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The recently proposed and experimentally validated Equilibrium Model provides the most detailed description of temperature's effect on enzyme catalytic activity to date. By introducing an equilibrium between Eact, the active form of enzyme, and Einact, a reversibly inactivated form of enzyme, the Equilibrium Model explains apparent enzyme activity loss at high temperatures that cannot be accounted for by irreversible thermal denaturation. The Equilibrium Model describes enzyme behavior in the presence of substrates and under assay conditions; thus its associated parameters, deltaHeq and Teq, may have physiological significance. The Equilibrium Model parameters have been determined for twenty-one enzymes of diverse origins. The results demonstrated the wide applicability of the Equilibrium Model to enzymes of different types and temperature affinity. The study has also established deltaHeq as the first quantitative measure of enzyme eurythermalism and demonstrated the relationship between Teq and optimal growth temperature of organisms. The Equilibrium Model is therefore a useful tool for studying enzyme temperature adaptation and its role in adaptations to thermophily and eurythermalism. Moreover, it potentially enables a description of the originating environment from the properties of the enzymes. The Equilibrium Model has been employed to characterize enzymes isolated from bacterial episymbionts of Alvinella pompejana. A. pompejana inhabits one of the most extreme environments known to science and has been proposed as an extremely eurythermal organism. A metagenomic study of the A. pompejana episymbionts has unveiled new information related to the adaptive and metabolic properties of the bacterial consortium; the availability of metagenomic sequences has also enabled targeted retrieval and heterologous expression of A. pompejana episymbiont genes. By inspecting enzymes derived from the unique episymbiotic microbial consortium intimately associated with A. pompejana, the study has shed light on temperature adaptations in this unique symbiotic relationship. The findings suggested that eurythermal enzymes are one of the mechanisms used by the microbial consortium to achieve its adaptations. By combining metagenomic and enzymological studies, the research described in this thesis has lead to insights on the eurythermalism of a complex microbial system from an enzymological aspect. The findings have enhanced our knowledge on how life adapts to extreme environments, and the validation of the Equilibrium Model as a tool for studying enzyme temperature adaptation paves the way for future studies.

Influenza B viruses are a significant cause of disease and influenza B antigens are present in all human vaccines. Achieving suitable yields of seed viruses is often difficult for vaccine manufacturers. With influenza A viruses increases in yields have been achieved by the preparation of reassortants between a high-yielding donor strain and an epidemic strain. However, reassortment of influenza B viruses for the preparation of seeds has not been usually undertaken due to the lack suitable donor strains. Such an approach, which formed the basis of this thesis, could improve vaccine yields, lower costs and introduce a further element of predictability to vaccine manufacture. Potential donor strains were prepared from B/Lee/40 (B/Lee) by two approaches involving the selection of stable cold- and high- temperature mutants. Initial passaging was undertaken in specific-pathogen-free (SPF) chicken embryo kidney (CEK) cultures and later passage in SPF embryonated chicken eggs. Both approaches were successful, although a smaller number of viable progeny could be isolated from plaques obtained at 38„aC. Potential donor strains, isolated by selection at either 25 or 38„aC and plaque-purified in SPF CEK cultures, were tested for haemagglutinin and infectious titre, in comparison with the original parental strain by three methods, and for differences in antigenicity by cross-haemagglutination-inhibition tests. Potential donor strains selected at temperatures of 25„aC (C25) and 38„aC (H38) produced haemagglutination titres of 320 units/50ƒÝL and infectivities of 8.57 and 8.39 50% egg infectious doses, respectively, when grown in eggs at the permissive temperature (34„aC). Reassorting experiments using the B/Lee-derived potential donor strains C25 and H38 and the epidemic strain, B/Johannesburg/5/99 (B/Johannesburg), showed that the preparation of reassortant progeny with both epidemic strain HA and NA was difficult. Only 1/24 of the resulting reassortants possessed both the HA and NA of the epidemic strain. None of the reassortant progeny produced in reassorting experiments using C25 and H38 and the epidemic strain B/Panama/45/90 (B/Panama) possessed the desired 6:2 gene constellation (i.e. genes for the two surface antigens of the epidemic strain and the remainder from the donor strain). The infectious titre of selected progeny from the reassortment experiments were determined by three methods and compared with their respective epidemic parents. Yields of several influenza B epidemic strains and potential donor strains were measured after growth in Madin-Darby canine kidney (MDCK) cells prepared in serum-containing (SC) and animal- and human-derived protein-free (AHPF) media. Optimal multiplicities of infection were determined for B/Panama, B/Johannesburg and C25 in MDCK cultures grown in SC medium. A series of experiments were then undertaken to determine the maximum virus yields in MDCK cells grown in SC medium, followed by a further experiment using C25, B/Panama, B/Johannesburg, and selected reassortants after preparation in AHPF medium. Cell culture yields from 5/6 viruses grown in MDCK cells prepared in AHPF medium were higher than in cells prepared in SC medium and approached those obtained in eggs.

