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In this research, important stress and redox regulatory genes present in Hydra vulgaris were isolated and characterized to facilitate our understanding of the evolution and mechanisms of stress response. H. vulgaris heat shock protein 70 (HvHSP70), extracellular copper zinc superoxide dismutase (HvECCuZnSOD), manganese superoxide dismutase (HvMnSOD), phospholipid peroxidase glutathione peroxidase (HvPHGPx) and monofunctional catalase (HvCatalase) were cloned and characterized with regard to stress response, phylogeny and molecular structure. The HSP70 gene isolated from H. vulgaris encodes a polypeptide of 650 amino acids (Mw=710,037) and is interrupted by three intron sequences. The 5' non-coding region of the HvHSP70 possessed the canonical heat shock elements. Phylogenetically HvHSP70 formed a distinct lineage. A molecular model generated for the N-terminal fragment of the HvHSP70 displayed the heat shock protein fold and domains of phosphotransferases. The EC-CuZnSOD cDNA isolated from H. vulgaris encodes a protein of 189 amino acids (Mw=20959.73); the first 19 amino acids constitute the presumed signal peptide. Phylogenetically HvEC-CuZnSOD is grouped with ECCuZnSODs from several organisms. A molecular model generated for the HvEC-CuZnSOD displayed the CuZnSOD (beta)-barrel fold. The MnSOD cDNA isolated from H. vulgaris encodes a protein of 219 amino acids (Mw=24348.75); the first 21 amino acids constitute the presumed mitochondria-targeting signal peptide. Phylogenetically HvMnSOD is clustered with mollusk and crustacean MnSODs. A molecular model generated for the HvMnSOD displayed the N-terminal long alpha antiparallel hairpin and the Cterminal mixed alpha/beta fold characteristic of MnSODs. The PHGPx gene isolated from H. vulgaris encodes a polypeptide of 168 amino acids (Mw=18746.51) including a TGA-encoded selenocysteine at residue 44 and lacks any intron. Phylogenetically HvPHGPx is grouped with PHGPxs from several organisms. A molecular model generated for the HvPHGPx displayed the thioredoxin fold. The 3'-end of a cDNA sequence encoding for 168 amino acids of the Cterminal end of a catalase was isolated from H. vulgaris. Phylogenetically HvCatalase is grouped with heme-containing monofunctional catalases. Hydrae exposed to thermal, starvation, oxidative and metal stress responded by regulating respective mRNA transcriptions suggesting that these genes are involved in stress and (anti)oxidative processes and may have potential as molecular biomarkers for assessing aquatic environment quality.

The whitefly Bemisia tabaci (Gennadius) (Hemiptera) complex is the sole arthropod vector of the genus, Begomovirus (family, Geminiviridae), which causes debilitating diseases of plants, worldwide. Virus-vector specificity is conferred through co-evolved, whitefly vector-viral capsid protein-protein interactions. Membrane-bound receptors are thought to facilitate virion passage across the gut-hemolymph and hemolymph-salivary gland interfaces, and virion circulation is expected to elicit innate defense and stress-related proteins. Our goal was to select and validate genes involved in whitefly-mediated transmission. Whitefly expressed sequence tags (ESTs) from a previous study were re-annotated, taking advantage of newly available insect EST, UniGene, and Protein sequences. Six whitefly genes and transcripts, actin, cyclophilin, GBLP, GAPDH 3, knottin, and whitefly endosymbiont HSP60, representing three gene ontology (GO) categories, were analyzed using PCR or RT- PCR, respectively, followed by cloning and DNA sequencing. Analysis confirmed the presence of all six whitefly genes and five transcripts, with the knottin transcript being undetectable.

