In two waters: contemporary evolution of lagoonal and marine white seabream (Diplodus sargus) populations

Brackish water ecosystems are often exposed to wide variations in environmental variables, including temperature and salinity, which may cause strong selective pressures on organisms modifying the genetic patterns of species. The aim of this work was to test whether there is a ‘divergence-with-gene flow’ in coastal lagoon populations of white seabream (Diplodus sargus) (Ria Formosa, S Portugal and Mar Menor, SE Spain) respect to four marine populations, by using partial sequences of cyt b mitochondrial gene and information from nine microsatellite loci. Genetic diversity was highest in both coastal lagoons (Mar Menor and Ria Formosa) considering mitochondrial and nuclear markers. Although some of FST population pairwise comparisons were not significant, analyses of molecular variance (AMOVAs) detected differences between groups (coastal lagoon and marine) close to significance. Also, only two haplotypes (Cytb-17 and Cytb-18) were detected in both coastal lagoon sampling sites and these localities (Mar Menor and Ria Formosa) showed the highest number of singletons, some of them with a high number of mutations, as has been already described for other Mar Menor populations (Pomatochistus marmoratus and Holothuria polii). Also, several tests detected significant positive and balancing selection considering mtDNA and microsatellite data. These data support the hypothesis of selection as one of the drivers of the genetic differences found between coastal lagoon and marine populations. The life strategy adopted by Diplodus sargus in coastal lagoons allows it to decrease its mortality rate and improve the heritability of its genes. Also, the increase time spent in coastal lagoons with different temperatures and salinities favours the fitness selection and the maintenance of exclusive haplotypes and genotypes in coastal lagoon inhabitants favouring the ‘divergence-with-gene-flow’.

Evolution of specific antibodies and proviral DNA in milk of small ruminants infected by small ruminant lentivirus

The diagnosis of Small Ruminant Lentivirus (SRLV) is based on clinical signs, pathological lesions and laboratory testing. No standard reference test for the diagnosis of maedi visna has been validated up to the present, and it is puzzling that tests which detect antibodies against the virus and tests which detect the proviral genome may render opposite results. The aim of this study was to evaluate the presence in milk throughout a lactation period of specific antibodies by ELISA and of SRLV proviral DNA by a PCR of the highly conserved pol region. A six-month study was conducted with the milk of 28 ewes and 31 goats intensively reared. The percentage of animals with antibodies against SRLV increased throughout the study period. Seroprevalence in sheep was 28% at the beginning of the study and by the end it had increased up to 52.4%. In goats, initial seroprevalence of 5.6% increased to 16%. The percentage of PCR positive ewes was stable throughout the study period. Of the positive sheep, 21.4% were PCR-positive before antibodies could be detected and most of them became PCR-negative shortly after the first detection of antibodies. This might suggest that antibodies have a neutralizing effect. In addition, an equal percentage of sheep were always PCR-negative but either became ELISA-positive or was always ELISA-positive, which might support this hypothesis. On the other hand, the PCR results in goats did not follow any pattern and oscillated between 35.3% and 55.6% depending on the month. Most goats positive by PCR failed to develop antibodies in the 6 months tested. We may conclude that the infection and the antibody response to it follow a different trend in sheep and goats.

The Herpesvirus Nuclear Egress Complex Component, UL31, is Recruited to Sites of DNA Damage Through Poly(ADP-RIBOSE) Binding

Thesis (Master, Biomedical & Molecular Sciences) -- Queen's University, 2016-08-23 15:03:30.807

“Acid-spike” effect in spurs/tracks of the low/high linear energy transfer radiolysis of water : potential implications for radiobiology and nuclear industry

