presence of a noncompetitive mGluR1 inhibitor YM298198. Co-expression of known downstream effectors of the agonist driven mGluR1 pathway such as PKC-γ, CaMKIIα, Grid2 had no effect on Kv1.2 surface expression or on the ability of mGluR1 agonist to modulate that expression. In contrast, the inverse agonist BAY 36-7620 significantly reduced the mGluR1 effect on Kv1.2 surface expression, as did pharmacological inhibition of PKA with KT5720. Therefore, mGluR1 is involved in regulation of surface Kv1.2 via dual mechanisms, the agonist dependent mechanism reduces surface Kv1.2 via PKC, while agonist independent constitutive mechanism increases surface Kv1.2 via PKA.

Rio de Janeiro, Brazil’s second biggest city is both seen as a leisurely paradise and a dangerous drug-warzone at the same time, two contra-dictory spatial images. In Rio de Janeiro, the urban conflict between the rich, formal city and the favela and the police and the favela has produced an abstract spatial image of the favela and its residents as being violent. In the same way in which the formal city and police have produced their abstract spatial image and social space of the fa-vela, those in the favela has produced their own abstract spatial imag-es of the police, the formal city and of themselves. This development in Rio de Janeiro is juxtaposed with the similar development in Los Angeles during their drug war in the 1980’s.This study analyzes, through narratives, how the spatial images in both Rio de Janeiro and Los Angeles have been constructed and shaped their urban landscapes into a fortified and exclusionary one.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

The task for this master thesis was to create a specification for a second prototype of Scandilumen´s handheld gated night vision system. Current prototype is analogue and is to be upgraded with a digital interface. The specification was to contain information about manufacturers and performance on critical components such as image intensifier tube, image sensor and display. Scandilumen have previous experience with CCD cameras and wanted to know if the CMOS technology were sensitive enough to work properly in gated systems where high sensitivity is critical. Different image processing techniques was analyzed to find out the possibility to enhance image quality with an FPGA built-in into the system. When the specification is implemented, Scandilumen will have a prototype up-to-date with a digital interface and real time image enhancement. / Uppdraget i denna magisteruppsats var att ta fram en specifikation för en andra prototyp av Scandilumens grindade mörkerkamera. Nuvarande prototyp är analog och skall uppgraderas till en digital variant med display och anslutningsmöjlighet till dator. Specifikationen skall innehålla uppgifter om vilka ingående komponenter som skall användas samt vilken prestanda de skall ha. Exempel på dessa komponenter är bildförstärkarrör, bildsensor och displayer. Stor vikt har lagts vid att avgöra vilken typ av bildsensor som skall ingå i systemet. Scandilumen har tidigare erfarenhet av CCD-kameror men ville undersöka om CMOS-tekniken var känslig nog för denna typ av applikation. En jämförelse gjordes mellan de olika teknikerna med fokus på de höga krav som ställs på känslighet. Dessutom analyserades olika typer av bildbehandling som är lämpliga för systemet och som också går att implementera i en FPGA på lämpligt sätt. Om specifikationen följs kommer Scandilumen ha en prototyp uppdaterad med ett digitalt format och den senaste tekniken.

The Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. It has been suggested that CNGCs contribute to a wide array of biological functions such as pollen tube growth and pathogen defence signalling. However, the precise mechanisms by which AtCNGCs act, and the extent of their biological roles, have yet to be fully elucidated. AtCNGC11/12, the chimeric CNGC that resulted from the fusion of AtCNGC11 and 12, induces a number of pathogen defence related phenotypes in the Arabidopsis mutant cpr22. Spontaneous lesion formation is one such phenotype. Interestingly, when AtCNGC11/12 is transiently expressed in N. benthamiana it causes cell death which was characterized in this study. Also, AtCNGC11/12 was used to investigate the structural features responsible for the proper function and regulation of AtCNGCs. Electron microscopic analysis of the AtCNGC11/12-induced cell death showed similar characteristics to programmed cell death (PCD), such as plasma membrane shrinkage and vesicle formation. Interestingly caspase-1 inhibitors and the silencing of vacuolar processing enzyme, a plant enzyme with caspase-1 activity, suppressed the induction of cell death. Additionally, pharmacological analyses indicated that the AtCNGC11/12-indiced cell death was also dependent on Ca2+. Furthermore, 3 amino acid residues, R190, A225, and G287, were demonstrated to be essential for AtCNGC11/12-induce cell death. Taken together, these results indicate that the cell death that develops in the cpr22 mutant is indeed PCD and that AtCNGC11/12, is at the point of, or up-stream of, the Ca2+ signal necessary for the development of HR. Furthermore, the functionality of AtCNGC11/12 as a model for AtCNGC structure-function analyses was demonstrated by the identification of several amino acids necessary for cell death development. Yoshioka et al. (2006) demonstrated that the loss of AtCNGC11 or 12 results in decreased resistance to avirulent isolates of the oomycete pathogen, H. arabidopsidis. Thus, the present biological role suggested for AtCNGC11 and 12 is in pathogen defence, specifically within effector triggered immunity (ETI). Like AtCNGC11 and 12, AtCNGC2 has been demonstrated to contribute to pathogen defence signalling but has also been implicated in other physiological responses such as ion stress and senescence. To better understand the roles of AtCNGC11 and 12 in both pathogen defence and other Ca2+ dependent signalling processes, I have investigated promoter:GUS reporter lines, as well as, AtCNGC11 and 12 KO and RNAi silenced lines subjected to various treatments. From this work, I have demonstrated that AtCNGC11 and 12 have similar expression patterns during pathogen defence, development, and dark-induced senescence. Additionally, the findings presented here further characterize AtCNGC11 and 12 as contributors to ETI rather than PAMP triggered immunity. Furthermore, I demonstrated that AtCNGC11 and 12 are likely involved in the endogenous movement of Ca2+, contributing to a range of Ca2+ associated signalling pathways including gravitropism and senescence. Taken together, these results have greatly improved the characterization of AtCNGC11 and 12; significantly contributing to the understanding of a large and increasingly important channel family.

This thesis asks the question: how can one describe the development of gated communities in Guatemala City? It collects and analyzes data on gated communities in Guatemala City in order to explore the nature of their development in a violent geographical region, which has also been neglected by the academic community. It argues that the development of gated communities in Guatemala City does not fit the mutually exclusive ‘security’ argument as scholars have made. Instead, a mixture of economic factors, social status, weak governance, and security concerns are involved as large private corporations draw upon security-related fears, unregulated development of real estate and weak governance, resulting in a disorganized model of spatial organization. This study adds to the growing body of literature on gated communities by laying the groundwork needed to fill the gap that currently exists in Central America.

This study aims to analyze the emergence and evaluation of a new type of suburbanization in T&uuml / rkiye, which are concomitant with the rise of new middle class having a high purchasing power. It examines different urbanization and suburbanization processes in various societies and demonstrates that the suburbanization of T&uuml / rkiye does not exactly fit in these models. Such a suburban expansion is taking place under the prevailing impact of political economy in the world and leads to a social segregation within metropolitan areas, which is argued to become permanent. Upper middle class have developed privatized, enclosed, and monitored exclusive spaces of residence, work, leisure, and consumption. The main focus of the study, therefore, is the gated communities, which is the last extension of suburbanization. In this thesis, the gated communities are mainly residential in character and offer a new concept of life-style, which is based upon the idea of total security and retreat from the illnesses of the urban core in terms of noise, dust, disorder, crowds and related issues. It is argued that, in the specific case of Angora Evleri-Kooperatif-18, gated communities can be viewed as a further theme of fragmentation of the city of Ankara. The new urban fragmentation indicates a dual process of increasing social and spatial polarization on the urban land. These dualities have been identified in society. One part of the society has experienced affluence, and success while the other has suffered degradation. The economic growth has been at the expense of sharp increases both at the top and bottom ends of the income distribution. Social inequality, in return, has manifested itself spatially.