Temperature is arguably the leading factor that drives adaptation of organisms and ecosystems. Remarkably, many sister species share the same habitat because of their different temporal or micro-spatial thermal adaptation. In this PhD, the underlying molecular mechanisms of the adaptation of closely related species to different temperatures are sought. A thermodynamic analysis was applied to a genome-scale metabolic model of S. cerevisiae at warm and cold temperatures to identify thermo-dependent reactions. Gene Ontology (GO) analysis of predicted cold-dependent reactions found that redox reactions were significantly enriched. A complementary large scale experimental approach was taken by competing 6,000 mutant strains at 16°C to identify genes that were responsible for the fitness at low temperatures. The experiment was carried out in three different nutritional conditions to test the plasticity of temperature dependency. A list of strains whose copy number significantly increased or decreased in all media conditions was constructed and analysed using Gene Ontology. Vitamin biosynthesis, lipid/fatty acid processes and oxido-reduction reactions were all found to be significantly affected by the cold condition. Combining the data from the two studies a list of candidate genes affected by temperature changes were generated. In particular, two genes, GUT2 and ADH3, were identified as potential cold favouring genes and studied in more detailed. Mutants for these two genes were created in a pair of natural sympatric cryotolerant and thermotolerant Saccharomyces yeasts, namely S. kudriavzevii CA111 and S. cerevisiae 96.2, representing an excellent ecological experimental model for differential temperature adaption. My results showed that when compared to the parental strains, both mutants showed lower fitness at cold temperatures as predicted, and in S. kudriavzevii CA111 these mutations significantly improve growth at warm temperatures. Results from all aspects of this work indicate that oxidation reduction reactions are important for cold acclimation. It is known that heat stress causes redox imbalances which are compensated by increasing glycerol production or cytosolic acetaldehyde. Since GUT2 and ADH3 are involved in these processes, mutations in these genes may not be able to compensate for temperature changes. My data also shows that vitamins may also play an important role in cold acclimation which would be an interesting line of investigation for future work. Overall this PhD thesis has incorporated in silico and in vivo work to identify potential processes and genes involved in the temperature adaptation of sister Saccharomyces yeast species. The approach and results provided in this study support the use of a systems biology framework to studying species adaptation to environmental changes, and show that such models can yield testable predictions that may lead to new biological discoveries.

One of the cellular responses to temperature fluctuations in plants is the adjustment in the degree of membrane unsaturation. Glycerolipids are major constituents of cellular membranes. In higher plants, glycerolipids are synthesized via two major metabolic pathways compartmentalized in the ER and chloroplast. Adaptive responses in membrane lipids include alterations in fatty acid desaturation, proportional changes in membrane lipids as well as molecular composition of each lipid species. In this study, I systematically explored the significance of glycerolipid pathway balance in temperature induced lipid composition changes in three plant species that have distinctive modes of lipid pathway interactions through a combination of biochemical and molecular approaches including lipidomics and RNA-seq analysis. In Arabidopsis thaliana, a 16:3 plant, low temperature induces an augmented prokaryotic pathway, whereas high temperature enhances the eukaryotic pathway. Atriplex lentiformis reduces its overall lipid desaturation at high temperature and switches lipid phenotype from 16:3 to 18:3 through drastically increasing the contribution of the eukaryotic pathway as well as suppression of the prokaryotic pathway. In sync with the metabolic changes, coordinated expression of glycerolipid pathway genes, as revealed by RNA-seq also occurs. In Triticum aestivum, an 18:3 plant, low temperature leads to a reduced glycerolipid flux from ER to chloroplast. Evidence of differential trafficking of diacylglycerol (DAG) moieties from ER to chloroplast was uncovered in three plant species as another layer of metabolic adaptation under different temperatures. Taken together, this study has established a biochemical basis that highlights the predominance and prevalence of lipid pathway interactions in temperature induced lipid compositional changes.

