Âncoras de glicosilfosfatidilinositol (GPI) são estruturas essenciais para a ancoragem de glicoconjugados e proteínas na superfície celular de protozoários. Trypanosoma cruzi produz uma gama de estruturas únicas de GPI, as quais ancoram mucinas e trans-sialidases, que participam de processos envolvidos na interação entre parasita e hospedeiro. Afim de estudar a biossíntese de âncora de GPI de T. cruzi e possivelmente utilizá-la como um potencial alvo anti-T.cruzi, este trabalho visa sintetizar análogos de âncoras de GPI e analisar o potencial destas moléculas como substratos da via biossintética de GPIs. Neste contexto, um pseudo-dissacarídeo 31 foi sintetizado através de O-glicosilação entre os doadores derivados de azido-glicopiranosídeo (32 ou 33a-d) e o acceptor de mio-inositol (34), preparados a partir de cloridrato de glucosamina (35) e metil-?-D-glucopiranósido (36), respectivamente, usando proteção/desproteção ortogonais. Cinco diferentes dadores de glicosídicos (32 e 33a-d) foram preparados para investigar a influcia dos seus grupos protetores na estereoselectividade da reações de O-glicosilação na presença de diferentes solventes para estudar o favorecimento da configuração ?, presente em GPIs. Ademais, a síntese do aceptor de mio-inositol 34 foi realizada em 12 etapas pela estratégia do rearranjo Ferrier para formar um derivado de ciclitol, além de diversas proteções/desproteções, funcionalizado que permite a introdução regiosselectiva da unidade de azido glicose (32-33a-d) e uma porção de fosfolípido no seu C-1 e posições C-6, respectivamente. Assim, O-glicosilação entre doador 33c e o acceptor 34, foi realizada utilizando TMSOTf como promotor para originar o composto 31c com boa estereoseletividade para ?, com elevado rendimento (~70%). Após a dealilação de 31c, a porção fosfodiéster contendo uma cadeia C-8 (87), preparada pela abordagem do H-fosfonato, foi anexada ao pseudo-dissacarídeo para gerar, após desprotecção global, o composto alvo 30a. A mesma estratégia sintética foi aplicada ao preparo do composto 91 contendo uma cadeia lateral alquil-naftil (90) que está em últmas etapas de desproteção para gerar o composto final 30c. Atualmente, o composto 30a está sendo testado como substrato da biossíntese de âncoras de GPI em membranas microssomais de Euglena gracilis, uma alga unicelular não patogênica, que pode potencialmente ser utilizada como modelo para parasitas humanos filogeneticamente relacionados. Após a incubação do potencial substrato de GPI 30a com membranas microssomais de E. gracilis para geração de metabólitos, será realizada análise do extrato por LC-MS e, eventualmente, isolamento dos produtos formados para posterior caracterização. Os produtos que apresentarem atividade como substrato ou como inibidores da biossíntese de GPI em E. gracilis serão também ensaiados na membrana microsomal do T. cruzi. / Glycosylphosphatidylinositol (GPI) anchors are essential molecules to attach glycoconjugates and proteins in protozoan\'s cell surface. Trypanosoma cruzi produces a range of unique GPI structures that anchor mucins and trans-sialidases which participate in important processes involved in the interaction between parasite and host. As an effort to study T. cruzi GPI anchor biosynthesis and possibly use it as a potential target for an antichagasic drug, this work aims to synthesize GPI anchor analogs (labelled or not) and analyze the potential of these molecules as substrates in the GPI biosynthetic pathway. In this context, a pseudo-disaccharide 31 was synthesized by O-glycosylation reaction between azide glycosyl donors (32 or 33a-d) and myo-inositol acceptor (34), prepared from glucosamine (35) hydrochloride and methyl ?-D-glucopyranoside (36), respectively, using orthogonal protection/ deprotection. Five different glycosyl donors (32 and 33a-d) were prepared to investigate the influence of their protective groups on the stereoselectivity of the O-glycosylation reaction in the presence of different solvents to afford the required GPI ?-linkage. In addition, the synthesis of the myo-inositol acceptor 34 was achieved using several protection/deprotection steps, besides the Ferrier rearrangement, to form a functionalized cyclitol derivative that enables the regioselective introduction of the azide glycoside unit and phospholipid moiety on its C-1 and C-6 positions, respectively. Then, O-glycosylation of acceptor 34 with donor 33c was accomplished in diethyl ether, using TMSOTf as promoter to give exclusively ?