<p>Fibroblast growth factor homologous factors (FHFs) are noncanonical members of the fibroblast growth factor family (FGFs, FGF11-FGF14) that bind directly to voltage gated sodium channels (VGSCs), thereby regulating channel activity and consequently neuronal excitability. Mutations in FGF14 cause spinocerebellar ataxia while FGF13 is a candidate for X-linked mental retardation. Since FGF13 and FGF14 are nearly identical within their respective VGSC-interacting domains, those distinct pathological consequences have generally been attributed to regional differences in expression. I have shown that FGF13 and FGF14 have non-overlapping subcellular distributions and biological roles even in hippocampal neurons where both are prominent. While both FHFs are abundant in the axon initial segment (AIS), only FGF13 is observed within the soma and dendrites. shRNA knockdown and rescue strategies showed that FGF14 regulates axonal VGSCs, while FGF13 only affects VGSCs in the somatodendritic compartment. Thus, FGF13 and FGF14 have nonredundant roles in hippocampal neurons, with FGF14 acting as an AIS-dominant positive regulator and FGF13 serving as a somatodendritic negative regulator. Both of these FHFs also perform important non-VGSC regulatory roles. FGF14 is a novel potassium channel regulator, which binds to KCNQ2 and regulates both localization and function. FGF14 is also capable of serving as a “bridge” between VGSCs and KCNQ2 thus implicating it as a broad organizer of the AIS. FGF13, on the other hand is involved in a new form of neuronal plasticity called axon initial segment structural plasticity. Knockdown of FGF13 impairs AIS structural plasticity and reduces L-type CaV current through channels known to be important to this new form of plasticity. Both of these novel non-VGSC roles are specific to the FHF in question because FGF13 does not regulate KCNQ2 whereas FGF14 knockdown does not affect AIS position. These data imply wider roles for FHFs in neuronal regulation that may contribute to differing roles in neural disease.</p> / Dissertation

Made available in DSpace on 2015-04-16T14:49:08Z (GMT). No. of bitstreams: 1 parte1.pdf: 14730236 bytes, checksum: 2ccdda99142dce312e725c851419b343 (MD5) Previous issue date: 2012-11-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The cities as they grow, has been closed between new walls, leading to worsening of issues the streets and occupation of urban space, the privatization of public spaces, the neighborhood relationship mandatory, the allusion of greater security and status. With this, the public spaces of the contemporary city are marked by selective appropriation and differentiated spaces that should be accessible to everyone. The gated alters the relationship between public and private from the privatization of public spaces. In the southern city of João Pessoa, only in the neighborhood of the Sun Portal, has today closed with seven condominiums, each with its large leisure areas. How cities can generate a variety of uses with an extension of breakaway walls for those who owe more purchasing power? The objective of this research is to analyze the areas of collective use of closed condominiums: Cabo Branco Residence Prive, Puerta del Sol and Orchid Grove, the morphology and sociospatial organization, noting planning rules, forms, uses and user behaviors . The methodology of this research is structured using as reference base: Lynch (1997), Kohlsdorf (2005) and Gehl (2006). Despite the formal and functional similarities visible in these collective spaces, there are fundamental differences in the spatial practices of its users. Know them in order to understand the dynamics and performance of these spaces and the social logic of segregation and production part of contemporary culture. / As cidades à medida que crescem, tem se fechado entre novos muros, levando ao agravamento das questões relativas as ruas e ocupação do espaço urbano, a privatização de espaços públicos, a relação de vizinhança obrigatória, a alusão de uma maior segurança e status. Com isso, os espaços públicos da cidade contemporânea são marcados pela apropriação seletiva e diferenciada dos espaços que deveriam ser acessíveis a todos. O condomínio fechado altera a relação entre público e privado a partir da privatização de espaços públicos. Na zona sul da cidade de João Pessoa, somente no Bairro Portal do Sol, conta-se hoje com sete condomínios horizontais fechados, cada um com as suas grandes áreas de lazer. Como as cidades podem gerar uma diversidade de usos com uma extensão de muros separatistas para aquele que deve mais poder aquisitivo? O objetivo desta pesquisa é analisar as áreas de uso coletivo dos condomínios horizontais fechados: Cabo Branco Residence Prive, Porta do Sol e Bosque das Orquídeas, quanto à morfologia e organização socioespacial, observando as regras de planejamento, as formas, usos e comportamentos dos usuários. A metodologia desta pesquisa será estruturada utilizando como referência base: Lynch (1997), Kohlsdorf (2005) e Gehl (2006). Apesar das semelhanças formais e funcionais visíveis nestes espaços coletivos, existem diferenças fundamentais nas práticas espaciais dos seus usuários. Conhecê-las para poder compreender as dinâmicas e desempenho destes espaços bem como a lógica social de produção da segregação e parte da cultura contemporânea.