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Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

L’adaptation des pathogènes à leurs hôtes et aux variations climatiques, particulièrement à la température est étudiée sur l’agent pathogène biotrophe obligatoire responsable de la rouille jaune du blé, Puccinia striiformis f. sp. tritici (Pst) au Moyen Orient. Cette étude s’est déroulée au Liban et en Syrie situés dans le berceau de la région de domestication du blé. Des gènes de résistance spécifique ont été postulés au stade plantule pour 87 lignées élites du programme d’amélioration de l’ICARDA,28 cultivars Libanais, et 23 landraces Libanaises en utilisant 11 pathotypes français disponibles à l’INRA-BIOGER. Un seul gène et une combinaison de gènes ont été postulés dans les lignées elites. Neuf gènes de résistance ont été identifiés dans les lignées élites ; plus de génotypes résistants figuraient parmi les lignées issues du programme d’amélioration. Les landraces sont les plus sensibles mais ont montré une ségrégation de réaction résistance parmi les plants sensibles.Pour la structuration de population pathogène du Liban et de la Syrie, un échantillonnage a été fait dans les deux pays sur du blé tendre, du blé dur et des repousses durant 2010-2011. Six isolats Libanais et 48 isolats Syriens ont été pathotypés avec une gamme de 43 hôtes différentiels. 275 échantillons ont été génotypés avec 20 marqueurs SSR. La population était clonale malgré avec la présence de l’hôte secondaire Berberis sp. dans la région, toutefois un nombre élevé de 50 MLG est observé était pour une population clonale. La présence de la race invasive PstS1/PstS2 caractérise cette région. Le profil de virulence Vr2, 6, 7, 9, 27 est le plus fréquent et typique du groupe génétique Méditerranéen (Bahri et al., 2009). La virulence Vr8 n’est pas fixée dans la population malgré sa présence dans la race invasive décrite depuis l’an 2000 (Milus et al., 2009). L’adaptation de la rouille jaune à la température a été décrite par Milus et al. (2009) et Mboup et al. (2012). Notre étude d’adaptation à la température a été faite sur un échantillon de 26 isolats provenant de zones froides et chaudes avec 4 isolats de référence. Nous avons testé deux paramètres d’agressivité, efficacité d’infection et période de latence sous quatre différents régimes de température (Chaud versus froid pour période de rosée et période d’incubation). Les isolats diffèrent pour leur réponse aux variations de température. Quelques isolats montrent une efficacité d’infection et une courte période de latence sous les différents régimes, d’autres sont efficaces au froid mais pas au chaud et vice versa. Pour l’efficacité d’infection, il n’y a pas d’adaptation mais par contre pour la période de latence on montre une adaptation à la température des isolats de la zone chaude ayant une efficacité d’infection. La température chaude de rosée a retardé la période de latence mais ce phénomène a été moins marqué pour les isolats d’origine chaude quand c’est incubé au chaud. Cette étude a montré que la population est clonale avec un haut nombre de pathotypes. Le germplasme n’est pas diversifié avec des gènes de résistance contre la rouille jaune. L’adaptation de l’agent de la rouille jaune à la température parmi les isolats testés a été décrite pour la période de latence pour les isolats provenant d’origine chaude. / The adaptation of fungal pathogen to its hosts and to the climate variation, in particular to the temperature, was investigated on wheat stripe (yellow) rust, caused by the biotroph fungus Puccinia striiformis f. sp. tritici (Pst) in the Middle East, focusing on Lebanon and Syria. This disease is a major problem for the crop in the region. Specific resistance genes were postulated in 138 wheat genotypes including elite lines, grown varieties and local landraces, using an array of 11 French pathotypes. Resistance gene diversity for yellow rust in wheat elite lines was higher than in current, commercial varieties grown in Lebanon, with nine Yr genes detected singly or in combination. Some varieties were resistant to all tested pathotypes and might provide interesting sources of resistance. Most of the Lebanese landraces were susceptible but also heterogeneous by their number of plants susceptible and resistant to a specific pathotype in a same landrace.A field survey was conducted in Lebanon and Syria in 2010-2011 and 275 Pst isolates were collected. The pathogen population was genotyped with 20 microsatellite markers and was found to be clonal, although the alternate host Berberis libanotica is present in the region. The dominant multilocus genotype shared similarity with the new invasive strain PstS1/PstS2 dispersed worldwide since 2000. The population was clonal with 10 pathotypes detected in Lebanon and Syria. 50 MLGs were detected considered high for clonal population. The virulence profiles combining Vr2, Vr6, Vr7, