Les protéines membranaires représentent environ le tiers des gènes dans les différents génomes séquencés. La prépondérance de ce type de protéines en terme de cibles thérapeutiques (50 % des médicaments) ainsi que leur implication dans beaucoup de phénomènes cellulaires tel que la transduction d'énergie, le transport de nutriments et la signalisation reflètent leur importance. Les interactions entre protéines membranaires jouent un rôle primordial dans leur structure, leurs fonctions et leur assemblage en complexes. La fonction de la plupart des protéines membranaires est liée à l'assemblage de leurs segments transmembranaires TMs dans la bicouche lipidique. Les segments TMs sont des morceaux de séquences majoritairement hydrophobes d'environ 20 résidus adoptant une structure en hélice alpha. En fait, les interactions entre hélices TMs sont essentielles pour le repliement des protéines membranaires et leur organisation dans la membrane. Pour cette raison, des interactions qualitatives entre domaines TMs de différentes protéines bitopiques ont été caractérisé en utilisant le système du double hybride bactérien (BACTH) basé sur une complémentation protéique de type adénylate cyclase. Ce système a révélé des interactions homo- et hétérologues entre des domaines TMs appartenant à deux familles de récepteurs humains, la famille des récepteurs du facteur de croissance épidermique à activité tyrosine kinase (EGFRs) et les Neuropilines. / Many cellular and biochemical processes/activities are actually carried out by the complexome, which is defined as a set of protein complexes. Identification and characterization of the complexome are essential for a comprehensive understanding and global visioning of cell functions since protein-protein interactions are the core of an entire interactomics system of any living cell. Membrane proteins make up to 30% of proteomes in eukaryotes and prokaryotes. They form a major class of proteins that are essentially involved in vital processes including bioenergetics, signal transduction, cell adhesion, catalysis and so on. Thus, they also represent more than 50% of all currently available drug targets. The function of most membrane proteins is inextricably linked to the proper packing and assembly of their transmembrane (TM) segments in the lipid bilayer. So, deciphering the contribution of TM domains interaction in the assembly of protein complexes will help to understand the dynamic assembly of membrane proteins complexes which are most important in cell signaling. For this reason, qualitative interactions between the TM domains of different bitopic proteins have been characterized using the bacterial adenylate cyclase complementation assay (BACTH). This system has been successfully adapted in the lab to study the homo- and heteromeric associations of selected TM sequences, using well characterized interactions as controls. Moreover, BACTH has revealed TM interactions of two major classes of mammalian membrane receptors, the family of epidermal growth factor receptors (EGFRs) which belongs to receptor tyrosine kinases (RTKs) superfamily and the neuropilins.

Es gibt tausende regulatorische Proteine in Eukaryoten, die spezifische cis-regulatorischen Elemente von Genen und/oder RNA-Transkripten binden und die Genexpession koordinieren. Auf DNA-Ebene modulieren Transkriptionsfaktoren (TFs) die Initiation der Transkription, während auf RNA-Ebene RNA-bindende Proteine (RBPs) viele Aspekte des RNA-Metabolismus und der RNA-Funktion regulieren. Für hunderte dieser regulatorischer Proteine wurden die gebundenen Gene beziehungsweise RNA-Transkripte, sowie deren etwaige Sequenzbindepräferenzen mittels in vivo oder in vitro Hochdurchsatz-Experimente bestimmt. Zu diesen Methoden zählen unter anderem Chromatin-Immunpräzipitation (ChIP) gefolgt von Sequenzierung (ChIP-seq) und Protein Binding Microarrays (PBMs) für TFs, sowie Cross-Linking und Immunpräzipitation (CLIP)-Techniken und RNAcompete für RBPs. In vielen Fällen kann die zum Teil hohe Bindespezifität für ein zumeist sehr kurzes Sequenzmotiv regulatorischer Proteine nicht allein durch die gebundene Primärsequenz erklärt werden. Um besser zu verstehen, wie verschiedene Proteine ihre regulatorische Spezifität erreichen, haben wir zwei Computerprogramme entwickelt, die zusätzliche Informationen in die Analyse von experimentell bestimmten Bindestellen einbeziehen und somit differenziertere Bindevorhersagen ermöglichen. Für Protein-DNA-Interaktionen untersuchen wir die Bindungsspezifität paraloger TFs (d.h. Mitglieder der gleichen TF-Familie). Mit dem Fokus auf der Unterscheidung von genomischen Regionen, die in vivo von Paaren eng miteinander verwandter TFs gebunden