Intricate interactions among constituent components are defining hallmarks of biological systems and sculpt physiology across different scales spanning gene networks to behavioural repertoires. Whereas interactions among channels and receptors define neuronal physiology, interactions among different cells specify the characteristic features of network physiology. From a single-neuron perspective, it is now evident that the somato-dendritic plasma membrane of hippocampus pyramidal neurons is endowed with several voltage-gated ion channels (VGICs) with varying biophysical properties and sub cellular expression profiles. Structural and physiological interactions among these channels define generation and propagation of electrical signals, thereby transforming neuronal dendrites to actively excitable membrane endowed with complex computational capabilities. In parallel to this complex network of plasma membrane channels is an elegantly placed continuous intraneuronal membrane of the endoplasmic reticulum (ER) that runs throughout the neuronal morphology. Akin to the plasma membrane, the ER is also endowed with a variety of channels and receptors, prominent among them being the inositol trisphosphate (InsP3) receptors (InsP3Rs) and ryanodine receptors (RyR), both of which are calcium release channels. Physiological interactions among these receptors transform the ER into a calcium excitable membrane, capable of active propagation of calcium waves and of spatiotemporal integration of neuronal signals. Thus, a neuron is endowed with two continuously parallel excitable membranes that actively participate in the bidirectional flow of intraneuronal information, through interactions among different channels and receptors on either membrane. Although the interactions among sets of channels and receptors present individually on either membrane are very well characterized, our understanding of cross-membrane interactions among channels and receptors across these two membranes has been very limited. Recent literature has emphasized the critical nature of such cross-membrane interactions and the several physiological roles played by such interactions. Such cross-channel interactions include ER depletion-induced signaling involving store-operated calcium channels, generation and propagation of calcium waves through interactions between plasma membrane and ER membrane receptors, and the plasticity of plasma membrane VGICs and receptors induced by ER Ca2+. Such tight interactions between these two membranes have highlighted several roles of the ER in the integration of intraneuronal information, in regulating signalling microdomains and in regulating the downstream signaling pathways that are regulated by these Ca2+ signals. Yet, our understanding about the functional interactions between the ion channels and receptors present on either of these membranes is very limited from the perspective of the combinatorial possibilities that encompass the span of channels and receptors across these two membranes. In this context, the first part of this thesis deals with two specific instances of such cross-membrane functional interactions, presented as two subparts with each probing different direction of impact. Specifically, whereas the first of these subparts deals with the impact of plasma membrane VGICs on the physiology of ER receptors, the second subpart presents an instance of the effect of ER receptor activation on plasma membrane VGIC. In the first subpart of the thesis, we establish a novel role for the A-type potassium current in regulating the release of calcium through inositol triphosphate receptors (InsP3R) that reside on the endoplasmic reticulum (ER) of hippocampus pyramidal neurons. Specifically, the A-type potassium current has been implicated in the regulation of several physiological processes including the regulation of calcium influx through voltage-gated calcium channels (VGCCs). Given the dependence of InsP3R open probability on cytosolic calcium concentration ([Ca2+]c) we asked if this regulation of calcium influx by A-type potassium current could translate into the regulation of release of calcium through InsP3Rs by the A-type potassium current. To answer this, we constructed morphologically realistic, conductance-based neuronal models equipped with kinetic schemes that govern several calcium signalling modules and pathways, and constrained the distributions and properties of constitutive components by experimental measurements from these neurons. Employing these models, we establish a bell-shaped dependence of calcium release through InsP3Rs on the density of A-type potassium current, during the propagation of an intraneuronal calcium wave initiated through established protocols. Exploring the sensitivities of calcium wave initiation and propagation to several underlying parameters, we found that ER calcium release critically depends on dendrite diameter and wave initiation occurred at branch points as a consequence of high surface area to volume ratio of oblique dendrites. Further, analogous to the role of A-type potassium channels in regulating spike latency, we found that an increase in the density of A-type potassium channels led to increases in the latency and the temporal spread of a propagating calcium wave. Next, we incorporated kinetic models for the metabotropic glutamate receptor (miler) signalling components and a calcium-controlled plasticity rule into our model and demonstrate that the presence of mGluRs induced a leftward shift in a BCM-like synaptic plasticity profile. Finally, we show that the A-type potassium current could regulate the relative contribution of ER calcium to synaptic plasticity induced either through 900 pulses of various stimulus frequencies or through theta burst stimulation. These results establish a novel form of interaction between active dendrites and the ER membrane and suggest that A-type K+ channels are ideally placed for inhibiting the suppression of InsP3Rs in thin-caliber dendrites. Furthermore, they uncover a powerful mechanism that could regulate biophysical/biochemical signal integration and steer the spatiotemporal spread of signalling micro domains through changes in dendritic excitability. In the second subpart, we turned our focus to the role of calcium released through InsP3Rs in regulating the properties of VGICs present on the plasma membrane, thereby altering neuronal intrinsic properties that are dependent on these VGICs. Specifically, the synaptic plasticity literature has focused on establishing necessity and sufficiency as two essential and distinct features in causally relating a signalling molecule to plasticity induction, an approach that has been surprisingly lacking in the intrinsic plasticity literature. Here, we complemented the recently established necessity of inositol trisphosphate (InsP3) receptors (InsP3R) in a form of intrinsic plasticity by asking if ER InsP3R activation was sufficient to induce plasticity in intrinsic properties of hippocampus neurons. To do this, we employed whole-cell patch-clamp recordings to infuse D-myo-InsP3, the endogenous ligand for InsP3Rs, into hippocampus pyramidal neurons and assessed the impact of InsP3R activation on neuronal intrinsic properties. We found that such activation reduced input resistance, maximal impedance amplitude and temporal summation, but increased resonance frequency, resonance strength, sag ratio, and impedance phase lead of hippocampus pyramidal neurons. Strikingly, the magnitude of plasticity in all these measurements was dependent upon [InsP3], emphasizing the graded dependence of such plasticity on InsP3R activation. Mechanistically, we found that this InsP3-induced plasticity depended on hyperpolarization-activated cyclic-nucleotide gated (HCN) channels. Moreover, this calcium-dependent form of plasticity was critically reliant on the release of calcium through InsP3Rs, the influx of calcium through N-methyl-D -aspartate receptors and voltage-gated calcium channels, and on the protein kinase A pathway. These results delineate a causal role for InsP3Rs in graded adaptation of neuronal response dynamics through changes in plasma membrane ion channels, thereby revealing novel regulatory roles for the endoplasmic reticulum in neural coding and homeostasis. Whereas the first part of the thesis dealt with bidirectional interactions between ER membrane and plasma membrane channels/receptors within a neuron, second part focuses on cross-cellular interactions, specifically