Agrobacterium is widely considered, when suitably modified, to be the most effective vector for gene transfer into plant cells. For a long time, many cereals crops (monocotyledonous plants) were recalcitrant species to genetic modification, mainly as a result of their recalcitrance to in-vitro regeneration and their resistance to Agrobacterium infection. However, recently Agrobacterium-mediated transformation has been used to transform monocot crops such as maize (Zea mays) but with severe restrictions on genotype suitability. This study was carried out to evaluate the transformation amenability of 2 Syrian maize varieties and 2 hybrids in comparison with the hybrid line Hi II by the Agrobacterium tumefaciens-mediated transformation technique using a callus induction based system from immature zygotic embryos IZEs. A. tumefaciens strains EHA101, harbouring the standard binary vector pTF102, and the EHA105 containing the pBINPLUS/ARS:PpCBF1 vector were used. The effects of genotypes and the size of IZEs explants on callus induction and development were investigated. Results showed that callus induction and subsequent callus growth were significantly affected by the initial explant size. Calli induction from IZEs explants sized 1.5-2.00mm was 76%. Callus weight however decreased to 8.2g, compared with 11.7g of callus derived from IZEs >2.00mm. Callus induction ranged between 73.6-78.9% for varieties and hybrids respectively. Calli derived from varieties weighed significantly more than those initiated from the hybrids. Results demonstrated that Syrian maize genotypes were efficiently transformed via the A. tumefaciens strains but there was variation in transformation frequency. A transformation frequency of 3.7-4.2% was achieved for hybrids and varieties respectively confirming that the transformation frequency was genotype-dependent. The transformation frequency averaged between 3.2-5.6% for the EHA105 and EHA101 respectively. Fertile transgenic plants were regenerated from mature somatic embryos with an average regeneration frequency of 59.2 and 17% respectively for varieties and hybrids. Transgenic seeds of R0 and R1 progenies were produced from 74% of the outcrosses attempted and more than 98% of transgenic plants were normal in morphology. Fertile transgenic maize plants carrying the transferred gene CBF were produced using the Agrobacterium EHA105/PpCBF1 and these plants were shown to be more salt tolerant. Transient expression of the GUS gene was confirmed in transgenic calli, shoots, leaves, roots and floral parts of transgenic R0 and R1 progenies using histochemical GUS assays. The presence of the introduced bar and CBF genes in the genomic DNA of the transformants was confirmed by the PCR amplification. Further, the stable expression of the CBF and bar transgenes in the maize genome of transgenic R1 progeny was confirmed by qRT-PCR. The transformation protocol developed using an A. tumefaciens standard binary vector system was an effective and reproducible method to transform Syrian maize with an anti-stress gene in which fertile salt-resistant transgenic plants were routinely produced. This approach has great potential for development of Syrian maize breeding programmes for abiotic stress resistance for application in many areas in Syrian maize production.

Le premier objectif de cette thèse était de déterminer les effets isolés et combinés de l'alpha thalassémie, des polymorphismes inducteurs (QTLs) de l'HbF et du génotype G6PD dans un contexte d'évolution naturelle de la drépanocytose (Etudes 1 et 2). L'étude 1 a permis d'évaluer pour la première fois les fréquences alléliques de ces gènes modificateurs chez 301 enfants sénégalais SS. Contrairement aux autres populations africaines, le Variant Betica de la G6PD était majoritaire par rapport au variant A(-). De plus, 12% de notre cohorte avait un déficit en G6PD combiné à une absence d'alpha-thalassémie. Ces patients-là seront à privilégier pour la réalisation d'un Doppler transcrânien. Les résultats obtenus dans l'étude 2 nous ont permis de conclure que l'alpha-thalassémie et les QTLs de l'HbF sont interdépendants et ne doivent pas être étudiés séparément pour une prédiction clinique précise. En effet, une combinaison d'alpha-thalassémie avec au moins 2 QTLs de l'HbF est nécessaire pour retarder de manière significative la première complication de la maladie. Cependant, une alpha-thalassémie homozygote, même associée à 3 à 6 QTLs de l'HbF, augmente la fréquence des CVO pendant