M.Tech. (Chiropractic) / Purpose: The aim of this study was to look at the effectiveness of massage with MYO₂ gel in conjunction with Spinal Manipulative Therapy (SMT) for the treatment of patients with posterior mechanical neck pain, with regards to pain, disability and cervical spine range of motion. The effect of these treatments was evaluated using a questionnaire consisting of Visual Analogue Scale (VAS) and a Vernon-Mior Neck Pain and Disability Index Questionnaire, and by measuring cervical spine range of motion using a cervical range of motion (CROM) measuring device. Method: 30 participants with posterior mechanical neck pain were randomly divided into two groups based on the order they start the study in. Group A – control group, received SMT to the cervical spine, followed by massage with ultrasound gel over the upper trapezius muscle and the area of the posterior neck musculature. Group B – experimental group, received SMT of the cervical spine, followed by massage with MYO₂ gel over the upper trapezius muscle and the area of the posterior neck musculature. Participants were treated six times out of a total of seven sessions, over a maximum three week period. Procedure: Subjective data was collected at the beginning of the first and fourth and seventh consultations. This was done by means of a Visual Analogue Scale (VAS) and a Vernon- Mior Neck Pain and Disability Index Questionnaire in order to assess pain and disability levels. Objective data was collected at the beginning of the first, fourth session, and seventh consultation by means of measuring cervical spine range of motion using a cervical range of motion (CROM) measuring device. Analysis of collected data was performed by a statistician from STATKON; a department of the University of Johannesburg. Results: When comparing the VAS of the initial with the final consultation it showed a statistically significant difference in both groups. This indicated that both interventions were effective in decreasing the perceived level of neck pain (VAS score). When comparing the Neck Pain and Disability Index of the initial with the final consultation it showed a statistically significant difference in both groups. This indicated that both interventions were effective in decreasing neck pain and disability of the cervical spine. When comparing the Cervical Range of Motion (CROM) of the initial consultation with the final consultation it showed a statistically significant improvement in both groups in all directions of motion. Both groups resulted in an increase in range of motion over time. Group B had a greater average CROM percentage increase of 28.60% average compared to Group A which had an average percentage increase of 22.25%.

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.

Thèse numérisée 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.

Development of hair cell stereocilia bundles involves three stages: elongation, thickening, and supernumerary stereocilia retraction. Although Myo-XVa is known to be essential for stereocilia elongation, its role in retraction/thickening remains unknown. We quantified stereocilia numbers/diameters in shaker-2 mice (Myo15sh2) that have deficiencies in “long” and “short” isoforms of myosin-XVa, and in mice lacking only the “long” myosin-XVa isoform (Myo15ΔN). Our data showed that myosin-XVa is largely not involved in the developmental retraction of supernumerary stereocilia. In normal development, the diameters of the first (tallest)/second row stereocilia within a bundle are equal and grow simultaneously. The diameter of the third row stereocilia increases together with that of taller stereocilia until P1-2 and then either decreases almost two-fold in inner hair cells (IHCs) or stays the same in outer hair cells (OHCs), resulting in a prominent diameter gradation in IHCs and less prominent in OHCs. Sh2 mutation abolishes this gradation in IHCs/OHCs. Stereocilia of all rows grow in diameters nearly equally in Myo15sh2/sh2 IHCs and OHCs. Conversely, ΔN mutation does not affect normal stereocilia diameter gradation until ~P8. Therefore, myosin-XVa “short” isoform is essential for developmental thinning of third row stereocilia, which causes diameter gradation within a hair bundle.

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.