between ER membrane on astrocytes and dendritic plasma membrane of neurons. Specifically, the universality of ER-dependent calcium signalling ensures that its critical influence extends to regulating the physiology of astrocytes, an abundant form of glial cells in the hippocampus. Due to the presence of calcium release channels on ER membrane, astrocytes are calcium excitable, whereby they respond to neuronal activity by increase in their cytosolic calcium levels. Specifically, astrocytes respond to the release of neurotransmitters from neuronal presynaptic terminals through activation of metabotropic receptors expressed on their plasma membrane. Such activation results in the mobilization of cytosolic InsP3 and subsequent release of calcium through InsP3 on the astrocytes ER membrane. These ER-dependent [Ca2+]c elevations in astrocytes then result in the release of gliotransmitters from astrocytes, which bind to corresponding receptors located on neuronal plasma membrane resulting in voltage-deflections and/or activation of signaling pathways in the neuron. Although it is well established that gliotransmission constitutes an important communication channel between astrocytes and neurons, the impact of gliotransmission on neurons have largely been centered at the cell body of the neurons. Consequently, the impact of the activation of astrocytic InsP3R on neuronal dendrites, and the role of dendritic active conductances in regulating this impact have been lacking. This lacuna in mapping the spatial spread of gliotransmission in neurons is especially striking because most afferent synapses impinge on neuronal dendrites, and a significant proportion of information processing in neurons is performed in their dendritic arborization. Additionally, given that active dendritic conductances play a pivotal role in regulating the impact of fast synaptic neurotransmission on neurons, we hypothesized that such active-dendritic regulation should extend to the impact of slower extrasynaptic gliotransmission on neurons. The second part of the thesis is devoted to testing this hypothesis using dendritic and paired astrocyte-neuron electrophysiological recordings, where we also investigate the specific roles of active dendritic conductances in regulating the impact of gliotransmission initiated through activation of astrocytic InsP3Rs. In testing this hypothesis, in the second part of the thesis, we first demonstrate a significantly large increase in the amplitude of astrocytically originating spontaneous slow excitatory potentials (SEP) in distal dendrites compared to their perisomatic counterparts. Employing specific neuronal infusion of pharmacological agents, we show that blocking HCN channels increased the frequency, rise-time and width of dendritically-recorded spontaneous SEPs, whereas blockade of A-type potassium channels enhanced their amplitude. Next, through paired neuron-astrocytes recordings, we show that our conclusions on the differential roles of HCN and A-type potassium channels in modulating spontaneous SEPs also extended to SEPs induced through infusion of InsP3 in a nearby astrocyte. Additionally, employing subtype-specific receptor blockers during paired neuron-astrocyte recordings, we provide evidence that GluN2B-and GluN2D-containing NMDARs predominantly mediate perisomatic and dendritic SEPs, respectively. Finally, using morphologically realistic conductance-based computational models, we quantitatively demonstrate that dendritic conductances play an active role in mediating compartmentalization of the neuronal impact of gliotransmission. These results unveil an important role for active dendrites in regulating the impact of gliotransmission on neurons, and suggest astrocytes as a source of dendritic plateau potentials that have been implicated in localized plasticity and place cell formation. This thesis is organized into six chapters as follows: Chapter 1 lays the motivations for the questions addressed in the thesis apart from providing the highlights of the results presented here. Chapter 2 provides the background literature for the thesis, introducing facts and concepts that forms the foundation on which the rest of the chapters are built upon. In chapter 3, we present quantitative analyses of the physiological interactions between A-type potassium conductances and InsP3Rs in CA1 pyramidal neurons. In chapter 4, using electrophysiological recordings, we investigate the role of calcium released through InsP3Rs in induction of plasticity of intrinsic response dynamics, and demonstrate that this form of plasticity is consequent to changes in neuronal HCN channels. In chapter 5, we systematically map the spatial dynamics of the impact of gliotransmission on neurons across the somato-apical trunk, also unveiling the role of neuronal HCN and A-type potassium channels in compartmentalizing such impact. Finally, chapter 6 concludes the thesis highlighting its major contributions and discussing directions of future research.

Made available in DSpace on 2015-03-26T13:36:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2100606 bytes, checksum: 5e91a2b8b12b7ee39c9e424e7736aa78 (MD5) Previous issue date: 2009-02-18 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The main objectives in this work were: i) to find the optimum conditions to disinfect tomato seeds (Solanum lycopersicum Mill.) and eggplant seeds (Solanum melongena L.); ii) to evaluate the influence of the type of sealing in the obtained seedlings and explants; iii) to evaluate the effect of sonication in the morphogenesis in vitro of the tomato explants and in the viability of Agrobacterium tumefaciens cells; iv) to establish the parameters that allow the genetic transformation mediate by A. tumefaciens aiming the genetic silencing mediate by RNAi from myo-inositol-phosphate synthase, using the GmMPIS1 gene. It was checked that the use of deionized water was more efficient to disinfect eggplant seeds than 0.13% v/v chlorine solution. The use of dry disinfection in chlorine cameras is not appropriate to clean the tomato and eggplant seeds due to the gas toxicity and that it also compromises their germination. It was observed that gas exchange helps the seedlings development and leads to a bigger number of explants and with better quality to be used in the genetic transformation via Agrobacterium tumefaciens. Using the SAAT technique (Sonication-assisted Agrobacterium-mediated transformation), the explants and the bacteria suspensions were exposed to ultrasound for 0, 3, 6 and 9 seconds. It was verified that the immersion time from 3 to 6 seconds was appropriate to be used in genetic transformation, since it shown the biggest transient expression areas evaluated by in situ histochemical analysis of the GUS gene, the biggest number of regenerated structures and less mortality in the A. tumefaciens cells. The process was optimized when the immersion of the A. tumefaciens suspension was made 24 hours after the exposition to ultrasound. To make possible to select the transformed plants it was established the dependence of the hygromycin agent lethality and the found concentration of the non-transformed selected cells was 7.5 mg.L-1 in cotyledonary hipocotyledonary and leaf tomato explants. It was found that concentrations above this value were toxic, showing chlorotic and necrotic areas in the explants. A genetic transformation in tomato and eggplant plants was successfully made to check the relation between the MIPS gene and the seeds development by A. tumefaciens containing plasmids with silencing construction by siRNA to the MIPS gene using a conserved sequence of the soy gene GmMIPS. The transgenic nature of the primary regenerators was confirmed by in situ histochemical tests of GUS and by PCR analysis with specific oligonucleotides initiators. The analysis of the genetic expression confirmed the MIPS gene silencing and the morphologic analysis of the fruits confirmed the hypothesis of the relationship between the myo-inositol- phosphate synthase and the seeds development. However, as shown by flow-citometry technique, the process of regeneration in vitro used in the tomato plants transformation protocol changed some transgenic plants to polypoids. This was not observed in eggplant plants. / Este trabalho teve como objetivo: i) a otimização das condições de desinfestação de seentes de tomateiros (Solanum lycopersicum Mill.) e berinjela (Solanum melongena L.); ii) a avaliação da influência do tipo de vedação sobre