l'enfance. Par conséquent, une alpha-thalassémie hétérozygote avec au moins deux QTL HbF constituerait le génotype le plus favorable relativement à la survenue des CVO. Le deuxième objectif de cette thèse était d'étudier les interrelations entre le stress oxydant (phénotype et génotype) et la sévérité clinique de la maladie (Etudes 3 à 4). La drépanocytose est caractérisée par un stress oxydatif élevé pouvant expliquer une partie des manifestations cliniques. Nos résultats ont montré que l'alpha-thalassémie homozygote semble diminuer le stress oxydatif, ce qui contribuerait à son effet protecteur sur certaines complications du sous-phénotype hémolytique. En outre, les patients qui ont le moins d'hospitalisations et de CVO semblent présenter une meilleure défense antioxydante (activités catalase et GPx augmentées). Dans l'étude 4 nous avons étudié 4 SNPs de gènes du stress oxydant (rs4880 du gène SOD2, rs207454 du gène XO, rs233322 du gène MPO et rs35652124 du gène NFE2L2). Le SNP rs4880 aurait un effet favorable au niveau biologique (réticulocytose moindre, activité GPx augmentée) mais sans traduction clinique associée. Il en est de même pour rs233322 qui serait associé à une hémolyse et à un stress oxydatif (AOPP) plus importants. En revanche, une tendance à un effet protecteur de rs207454 vis-à-vis de certaines complications (hospitalisations, ostéonécrose, sepsis, STA) a été observée. Notre travail contribue à la compréhension de l'impact des gènes modificateurs dans la drépanocytose. Il pourrait donc permettre, via une sélection positive des patients à risque, d'améliorer la prise en charge de la maladie dans les pays où les traitements de fond (hydroxyurée, doppler transcrânien, échanges transfusionnels) ne peuvent être proposés à tous / The primary objective of this thesis was to determine the isolated and combined effects of alpha-thalassemia, inductors polymorphisms (QTLs) of HbF and genotype G6PD in a context of natural progression of sickle cell disease (Studies 1 and 2). Study 1 was undertaken to evaluate for the first time the allelic frequencies of these modifiers genes in 301 Senegalese SS children. Unlike other African populations, the G6PD Betica Variant was predominant over the A (-) variant. In addition, 12% of our cohort had G6PD deficiency combined with no alpha-thalassemia. These patients will be favoured for the realization of a transcranial doppler. The results obtained in Study 2 allowed us to conclude that alpha thalassemia and QTLs of HbF are interdependent and should not be studied separately for accurate clinical prediction. Indeed, a combination of alpha thalassemia with at least 2 QTLs of HbF is required to significantly delay the first complication of the disease. However, a homozygous alpha thalassemia, even associated with 3 to 6 QTLs of HbF, increases the frequency of CVOs during childhood. Therefore, a heterozygous alpha-thalassemia with at least two QTL HbFs would be the most favourable genotype for the occurrence of CVOs. The second objective of this thesis was to study the interrelationships between oxidative stress and the clinical severity of the disease (Studies 3 to 4). Sickle cell disease is characterized by high oxidative stress that may explain some of the clinical manifestations. Our results showed that homozygous alpha-thalassemia appears to reduce oxidative stress, which would contribute to its protective effect on certain complications of the hemolytic sub-phenotype. In addition, patients with the least hospitalization and CVO appear to have better antioxidant defense (catalase and GPx activities increased). In Study 4 we studied 4 SNPs of oxidative stress genes (rs4880 of the SOD2 gene, rs207454 of the XO gene, rs233322 of the MPO gene and rs35652124 of the NFE2L2 gene). The rs4880 SNP would have a favourable effect on the biological level (less reticulocytosis, increased GPx activity) but without associated clinical translation. The same is true for rs233322, which is associated with greater haemolysis and oxidative stress (AOPP). On the other hand, a tendency to a protective effect of rs207454 for some complications (hospitalizations, osteonecrosis, sepsis, STA) was observed. Our work contributes to the understanding of the impact of modifiers genes in sickle cell disease. It could therefore, through a positive selection of at-risk patients, improve the management of the disease in countries where the basic treatments (hydroxyurea, transcranial doppler, blood transfusion) cannot be offered to all