L’étude de l’impact des protéines non structurales NS1 et NS2 du virus respiratoire syncitial sur la réponse immunitaire innée

Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale, nous avons investigué le mécanisme de reconnaissance de l’hôte vis-à-vis de RSV. Nous démontrerons, pour la première fois, que le senseur cytosolique MDA5 est impliqué dans la réponse antivirale contre le RSV. Nous présenterons des résultats préliminaires qui suggèrent que le rôle de MDA5 est non redondant à RIG-I. À l’aide d’ARN interférant dirigé contre RIG-I et de transfection de MDA5, nous démontrerons que MDA5 ne contribue pas à la phosphorylation d’IRF-3, mais plutôt qu’elle régit la stabilité du facteur de transcription. Nous démontrerons aussi que, contrairement à l’hypothèse actuelle sur le fonctionnement de NS1 et NS2, l’inhibition de ces derniers ne provoque pas une augmentation de la cytokine antivirale IFN−β. Cependant, l’expression ectopique de NS1 et NS2 réduit l’activité du promoteur de l’IFN-β et de la protéine cytoplasmic antivirale ISG56 lorsqu’elle est mesurée par essai luciférase.

Not the outside but the inside matters : functional analysis of Capricious and Tartan during Drosophila tracheal morphogenesis

The tubular structures, which transport essential gases, liquids, or cells from one site to another, are shared among various divergent organisms. These highly organized tubular networks include lung, kidney, vasculature and mammary gland in mammals as well as trachea and salivary gland in Drosophila melanogaster. Many questions regarding the tubular morphogenesis cannot be addressed sufficiently by investigating the mammalian organs because their structures are extremely complex and therefore, systematic analyses of genetic and cellular programs guiding the development is not possible. In contrast, the Drosophila tracheal development provides an excellent model system since many molecular markers and powerful tools for genetic manipulations are available. Two mechanisms were shown to be important for the outgrowth of tracheal cells: the FGF signaling pathway and the interaction between the tracheal cells and the surrounding mesodermal cells. The Drosophila FGF ligand encoded by branchless (bnl) is localized in groups of cells near tracheal metameres. The tracheal cells expressing the FGF receptor breathless (btl) respond to these sources of FGF ligand and extend towards them. However, this FGF signaling pathway is not sufficient for the formation of continuous dorsal trunk, the only muticellular tube in tracheal system. Recently, it was found out that single mesodermal cells called bridge-cells are essential for the formation of continuous dorsal trunk as they direct the outgrowth of dorsal trunk cells towards the correct targets. The results in this PhD thesis demonstrate that a cell adhesion molecule Capricious (Caps), which is specifically localized on the surface of bridge-cells, plays an essential role in guiding the outgrowing dorsal trunk cells towards their correct targets. When caps is lacking, some bridge-cells cannot stretch properly towards the adjacent posterior tracheal metameres and thus fail to interconnect the juxtaposing dorsal trunk cells. Consequently, discontinuous dorsal trunks containing interruptions at several positions are formed. On the other hand, when caps is ectopically expressed in the mesodermal cells through a twi-GAL4 driver, these mesodermal cells acquire a guidance function through ectopic caps and misguide the outgrowing dorsal trunk cells in abnormal directions. As a result, disconnected dorsal trunks are formed. These loss- and gain-of-function studies suggest that Caps presumably establishes the cell-to-cell contact between the bridge-cells and the tracheal cells and thereby mediates directly the guidance function of bridge-cells. The most similar protein known to Caps is another cell adhesion molecule called Tartan (Trn). Interestingly, trn is expressed in the mesodermal cells but not in the bridge-cells. When trn is lacking, the outgrowth of not only the dorsal trunks but also the lateral trunks are disrupted. However, in contrast to the ectopic expression of caps, the misexpression of trn does not affect tracheal development. Whereas Trn requires only its extracellular domain to mediate the matrix function, Caps requires both its extracellular and intracellular domains to function as a guidance molecule in the bridge-cells. These observations suggest that Trn functions differently from Caps during tracheal morphogenesis. Presumably, Trn mediates a matrix function of mesodermal cells, which support the tracheal cells to extend efficiently through the surrounding mesodermal tissue. In order to determine which domains dictate the functional specificity of Caps, two hybrid proteins CapsEdTrnId, which contains the Caps extracellular domain and the Trn intracellular domain, and TrnEdCapsId, which consists of the Trn extracellular domain and the Caps intracellular domain, were constructed. Gain of function and rescue experiments with these hybrid proteins suggest on one hand that the extracellular domains of Caps and Trn are functionally redundant and on the other hand that the intracellular domain dictates the functional specificity of Caps. In order to identify putative interactors of Caps, yeast two-hybrid screening was performed. An in vivo interaction assay in yeast suggests that Ras64B interacts specifically with the Caps intracellular domain. In addition, an in vitro binding assay reveals a direct interaction between an inactive form of Ras64B and the Caps intracellular domain. ras64B, which encodes a small GTPase, is expressed in the mesodermal cells concurrently as caps. Finally, a gain-of-function study with the constitutively active Ras64B suggests that Ras64B presumably functions downstream of Caps. All these results suggest consistently that the small GTPase Ras64B binds specifically to the Caps intracellular domain and may thereby mediate the guidance function of Caps.