a qualidade das plântulas e explantes oriundos destas; iii) a avaliação do efeito da sonicação sobre a morfogênese in vitro de explantes de tomateiros e sobre a viabilidade de células de Agrobacterium tumefaciens; iv) o estabelecimento de parâmetros para possibilitar a transformação genética mediada por A. tumefaciens visando ao silenciamento gênico mediado por RNAi da sintase do mio-inositol-fosfato, utilizando-se o gene GmMIPS1. Na desinfestação das sementes de berinjela, comprovou-se que tratamentos utilizando imersão em água deionizada são mais eficientes que imersão em solução de 0,13% v/v de cloro. A utilização de desinfestação a seco, em câmara de gás cloro, não é indicada para a assepsia de sementes de tomate e berinjela, pela toxicidade do gás às sementes, comprometendo sua germinação. Observou-se que as trocas gasosas favorecem o desenvolvimento das plântulas e geram explantes em maior número e de melhor qualidade para utilização de transformação genética via Agrobacterium tumefaciens. Ao se utilizar a técnica de SAAT ( Sonication-assisted Agrobacterium-mediated transformation ), os explantes e a suspensão bacteriana foram expostos a tempos de exposição ao ultra-som (0, 3, 6 e 9 segundos). Verificou-se que o intervalo de 3 a 6 segundos é o indicado para se utilizar em transformação genética, pois resultou nas maiores áreas de expressão transiente avaliada pela análise histoquímica in situ do gene GUS, maior número de estruturas regeneradas e menor mortalidade nas células de A. tumefaciens. O processo foi otimizado quando a imersão em suspensão de A. tumefaciens foi realizado após 24 horas de exposição ao ultra-som. Para ser possível a seleção de transformantes foi estabelecida a curva de letalidade ao agente higromicina e a concentração encontrada para seleção de células não transformadas foi de 7,5 mg.L-1 em explantes cotiledonares, hipocotiledonares e foliares de tomateiro. Dosagens acima de 7,5 mg.L-1 mostratam-se tóxicas, resultando em explantes com áreas cloróticas e necróticas. A fim de verificar a relação do gene MIPS com o desenvolvimento de sementes, a transformação genética foi realizada com sucesso em tomateiro e berinjela, via A. tumefaciens contendo plasmídeo com construção de silenciamento por siRNA para o gene MIPS, utilizando uma seqüência conservada do gene de soja GmMIPS. A natureza transgênica dos regenerantes primários foi confirmada mediante o teste histoquímico in situ de GUS e análise de PCR com oligonucleotídeos iniciadores específicos. A análise de expresão gênica confirmou o silenciamento do gene MIPS, e a análise morfológica dos frutos confirmou a hipótese do relacionamento da mio-inositol-fosfato-sintase com o desenvolvimento de sementes. Porém, conforme detectado pela técnica de citometria de fluxo, o processo de regeneração in vitro adotado no protocolo de transformação de tomateiro, ao contrário de berinjela, induziu poliploidia em algumas plantas transgênicas.

As paredes celulares vegetais são estruturas essenciais para o crescimento e desenvolvimento das plantas. Além das suas diversas funções biológicas, os componentes polissacarídicos constituintes das paredes celulares (celulose, hemiceluloses e pectinas) são de vital importância como fonte natural de fibras para a nutrição humana e animal e são considerados os principais recursos renováveis do planeta, utilizados como matéria-prima para diversos processos industriais, por exemplo nos processos de produção de polpa celulósica. Todos esses fatores têm despertado grande interesse no estudo da composição e biossíntese das paredes celulares. A biossíntese dos seus polímeros se inicia no citoplasma das células, onde ocorre a formação dos precursores por uma rota metabólica complexa de biossíntese de açúcares-nucleotídeo. O entendimento da regulação dessa rota metabólica é fundamental para modular a dinâmica de biossíntese desses açúcares e assim tentar manipular as propriedades bioquímicas das paredes celulares. Nesse contexto, o presente projeto de pesquisa teve como objetivo avaliar o efeito da superexpressão do gene miox2 de Arabidopsis thaliana em plantas de Nicotiana tabacum. O produto desse gene é a enzima mio-inositol oxigenase (E.C. 1.13.99.1), cuja função é converter o mio-inositol em ácido D-glucurônico, composto central da rota de biossíntese de açúcares-nucleotídeo. Foram determinadas quatro isoformas tecido-específicas para o gene miox (miox1, miox2, miox4 e miox5) em Arabidopsis, sendo que a isoforma miox2 é a predominante em caules. Esse gene foi clonado em trabalhos anteriores realizados no laboratório e no presente trabalho, o cDNA do gene miox2 foi superexpresso em plantas de tabaco (Nicotiana tabacum) a fim de se avaliar o efeito da superexpressão na composição de carboidratos de parede celular secundária. As linhagens de plantas transgênicas obtidas, não mostraram diferenças visualmente perceptíveis em comparação aos controles, indicando ausência de alterações fisiológicas e morfológicas. Foram quantificados os monossacarídeos de paredes celulares secundárias (arabinose, ramnose, galactose, glicose, xilose, manose), os ácidos urônicos (ácido galacturônico e glucurônico) e as ligninas (solúvel e insolúvel), a partir de tecido xilemático e parênquima medular do caule. A ausência de modificações significativas nas proporções desses metabólitos, indica que as plantas exercem um estrito controle na regulação da biossíntese de paredes celulares secundárias de forma que a superexpressão do gene miox2 não provocou nenhuma alteração altamente significativa. Outros genes candidatos e os mecanismos envolvidos na sua regulação deverão ser testados quanto ao nível de transcrição, modificações pós-trancricionais e pós-traducionais a fim de entender a regulação do fluxo de carbono para a biossíntese de paredes celulares. / Cell-walls are essential structures for plant development and growth. Apart from its biological functions, the polyssacharides that make cell-walls (cellulose, hemicellulose and pectins) are the principal natural fibrous materials used for human and animal nutrition. They are also considered the most important renewable resource on earth and their use as industrial raw material is inevitable. An example is the use of wood in the production of pulp and paper. For all these reasons, the study of molecular composition and biosynthesis of plant cell-walls has been a matter of great interest for researchers over the past few years. Cell-wall polyssacharides biosynthesis begins at the cytoplasm, where a pool of UDP-glucose and other activated sugar nucleotide precursors are generated by multiple and complex interconvertion reactions. Understanding how cells control the metabolic pathways responsible for sugar nucleotide precursors synthesis, would be a primary requirement for manipulating them in an attempt to generate plants with improved properties for human use. In that context, tha aim of this research work was to analyze the effects of Arabidopsis thaliana miox2 gene overexpression in a plant model system (Nicotiana tabacum). The product of miox2 gene is myo-inositol oxygenase enzyme 2 (E.C.1.13.99.1) which converts D-glucuronic acid, an important sugar nucleotide precursor, from its substrate myo-inositol. Four isoforms of miox gene, with apparent tissue specific expression (miox1, miox2, miox4 and miox5) were already determined, but miox2 is the one primarily expressed in stems. Its cDNA was cloned from Arabidopsis thaliana in previous works and overexpressed in tobacco plants. Five normal transgenic lines were obtained, showing no phenotypically differences relative to the control line. This fact implied that miox2 overexpression did not alter any physiological nor morphological aspect of plant development. The cell-wall monossacharides (arabinose, rhamnose, galactose, glucose, xylose and mannose), uronic acids (galacturonic and glucuronic acid) and lignins (soluble and insoluble) from stem xylem and parenchymal tissue were quantified. The absence of major changes in any of the compounds measured for the transgenic lines indicated that they were able to adjust their level of carbohydrate composition. Plants seem to regulate the proportions of sugar nucleotide precursors through highly complex metabolic pathways that establish strong compensatory mechanisms. It will be necessary to study other candidate genes and some aspects of their regulation at transcriptional, postranscriptional and postransaltional level, as an attempt to understand the cell-wall carbohydrate flux.

