Die Atyidae sind eine artenreiche Familie weit verbreiteter Süßwassergarnelen, zu der sowohl landumschlossene als auch amphidrome Arten gehören. Ein großer Teil der Atyidae ist im Indopazifik verbreitet, einer Region, die für ihre geologisch komplexe Geschichte bekannt ist. Die Evolution der Atyidae in Kombination mit der Taxonomie, die bis ins frühe 19. Jahrhundert zurückreicht, führt zu einem hohen Maß an taxonomischer Unsicherheit. Das zeigt sich darin, dass über 70 % der Artenvielfalt in einer Gattung – aus insgesamt 54 der gesamten Familie – enthalten sind. Diese Gattung, Caridina, ist nicht monophyletisch. Eine vollständige Überarbeitung fehlt jedoch noch aus mehreren Gründen; darunter unvollständige Kenntnisse über entsprechende Verwandtschaftsverhältnisse, das Abwägen zwischen der Arbeit mit Museumsexemplaren und dem Sammeln im weiten Verbreitungsgebiet der Familie sowie die Plastizität ihrer Morphologie. Ich führe eine Meta-Analyse der Biogeographie der Landmassen innerhalb der Grenzen des Indischen Ozeans durch. Die bei mehreren Taxa festgestellten Tendenzen belegen, dass die physische Verbindung zwischen den Landmassen für die Ausbreitung weniger wichtig war als der durch ökologische Bedingungen ausgeübte Druck. Dann vergleichen wir Extraktionsmethoden, abwägend zwischen DNA-Ausbeute und -Integrität sowie Probenkonservierung. Wir zeigen, dass die Maximierung der DNA-Ausbeute durch Ganzkörperlyse jedoch möglich ist, ohne die Merkmale im Exoskelett zu beschädigen. Abschließend verwenden wir einen Exon-Capture-Ansatz, um eine zeitkalibrierte Phylogenie der artenreichsten Gruppe innerhalb der Atyidae, d. h. Caridina und ihrer Verwandten, zu erstellen. Wir erläutern Details über die Evolution lebensgeschichtlicher Merkmale sowie der Biogeographie und zeigen, dass die Geschichte der Familie eng mit dem Indopazifik verbunden ist und schaffen einen robusten phylogenomischen Rahmen, der zukünftige Bemühungen zur Überarbeitung der Familie erleichtern wird. / The Atyidae is a speciose family of widely distributed freshwater shrimps that includes landlocked and amphidromous species. A large portion of the atyid diversity is distributed in the Indo-Pacific, a region known for its geologically complex history. Its evolution combined with a vast distribution range and a taxonomy that dates back to the early nineteenth century amounts to a high degree of taxonomic uncertainty, which is illustrated by the fact that over 70% of its diversity is contained within one genus out of the 54 in the family. This genus, Caridina, is not monophyletic, but a full revision is still lacking for several reasons, including incomplete knowledge about its relationships, the compromise between working with museum specimens and collecting new specimens along its wide distribution, and the plasticity of its morphology. My coauthors and I conduct a meta-analysis of the historical biogeography of the landmasses within the boundaries of the Indian Ocean. Trends shown by multiple taxa demonstrate that physical connectivity between said landmasses is less important to dispersal than the pressure exerted by ecological conditions. Then, we compare extraction methods in terms of the trade-off between DNA yield and integrity and specimen preservation. We show that, whereas traditional extraction methods are still useful to obtain viable DNA from museum samples a few decades old, maximising the DNA yield through whole-body lysis is possible without damaging characters in the exoskeleton. Finally, we proceed to use an exon-capture approach to produce a time-calibrated phylogeny of the most diverse group in Atyidae, i.e., Caridina and their kin. We reveal details about the evolution of life history traits as well as biogeography, showing that the history of the family is intimately linked to the Indo-Pacific and constructing a robust phylogenetic framework that will facilitate future endeavours to revise the family.

Objetivo El objetivo principal de este estudio fue el de evaluar los efectos de la terapia fotodinámica (TFD) y el chitosan sobre la candidiasis oral en ratones y estudiar el posible efecto positivo sobre el fotosensibilizante azul de metileno usado en este tratamiento. Los objetivos específicos fueron: 1. Comparar la colonización de cándida albicans en tres cepas diferentes de ratones (DBA/2, Balb/c y Swiss) para determinar la más indicada como modelo de experimentación. 2. Valorar la efectividad de la los diferentes tratamientos comparándolos con el grupo control. 3. Analizar macroscópicamente las lenguas de ratones DBA/2 inmunodeprimidos afectados de candidiasis oral con los diferentes tratamientos y compararlo con un grupo control. 4. Estudiar las alteraciones epiteliales microscópicas de las lenguas de ratones DBA/2 inmunodeprimidos afectados de candidiasis oral con los diferentes tratamientos y compararlo con un grupo control. 5. Observar la respuesta inflamatoria microscópica en el tejido conectivo de las lenguas de ratones DBA/2 inmunodeprimidos afectados de candidiasis oral con los diferentes tratamientos y compararlo con un grupo control. Material y método Un total de 41 ratones (n= 37 DBA/2, n=2 Balb/c y n=2 Swiss) fueron utilizados. Para la consecución del primer objetivo específico se incluyeron 6 ratones: - Grupo 1: 2 ratones consanguíneos de la cepa DBA/2. - Grupo 2: 2 ratones consanguíneos de la cepa Balb/c. - Grupo 3: 2 ratones de cruzamiento libre de la cepa Swiss. Un ratón de cada grupo fue inoculado con una solución de Candida Albicans de 109 cels/mL y el otro con una solución de 1010 cels/mL, posteriormente a administrarles fármacos inmunosupresores (dexametasona). Las muestras fueron recogidas mediante frotis bucal para su posterior estudio. Para la consecución del segundo, tercero, cuarto y quinto objetivos específicos fueron incluidos un total de 35 ratones DBA/2. Todos los animales fueron inoculados con una suspensión de 109 células/mL, posteriormente a la administración de inmunosupresores (dexametasona). Los animales fueron divididos en 7 grupos de tratamiento (n=5 animales por grupo) en relación al tratamiento empleado para la candidiasis oral: Grupo 1 (control), Grupo 2 (nistatina), Grupo 3 (TFD), Grupo 4 (chitosan 1,5 mg/mL), Grupo 5 (chitosan 3 mg/mL), Grupo 6 (TFD + chitosan 1,5 mg/mL) y Grupo 7 (TFD + chitosan 3 mg/mL). Resultados Al comparar la colonización cándida albicans en tres cepas diferentes (DBA/2, Balb/c y Swiss) para determinar la más indicada en los modelos de candidiasis oral en ratones inmunodeprimidos, observamos que el mayor número de UFC/mL se produjo en la cepa DBA/2. Al valorar la efectividad de la TFD, nistatina, aplicación tópica de chitosan a 1,5 y 3 mg/mL y el tratamiento combinado de TFD + chitosan tópico a 1,5 y 3 mg/mL y compararlo con un grupo control (a los 3, 5, 7 y 11 días de tratamiento) observamos que el recuento de UFC/mL fue similar en todos los grupos a los 3 y 5 días de finalizar la inoculación. Al finalizar el experimento, todos los grupos de estudio presentaban un recuento de UFC/mL mucho más bajo que el grupo control, con diferencias estadísticamente significativas (p≤0,05). Al analizar macroscópicamente la lengua de los animales al finalizar el experimento sólo observamos la presencia de placas blancas en 2 dorsos linguales de los 35 animales y ambos pertenecían a animales del grupo control. Histológicamente, sólo la presencia de levaduras e hifas en queratina y la espongiosis fueron significativamente más altas en el control que en el resto de los grupos de tratamiento (p=0,024 y p=0,018 respectivamente). Finalmente, la presencia de células inflamatorias en la lámina propia fue menor en los animales tratados con TFD (Grupo 3) que en el resto de grupos, con diferencias estadísticamente significativas (p=0,024). / Objective The main objective of this study was to evaluate the effects of photodynamic therapy (PDT) and chitosan on oral candidiasis in mice and study the possible positive effect on the photosensitizer methylene blue used in this treatment. The specific objectives were: 1. Compare the colonization of Candida albicans in three different strains of mice (DBA / 2, BALB / c and Swiss) to determine the most appropriate as a model of experimentation. 2. Assess the effectiveness of different treatments compared to the control group. 3. Macroscopic analysis of the tongues of immunosuppressed DBA / 2 mice affected with oral candidiasis and different treatments and compared to a control group. 4. To study the microscopic epithelial disruption of the tongues of immunosuppressed DBA / 2 oral mice affected with oral candidiasis and different treatments and compared with a control group. 5. Observe the microscopic inflammatory response in the connective tissue of the tongues of immunosuppressed DBA / 2 mice afeccted with oral candidiasis and different treatments and compared with a control group. Materials and methods A total of 41 mice (n = 37 DBA / 2, n = 2 Balb / c and n = 2 Swiss) were used. To achieve the first specific objective 6 mice were included: - Group 1: 2 inbred mice of the DBA / 2 strain. - Group 2: 2 inbred mice of the Balb / c strain. - Group 3: 2 free crossing mice of Swiss strain. One mouse from each group was inoculated with Candida albicans solution of 109 cells / ml and the other with a solution of 1010 cells / mL, then to administer immunosuppressant drugs (dexamethasone). To achieve the remaining specific objectives were included a total of 35 DBA / 2 mice. All animals were inoculated with a suspension of 109 cells / mL, after the administration of immunosuppressants (dexamethasone). The animals were divided into 7 treatment groups (n = 5) in relation to the treatment used for oral candidiasis: G1 (control), G2 (Nystatin), G3 (TFD), G4 (chitosan 1 , 5 mg / mL), G5 (chitosan 3 mg / mL), G6 (PDT + chitosan 1.5 mg / mL) and G7 (PDT + chitosan 3 mg / mL). Results Comparing candida albicans colonization in three different strains (DBA / 2, BALB / c and Swiss) to determine the most appropriate models of oral candidiasis in immunocompromised mice, we found that the largest number of CFU / mL occurred in the DBA strain /2. In assessing the effectiveness of PDT, nystatin, topical application of chitosan 1.5 and 3 mg / mL and the combined treatment of topical PDT chitosan + 1.5 and 3 mg / mL and compared with a control group, note that the count of CFU / mL was similar in all groups at 3 and 5 days of inoculation end. At the end of the experiment, all the study groups presents a count of CFU / mL much lower than the control group, with statistically significant differences (p ≤ 0.05). In macroscopic analysis of the tongues at the end of the experiment, we observed the presence of white patches on the backs of two tongues of the 35 animals and both belonged to control animals. Histologically, only the presence of yeast and hyphae in keratin and spongiosis were significantly higher in the control than in the other treatment groups (p = 0.024 and p = 0.018 respectively). Finally, the presence of inflammatory cells in the lamina propria was lower in animals treated with PDT (Group 3) than in the other groups, with statistically significant differences (p = 0.024).

Modern humans originated in Africa around 200,000 years ago and today have settled in nearly every corner of earth. During migrations humans became exposed to new pathogens, food sources and have encountered vastly different environments. Natural selection likely contributed to the survival under such diverse conditions by promoting the raise in frequency of advantageous alleles. Thereby natural selection leaves genetic footprints that we can identify. The thesis at hand is about understanding how natural selection has shaped different human populations by analyzing these genetic footprints. In the first study, I infer the evolutionary history of an insertion-substitution variant using present-day human genomic data. This variant is interesting because the ancestral allele encodes a previously unannotated open-reading frame for a gene with antiviral ac- tivity (IFNL4 ), while the derived allele truncates this open-reading frame and is strongly associated with improved clearance of Hepatitis C, a major health care problem. Using an approximate bayesian computation approach I infer a complex evolutionary history, where the derived, truncating allele evolved under weak positive selection in Africa, with selection strength increasing in non-African populations, especially in East Asian popu- lations where the truncating allele is nearly fixed today. Hence, the changes in selection and resulting population differences in allele frequency contribute to the variation in Hep- atitis C clearance observed across human populations today. In the second study, I use ancient human genomes to estimate genome-wide allele frequencies in the past to understand present-day population differentiation. I develop a new statistic and incorporate the genome of Ust’-Ishim, a modern human that lived 45,000 year ago in Siberia, to study to what extent natural selection and drift have contributed to human population differentiation. The results suggest that European populations carry high frequency alleles in protein-coding (genic) regions that evolved under strong, recent positive selection. Further, the genic alleles that rose in frequency recently in Europeans were already present in ancient hunter-gatherers more often than in ancient farmers. This suggests that during the colonization of Europe local, positive selection changed the frequency of advantageous alleles in hunter-gatherer populations prior to the influx of farming individuals and those alleles remained beneficial also in the later admixed populations.