Legumes regulate Rhizobium bacteroid development and persistence by the supply of branched-chain amino acids

One of the largest contributions to biologically available nitrogen comes from the reduction of N-2 to ammonia by rhizobia in symbiosis with legumes. Plants supply dicarboxylic acids as a carbon source to bacteroids, and in return they receive ammonia. However, metabolic exchange must be more complex, because effective N-2 fixation by Rhizobium leguminosarum bv viciae bacteroids requires either one of two broad-specificity amino acid ABC transporters (Aap and Bra). It was proposed that amino acids cycle between plant and bacteroids, but the model was unconstrained because of the broad solute specificity of Aap and Bra. Here, we constrain the specificity of Bra and ectopically express heterologous transporters to demonstrate that branched-chain amino acid (LIV) transport is essential for effective N-2 fixation. This dependence of bacteroids on the plant for LIV is not due to their known down-regulation of glutamate synthesis, because ectopic expression of glutamate dehydrogenase did not rescue effective N-2 fixation. Instead, the effect is specific to LIV and is accompanied by a major reduction in transcription and activity of LIV biosynthetic enzymes. Bacteroids become symbiotic auxotrophs for LIV and depend on the plant for their supply. Bacteroids with aap bra null mutations are reduced in number, smaller, and have a lower DNA content than wild type. Plants control LIV supply to bacteroids, regulating their development and persistence. This makes it a critical control point for regulation of symbiosis. MICROBIOLOGY

PtdIns(5)P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns(5) P). We show that PtdIns(5) P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns(5) P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns(5) P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns(5) P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns(5) P production that plays an important role in host cell responses such as survival.

Genetic diversity of red-fleshed apples (Malus)

Anthocyanins are flavonoid pigments imparting red, blue, or purple pigmentation to fruits, flowers and foliage. These compounds are powerful antioxidants in vitro, and are widely believed to contribute to human health. The fruit of the domestic apple (Malus x domestica) is a popular and important source of nutrients, and is considered one of the top ‘functional foods’—those foods that have inherent health-promoting benefits beyond basic nutritional value. The pigmentation of typical red apple fruits results from accumulation of anthocyanin in the skin. However, numerous genotypes of Malus are known that synthesize anthocyanin in additional fruit tissues including the core and cortex (flesh). Red-fleshed apple genotypes are an attractive starting point for development of novel varieties for consumption and nutraceutical use through traditional breeding and biotechnology. However, cultivar development is limited by lack of characterization of the diversity of genetic backgrounds showing this trait. We identified and cataloged red-fleshed apple genotypes from four Malus diversity collections representing over 3,000 accessions including domestic cultivars, wild species, and named hybrids. We found a striking range of flesh color intensity and pattern among accessions, including those carrying the MYB10 R 6 allele conferring ectopic expression of a key transcriptional regulator of anthocyanin biosynthesis. Although MYB10 R 6 was strongly associated with red-fleshed fruit among genotypes, this allele was neither sufficient nor required for this trait in all genotypes. Nearly all red-fleshed accessions tested could be traced back to ‘Niedzwetzkyana’, a presumed natural form of M. sieversii native to central Asia.

Overexpression of coconut AINTEGUMENTA-like gene, CnANT, promotes in vitro regeneration in transgenic Arabidopsis

Knowledge of the molecular biological changes underlying the process of embryogenesis is important for the improvement of somatic embryogenesis of coconut. Among the transcription factors that control the transition from vegetative to embryogenic growth, members of APETALA2/Ethylene-responsive element binding protein domain family play an important role in promoting embryo development. Significant insights into the role of AP2 genes have been obtained by the ectopic expression of AP2 sub family genes in transgenic Arabidopsis. A homolog of the AINTEGUMENTA-like gene that encodes the two AP2 domains and the linker region was identified in the coconut genome. Phylogenetic analysis showed that this gene, CnANT, encodes a protein that branched with BABY BOOM/PLETHORA clade in the AINTEGUMENTA-like major clade and was similar to the oil palm EgAP2-1 protein. According to real time RT-PCR results, higher expression of CnANT was observed in more mature zygotic embryos. Also, high CnANT expression was recorded in embryogenic callus compared to other stages of somatic embryogenesis. We examined the effect of ectopic CnANT expression on the development and regenerative capacity of transgenic Arabidopsis. Overexpression of CnANT in Arabidopsis induced hormone free regeneration of explants. Furthermore, ectopic expression of CnANT enhanced regeneration in vitro and suggested a role for this gene in cell proliferation during in vitro culture.

The stability and aggregation properties of the GTPase domain from human SEPT4

The septins are a family of conserved proteins involved in cytokinesis and cortical organization. An increasing amount of data implicates different septins in diverse pathological conditions including neurodegenerative disorders, neoplasia and infections. Human SEPT4 is a member of this family and its tissue-specific ectopic expression profile in colorectal and urologic cancer makes it a useful diagnostic biomarker. Thermal unfolding of the GTPase domain of SEPT4 (SEPT4-G) revealed an unfolding intermediate which rapidly aggregates into amyloid-like fibers under physiological conditions. In this study, we examined the effects of protein concentration, pH and metals ions on the aggregation process of recombinant SEPT4-G using a series of biophysical techniques, which were also employed to study chemical unfolding and stability. Divalent metal ions caused significant acceleration to the rate of SEPT4-G aggregation. Urea induced unfolding was shown to proceed via the formation of a partially unfolded intermediate state which unfolds further at higher urea concentrations. The intermediate is a compact dimer which is unable to bind GTR At 1 M urea concentration, the intermediate state was plagued by irreversible aggregation at temperatures above 30 degrees C. However, higher urea concentration resulted in a marked decay of the aggregation, indicating that the partially folded structures may be necessary for the formation of these aggregates. The results presented here are consistent with the recently determined crystal structure of human septins and shed light on the aggregation properties of SEPT4 pertinent to its involvement in neurodegenerative disease. (C) 2008 Elsevier B.V. All rights reserved.

Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp citri: targeting the division septum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Interação entre o gene TKN2 (KNOX-type I) e o miR156 node durante a transição de fase vegetativa para reprodutiva em tomateiro (Solanum lycopersicum)

Pós-graduação em Ciências Biológicas (Genética) - IBB

The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Análise do polimorfismo da região 3’não traduzida (3’NT) do gene HLA-G de pacientes com esclerose múltipla e neuromielite óptica da região noroeste do estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Impact of complex NOTCH1 mutations on survival in paediatric T-cell leukaemia

Background: Molecular alterations occur frequently in T-ALL and the potential impact of those abnormalities on outcome is still controversial. The current study aimed to test whether NOTCH1 mutations and additional molecular abnormalities would impact T-ALL outcome in a series of 138 T-ALL paediatric cases. Methods: T-ALL subtypes, status of SIL-TAL1 fusion, ectopic expression of TLX3, and mutations in FBXW7, KRAS, PTEN and NOTCH1 were assessed as overall survival (OS) and event-free survival (EFS) prognostic factors. OS and EFS were determined using the Kaplan-Meier method and compared using the log-rank test. Results: The frequencies of mutations were 43.5% for NOTCH1, while FBXW7, KRAS and PTEN exhibited frequencies of 19.1%, 9.5% and 9.4%, respectively. In 78.3% of cases, the coexistence of NOTCH1 mutations and other molecular alterations was observed. In multivariate analysis no statistical association was revealed between NOTCH1 mutations and any other variable analyzed. The mean length of the follow-up was 68.4 months and the OS was 50.7%. SIL-TAL1 was identified as an adverse prognostic factor. NOTCH1 mutation status was not associated with outcome, while the presence of NOTCH1 complex mutations (indels) were associated with a longer overall survival (p = 0.031) than point mutations. Conclusion: NOTCH1 mutations alone or in combination with FBXW7 did not impact T-ALL prognosis. Nevertheless, complex NOTCH1 mutations appear to have a positive impact on OS and the SIL-TAL1 fusion was validated as a negative prognostic marker in our series of T-ALL.