One key to the pathogenic potential of the mycobacteria lies in their capacity to resist destruction by infected macrophages and dendritic cells. Robust host immune responses during mycobacterial infection often involve a potent CD4, CD8 and gamma delta T cell mediated effector responses including lysis of mycobacteria infected host cells, secretion of variety of cytokines like IFN-γ etc. However, pathogenic mycobacteria survives for prolonged periods in the phagasomes of infected macrophages within the host in an asymptomatic, latent state and can reactivate years later if the host’s immune system wanes. One of the most devastating consequences of infection with mycobactreia is the formation of caseating granulomas followed by tissue destruction with liquefaction causing cavity formation. Pathogenic mycobacteria reside in these granulomas, which are formed by the accumulation of monocytes, epithelioid and foamy macrophages as well as cytolytic lymphocytes including CD8 T cells around the infection focus. In this regard, rigid balance as well as modulation of inflammatory immune responses by the host upon infection of pathogenic microbes is one of the crucial steps not only in controlling the spread of pathogen from the site of infection to reminder of host organs, but also in mounting an effective memory response so that future exposures/infections by similar pathogen can be effectively controlled. Significantly, despite this complex host response, it remains unclear, that why the immune response controls mycobacteria but does not eradicate infection. Both human and mouse studies have provided ample evidence that even in the face of an adequate immune response, mycobacteria are able to persist inside macrophages. These findings have suggested series of survival strategies employed by Mycobacterium sp. during its infection of host macrophages/dendritic cells which include, blockade of phagosome-lysosome fusion, secretion of ROI antagonistic proteins like superoxide dismutase & catalase, inhibition of processing of its antigens for presentation to T cells, decrease in secretion of proinflammatory cytokines by inducing secretion of immunosuppressive cytokines like IL-10 and TGF-β etc. In view of above-mentioned observations, graulomas in response to pathogenic mycobacterial infections have long been considered host-protective structures formed to contain infection. In this perspective, Matrix metalloproteinase-9 (MMP-9), an important member of Zn2+ and Ca2+ dependent endopeptidases, participates in a significant manner in several aspects of host immune responses to mycobacterial infection such as graunloma formation, matrix (ECM) reorganization, lymphocytes trafficking and infiltrations, inflammation etc. MMP-9 is expressed at various clinical categories of tuberculosis disease like active cavitary tuberculosis, meningitis and pleuritis. Notably, in case of pulmonary tuberculosis, breakdown of ECM by MMP-9 forms an integral part of the granuloma formation. Importantly, Mycobacterium tuberculosis infection in MMP-9 deficient mice revealed defective bacterial proliferation, reduced bacterial burden and reduced lung macrophages recruitment compared to wild-type, in addition, to reduced ability to initiate or maintain well-formed granulomas. In this context, we explored the signaling events modulated by Mycobacterium bovis bacillus Calmette-Gue´rin (BCG) or its novel cell wall antigens during induced expression of MMP-9 or SPI6 in macrophages. Our studies clearly demonstrate that NO, a product of iNOS activity, is responsible for M. bovis BCG-triggered activation of Notch1 in macrophages through direct regulation of Jagged1 expression as well as in generation of activated Notch1. We present the evidence that iNOS activity is a critical factor in TLR2 mediated Notch1 activation as macrophages derived from iNOS knockout (iNOS-/-), but not from wild-type (WT) mice failed to activate Jagged1 expression as well as Notch1 signaling upon M. bovis BCG infection. The loss of TLR2-mediated Jagged1 expression or Notch1 activation in iNOS-/-macrophages could be rescued by treatment with NO donor 3-morpholinosydnonimine (SIN1) or S-nitroso-Nacetylpenicillamine (SNAP). Signaling perturbations strongly implicated the role for cross talk among members of Notch1-PI3 Kinase and MAPK cascades in M. bovis BCG-TLR2– mediated activation of Notch1 target genes MMP-9 or Hes1. Chromatin immunoprecipitation experiments demonstrate that M. bovis BCG’s ability to trigger increased binding of CSL/RBP-Jk to MMP-9 promoter was severely compromised in macrophages derived from iNOS-/-mice compared to WT mice. These results are consistent with the observation that NO-triggered Notch1 signaling-mediated CSL/RBP-Jk recruitment has a positive regulatory role in M. bovis BCG-induced MMP-9 transcription. We show the correlative evidence that this mechanism operates in vivo by immunohistochemical expression analysis of activated Notch1 or its target gene products Hes1 or MMP-9 in brains of WT or iNOS-/-mice that were intracerebrally infected with M. bovis BCG. Further, activation of Notch1 signaling in vivo could be demonstrated only in granulomatous lesions in brains derived from human patients with tuberculous meningitis (TBM) as opposed to healthy individuals, validating the role of Notch1 signaling in mycobacterial pathogenesis. Briefly, we have identified NO as the pathological link between TLR2 and Notch1 signaling, which regulates the relative abundance of various immunopathological parameters including MMP-9 in macrophages. Synopsis Despite mycobacteria elicits robust host T cell responses as well as production of NO, ROI or cytokines like interferon-γ (IFN-γ) that are essential for the control of infection, the mounted immune response contain, but does not eliminate the infection. These findings clearly advocate roles for mycobacteria mediated various immune evasion strategies to modulate the signaling cascades thus leading to macrophage activation. Importantly, TLR2 triggering by mycobacteria elicits the activation of divers sets of anti or pro-apototic genes expression, a balance of which will have strong bearing on the overall cell-fate decisions across many cell types. In this regard, a novel granzyme B inhibitor, SPI6/PI9, can exhibit robust resistance to various cells including dendritic cells or tumor cells from lysis by CD8 cytotoxic T cells (CTL). SPI6/PI9 predominantly functions by inhibiting Granzyme B, an effector protease of cytotoxic granules released by CTL upon its TCR recognition of infected cells such as macrophages, dendritic