El disseny i la producció de zimògens de ribonucleases que puguin ser específicament activats per la proteasa d’un patogen, constitueix un enginyós mecanisme per controlar la seva activitat enzimàtica i, conseqüentment, la seva citotoxicitat. En aquest treball es descriu la construcció de 12 variants de zimògens d’Onconasa® (ONC) i de ribonucleasa pancreàtica humana (HP-RNasa), activables per la proteasa del Virus de la Immunodeficiència Humana (HIV-1 PR). Els zimògens d’ONC i HP-RNasa s’activen in vitro amb la proteasa, mantenint una elevada estabilitat sobretot els zimògens d’ONC. L’augment d’activitat ribonucleolítica de la forma activada respecte el precursor, que resulta baix per les variants d’ONC, és d’unes 150 vegades per les d’HP-RNasa. Els zimògens d’ONC presenten una molt bona internalització en limfòcits-T humans, a més de no presentar, en cap dels casos, citotoxicitat en cèl·lules Jurkat en cultiu abans de l’activació. Finalment, s’ha estudiat l’estructura i dinàmica de la forma intacta d’un dels zimògens d’ONC per Ressonància Magnètica Nuclear (NMR), permetent el desenvolupament i optimització futura de nous zimògens més efectius com a agents antivirals. / Design and production of ribonuclease zymogens that could be specifically activated by proteases of pathogens, would provide an ingenious mechanism to control their activity and, consequently, their cytotoxicity. Here we report the construction of 12 variants of Onconase® (ONC) and Human Pancreatic Ribonuclease (HP-RNase) zymogens that are recognized and activated specifically by the Human Immunodeficiency Virus Protease (HIV-1 PR). ONC- and HP-RNase- zymogens are activated by the HIV-1 PR in vitro, and mantain a high conformational stability mainly in ONC variants. The increase of catalytic efficiency of the activated form compared to the precursor has been low in ONC zymogens, but 150-fold for those of HP-RNase. ONC zymogens can internalize in human T-lymphocyte Jurkat cells efficiently, and all of them do not show any cytotoxicity in this type of cells before their activation. Finally, the structure and dynamics of intact form of one of ONC zymogens has been determined by Nuclear Magnetic Resonance spectroscopy (NMR). This represents a first step toward the development and optimization of more effective zymogens, as antiviral agents.

No description available.

In the present Thesis, we observed that vitrifying-warming porcine embryos had detrimental effects. Indeed, vitrification and warming were seen to reduce developmental ability and quality of in vitro produced porcine blastocysts, with increased levels of DNA fragmentation, raised levels of reactive oxygen species and alterations in the expression of pluripotency and thermal shock-related genes. This suggested that establishing effective cryopreservation methods was required. Following this, we found that embryo cryotolerance did not only depend on the cryopreservation procedures but also on the composition of the culture medium. On the other hand, beneficial effects were also obtained from exposing embryos to antioxidants. Specifically, L-ascorbic acid addition during culture and/or vitrification and warming enhanced embryo survival, redox status and thermal stress response / En aquesta tesi s’ha observat que els processos de vitrificació i escalfament sotmeten els embrions porcins produïts in vitro a situacions d’estrès. Els resultats obtinguts quant a la capacitat dels embrions a l’hora de sobreviure a la criopreservació i a la qualitat dels embrions vitrificats, amb augments en els nivells de fragmentació del DNA i en les espècies reactives d’oxigen i amb alteracions en el perfil d’expressió de gens involucrats en la pluripotencialitat i en la resposta al xoc tèrmic, suggereixen la necessitat d’incrementar l’efectivitat dels mètodes de criopreservació. S’ha determinat, a més, que la criotolerància embrionària no només depèn dels processos de criopreservació sinó també de la composició dels medis de cultiu. Finalment, s’ha observat que l’exposició dels embrions a molècules antioxidants desencadena efectes beneficiosos. Concretament, l’addició d’àcid ascòrbic en els medis de cultiu i vitrificació-escalfament millora la supervivència, l’estat d’oxidació/reducció i la resposta a l’estrès tèrmic

Durante la presente Tesis Doctoral se han estudiado los efectos inmunotoxicológicos de la exposición mediante baño a arsénico, cadmio y mercurio en la dorada (Sparus aurata L.), la cual es una especie que posee la mayor tasa de producción en la acuicultura mediterránea. Por otra parte, también se han estudiado los parámetros inmunológicos y físico-químicos del moco de la piel de cinco especies de peces teleósteos marinos. En el caso de la dorada los parámetros inmunológicos en el moco se compararon con los presentes en el suero. Por último, se evaluaron los efectos de estos metales pesados en la inmunidad de la mucosa de la piel de la dorada. Por otra parte, los peces ocupan una posición filogenética clave en la evolución de los vertebrados representando el primer grupo animal que tiene un sistema inmunitario innato y adaptativo bien estructurado, en este sentido, el estudio de la inmunología de este grupo de vertebrados tiene un doble interés científico y filogenético básico. En primer lugar, hemos evaluado los efectos de la exposición mediante baño a concentraciones sub-letales de arsénico (As), cadmio (Cd) y mercurio (Hg) en la dorada, con especial énfasis en la respuesta inmunitaria innata. En el caso del As, se observaron alteraciones histológicas en el