Mechanisms of cell senescence: p21 and DNA damage response in aging and longevity

Theory of aging postulates that aging is a remodeling process where the body of survivors progressively adapts to internal and external damaging agents they are exposed to during several decades. Thus , stress response and adaptation mechanisms play a fundamental role in the aging process where the capability of adaptating effects, certainly, also is related the lifespan of each individual. A key gene linking aging to stress response is indeed p21, an induction of cyclin-dependent kinase inhibitor which triggers cell growth arrest associated with senescence and damage response and notably is involved in the up-regulation of multiple genes that have been associated with senescence or implicated in age-related . This PhD thesis project that has been performed in collaboration with the Roninson Lab at Ordway Research Institute in Albany, NY had two main aims: -the testing the hypothesis that p21 polymorphisms are involved in longevity -Evaluating age-associated differences in gene expression and transcriptional response to p21 and DNA damage In the first project, trough PCR-sequencing and Sequenom strategies, we we found out that there are about 30 polymorphic variants in the p21 gene. In addition, we found an haplotpype located in -5kb region of the p21 promoter whose frequency is ~ 2 fold higher in centenarians than in the general population (Large-scale analysis of haplotype frequencies is currently in progress). Functional studies I carried out on the promoter highilighted that the ―centenarian‖ haplotype doesn’t affect the basal p21 promoter activity or its response to p53. However, there are many other possible physiological conditions in which the centenarian allele of the p21 promoter may potentially show a different response (IL6, IFN,progesterone, vitamin E, Vitamin D etc). In the second part, project #2, trough Microarrays we seeked to evaluate the differences in gene expression between centenarians, elderly, young in dermal fibroblast cultures and their response to p21 and DNA damage. Microarray analysis of gene expression in dermal fibroblast cultures of individuals of different ages yielded a tentative "centenarian signature". A subset of genes that were up- or downregulated in centenarians showed the same response to ectopic expression of p21, yielding a putative "p21-centenarian" signature. Trough RQ-PCR (as well Microarrays studies whose analysis is in progress) we tested the DNA damage response of the p21-centenarian signature genes showing a correlation stress/aging in additional sets of young and old samples treated with p21-inducing drug doxorubicin thus finding for a subset of of them , a response to stress age-related.

Ruolo della fosfolipasi C-β1 nel differenziamento miogenico: identificazione di nuovi target nucleari

The expression of phospholipase C-β1 (PLC-β1) and cyclin D3 is highly induced during skeletal myoblast differentiation. We have previously shown that PLC-β1 activates cyclin D3 promoter during the differentiation of myoblasts to myotubes, indicating that PLC-β1 is a crucial regulator of mouse cyclin D3 gene. Here we report that PLC-β1 catalytic activity plays a role in the increase of cyclin D3 levels and in the induction of differentiation of C2C12 skeletal muscle cells. PLC-β1 mutational analysis revealed the importance of His331 and His378 for the catalytic activity. We show that following insulin administration, cyclin D3 mRNA levels are lower in cells overexpressing the PLC-β1 catalytically inactive form, as compared to wild type cells. We describe a novel signaling pathway elicited by PLC-β1 that modulates Activator Protein-1 (AP-1) activity. Indeed, gel mobility shift assays indicate that there is a c-jun binding site located in cyclin D3 promoter region specifically regulated by PLC-β1 and that c-jun binding activity is significantly increased by insulin stimulation and PLC-β1 overexpression. Moreover, mutation of c-jun/AP-1 binding site decreases the basal cyclin D3 promoter activity and eliminates its induction by insulin and PLC-β1 overexpression. Interestingly, we observed that the ectopic expression of the Inositol Polyphosphate Multikinase (IPMK) in C2C12 myoblasts enhances cyclin D3 gene expression and that the mutation of c-jun site in cyclin D3 promoter determines an impairment of IPMK-dependent promoter induction. These results indicate that PLC-β1 activates a c-jun/AP-1 target gene, i.e. cyclin D3, during myogenic differentiation through IPMK signaling.