cells etc. In this context, current investigation attempted to investigate molecular details involved in M. bovis BCG triggered SPI6 expression as well as the involvement of TLR2NO-Notch1 signaling axis in driving induced expression of SPI6, akin to that of MMP-9 expression. We demonstrate that M. bovis BCG trigger SPI6 expression in macrophages and requires critical participation of TLR2-MyD88 dependent NO-Notch1 signaling events. More importantly, signaling perturbations data suggest the involvement of cross talk among the members of PI3 Kinase and MAPK cascades with Notch1 signaling in SPI6 expression. In addition, SPI6 expression requires the Notch1 mediated recruitment of CSL/RBP-Jk and NF-κB to the SPI6 promoter. Functional studies strongly attribute critical involvement of SPI6 and MMP-9 in imparting protection to M.bovis BCG infected macrophages from lysis effectuated by CTL. Macrophages are principal mediators of initiation as well as activation of host inflammatory responses to pathogenic mycobacterial infection. Albeit mycobacteria reside within phagolysosomes of the infected macrophages, envelope glycoconjugates like Lipoarabinomannan (LAM), phosphatidyl-myo-inositol mannosides (PIM), Trehalose 6,6′dimycolate (TDM; cord factor) etc. are released and traffic out of the mycobacterial phagosome into endocytic compartments as well as can gain access to the extracellular environment in the form of exocytosed vesicles. In this perspective, PIM represent a variety of phosphatidyl-myo-inositol mannosides (PIM) 1-6 containing molecules and are integral component of the mycobacterial envelope. A number of biological functions have been credited to PIM2. PIM2 was shown to trigger TLR2 mediated activation of macrophages that resulted in activation of NF-κB, AP-1, and mitogen-activated protein (MAP) kinases. In addition to pulmonary granuloma-forming activities, PIM2 was shown to recruit NKT cells into granulomas. Further, surface associated PIM was suggested to act as adhesins mediating attachment of M. tuberculosis bacilli to non-phagocytic cells. Accordingly, mycobacterial envelope antigen PIM2 could initiate or affect the inflammatory responses similar to mycobacteria bacilli. In this perspective, we explored whether novel cell surface antigen PIM2 similar to whole M. bovis BCG bacilli can contribute to molecular signaling events leading to MMP-9 expression in macrophages. Our current study provides the evidence that PIM2 driven activation of signaling cascades triggers the expression of MMP-9. TLR stimulation by various agonists has been shown to activate Notch signaling resulting in modulation of diverse target genes involved in pro-inflammatory responses in macrophages. In this regard we demonstrated that PIM2 induced expression of MMP-9 involved Notch1 upregulation and activation of Notch1 signaling pathway in a TLR2-MyD88 manner. Enforced expression of the cleaved Notch1 in macrophages induced the expression of MMP-9. Further, PIM2 triggered significant p65 nuclear factor-κB (NF-κB) nuclear translocation that was dependent on activation of PI3 Kinase or Notch1 signaling. Furthermore, MMP-9 expression requires Notch1 mediated recruitment of Suppressor of Hairless (CSL) and NFκB to MMP-9 promoter. Taken together, our observations clearly describe involvement of TLR2/iNOS in activating Notch1 and PI3 Kinase signaling during infection of macrophages with M. bovis BCG, thus effectuating the regulation of specific effector gene expressions, such as SPI6 and MMP-9. These results clearly describe the cross talk of Notch1 signaling with PI3 Kinase and MAPK pathways, thus leading to differential effects of Notch1 signaling. Overall, we believe that our work will extend the current understanding of inflammatory parameters associated with host-mycobacteria interactions which might lead to better design as well as evaluation of therapeutic potential of novel agents targeted at diverse mycobacterial diseases.

1. The effect of urotensin II (U-II) on spontaneous transmitter release was examined in the frog to see if the biological activity of this vasoactive peptide extended to neural tissues. 2. In normal Ringer solution, frog and human U-II (fU-II and hU-II, respectively) caused concentration-dependent, reversible increases in miniature endplate potential (MEPP) frequency, with hU-II about 22 times more potent than fU-II. hU-II caused a dose-dependent increase in MEPP amplitude, whereas fU-II caused an increase, followed by a decrease with higher concentrations. 3. Increasing extracellular Ca 2+ three-fold had no effect on the MEPP frequency increase to 25 μM hU-II. Pretreatment with thapsigargin to deplete endoplasmic reticulum Ca 2+ caused a 61% reduction in the MEPP frequency increase to 25 μM hU-II. 4. Pretreatment with the phospholipase C inhibitor U-73122 caused a 93% reduction in the MEPP frequency increase to 25 μM hU-II and a 15% reduction in the increase in MEPP amplitude. Pretreating with antibodies against the inositol 1,4,5-trisphosphate (IP 3) type 1 receptor using liposomal techniques reduced the MEPP frequency increase by 83% but had no effect on MEPP amplitude. 5. Pretreating with protein kinase C inhibitors (bisindolylmaleimide I and III) had no effect on the response to 25 μM hU-II, but pretreating with protein kinase A inhibitors (H-89 and KT5720) reduced the MEPP frequency increase by 88% and completely abolished the increase in MEPP amplitude. 6. Our results show that hU-II is a potent stimulator of spontaneous transmitter release in the frog and that the effect is mediated by IP 3 and cyclic AMP/protein kinase A.

Pleckstrin homology (PH) domains, which play an essential role in membrane trafficking and signal transduction, recognize phosphoinositides with a diverse range of affinities and specificities. The PH domains of the Grp1 family of Arf GTPase exchange factors recognize a select group of phosphoinositides with dramatic differences in specificity, despite 90% sequence identity. The work described in this thesis has focused on the structural basis for these differences. The structure of the Grp1 PH domain revealed structural determinants for phosphoinositide recognition. Through a wide range of crystallographic and biochemical means, the structural basis that accounts for the differential binding affinities amongst the Grp1 family PH domains has also been determined. Furthermore, examination of the structural details of these PH domains bound to different inositol phosphate groups have aided in understanding the structural mechanisms by which all PH domains recognize phosphoinositides.