sind, haben wir ein Klassifikationsframework entwickelt, das potenzielle Co-Faktoren identifiziert, die zur Spezifität paraloger TFs beitragen. Für Protein-RNA-Interaktionen untersuchen wir die Rolle von RNA-Sekundärstruktur und ihre Auswirkung auf die Auswahl von Bindestellen. Wir haben einen Motif-Finding-Algorithmus entwickelt, der Sekundärstruktur und Primärsequenz integriert, um Bindungspräferenzen der RBPs besser zu bestimmen. / There are thousands of eukaryotic regulatory proteins that bind to specific cis regulatory regions of genes and/or RNA transcripts and coordinate gene expression. At the DNA level, transcription factors (TFs) modulate the initiation of transcription, while at the RNA level, RNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. The DNA or RNA targets and/or the sequence preferences of hundreds of eukaryotic regulatory proteins have been determined thus far using high-throughput in vivo and in vitro experiments, such as chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) and protein binding microarrays (PBMs) for TFs, or cross-linking and immunoprecipitation (CLIP) techniques and RNAcompete for RBPs. However, the derived short sequence motifs do not fully explain the highly specific binding of these regulatory proteins. In order to improve our understanding of how different proteins achieve their regulatory specificity, we developed two computational tools that incorporate additional information in the analysis of experimentally determined binding sites. For protein-DNA interactions, we investigate the binding specificity of paralogous TFs (i.e. members of the same TF family). Focusing on distinguishing between genomic regions bound in vivo by pairs of closely-related TFs, we developed a classification framework that identifies putative co-factors that provide specificity to paralogous TFs. For protein-RNA interactions, we investigate the role of RNA secondary structure and its impact on binding-site recognition. We developed a motif finding algorithm that integrates secondary structure together with primary sequence in order to better identify binding preferences of RBPs.

More human lives have been lost to tuberculosis than to any other disease and despite the availability of effective short course chemotherapy (DOTS) as well as the Bacilli Calmette Guerin (BCG) vaccine, tuberculosis continues to claim more than a million lives annually. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is one of the most successful and scientifically challenging pathogens of all time. However in the last two decades, the ability to perform molecular genetic analysis of M. tuberculosis has resulted in powerful new research tools, while the availability of the complete genome sequence has provided us with a wealth of new information and understanding of the biology of this major pathogen. One of the major challenges, however, is to analyze the properties and functions of those genes that are unique to M. tuberculosis genome. The identification and characterization of such genes which impart various survival strategies employed by M. tuberculosis for successful infection will be of particular significance. One of the important outcomes from the complete genome sequence of M. tuberculosis is the discovery of two multigene families designated PE (99 members) and PPE (69 members) named respectively for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. In addition to these motifs, proteins of the PE family possess highly homologous N-terminal domains of approximately 100 amino acids (PE domain), whereas the PPE proteins possess a highly homologous N-terminal domain of about 180 amino acids (PPE domain). Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. The current study focuses on Rv3097c gene of M. tuberculosis, a PE family gene that was bioinformatically predicted to be a triacylglycerol hydrolase (lipase). In order to decipher the role of the PE domain, we have carried out functional characterization of the Rv3097c gene (also named lipY) as it was, initially, the only known PE protein for which an enzymatic function (i.e. lipase activity) had been predicted. Further, to understand the function of PE family proteins, an important question that needs to be answered is; whether the PE domain of different PE family proteins has similar or different functions? In this context, our studies were focused on studying the functional role of the PE domain in LipY, as outlined below. In general, the in vivo function and subcellular localization of any protein are integrally connected. PE domain has been reported to be essential for cell wall localization of PE_PGRS33, another PE family protein. Therefore we