hígado, así como acumulación de este metal en este órgano después de 30 días de exposición. Los parámetros inmunitarios humorales (niveles de IgM, actividades del complemento y la peroxidasa) no se vieron afectados, mientras que los parámetros innatos celulares del riñón cefálico (peroxidasa, explosión respiratoria y las actividades fagocíticas en los leucocitos) se incrementaron significativamente después de 10 días de exposición en comparación con los peces control. Del mismo modo, con respecto a la exposición a Cd, se observaron alteraciones morfológicas progresivas en el hígado y en el páncreas exocrino que se correlacionaron con la acumulación hepática de Cd, la reducción en la actividad del complemento en el suero y la explosión respiratoria en los leucocitos en un grado significativo después de 10 y 30 días de exposición. La actividad de la peroxidasa en el suero y la fagocitosis de los leucocitos aumentaron en diferentes momentos del muestreo, mientras que los niveles de IgM en el suero y la actividad de la peroxidasa en los leucocitos resultaron inalterados. Finalmente, se confirmaron los efectos toxicológicos del Hg porque los ejemplares de dorada expuestos mediante baño mostraron un aumento de las enzimas antioxidantes en el hígado después de 2 días, alteraciones histopatológicas en el hígado y la piel, así como la sobre-expresión de genes relacionados con el metabolismo de xenobióticos, el estrés celular y la apoptosis en la piel. Además, las actividades del complemento y la peroxidasa en el suero se incrementaron, así como la peroxidasa, la explosión respitaroria y las actividades fagocíticas en los leucocitos de riñón cefálico que se incrementaron en diferentes tiempos de exposición. En segundo lugar, se identificaron y caracterizaron los diferentes mecanismos de defensa humorales constitutivos de la mucosa de la piel de la dorada (Sparus aurata) y se compararon con los presentes en el suero. Por lo tanto, se demostró que el moco de la piel de la dorada contiene niveles más bajos de IgM, niveles similares de lisozima, fosfatasa alcalina y proteasas, y más altos en actividades como la esterasa, peroxidasa y antiproteasa que en el suero. Asimismo, el moco de la piel reveló una fuerte actividad bactericida contra bacterias patógenas de peces testadas en comparación con la actividad encontrada en el suero, mientras que las bacterias no patógenas pueden incluso crecer mejor en la presencia de moco. Además, se evaluaron los mecanismos de defensa humoral y parámetros físico-químicos en el moco de la piel de cinco especies de teleósteos marinos. Se observaron varias correlaciones entre los parámetros como el pH, la conductividad y el potencial redox, así como, entre la densidad y la osmolaridad. Centrándose en la respuesta inmunitaria, los resultados mostraron que mientras que algunas de las actividades inmunitarias fueron muy similares en los peces estudiados, otras, tales como las actividades de la proteasa, antiproteasa, fosfatasa alcalina, esterasa y peroxidasa variaron dependiendo de las especies de peces. Por último, se investigó si los parámetros de la inmunidad innata de las mucosas de la piel determinados en la dorada se ven afectados o no por los metales pesados ensayados anteriormente. De este modo, se detectaron algunos cambios en la composición del moco y en las funciones inmunitarias después de la exposición a estos metales. En general, el perfil de carbohidratos sufrió pequeñas variaciones y la mayoría de las actividades enzimáticas se incrementaron después de la exposición. Curiosamente, el Hg provocó los incrementos más importantes en la mucosa de la piel, mientras que los perfiles de proteínas obtenidos por SDS-PAGE y HPLC mostraron poca variación en el moco de la dorada después de la exposición a estos metales. En conclusión, estos resultados podrían ser útiles para una mejor comprensión de los efectos de la exposición a metales pesados en el sistema inmunitario de la dorada y como podrían afectar a la biología marina, la acuicultura o a los consumidores humanos. Por otra parte, el estudio de la función y el comportamiento de la inmunidad de las mucosas en la piel, como un componente clave del sistema inmunitario innato, podría ayudar a comprender la resistencia de los peces, así como la presencia y distribución de los patógenos y la magnitud de las infecciones y el uso de los parámetros como biomarcadores en la toxicología de los peces, los cuales serían los aspectos de mayor importancia para la acuicultura / During the present PhD Thesis, we have studied the immunotoxicological effects of waterborne exposure to arsenic, cadmium and mercury in the gilthead seabream (Sparus aurata L.), which is a species with the highest rate of production in Mediterranean aquaculture. Furthermore, the immunological and physico-chemical parameters of the skin mucus from five marine teleost fish species have been also studied. In the case of gilthead seabream the immunological parameters in mucus were compare with those present in serum. Finally, the effects of these heavy metals in the skin mucosal immunity of gilthead seabream were evaluated. Moreover, fish occupy a key phylogenetic position in the evolution of vertebrates representing the first animal group that has a well structured innate and adaptive immune system; in this sense, the study of the immunology of this group of vertebrates has a dual basic scientific and phylogenetic interest. Firstly, we have evaluated the effects of waterborne exposure to sub-lethal concentrations of arsenic (As), cadmium (Cd) and mercury (Hg) in the teleost fish gilthead seabream, with special emphasis in the innate immune response. In the case of As, we observed histological alterations in the liver as well as As-accumulation in this organ after 30 days of exposure. The humoral immune parameters (seric IgM, complement and peroxidase activities) were not affected while the cellular innate parameters of head-kidney (leucocyte peroxidase, respiratory burst and phagocytic activities) were significantly increased after 10 days of exposition compared to the control fish. Similarly, regarding Cd exposure, it was observed progressive deleterious morphological alterations in liver and exocrine pancreas that correlated with the hepatic Cd-accumulation, reduction in the serum complement activity and leucocyte respiratory burst to a significant extent after 10 and 30 days of exposure. The serum peroxidase activity and leucocyte phagocytosis were increased at different sampling times while serum IgM levels and leucocyte peroxidase activity resulted unaltered. Finally, toxicological effects were confirmed for Hg because waterborne-exposed seabream specimens showed increased liver antioxidant enzymes after 2 days, histopathological alterations in the liver and skin as well as up-regulation of the expression of genes related to xenobiotic metabolism, cellular stress and apoptosis in the skin. Further, serum complement and peroxidase activities were increased, as well as head-kidney leucocyte peroxidase, respiratory burst and phagocytic activities were increased in different exposure time. Secondly, different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata) were identified and characterized and compared with those present in the serum. Thus, it was demonstrated that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. Skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, whilst non-pathogen bacteria can even grow better in the presence of mucus. In addition, humoral defence mechanisms and physico-chemical parameters in the skin mucus of five species of teleosts were evaluated. Several correlations were observed between parameters as pH, conductivity and redox potential, as well as, density and osmolarity. Focusing on the immunological response, results showed that while some immune activities were very similar in the studied fish other such as protease, antiprotease, alkaline phosphatase, esterase and peroxidase activities varied depending on the fish species. Finally, we investigated whether the skin mucus innate immune parameters determined in the gilthead seabream are affected or not by the heavy metals assayed above. Thus, some changes in the mucus composition and immune functions after heavy metal exposure were detected. Overall, carbohydrate profile suffered little changes and most of the enzymatic activities were increased after exposition. Interestingly, Hg evoked the most important increments in the skin mucus and protein profiles obtained by SDS-PAGE and HPLC showed little variations in the seabream mucus after exposure to heavy metals. In conclusions, these results could be useful for better understanding the effects of exposition to heavy metals in the seabream immune system and as could interfere with fish biology, aquaculture management or human consumers. Moreover, the study of the role and behaviour of the mucosal immunity in skin, as a key component of the innate immune system, could help to understand the fish resistance as well as the presence and distribution of pathogens and magnitude of infections and use of parameters as biomarkers in fish toxicology, which would be aspects of major importance for the aquaculture industry.

Microorganisms have evolved in a low Reynolds number environment and have adapted their behaviour to its viscosity. Here, we consider some features of behaviour observed in microorganisms and use hydrodynamic models to show that these behaviours emerge from physical interactions, including hydrodynamic friction, hydrodynamic interactions and mechanical constraints. Swimming behaviour is affected by surfaces and observations of Vibrio cholerae show that it swims near a surface with two distinct motility modes. We develop a model which shows that friction between pili and the surface gives the two motility modes. The model is extended to study the behaviour of bacteria which are partially attached to a surface. Observations of Shewanella constrained by a surface show several different behaviours. The model shows that different degrees of surface constraint lead to different types of behaviour; the flexibility of the flagellar hook and the torque exerted by the flagellar motor also cause different behaviours. Near surface behaviour is important for understanding the initial stages of biofilm formation. Chlamydomonas swims using synchronous beating of its two flagella. A simple model of Chlamydomonas is developed to study motility and synchronisation. This model shows that the stability of synchronisation is sensitive to the beat pattern. Run-and-tumble behaviour emerges when we include intrinsic noise, without the need for biochemical signalling. The model is also used to show how observed responses of the flagella to light stimuli produce phototaxis. Finally we study hydrodynamic synchronisation of many cilia and consider the stability of metachronal waves in arrays of hydrodynamically coupled cilia. This thesis shows that physical interactions are responsible for many behavioural features and that physical models provide a useful technique for exploring open questions in biology.