Résumé : Les ions hydronium (H3O + ) sont formés, à temps courts, dans les grappes ou le long des trajectoires de la radiolyse de l'eau par des rayonnements ionisants à faible transfert d’énergie linéaire (TEL) ou à TEL élevé. Cette formation in situ de H3O + rend la région des grappes/trajectoires du rayonnement temporairement plus acide que le milieu environnant. Bien que des preuves expérimentales de l’acidité d’une grappe aient déjà été signalées, il n'y a que des informations fragmentaires quant à son ampleur et sa dépendance en temps. Dans ce travail, nous déterminons les concentrations en H3O + et les valeurs de pH correspondantes en fonction du temps à partir des rendements de H3O + calculés à l’aide de simulations Monte Carlo de la chimie intervenant dans les trajectoires. Quatre ions incidents de différents TEL ont été sélectionnés et deux modèles de grappe/trajectoire ont été utilisés : 1) un modèle de grappe isolée "sphérique" (faible TEL) et 2) un modèle de trajectoire "cylindrique" (TEL élevé). Dans tous les cas étudiés, un effet de pH acide brusque transitoire, que nous appelons un effet de "pic acide", est observé immédiatement après l’irradiation. Cet effet ne semble pas avoir été exploré dans l'eau ou un milieu cellulaire soumis à un rayonnement ionisant, en particulier à haut TEL. À cet égard, ce travail soulève des questions sur les implications possibles de cet effet en radiobiologie, dont certaines sont évoquées brièvement. Nos calculs ont ensuite été étendus à l’étude de l'influence de la température, de 25 à 350 °C, sur la formation in situ d’ions H3O + et l’effet de pic acide qui intervient à temps courts lors de la radiolyse de l’eau à faible TEL. Les résultats montrent une augmentation marquée de la réponse de pic acide à hautes températures. Comme de nombreux processus intervenant dans le cœur d’un réacteur nucléaire refroidi à l'eau dépendent de façon critique du pH, la question ici est de savoir si ces fortes variations d’acidité, même si elles sont hautement localisées et transitoires, contribuent à la corrosion et l’endommagement des matériaux.

Cartographie des cassures bicaténaires du remodelage chromatinien du spermatide et développement des outils techniques associés.

Résumé : La phase haploïde de la spermatogenèse (spermiogenèse) est caractérisée par une modification importante de la structure de la chromatine et un changement de la topologie de l’ADN du spermatide. Les mécanismes par lesquels ce changement se produit ainsi que les protéines impliquées ne sont pas encore complètement élucidés. Mes travaux ont permis d’établir la présence de cassures bicaténaires transitoires pendant ce remodelage par l’essai des comètes et l’électrophorèse en champ pulsé. En procédant à des immunofluorescences sur coupes de tissus et en utilisant un extrait nucléaire hautement actif, la présence de topoisomérases ainsi que de marqueurs de systèmes de réparation a été confirmée. Les protéines de réparation identifiées font partie de systèmes sujets à l’erreur, donc cette refonte structurale de la chromatine pourrait être génétiquement instable et expliquer le biais paternel observé pour les mutations de novo dans de récentes études impliquant des criblages à haut débit. Une technique permettant l’immunocapture spécifique des cassures bicaténaires a été développée et appliquée sur des spermatides murins représentant différentes étapes de différenciation. Les résultats de séquençage à haut débit ont montré que les cassures bicaténaires (hotspots) de la spermiogenèse se produisent en majorité dans l’ADN intergénique, notamment dans les séquences LINE1, l’ADN satellite et les répétions simples. Les hotspots contiennent aussi des motifs de liaisons des protéines des familles FOX et PRDM, dont les fonctions sont entre autres de lier et remodeler localement la chromatine condensée. Aussi, le motif de liaison de la protéine BRCA1 se trouve enrichi dans les hotspots de cassures bicaténaires. Celle-ci agit entre autres dans la réparation de l’ADN par jonction terminale non-homologue (NHEJ) et dans la réparation des adduits ADN-topoisomérase. De façon remarquable, le motif de reconnaissance de la protéine SPO11, impliquée dans la formation des cassures méiotiques, a été enrichi dans les hotspots, ce qui suggère que la machinerie méiotique serait aussi utilisée pendant la spermiogenèse pour la formation des cassures. Enfin, bien que les hotspots se localisent plutôt dans les séquences intergéniques, les gènes ciblés sont impliqués dans le développement du cerveau et des neurones. Ces résultats sont en accord avec l’origine majoritairement paternelle observée des mutations de novo associées aux troubles du spectre de l’autisme et de la schizophrénie et leur augmentation avec l’âge du père. Puisque les processus du remodelage de la chromatine des spermatides sont conservés dans l’évolution, ces résultats suggèrent que le remodelage de la chromatine de la spermiogenèse représente un mécanisme additionnel contribuant à la formation de mutations de novo, expliquant le biais paternel observé pour certains types de mutations.