Ziele: Mittels absolutquantifizierender Protonen-Magnet-Resonanz-Spektroskopie (1H-MRS) wollten wir das Ergebnis einer Vorstudie bestätigen, die im Frontallappen einen reduzierten Quotienten von myo-Inositol/Gesamtcreatin (mI/tCr) bei Depressiven fand. Darüber hinaus testeten wir den antidepressiven Effekt von Inositol als Add-on-Therapie. Methodik: Wir untersuchten Einzelvoxel (2 x 2 x 2 cm3) in der weißen Substanz der rechten und linken Präfrontalregion mit Hilfe eines 3-Tesla Bruker Medspec Systems (STEAM Sequenz, TR/TE/TM = 6000/20/30 ms). Die einzelnen Metabolite wurden anhand des cerebralen Wassers als internem Standard quantifiziert (nach dem LCModell). Es wurden 24 unmedizierte Patienten mit unipolaren depressiven Episoden mit 24 alters- und geschlechtsgematchten gesunden Kontrollen verglichen. In doppelblindem, Plazebo-kontrollierten Parallelgruppen-Design erhielten die Patienten täglich 18 Gramm Inositol oder Plazebo zusätzlich zu Citalopram über vier Wochen. Ergebnisse: An der Baseline unterschieden sich die mI-, Cholin- und N-Acetyl-Aspartat-Konzentrationen der Patienten nicht von jenen der Kontrollen. Es fanden sich keine sich keine signifikanten Unterschiede zwischen Inositol- und Plazebo-Gruppe. Überraschenderweise zeigten die depressiven Patienten an der Baseline gegenüber den Kontrollen signifikant höhere tCr-Konzentrationen (mmol/kg) links (5,57 ± 0,96 vs. 4,87 ± 0,63; + 15 %, p < 0,01) und rechts präfrontal (5,29 ± 0,92 vs. 4,46 ± 0,41; + 17 %, p < 0,01). Nach der Behandlung ergab sich eine Reduktion der tCr-Konzentration links- (Tag 28: 5,05 ± 1,16; – 12 %, p = 0,08) und rechtsfrontal (Tag 28: 4,61 ± 1,07; – 9 %, p = 0,09). Die tCr-Konzentrationen der Patienten am Tag 28 unterschieden sich nicht mehr von jenen der Kontrollen. Zusammenfassung: Wir zeigten eine reversible Steigerung der tCr-Konzentration der Patienten im Vergleich zu Kontrollen, die auf Veränderungen des Creatin-Transports oder der ATP-Synthese bei unmedizierter unipolarer Depression hinweisen könnte. / Objectives: By means of proton magnetic resonance spectroscopy (1H-MRS) with absolute quantification we wanted to confirm our previous finding of decreased ratios of the metabolites myo-Inositol/total creatine (mI/tCr) in the right frontal brain of depressives. Moreover, we tested the antidepressive effect of oral Inositol ingestion as add-on-therapy. We measured concentrations (mmol/kg ww) of mI, tCr (= Creatine + Phosphocreatine), Choline (Cho) and N-Acetyl-Aspartate (NAA) in the frontal brain. Methods: Single voxels (2x2x2 cm3) in the white matter of the left and right prefrontal region were examined in a three Tesla Bruker Medspec System (STEAM sequence, TR/TE/TM = 6000/20/30 ms). Metabolites were quantified using the LCModel. At baseline, 24 drug-free patients with unipolar depressive episodes were compared to 24 age and sex matched healthy controls. In a double blind, placebo controlled parallel-group design patients received daily 18 grams Inositol or placebo as an add on therapy to Citalopram over four weeks. Results: At baseline, mI, Cho and NAA concentrations showed no significant differences between patients and controls. The treatment with Inositol did not result in any significant differences to the treatment with placebo. Surprisingly the patients showed significant higher tCr concentrations in the left (5.57 ± 0.96 vs. 4.87 ± 0.63; + 15 %, p < 0.01) as well as in the right prefrontal region (5.29 ± 0.92 vs. 4.46 ± 0.41; + 17 %, p < 0.01) compared to controls. The treatment caused a trend towards a decrease of tCr in the left (day 28: 5.05 ± 1.16; – 12 %, p = 0.08) and in the right frontal hemisphere (day 28: 4.61 ± 1.07; – 9 %, p = 0.09) compared to baseline. The differences between the patients’ tCr at day 28 and the tCr of controls were no more significant. Conclusion: We have found a state dependent increase of tCr concentration indicating bifrontal deviations in Creatine transport or ATP synthesis in drug free unipolar depressives.

Chez les végétaux supérieurs, lembryogenèse est une phase clé du développement au cours de laquelle lembryon établit les principales structures de la future plante. La compréhension des processus moléculaires et physiologiques menant à la formation de la graine est donc dun intérêt agronomique majeur. Chez Phaseolus la caractérisation moléculaire de lembryogenèse permet de mieux comprendre les mécanismes du développement embryonnaire et de son dysfonctionnement observé chez les hybrides interspécifiques. Cette thèse sinscrit dans ce cadre et vise à identifier et caractériser des gènes clés impliqués dans le développement de l'embryon chez Phaseolus. Des hybridations interspécifiques ont été réalisées entre lespèce P.vulgaris L. (cultivar NI637) utilisée comme parent mâle et lespèce P. coccineus L. (cultivar NI16) utilisée comme parent femelle. Des analyses ont aussi été effectuées sur un mutant obtenu par mutagenèse chimique à l'EMS (Ethyl Méthyl Sulfonate) de graines de la variété BAT93 de P.vulgaris. Une étude histologique comparative a permis de suivre la dynamique de lembryogenèse du haricot commun à partir dembryons prélevés 3 à 12 jours après la pollinisation et provenant de plantes normales et déficients dans la production de graines. Les embryons de P. vulgaris se développent plus rapidement par rapport à ceux issus du mutant EMS. Ces derniers présentent des anomalies au niveau de lembryon et du suspenseur. La caractérisation fonctionnelle de deux gènes candidats MIPS (myo-inositol phosphate synthase) et Sus (sucrose synthase) a été réalisée par RT-PCR quantitative et hybridation in situ suite à une étude spatio-temporelle dexpression de ces deux gènes candidats au cours de développement embryonnaire chez Phaseolus. Lanalyse du profil dexpression de ces deux gènes montre quils sont exprimés différemment au niveau des tissus de lembryon et du suspenseur. Lanalyse in silico nous a permis de sélectionner 22 gènes candidats dont nous avons vérifié l'expression au cours de développement de la graine chez Phaseolus. Des variations au niveau de la méthylation de lADN ont été déterminées chez les hybrides interspécifiques comparativement à leurs parents. La technique de lHSS a permis disoler des fragments dADNs complémentaires différemment exprimés au cours de développement de la graine chez Phaseolus. Lanalyse des séquences de ces ADNs complémentaires montre quils codent pour plusieurs protéines intervenant dans le développement cellulaire et embryonnaire, en particulier le "storage protein activator" (SPA), le "pentatricopeptide repeat-containing protein" (PPR) et lacetyl-CoA carboxylase (ACCase). La caractérisation de ces différents gènes exprimés au cours du développement de la graine, fournit de nouveaux outils susceptibles de mettre en évidence des mécanismes de dysfonctionnement embryonnaire chez le genre Phaseolus.