La compréhension de l'apparition d'espèces nouvelles est depuis longtemps un problème majeur en biologie évolutive. Dans le cadre de la spéciation, une attention particulière est portée sur l'étude de la structure des populations c'est-à-dire l'agencement spatio-temporel de la diversité biologique permettant de mesurer et d'identifier la nature de la divergence. Cette divergence est dépendante de quatre forces: la dérive génétique, le flux de gène, la mutation et la sélection et a largement été étudié au sein des milieux insulaires. En effet, ces milieux ont toujours occupé une place de choix dans l'étude de la diversification du vivant car i) ils fournissent un cadre idéal pour étudier les mécanismes impliqués dans la diversification et ii) ils renferment une faune et une flore originales composées de nombreuses espèces endémiques. Ces espèces endémiques montrent une fragilité singulière de part leur faible effectif et leur aire de distribution restreinte. La Grive à Pieds Jaunes, Turdus lherminieri, est un oiseau forestier et endémique de quatre îles des Petites Antilles : Montserrat, la Guadeloupe, la Dominique et Sainte-Lucie. Quatre-sous espèces ont été décrites sur chacune des îles sur des critères de coloration du plumage mais aucune étude n'a réellement quantifié la divergence entre ces sous-espèces. De plus, cet oiseau voit ses effectifs déclinés depuis une quinzaine d'années ce qui lui vaut d'être protégé sur trois îles alors qu'il est encore chassé en Guadeloupe. Les objectifs dégagés dans cette étude sont: i) d'étudier la structure spatiale des grives et d'en inférer les causes, ii) de retracer l'évolution temporelle récente de la diversité génétique ces populations, iii) de déterminer si les parasites peuvent exercer des pressions de sélection contrastées entre ces populations. Notre étude a permis d'identifier trois clades et quatre îles très différenciées sur le plan génétique et morphologique à confronter aux quatre sous-espèces définies auparavant sur des critères de coloration du plumage. A une échelle spatiale plus fine, la Guadeloupe seule, nous avons constaté des niveaux de différentiation peu commun chez des oiseaux. Enfin, le contexte parasitaire semble lui aussi structuré spatialement et présente des éléments de congruence avec les structurations morphologiques et génétiques. A la suite de cette étude, trois unités de gestion peuvent être définies afin de préserver les populations de Grives à pieds jaunes: une première incluant Sainte-Lucie, une seconde incluant la Dominique et une troisième incluant l'ensemble Guadeloupe et Montserrat / Understanding the mechanisms underlying population structure is a crucial issue in evolutionary biology and ecology. Identifying the processes driving biodiversity is also of prime interest to draw up guidelines for population management and conservation planning. Level of differentiation depends on a subtle balance between gene flow, genetic drift and natural selection and insight into the relative contribution of these factors can be provided by spatial and temporal patterns of intra-specific phenotypic and genetic variations. Differentiation has mainly been studied in insular systems because i) islands give ideal framework to study divergence in species ii) islands enclosed endemic fauna and flora. These endemic species are characterised by small population sizes and small distribution areas. They are consequently more fragilized by strong environmental changes. The Forest thrush Turdus lherminieri is an endemic and understorey bird present in four islands of Lesser Antilles: Montserrat, Guadeloupe, Dominica and Saint-Lucia. Four subspecies have been described on each island on the basis of feathers coloration but no study has really quantified the divergence between these subspecies. In addition, bird numbers have decline since 15 years and now Forest thrushes are protected on three islands but are always hunted in Guadeloupe. Aims proposed in this study are i) to study the spatial structure in Forest thrush in order to deduce potential causes of this structure ii) to describe the temporal evolution of genetic diversity, iii) to determine if parasites can exert contrasted selection pressures which follow gentic and morphological pattern in Forest thrushes populations. In this study, we have identified three clades and subspecies on each island which are differentiated morphologically and genetically. At fine scale i.e Guadeloupe only, we have revealed an uncommon level of differentiation. In last, the parasites’ structure seems to follow the morphological and genetic structure observed in Forest thrushes. To conclude, three management units can be defined: a first in Saint- Lucia, a second in Dominica and a third including Montserrat and Guadeloupe