investigated the subcellular localization of LipY and the influence of the PE domain on subcellular localization of LipY. LipY and a truncated form of LipY lacking the PE domain [LipY(ΔPE)] were expressed in mycobacteria(M. smegmatis and M. bovis BCG). Subcellular fractionation and western blot demonstrated that both LipY and LipY(ΔPE) were predominantly detected in the cell wall fraction, indicating that LipY is localized to the cell wall and the PE domain of LipY was not required for translocation of LipY to cell wall. This result is in contrast to the findings for PE_PGRS33, where the absence of the PE domain caused the cell wall associated protein to localize to the cytosol. Furthermore, immuno-electron microscopy of M. bovis BCG expressing LipY(ΔPE) clearly showed a cell surface localization of LipY(ΔPE). These results signify that the function of the PE domain might not always be similar amongst different PE family proteins. In order to further investigate the role of the PE domain in LipY, we studied the lipase activity of LipY and the influence of the PE domain on lipase activity. Bioinformatic analysis confirmed the presence of a lipase domain containing a GDSAG active site motif characteristic of lipases. Overexpression of LipY in mycobacteria (M. smegmatis and M. bovis BCG) resulted in a significant reduction in the pool of triacylglycerols (TAG), consistent with the lipase activity of this enzyme. Interestingly, this reduction was more pronounced in mycobacteria overexpressing LipY(ΔPE), suggesting that the presence of the PE domain diminishes the lipase activity of LipY. In vitro lipase assays also confirmed LipY(ΔPE) as a more efficient lipase compared to the wild-type LipY. Together these results suggest that the PE domain of LipY might be involved in the modulation of lipase activity. Surprisingly, M. marinum, another pathogenic mycobacteria, possesses a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain. The overexpression of LipYmar in M. smegmatis significantly reduced the TAG pool suggesting that it is a triacylglycerol hydrolase/lipase. Interestingly, similar to the removal of the PE domain of LipY, this reduction in the TAG pool was further pronounced when the PPE domain of LipYmar was removed. This suggests that PE and PPE domains might share similar functional roles in modulating the enzymatic activities of these lipase homologs. In order to assess the in vivo relevance of LipY expression during M. tuberculosis infection, we examined the humoral immune responses against LipY in sera derived from various clinical categories of tuberculosis patients. The presence of specific antibodies against any protein is suggestive of expression of the protein during infection and could potentially be used to differentiate between healthy individuals and infected patients (serodiagnosis of tuberculosis). The cell wall localization suggested that LipY may be accessible for interaction with the host immune system during infection. Moreover, humoral responses were observed against LipY in mice immunized with DNA constructs expressing LipY, indicating that LipY could be an effective B-cell antigen. Accordingly, a strong humoral response against LipY and LipY(ΔPE) was observed in tuberculosis patients compared to healthy individuals, suggesting that LipY is expressed during infection by clinical strains of M. tuberculosis and might represent an immunodominant antigen of M. tuberculosis with potential use in serodiagnosis of tuberculosis.

ΔPhe an analogue of a coded amino acid phenylalanine (Phe) residue but with double bond between Cα and Cβ atoms, is one of the well studied residue among all the dehydro amino acids, as a conformation constraining amino acid. Due to the presence of double bond Cα=Cβ, and consequent conjugation of ΔPhe ring electrons with Cα=Cβ double bond, ΔPhe gains conformation restricting (constraining) characteristics compared to coded amino acid Phe. ΔPhe which assumes an achiral residue has all its atoms restricted to an approximate plane. Apart from the conformation constraining property, the designer friendly ΔPhe residue has its ability to i) engage in side chain aromatic interactions ii) act as nuclei for C-HLO/N-HLπ weak interactions involving the side chain and/ or backbone atoms, and iii) acquire ambidextrous conformation as observed in many model peptides. It is these properties, which makes ΔPhe, a residue of intense research in the field of de novo protein secondary and super secondary design. Analysis of solid state and solution state structures of containing ΔPhe residues suggests that ΔPhe, in general induces β-bend in short peptides and 310-helical conformation in longer peptides (>4).