-anomer 31c in high yield. After deallylation of 31c, the phosphodiester moiety bearing an octyl chain (87), prepared by the H-phosphonate approach, was appended to the pseudo-disaccharide to yield, after deprotection, target compounds 30a. The same synthetic strategy was applied to the preparation of 30c, even though in the protective form, compound 91 bearing an alkyl-naphthyl side chain (90). Currently, compound 30a is being tested as substrates of GPI anchor biosynthesis in Euglena gracilis cell membranes, a non-pathogenic unicellular algae, which may potentially be used as a model for phylogenetically related human parasites. After incubation of the potential GPI substrate 30a with E. gracilis microsomal membranes for generation of metabolites, the analysis by LC-MS and, eventually, isolation of the products will be performed for further characterization. Products that show any substrate or inhibitory activities will be also assayed in T. cruzi microsomal membrane.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (InsP6), also called phytic acid, is ubiquitous in eukaryotic cells and the most abundant inositol phosphate derivative. Loblolly pine (LP, Pinus taeda) constitutes the primary commercial species in the southern forest of U.S. Somatic embryogenesis (SE) is an effective technique to maintain the desirable genetic composition of the progeny and to accomplish the efficiency of propagation. SE can also serve as a tool for study of plant development. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). In LP SE, FG tissue is absent in the embryogenic tissue culture. We found that extracts from early-stage FG stimulate growth and multiplication of early-stage somatic embryos, whereas FG water extracts from late stage contain substance(s) inhibitory to early-stage somatic embryo growth (DeSilva et al., 2007). We now present the isolation and identification of the inhibitory substance as InsP6 by means of water extraction, two gel filtrations and two ion exchange FPLC chromatographies. The results represent the first complete structural characterization of InsP6 from a natural product using LC/MS, LC/MS/MS, exact MS, 1D- and 2D-NMR analyses. We also report that there is a good correlation between the amount of InsP6 purified from FG tissue (1.3 nmoles per full-term FG) and the amount of InsP6 which inhibits somatic embryo growth. This novel approach of isolating and characterizing InsP6 from plant tissue, and investigating its role on SE can allow us to improve SE technology by circumventing current bottleneck, to elucidate enigmatic functions of InsP6 in plants, and most importantly, to utilize this molecule properly.

Introduktion: Polycystiskt ovarialsyndrom ( PCOS)  är en av de vanligaste orsakerna till infertilitet och metabola störningar bland kvinnor i fertil ålder. Diagnosen vid PCOS karakteriseras av tre relaterade hälsotillstånd: ovulationsstörningar, hyperandrogenism och polycystiskt ovarial morfologi (PCOM). Oberoende av ovanstående huvuddiagnoser verkar insulinresistens (IR) och kompensatorisk hyperinsulinism spela en viktig roll för patogenesen av PCOS. Till följd av detta har ämnen som ökar insulinkänsligheten, såsom myo-inositol, hamnat i allt större fokus som en potentiell behandling av PCOS. Syftet med detta arbete var att undersöka effekten av ett kosttillskott med MI hos kvinnor som lider av infertilitet och ovulationsstörningar, med PCOS som bakomliggande sjukdom.   Metod: Detta är en litteraturstudie där litteratur sökts via databaserna PubMED och MEDLINE. Sökningen genomfördes med sökorden “PCOS” “Inositol PCOS” “myo-inositol PCOS” “myo-inositol metabolic effects PCOS” and “myo-inositol endocrine effects”. Totalt åtta originalstudier svarade mot utvalda urvalskriterier. Utifrån de utvalda studierna analyserades och sammanställdes vidare relevanta effekter av MI tillskott på infertilitet och ovulationsstörningar.  Resultat: Alla de undersökta studierna visade statistiskt signifikanta förbättringar när det gällde ovulationsstörningar. Detta indikerar även en förbättrad fertilitet hos patienterna. Signifikanta minskningar av androgena nivåer (särskilt testosteron) samt en ökad insulinkänslighet rapporterades därtill i ytterligare en majoritet av studierna (6 av 8 resp 5 av 8 studier).  Slutsats: Resultaten visar att Mi som tillskott verkar lindra ovulationsstörningar och förbättra fertilitet, vilket därtill stärker hypotesen om MI som en möjlig behandling av reproduktiva störningar hos kvinnor med PCOS. Fler studier bör genomföras för att fastställa effekten av MI-tillskott vid PCOS. / Introduction: Polycystic ovarian syndrome, PCOS is one of the most common causes of infertility and metabolic disturbances among women in reproductive age. The diagnosis of the syndrome assumes the occurrence of three interrelated health stages:  ovulation disorders, hyperandrogenism and polycystic ovarian morphology (PCOM). In addition to the main diagnoses, insulin resistance (IR) and compensatory hyperinsulinemia seems to  play an important role in the pathogenesis of PCOS. Recent studies have furthermore investigated insulin sensitizers, such as myo-inositol (MI), as a potential alternative treatment of PCOS. The aim of this study was thereby to investigate the effect of MI- supplement on PCOS patients suffering from infertility and ovulation disorders.  Method: This is a literature study relevant literature was gathered through PubMed and MEDLINE. The keywords used for the search were “PCOS” “Inositol PCOS” “myo-inositol PCOS” “myo-inositol metabolic effects PCOS” and “myo-inositol endocrine effects”. A total of 8 studies were selected based on previously determined selection criteria. Among the selected studies, relevant effects of MI-supplement on infertility and ovulation disorders in PCOS patients were further analyzed and compiled. Results: A majority of the studies reported either statistic significant ameliorated or normalized ovulation disorders, indicating an increased fertility among the patients. Likewise, a statistically significant amelioration in androgenic levels (especially testosterone) and insulin levels were also reported in a majority of the studies.  Conclusion: According to the compiled results, there seems to be an enhancing effect of MI-supplement on ovulation disorders and infertility, furthermore strengthening the hyphothesis of MI as a potential treatment for reproductive disorders in PCOS patients. Finally, more studies should be conducted to determine the effect of MI supplements on PCOS.

To survive, an organism must constantly adjust its internal state to changes in the environment from which it receives signals. The signals set off a chain of events referred to signal transduction. Signal transduction systems are especially important in multicellular organisms, such as plants and animals, because of the need to coordinate the activities of hundreds to trillions of cells. Plants, in particular, have a special need for perceiving signals from their environment because of their static nature. As in the animal cell, the first steps in perception of a signal include signal interaction with a receptor, signal amplification through second messenger production, and signal termination through second messenger hydrolysis. Myo-inositol polyphosphate 5-phosphatases (5PTases) (EC 3.1.3.56) have unique signal terminating abilities toward the second messenger inositol trisphosphate (Ins (1,4,5)P3, InsP3). In Arabidopsis thaliana there are 15 members of the 5PTase family, the majority of which contain a single 5PTase catalytic domain. Four members of the Arabidopsis 5PTase family, however, have a unique protein domain structure, with additional N-terminal WD40 repeats that are implicated in protein-protein interactions. The research presented here focused on the identification of 5PTase interacting proteins and the characterization of their functional role in Arabidopsis. To accomplish this goal, I examined a 5PTase13-interacting protein, the sucrose (Suc) nonfermenting-1-related kinase, SnRK1.1, an important energy sensor that is highly conserved among eukaryotes. My identification of a 5PTase13:SnRK1.1 complex points to the novel interaction of this metabolic modulator and inositol signaling/metabolism. 5PTase13 , however, plays a regulatory role in other plant specific processes as well, since I also identified the Arabidopsis homolog (Atp80) of the human WDR48 (HsWDR48, Hsp80) as a novel protein interactor of 5PTase13. My results indicate that Atp80 is important for leaf emergence, development and senescence likely via a regulatory interaction with 5PTase13 and PINOID â binding protein (PBP1). / Ph. D.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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.

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.

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.