L'une des infections parasitaires les plus courantes chez les animaux à travers le monde est la babésiose ou piroplasmose. Causée par le développement intraérythrocytaire d'un parasite du genre Babesia, elle présente de nombreux signes cliniques semblables à ceux du paludisme. Ce parasite, du phylum des Apicomplexes, est transmis via le vecteur tique et effectue son cycle de reproduction dans les cellules rouges du sang de l'hôte vertébré. En Europe B. divergens et B. canis sont les espèces majoritairement responsables respectivement de la babésiose bovine et la babésiose canine. Dans une stratégie de recherche vaccinale, l'étude de protéines parasitaires en contact avec la circulation sanguine est primordiale pour comprendre les interactions hôte-parasite et identifier des candidats vaccins à haut potentiel. Les protéines à ancrage GPI (glycosylphosphatidylinositol) font partie de ces protéines. La première protéine à ancrage GPI décrite chez B. divergens est Bd37.1. Elle induit une protection totale contre une infection à B. divergens à la condition qu'une séquence hydrophobe soit ajoutée en C-terminale. La résolution de la structure RMN de cette protéine a permis de mettre en évidence un probable mécanisme de changement conformationnel en fonction du pH. La structure composée de 3 sous domaines montre que celle-ci n'est maintenue que par des ponts salins qui peuvent se rompre en milieu acide. Or l'environnement membranaire dans lequel évolue Bd37.1 ancrée à la surface du parasite et/ou à l'approche du globule rouge lors de l'invasion est acide. Cette dynamique conformationnelle de la protéine Δ-Bd37, liée à l'environnement membranaire, pourrait être à l'origine du mécanisme qui confère une immunité en fonction de la présence ou non de la séquence hydrophobe en C-terminale de Bd37.1. Nous avons cherché à estimer les implications d'une telle dynamique dans les interactions hôtes-parasites à travers l'étude structurale de 2 protéines parasitaires (Bd37.1 et Bc28.1). Dans le premier cas nous étudions la dynamique conformationnelle de la protéine d'adhésion Bd37.1. Nous avons exploré les différentes conformations que pourrait adopter la protéine Bd37.1 par une approche de biophysique et nous avons stabilisé ces différentes conformations en solution par le biais de mutations pour les étudier. Parmi ces mutants, le mutant EDK-Δ-Bd37 dont les ponts salins ont été rompus montre des caractéristiques différentes de Δ-Bd37. Les données enregistrées sur ce mutant nous ont amené à résoudre sa structure et à tester son pouvoir vaccinant. Dans une seconde partie, nous caractérisons biochimiquement et fonctionnellement une autre protéine Bc28.1, l'orthologue de Bd37.1. chez B. canis, accompagnée de la résolution de sa structure. Nous montrons que Bc28.1 est une protéine d'adhésion localisée à la surface du parasite et nous comparons les structures de Bd37.1 et Bc28.1. Ces deux structures sont finalement très différentes tandis que localisation et fonction sont similaires. / One of the most common parasitic infections in animals worldwide is babesiosis or piroplasmosis. Caused by the intraerythrocytic development of Babesia parasite, it has many clinical signs similar to those of malaria. This parasite of the phylum Apicomplexa, is transmitted via the tick vector and performs its reproductive cycle in red blood cells of the vertebrate host. B. In Europe divergens and B. canis species are mainly responsible respectively for bovine babesiosis and canine babesiosis. A strategy of vaccine research, the study of parasite proteins in contact with the bloodstream is essential for understanding host-parasite interactions and identify vaccine candidates with high potential. Anchored protein GPI (glycosylphosphatidylinositol) are part of these proteins. The first protein GPI anchors described in B. divergens is Bd37.1. It induces complete protection against infection with B. divergens provided a hydrophobic sequence is added at the C-terminus. Resolution NMR structure of this protein has highlighted a probable mechanism of conformational change as a function of pH. The structure consists of three sub areas shows that it is only maintained by salt bridges which can break in acidic medium. However, the environment within which Bd37.1 membrane anchored to the surface of the parasite and / or approach the red blood cell during the invasion is acidic. This conformational dynamics of the protein-Δ Bd37 linked to the membrane environment, could be at the origin of the mechanism that confers immunity depending on the presence or absence of the hydrophobic sequence at the C-terminus of Bd37.1. We sought to assess the implications of such dynamics in host-parasite interactions through structural study of two parasite proteins (Bd37.1 and Bc28.1). In the first case we study the conformational dynamics of the adhesion protein Bd37.1. We explored the different conformations that may be adopted by a protein Bd37.1 biophysical approach and we have stabilized in different conformations in solution through mutations to study. Among these mutants, the mutant Δ-Bd37-EDK including salt bridges were broken shows different characteristics Δ-Bd37. The data on this mutant led us to solve the structure and to test its power vaccinating. In a second part, we characterize biochemically and functionally Bc28.1 another protein, the ortholog Bd37.1. in B. canis, accompanied with the resolution of its structure. We show that Bc28.1 is an adhesion protein localized to the parasite surface and compare the structures and Bd37.1 Bc28.1. These two structures are ultimately very different while location and function are similar.