One-third of the world’s population is latently infected with Mycobacterium tuberculosis, which causes over 2 million deaths every year. The current live attenuated vaccine, Bacille Calmette-Guerin (BCG), protects against miliary tuberculosis in children, but fails to consistently protect against pulmonary tuberculosis in adults. The global resurgence of tuberculosis, together with the HIV pandemic and emerging multi-drug resistance, has heightened the need for an effective vaccine. Completion of the M. tuberculosis genome sequence paved way for identification of many new candidate antigens for protective vaccine against tuberculosis. This includes the discovery of two multigene families of proteins PE and PPE which constitute 10% of the coding capacity of the M. tuberculosis genome. Members of the PE and PPE protein families are characterized by highly conserved N-terminal domains and the C-terminus, however, exhibit considerable variation in the number of residues as well as in the sequence. Till date, little is known about the functional role of the proteins of PPE or PE family in the biology of M.tuberculosis. Some of the PE_PGRS proteins have been found to be associated with the cell wall and influence interactions with other cells. PE and PPE family of proteins are of potential interest from the point of view of immune response, since they show antigenic variation which may play a role in immune evasion. Very little is known about the immunogenecity of these two classes of proteins and only few proteins have been shown to be potent B or T cell antigens, like Rv3873, Mtb39 and Rv0915c. Two proteins from PE_PGRS subfamily, Rv1759c and Rv3367 are expressed during infection and show antibody response in humans and rabbits, respectively. Rv1196 and Rv0915c from PPE family have been shown to be good T cell antigens. Another study has shown that the PE domain of PE_PGRS protein Rv1818c upon immunization into mice induces good cell mediated immune response in mice, whereas the PGRS domain is responsible for good humoral response. In humans there is increasing evidence to suggest that CD8+ T cells are elicited in response to infection with mycobacteria. CD8+ CTL may play an important role through several mechanisms. They produce potent anti-bacterial cytokines such as IFN-γ and TNF-α in response to antigenic stimulation and IFN-γ is critical for immunity to TB. Thus, identification of antigens and peptides that induce T cell responses could be useful for designing new vaccines to protect against TB. Relatively few epitopes in mycobacterial antigens have so far been identified for human CD8 T cells. In this regard, release of genome sequences of M. tuberculosis has provided an opportunity to identify proteins with vaccine potential that could give immune protection in individuals with different HLA backgrounds. Objectives and scope of the present work 1. Prediction of putative T cell antigens in PE and PPE family of proteins of Mycobacterium tuberculosis through immuno-informatics approach 2. Evaluation of immune response to three of the PE and PPE proteins in mouse model. 3. Evaluation of immune response against chosen PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive healthy donors and TB patients. 4. Immune response to multi-epitope DNA vaccine construct for Mycobacterium tuberculosis. Prediction of MHC class I peptides from PE and PPE proteins. In an effort to identify potential T cell antigens from PE and PPE family of proteins, we have carried out a systematic in silico analysis of the 167 different PE and PPE proteins. Employing immuno-informatics approach, a set of HLA class I binding peptides have been identified from these proteins. Further, their binding abilities have been ascertained using independent methods such as molecular modeling and structural analysis methods. The nonameric sequences from PE and PPE families of proteins were predicted to contain high percentage of binding peptides to human class I HLA, whereas PE_PGRS proteins show relatively low level of binding. This difference is seen in spite of PE and PE_PGRS being Sub-families of the same family, PE. Seventy-one high- as well as low-affinity peptides from both PE and PPE proteins have been analyzed for structural compatibility with crystal structures of HLA in terms of intermolecular energies and were found to correlate well with the corresponding affinities predicted by the BIMAS algorithm. Most of the peptides binding to HLA are specific with very few promiscuous binders. Identification of T cell epitopes from three of the PE/PPE proteins using DNA immunization This work describes the evaluation of immune responses to three of the PE and PPE proteins in mouse model. Three of PE and PPE proteins, coded by Rv1818c, Rv3812 and Rv3018c genes were chosen based on immuno-informatics approach. They were cloned, expressed in prokaryotic and mammalian expression vectors and recombinant protein expressing stable cell lines were made. T lymphocytes from DNA immunized mice recognize synthetic peptides from chosen proteins in vitro, indicating that these peptides are being processed and presented by MHC molecules to T cells. By MHC stabilization assay, 5 of the synthetic peptides were found to stabilize the MHC class I molecules on the cell surface for more than 6 hrs, validating the computational prediction. Recognition of T cell epitopes derived from PE/PPE proteins by human PBMCs This work describes the evaluation of immune response against three of PE and PPE proteins of Mycobacterium tuberculosis with Human Peripheral Blood Mononuclear Cells (PBMCs) from PPD positive Healthy donors and TB patients. Proliferation response of PBMCs from ten PPD positive healthy donors as well as from ten TB patients, indicated that the peptides from PE and PPE proteins of Mtb can sensitize naive T cells and induce peptide specific IFN-γ and also the T cell response to the chosen peptides was both HLA class I restricted and CD8 mediated. After the peptide specific expansion, significant percentage of CD8+ T cells were shown to secrete IFN-γ and stained positive for perforin. Antigen specific CD8+ T cells were found to have cytolytic potential in addition to their cytokine function. Immune response to a multiepitope DNA vaccine in mouse model Minigene poly-epitope vaccine constructs coding for nine peptides derived from identified T cell antigens of PE and PPE proteins and three of the experimentally mapped epitopes from M tuberculosis was designed and constructed. The minigene was used to immunize mice and the immune response was tested. The DNA primed splenocytes recognized the full length poly-epitope protein as well as the individual peptides. T cell response to epitopes was enhanced by mere presence in multi-epitope construct compared to full length antigens. Human PBMCs derived from both PPD+ve and TB patients also recognized the peptides in vitro. It is thus obvious that a large cocktail of proteins are required to achieve reasonable population coverage. Besides, this work suggests the feasibility of designing haplotype specific subunit vaccine, which can be given to individuals with known HLA haplotype. The haplotype specific vaccines can be combined to target a population where the distribution of HLA alleles is known. This work also indicates that use of single or limited number of genes in a DNA vaccine may not be suitable to cover a given population.

This study has attempted to show the overarching significance of the conquering motif in relation to discourse dynamics of the entire book of Revelation and the significance of salvific history for its syntagmatic understanding. Based on language-in-use as a whole between the model author and the model audience, syntagmatic analysis (i.e., SVU analysis) and associative analysis (i.e., sign-intertextual reading) are eclectically and concertedly utilized by means of sampling analysis. Utilizing this integrative method, the findings are as follows: (1) the interwoven network of the prologue (Rev 1:1-8) programmatically provides the paradigmatic reading strategy for understanding the key paraenetic motif in the rest of the book against the background of salvific history; (2) by summarizing the churches’ earthly prophetic roles – withdrawal and witness through martyrdom – in terms of conquering, the model author alerts his audience to the military significance of their daily actions or choices in their ordinary earthly lives through visionary communication; (3) just as the prologue preliminarily guides, the ever-forward-moving historical framework serves as an incentive device for the paraenetic-imperative in Rev 2-3 and 4-22.

No description available.

No description available.