Convergent evolution of PICIs-mediated phage interference

Staphylococcal pathogenicity islands (SaPIs), the prototype members of the family of phage inducible chromosomal islands (PICIs), are extremely mobile phage satellites, which are transferred between bacterial hosts after their induction by a helper phage. The intimate relationship between SaPIs and their helper phages is one of the most studied examples of virus satellite interactions in prokaryotic cells. SaPIs encode and disseminate virulence and fitness factors, representing a driving force for bacterial adaptation and pathogenesis. Many SaPIs encode a conserved morphogenetic operon, including a core set of genes whose function allows them to parasitize and exploit the phage life cycle. One of the central mechanisms of this molecular piracy is the specific packaging of the SaPI genomes into reduced sized capsid structures derived from phage proteins. Pac phages were classically thought to be the only phages involved in the mobilisation of phage-mediated virulence genes, including the transfer of SaPIs within related and non-related bacteria. This study presents the involvement of S. aureus cos phages in the intra- and intergeneric transfer of cos SaPIs for the first time. A novel example of molecular parasitism is shown, by which this newly characterised group of cos SaPIs uses two distinct and complementary mechanisms to take over the helper phage packaging machinery for their own reproduction. SaPIbov5, the prototype of the cos SaPIs, does not encode the characteristic morphogenetic operon found in pac SaPIs. However, cos SaPIs features both pac and cos phage cleavage sequences in their genome, ensuring SaPI packaging in small- and full-sized phage particles, depending on the helper phage. Moreover, cos-site packaging in S. aureus was shown to require the activity of a phage HNH nuclease. The HNH protein functions together with the large terminase subunit, triggering cleavage and melting of the cos-site sequence. In addition, a novel piracy strategy, severely interfering with the helper phage reproduction, was identified in cos SaPIs and characterised. This mechanism of piracy depends on the cos SaPI-encoded ccm gene, which encodes a capsid protein involved in the formation of small phage particles, modifying the assembling process via a scaffolding mechanism. This strategy resembles the ones described for pac SaPIs and represents a remarkable example of convergent evolution. A further convergent mechanism of capsid size-reduction was identified and characterised for the Enterococcus faecalis EfCIV583 pathogenicity island, another member of the PICI family. In this case, the self-encoded CpmE conducts this molecular piracy through a putative scaffolding function. Similar to cos SaPIs, EfCIV583 carries the helper phage cleavage sequence in its genome enabling its mobilisation by the phage terminase complex. The results presented in this thesis show how two examples of non-related members of the PICI family follow the same evolutionary convergent strategy to interfere with their helper phage. These findings could indicate that the described strategies might be widespread among PICIs and implicate a significant impact of PICIs mediated-virulence gene transfer in bacterial evolution and the emergence of pathogenic bacteria.

Investigation of RuO2/Ta2O5 thin film evolution by thermogravimetry combined with mass spectrometry

The thermal evolution process of RuO2–Ta2O5/Ti coatings with varying noble metal content has been investigated under in situ conditions by thermogravimetry combined with mass spectrometry. The gel-like films prepared from alcoholic solutions of the precursor salts (RuCl3·3H2O, TaCl5) onto titanium metal support were heated in an atmosphere containing 20% O2 and 80% Ar up to 600 °C. The evolution of the mixed oxide coatings was followed by the mass spectrometric ion intensity curves. The cracking of retained solvent and the combustion of organic surface species formed were also followed by the mass spectrometric curves. The formation of carbonyl- and carboxylate-type surface species connected to the noble metal was identified by Fourier transform infrared emission spectroscopy. These secondary processes–catalyzed by the noble metal–may play an important role in the development of surface morphology and electrochemical properties. The evolution of the two oxide phases does not take place independently, and the effect of the noble metal as a combustion catalyst was proved.