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal. / L'anion superoxyde peut agir comme une molécule de signalisation ou comme un facteur préjudiciable selon sa concentration, l'organe cible, et selon la présence ou non d'antioxydants neutralisants. Actuellement, dans les cellules musculaires lisses (CMLs) vasculaires, les effets de l'anion superoxyde sur les différentes voies de transduction du signal et sur les interactions croisées entre ces voies ne sont pas encore définies. Par conséquent, une meilleure connaissance des effets de l'anion superoxyde sur les différentes voies de signalisation pourrait fournir une meilleure compréhension des mécanismes sous-jacents aux fonctions altérées des CMLs vasculaires observées dans des conditions pathologiques. L'objectif général de cette étude était de caractériser et d'évaluer le rôle modulateur de l'anion superoxyde, produit par la réaction de l'hypoxanthine avec la xanthine oxidase, sur les activités de différentes voies de signalisation dans les CMLs vasculaires, et de déterminer si la sensibilité de différentes voies de signalisation à l'anion superoxyde était altérée dans l'hypertension artérielle. Le projet de ce programme de recherche était basé sur les principaux postulats suivants : (1) l'anion superoxyde pourrait affecter sélectivement la production d'inositol 1,4,5-triphosphates (IP3), de GMPc, ou d'AMPc dans les CMLs vasculaires; (2) le rôle modulateur de l'anion superoxyde pourrait être dû à une altération des interactions croisées entre différentes voies de signalisation; et (3) les anomalies observées dans les CMLs vasculaires chez le rat spontanément hypertendu (SHR) pourraient être reliées à des altérations des différentes voies de signalisation induites par l'anion superoxyde. Une production augmentée d'1P3induite par l'anion superoxyde dans les CMLs d'aorte de rat ou d'artère mésentérique en culture a été démontrée pour la première fois dans cette étude. L'anion superoxyde a augmenté la formation d'IP3d'une manière concentration-dépendante et temps-dépendante. La superoxyde dismutase (SOD), mais non la catalase, a inhibé significativement la formation d'IP3 induite par l'anion superoxyde. L'inhibition de la phospholipase C (PLC) a aboli l'effet de l'anion superoxyde sur la formation d'1P3. La génistéine et la tyrphostine A25, deux inhibiteurs de la tyrosine kinase, ont aussi inhibé significativement la formation d'IP3induite par l'anion superoxyde. L'utilisation d'anticorps anti-PLCy a atténué significativement la formation d'1P3induite par l'anion superoxyde. De plus, le taux d'expression des protéines de la PLCy a été augmenté après l'exposition des CMLs à l'anion superoxyde. Ces observations suggèrent donc que dans les CMLs vasculaires la formation d'1P3 induite par l'anion superoxyde pourrait être en grande partie secondaire à une augmentation de l'activité de la tyrosine kinase liée aux voies de signalisation de la PLCy. En ce qui concerne la voie du GMPc, l'anion superoxyde a diminué significativement les niveaux de base de GMPc et supprimé aussi l'augmentation des niveaux de GMPc induite par des stimulateurs de la guanylyl cyclase, le nitroprussiate de sodium (NPS) ou la s-nitroso-nacétylpénicillamine (SNAP). La formation d'1P3stimulée par l'anion superoxyde a été significativement inhibée par le NPS ou la SNAP, mais potentialisée de façon importante par un inhibiteur de la guanylyl cyclase l'ODQ ou par le KT5823 (un inhibiteur de la protéine kinase dépendant du GMPc). Cependant, l'anion superoxyde n'a pas eu d'effet sur les niveaux de base d'AMPc ou sur la production d'AMPc induite par la forskoline et de plus, l'inhibition de l'adénylyl cyclase ou de la protéine kinase dépendante de l'AMPe n'a pas affecté la formation d'lP3stimulée par l'anion superoxyde. Ces données, par conséquent, suggèrent que l'inhibition de la formation de GMPc par l'anion superoxyde contribue probablement à l'activation de la formation d'1P3induite par l'anion superoxyde en atténuant le rétrocontrôle inhibiteur du GMPc sur les voies de signalisation liées à la PLC, tandis que la voie de signalisation de l'AMPc ne serait pas impliquée dans la formation d'EP3induite par l'anion superoxyde. Dans les CMLs vasculaires de rat SHR, les effets de l'anion superoxyde ont été plus puissants que dans les CMLs de rat WKY, en ce qui concerne l'augmentation de formation d'1P3, la diminution des taux de GMPc et la facilitation induite par l'anion superoxyde des interactions croisées entre les voies du GMPc et de 1'IP3. Dans les CMLs vasculaires des deux souches de rat, la formation d'IP3induite par l'anion superoxyde a été inhibée par une variété d'antioxydants, dont la N-acétylcystéine, l'acide a-lipoïque, la mélatonine et la SOD. Il apparaît donc vraisemblable que l'hypersensibilité à l'anion superoxyde des voies de 1'IP3et du GMPc puissent contribuer à l'augmentation du tonus vasculaire et de la réactivité des CMLs dans l'hypertension artérielle. Nous avons aussi investigué si l'effet de la mélatonine était dû à ses propriétés antioxydantes. Un effet inhibiteur plus important de la mélatonine sur la contraction aortique induite par la norépinéphrine (NE) a été observé chez les rats SHR en comparaison avec les rats Wistar-Kyoto (WKY). L'inhibition par la mélatonine de la formation d'IP induite par la NE a été aussi plus importante dans les CMLs aortiques de rat SHR que dans celles de rat WKY. Les effets plus puissants de la mélatonine chez le rat SHR, qui ont été aussi observés avec la SOD, mais non avec la catalase, ne sont pas dûs à l'activation des récepteurs à la mélatonine ou des récepteurs a-adrénergiques. Ces résultats indiquent que les effets anti-hypertenseurs de la mélatonine sont largement dûs à l'inactivation de l'anion superoxyde, et que les niveaux endogènes d' antioxydants ne parviennent pas à contrecarrer les niveaux accrus d'anion superoxyde produits chez le rat SHR. En conclusion, cette étude révèle une variété de nouveaux mécanismes de signalisation de l'anion superoxyde. Pour la première fois, il a été démontré que l'anion superoxyde active l'hydrolyse des phosphoinositides et augmente les taux d'IP3dans les CMLs vasculaires, principalement par la stimulation de la tyrosine kinase liée à la voie de signalisation de la PLCy. Il a aussi été observé que l'anion superoxyde réduit la formation de GMPc et supprime l'inhibition croisée de 1'1P3par le GMPc, facilitant ainsi la formation d'1P3. Les effets sélectifs de l'anion superoxyde sur les voies de 1'IP3et du GMPc, ainsi que l'existence d'une inhibition croisée de la formation d'1P3par la voie du GMPc, révèlent des