Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

Background: Heat-shock proteins of the 70 kDa family (Hsp70s) are essential chaperones required for key cellular functions. In eukaryotes, four subfamilies can be distinguished according to their function and localisation in different cellular compartments: cytosol, endoplasmic reticulum, mitochondria and chloroplasts. Generally, multiple cytosol-type Hsp70s can be found in metazoans that show either constitutive expression and/or stress-inducibility, arguing for the evolution of different tasks and functions. Information about the hsp70 copy number and diversity in microbial eukaryotes is, however, scarce, and detailed knowledge about the differential gene expression in most protists is lacking. Therefore, we have characterised the Hsp70 gene family of Paramecium caudatum to gain insight into the evolution and differential heat stress response of the distinct family members in protists and to investigate the diversification of eukaryotic hsp70s focusing on the evolution of heat-inducibility. Results: Eleven putative hsp70 genes could be detected in P. caudatum comprising homologs of three major Hsp70-subfamilies. Phylogenetic analyses revealed five evolutionarily distinct Hsp70-groups, each with a closer relationship to orthologous sequences of Paramecium tetraurelia than to another P. caudatum Hsp70-group. These highly diverse, paralogous groups resulted from duplications preceding Paramecium speciation, underwent divergent evolution and were subject to purifying selection. Heat-shock treatments were performed to test for differential expression patterns among the five Hsp70-groups as well as for a functional conservation within Paramecium. These treatments induced exceptionally high mRNA up-regulations in one cytosolic group with a low basal expression, indicative for the major heat inducible hsp70s. All other groups showed comparatively high basal expression levels and moderate heat-inducibility, signifying constitutively expressed genes. Comparative EST analyses for P. tetraurelia hsp70s unveiled a corresponding expression pattern, which supports a functionally conserved evolution of the Hsp70 gene family in Paramecium. Conclusions: Our analyses suggest an independent evolution of the heat-inducible cytosol-type hsp70s in Paramecium and in its close relative Tetrahymena, as well as within higher eukaryotes. This result indicates convergent evolution during hsp70 subfunctionalization and implies that heat-inducibility evolved several times during the course of eukaryotic evolution.

Les personnes qui vivent avec le VIH (PVVIH) doivent prendre un traitement d’antirétroviraux combinés (ART) pour contrôler la réplication virale et empêcher le développement d’une immunodéficience dont l’issue est fatale. Les ART protègent les cellules saines de l’infection et permettent ainsi de rendre la charge virale plasmatique indétectable. Cependant, l’arrêt des ART entraine presque inévitablement un rebond de la charge virale, puisque le virus n’est jamais complètement éliminé par le système immunitaire. En effet, de multiples cellules infectées, où le virus s’est intégré et demeure dans un état de latence, restent présentes tout au long de la vie des PVVIH. Une partie de ces cellules infectées forme le réservoir compétent pour la réplication. Les provirus responsables du rebond de la charge virale possèdent trois caractéristiques : ils gardent la capacité d’être réactivés (sortie de latence), ils sont génétiquement intacts, et ils peuvent produire de nouvelles particules virales infectieuses. Afin de guérir les PVVIH de l’infection, il faut donc cibler les quelques rares cellules portant un provirus intact et inductible. Pour ce faire, il est impératif de comprendre comment ces cellules sont maintenues pendant les années de ART, de les localiser dans tout l’organisme, et d’identifier ce qui peut les distinguer des autres. Ce sont ces trois aspects que nous avons abordés au cours des travaux de recherche présentés dans cette thèse, autant à l’échelle de la cellule unique que de l’organisme entier. Nos résultats montrent que les provirus compétents pour la réplication persistent dans des lymphocytes T CD4+ mémoires exprimant l’intégrine VLA-4 en grande quantité, que les provirus intacts peuvent subsister au sein de différents compartiments anatomiques, que les provirus inductibles et compétents pour la traduction de la protéine virale p24 sont majoritairement défectifs, et que l’expansion clonale est un mécanisme important qui favorise le maintien du réservoir viral dans le sang et dans les tissus tout en favorisant la diversité phénotypique de ces cellules. / People with HIV (PWH) must take combinational antiretroviral therapy (ART) to control viral replication and avoid developing fatal immunodeficiency. ART allows achieving undetectable plasma viral load, and thus protects uninfected cells from HIV. However, ART interruption will almost inevitably result in a viral rebound since HIV is never completely cleared by the immune system. Indeed, a group of infected cells, where the virus has integrated and remains in a latent state, persists throughout the life course of PWH, and some of these cells form the replicationcompetent reservoir. Proviruses responsible for viral rebound have three characteristics: they can be induced to exit their latent state, they are genetically intact, and they are able to produce new infectious viral particles. Therefore, in order to cure PWH, it is essential to target the few cells with intact and inducible provirus, and to be able to do so, it is imperative to understand how these cells are maintained during years of ART, to localize them throughout the body, and to identify what distinguishes them from other cells. These three aspects are the focus of the work presented in this thesis, whether at the single-cell level or looking through the whole body. Our results show that replication-competent proviruses persist in memory CD4+ T cells expressing high levels of the integrin VLA-4, that intact proviruses can persist among various anatomical compartments, that inducible and translation-competent proviruses are predominantly defective, and that clonal expansion is an important mechanism that favors the maintenance of reservoir cells both in the blood and in deep tissues in addition to diversify phenotypically those cells.