Les canaux ioniques pentamériques activables par un ligand (pLGIC) sont l'une des principales familles de canaux transmembranaires. Ils permettent la transduction rapide du signal dans le système nerveux central et périphérique via la liaison de neurotransmetteurs. Les pLGIC sont également présents chez les archées et les bactéries. Seuls deux pLGIC bactériens ont été caractérisés biochimiquement et structurellement jusqu'à présent (GLIC et ELIC). Ils servent de modèle d’étude à de nombreux scientifiques et ont été largement étudiés aussi bien au niveau fonctionnel que structural. Dans la première partie de mon travail de thèse, j'ai purifié, cristallisé et résolu la structure cristalline d'un nouveau pLGIC originaire d'un symbiote de gamma-protéobactérie de Tevnia jerichonana (sTeLIC). Des expériences fonctionnelles montrent que sTeLIC est activé par un pH alcalin, est sélectif pour les ions cationiques monovalents et inhibé par les cations divalents. La structure cristalline résolue à pH 8,0 présente un pore largement ouvert qui est le premier de ce type à être caractérisé dans cette famille pLGIC. De plus, nous avons identifié un modulateur fortement positif qui se lie au "site vestibulaire" dans le domaine extracellulaire, et nous avons résolu la structure cristalline de ce complexe. Des expériences fonctionnelles montrent également que sTeLIC partage de nombreuses fonctionnalités avec ELIC. ELIC et sTeLIC constitutent les archétypes d’une nouvelle classe de pLGICs, dont la forme active se caractérise par un pore largement plus ouvert que les autres pLGICs.Dans la deuxième partie de mon travail de thèse, les résidus senseurs de protons dans GLIC ont été cartographiés, afin de déterminer comment la liaison du proton stabilise l'état ouvert de GLIC. Tous les résidus titrables de GLIC ont été cartographiés par mutagenèse dirigée afin de découvrir des capteurs de protons impliqués dans le processus de déclenchement. Nous avons ainsi démontré que la résidu E35 est un résidu clé, dont la forme chargée stabilise l’état de repos, et la forme protonée l'état actif. Nous avons également démontré que la réponse au proton dépend de deux réseaux distincts à l'interface ECD-TMD qui stabilisent l'état ouvert de GLIC. Dans la troisième partie, j'ai cloné, purifié, cristallisé et déterminé les structures cristallines des formes ouvertes et fermées de DeCLIC, un pLGIC de la protéobactérie Desulfofustis. Chaque sous-unité contient un grand domaine additionnel N-terminal constitué de deux sous-domaines (NTD1 et NTD2). Il s’agit de la première structure d’un pLGIC qui contient un domaine supplémentaire extracellulaire non-canonique. / Ligand-gated pentameric ion channels (pLGIC) are one of the major families of transmembrane receptors. They allow rapid signal transduction in the central and peripheral nervous systems via neurotransmitters binding. PLGICs are also present in archaea and bacteria. Only two bacterial pLGICs have been biochemically and structurally characterized so far (GLIC and ELIC). They serve as working models for many scientists and have been extensively studied both at the functional and structural levels. In the first part of my thesis, I purified, crystallized and solved the crystal structure of a new pLGIC from gamma-proteobacterial symbionts of Tevnia jerichonana (sTeLIC). Functional experiments show that sTeLIC is activated by alkaline pH, and is selective for monovalent cationic ions and inhibited by divalent cations. The crystal structure solved at pH 8.0 displays a widely open pore that is the first of this kind to be characterized in the pLGIC family. In addition, we identified a strongly positive modulator that binds to the "vestibule site" in the extracellular domain, and we solved the crystal structure of this complex. Functional experiments show that sTeLIC shares many features with ELIC. ELIC and sTeLIC are the archetypes of a new class of pLGICs, whose active form is characterized by a much more open pore than other pLGICs. In the second part of my thesis, the proton sensor residues in GLIC have been mapped. All titratable GLIC residues were tested by site-directed mutagenesis to discover proton sensors involved in the triggering process. We have demonstrated that the residue E35 is a key residue, whose charged form stabilizes the resting state, and the protonated form the active state. We have also demonstrated that the proton response is dependent on two distinct networks at the ECD-TMD interface, which stabilize the open state of GLIC.In the third part of my thesis, I cloned, purified, crystallized and determined the crystal structures of the open and closed forms of DeCLIC, a pLGIC of Desulfofustis proteobacterium. Each subunit contains a large N-terminal additional domain consisting of two subdomains (NTD1 and NTD2). This is the first structure of a pLGIC which contains a non-canonical additional extracellular domain.