mécanismes nouveaux pour expliquer le rôle modulateur de l'anion superoxyde sur les voies de signalisation dans les CMLs. Par conséquent, les effets plus puissants de l'anion superoxyde sur la signalisation de la voie de 1'IP3et de la voie du GMPc dans les CMLs vasculaires de rat SHR, effets qui ont été démontrés pour la première fois dans cette étude, pourraient être responsables des altérations des mécanismes de transduction du signal cellulaire chez le rat SHR et ainsi contribuer au développement et/ou au maintien de l'hypertension artérielle. Ces observations permettent donc d'imaginer de nouvelles orientations pour le développement de nouvelles stratégies pour la prévention ou le traitement de l'hypertension artérielle. / Superoxide anion can act as a signalling molecule or a detrimental factor depending on its concentration, the targeted organ, and the presence of counteracting antioxidants. The effects of superoxide on different signal transduction pathways and on the cross-talk interactions among these pathways in vascular smooth muscle cells (SMCs) are presently still unsettled. Therefore, a better knowledge on the effects of superoxide on different signalling pathways may provide a better understanding of the mechanisms underlying the altered functions in vascular SMCs observed in pathological conditions. The general objective of this study was to characterize and evaluate the modulating role of superoxide generated by the hypoxanthine and xanthine oxidase reaction on the activities of different signalling pathways in vascular SMCs and to investigate whether the sensitivities of different signalling pathways to superoxide were altered in hypertension. The design of the present research program was based on the following major postulates. (1) superoxide might selectively affect the generation of inositol 1,4,5-triphosphates (IP3), cGMP, or cAMP in vascular SMCs; (2) the modulating role of superoxide might be mediated by alteration in the cross-talk interactions among different signalling pathways; and (3) the abnormalities observed in vascular SMCs from spontaneously hypertensive rats (SHR) might be related to the alterations induced by superoxide on different signalling pathways. An enhanced production of 1P3induced by superoxide in cultured SMCs from rat aorta or mesenteric artery was demonstrated, for the first time, in this study. Superoxide increased 1P3 formation in a concentration- and time-dependent manner. Superoxide dismutase (SOD), but not catalase, significantly inhibited the superoxide-increased 1P3formation. The inhibition of phospholipase C (PLC) abolished the effect of superoxide on IP3formation. Genistein and tyrphostin A25, two tyrosine kinase inhibitors, also significantly inhibited the superoxideinduced IP3formation. The application of antibody against PLCI, significantly attenuated the superoxide-induced 1P3formation. Moreover, the expression level of PLC7proteins was increased after exposing SMCs to superoxide. These observations thus suggest that superoxideinduced IP3 formation may be in a great part secondary to an increase in the activity of tyrosine kinase-link PLCy signalling pathways in vascular SMCs. Concerning the cGMP pathway, superoxide significantly decreased the basal levels of cGMP and also suppressed the rise in cGMP levels induced by guanylyl cyclase stimulator sodium nitroprusside (SNP) or s-nitroso-n-acetylpenicillamine (SNAP). The superoxide-induced IP3 formation was significantly inhibited by SNP or SNAP, but markedly potentiated by a guanylyl cyclase inhibitor ODQ or KT5823 (a cGMP-dependent protein kinase inhibitor). However, superoxide had no effect on the basal levels of cAMP or the forskolin-induced cAMP production and moreover, the inhibition of adenylyl cyclase or cAMP-dependent protein kinase did not affect the superoxide-enhanced IP3formation. These data, therefore, suggest that the reduced cGMP formation by superoxide probably contributes to the superoxide induced activation of 1P3 formation by lifting the inhibitory feedback of cGMP on the PLC pathway(s), whereas, the cAMP pathway may not be involved in the superoxide-induced IP3formation. In vascular SMCs from SHR, the effects of superoxide were more potent than in SMCs from WKY, including the increase in 1P3 formation, the decrease in cGMP levels, and the superoxide-induced facilitation of the cross-talk interaction between cGMP and IP3pathways. The superoxide-induced 1P3formation was inhibited by a variety of antioxidants, including nacetylcysteine, cc-lipoic acid, melatonin and SOD, in vascular SMCs from both strains. It thus appears that the hypersensitivity of 1P3and cGMP pathways to superoxide is likely to contribute to the increased vascular tone and reactivity of SMCs in hypertension. Whether the effect of melatonin is due to its antioxidant properties was also explored. A greater inhibitory effect of melatonin on the norepinephrine (NE)-induced aortic contraction was observed in SHR than in Wistar-Kyoto rats (WKY). The inhibition of the NE-induced IP formation by melatonin was also greater in aortic SMCs from SHR than that from WKY. The enhanced effects of melatonin in SHR, which were found to be similarly enhanced with SOD but not with catalase, were not mediated by melatonin receptors or oc-adrenoceptors. These results indicate that the anti-hypertensive effects of melatonin are largely due to the scavenging of superoxide, and that the levels of endogenous antioxidants may not counteract the levels of overproduced superoxide in SHR. In conclusion, this study reveals a variety of novel signalling mechanisms for superoxide. For the first time, it was demonstrated that superoxide activates the hydrolysis of phosphoinositides and increases IP3levels in vascular SMCs mainly through the stimulation of tyrosine kinase-link PLCy signal pathway. It was also found that superoxide reduces cGMP formation and suppresses the cross-inhibition of IP3by cGMP, thus facilitating 1133formation. The selective effects of superoxide on 1133and cGMP pathways as well as the existence of a cross-inhibition of IP3formation by cGMP pathway provide novel mechanisms for the signalling role of superoxide in vascular SMCs. Therefore, the altered signalling effects of superoxide on the IP3pathway and the cGMP pathway, which were demonstrated in vascular SMCs from SHR for the first time in this study, could thus be responsible for the alterations in cellular signal transduction mechanisms in SHR and might contribute to the development and/or maintenance of hypertension. These observations could provide new avenues for the development of new strategies for the prevention or treatment of hypertension.

In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.