Plusieurs monographies récentes se sont intéressées à la traduction des littératures africaines europhones (Gyasi 2006, Bandia 2008, Batchelor 2009), faisant valoir le concept d’autotraduction (au sens métaphorique) et insistant sur le fait que ces écritures sont porteuses d’une oralité ou de marques linguistiques issues des langues parlées par les écrivains. Toutefois, la question de l’hybridité comme point de jonction entre littératures orales et écrites a encore rarement été examinée sous un angle poétique et c’est précisément dans cet esprit que cette recherche a été entreprise. Dans un premier temps, à partir des ouvrages originaux de six auteurs, trois d’expression littéraire anglaise (Farah, Hove et Armah) et trois d’expression littéraire française (Waberi, Adiaffi et Djebar), je montre en quoi ces écritures méritent d’être qualifiées de poétiques avant de mettre cette esthétique en relation avec le patrimoine littéraire de chacun des auteurs du corpus; ponctuellement, d’autres affiliations littéraires sont mises en évidence. Cette poétique est examinée dans sa dimension mélopoéique (Pound 1954), c’est-à-dire sous l’angle des structures audibles, appelées aussi figures de style jouant sur la forme phonétique des mots (Klein-Lataud 2001). Dans un second temps, j’examine comment cette poétique sonore a été recréée, tant de manière qualitative que quantitative, dans les traductions de Bardolph, de Richard et de J. et R. Mane (pour les auteurs d’expression anglaise) et de Garane, de Katiyo et de Blair (pour les auteurs d’expression française). Les enjeux associés à la réactivation des structures poétiques sonores sont mis en évidence dans le dernier chapitre qui propose un tour d’horizon des modalités de « consommation » de l’objet littéraire et qui s’achève sur les questions soulevées par la progression du livre audio. La méthodologie élaborée dans ce cadre s’inspire essentiellement de Berman (1995) et de Henry (2003). La conceptualisation de la poétique sonore, telle que mise en œuvre dans le contexte particulier de ces littératures, fait appel aux paradigmes de valence traductive (Folkart 2007) et de traduction métonymique (Tymoczko 1999). Par ailleurs, cette recherche s’appuie sur la récente thèse de doctorat de Fraser (2007) consacrée à la théorisation du sonore en traduction. / Recent publications explore Europhone African literatures as translation and in translation (Gyasi 2006, Bandia 2008, Batchelor 2009) insisting that these texts are better understood as a form of self-translation through oral subtexts, showing evidence of linguistic interplay by drawing on the writers’ native language(s). Yet hybridity as an encounter between oral and written literatures has seldom been explored in its poetic dimension. This lack of attention shapes the blueprint of this dissertation. Drawing on six original texts from African writers publishing in English (Farah, Hove and Armah) or in French (Waberi, Adiaffi and Djebar), I show in which extent these writings deserve to be labelled as poetic and how they are informed by the authors’ native literary background; occasionally I discuss other literary affiliations. In this specific context, I explore poetry in its melopoeic actualization (Pound 1954) as it relates to aural poetic devices (i.e. relying on their audible features). I then analyze, qualitatively and quantitatively, how this audible poetry has been reactivated by the translators: Bardolph, Richard, J. and R. Mane (translating Farah, Hove and Armah, respectively); Garane, Katiyo, Blair (translating Waberi, Adiaffi and Djebar, respectively). The last chapter suggests how reactivating sonorous poetic devices in translation relates to different literary modalities, especially audiobooks, as they represent a rapidly growing trend. The methodology draws on Antoine Berman’s translation project (1995) and Jacqueline Henry’s Traduire les jeux de mots (2003). Approaching the translation of aural/audible poetry in the specific context of these texts was facilitated by calling upon paradigms such as translational valency (Folkart 2007) and metonymy (Tymoczko 1999). Last but not least, this dissertation benefited from Fraser’s recent doctoral thesis (2007) dedicated to theorizing sound translation.

This research is focused on the extraction of visual set patterns in digital images, using relational properties like nearness and similarity measures, as well as descriptive properties such as texture, colour and image gradient directions. The problem considered in this thesis is application of topology in visual set pattern discovery, and consequently pattern generation. A visual set pattern is a collection of motif patterns generated from different unique points called seed motifs in the set. Each motif pattern is a descriptive neighbourhood of a seed motif. Such a neighbourhood is a set of points that are descriptively near a seed motif. A new similarity distance measure based on dot product between image feature vectors was introduced in this research, for image classification with the generated visual set patterns. An application of this approach to pattern generation can be useful in content based image retrieval and image classification.