Mechanismen der Caveolin-Genregulation in neuralen Zellsystemen

Caveolae sind vesikuläre Invaginationen der eukaryontischen Zellmembran, die bei einer Vielzahl zellbiologischer Prozesse eine bedeutende Rolle spielen. Die strukturellen und funktionellen Hauptbestandteile der Caveolae sind die Caveolin-Proteine, welche von drei homologen Genen (Caveolin-1,-2,-3) kodiert werden. Die Caveoline stellen die Struktur-Organisatoren der Caveolae dar, und regulieren direkt die Aktivität von zahlreichen Caveolae-assoziierten Rezeptorproteinen und Signalmolekülen. Oftmals werden die pleiotropen Effekte der Caveoline über eine Veränderung der Caveolin-Genexpressionsstärke moduliert. In der vorliegenden Arbeit wurden drei unterschiedliche biologische Steuerfaktoren identifiziert, unter deren Kontrolle die Caveolin-Genexpression in neuralen Zellsystemen steht. Bei diesen Faktoren handelt es sich um das Steroidhormon Oestrogen und seine Rezeptoren, den Wachstumsfaktor TGFa und den sekundären Botenstoff zyklisches AMP (cAMP). Oestrogen wirkt über die Aktivierung von Oestrogen-Rezeptoren (ERs) im zentralen Nervensystem in der Regel als neurotropher Faktor. In der vorliegenden Arbeit konnte erstmalig gezeigt werden, daß in humanen Neuroblastom-Zellen (SK-N-MC) die stabile, rekombinante Expression des ERa-Subtyps zu einer drastischen Reduktion der Caveolin-1/-2-Transkription führt, und daß in der Folge die zelluläre Caveolin-Biosynthese eingestellt wird. Eine Analyse des Caveolin-1-Gens ergab, daß einhergehend mit der Inaktivierung der Caveolin-1-Transkription eine Vielzahl der im Promoter enthaltenen CpG-Dinukleotide methyliert vorliegen. Durch pharmakologische Inhibition der nukleären DNA-Methyltransferasen sowie der Histon-Deacetylasen konnte die Caveolin-1-Transkription teilweise wiederhergestellt werden. Diese Befunde lassen auf die Existenz eines DNA-Methylierungs-abhängigen Stilllegungsmechanismus der Caveolin-Genexpression durch ERa schließen. Dagegen führte die Überexpression des ERb-Subtyps in SK-N-MC-Zellen zu keiner Veränderung der Caveolin-1/-2-Expression. Interessanterweise wurde die supprimierende Wirkung des ERa durch die gleichzeitige Überexpression des ERb vollständig aufgehoben. Der mitogene Wachstumsfaktor TGFa wurde als zweites extrazelluläres Signalmolekül identifiziert, welches eine Reduktion der Caveolin-1/-2-Genexpression bewirkt. In primären kortikalen Astrozyten konnte gezeigt werden, daß TGFa seine supprimierende Wirkung auf die Caveolin-1-Expression partiell über die Aktivierung des PI3-Kinase-abhängigen Signalweges vermittelt. Zudem wurde die supprimierende Wirkung von TGFa durch einen Inhibitior der Histon-Deacetylasen relativiert. Daher scheinen sowohl für den ERa als auch für TGFa epigenetische Prozesse bei der Suppression der Caveolin-1-Genexpression eine entscheidende Rolle zu spielen. Intrazellulär wirkte neben der PI3-Kinase auch der Botenstoff cAMP in kortikalen Astrozyten als Suppressor der Caveolin-Genexpression. Es wäre denkbar, daß die Caveolin-Suppression funktioneller Bestandteil des seit langem etablierten Effekts der cAMP-induzierten Astrozyten-Differenzierung ist. Desweiteren wiesen der cAMP- und TGFa-abhängige Signalweg ein überlappendes, Gehirnregion-spezifisches Regulationsprofil der Caveolin-Expression in Astrozyten auf: während in Kortex und Striatum eine Regulation durch cAMP und TGFa erfolgte, blieb diese in Klein- und Zwischenhirn aus. Somit bewirken drei zentrale regulatorische Faktoren der Proliferation und Differenzierung neuraler Zellen eine Reduktion in der Konzentration der pleiotrop funktionellen Caveoline. Zukünftige Studien müssen zeigen, inwieweit die reduzierte Caveolin-Expression für die morphologischen und biochemischen Primärwirkungen dieser Faktoren während der Entwicklung und im Zuge der Tumorgenese mitverantwortlich ist. Außerdem könnten über die Beobachtungen der zellbiologischen Auswirkungen reduzierter Caveolin-Spiegel neue Erkenntnisse über die Funktion dieser Proteine gewonnen werden.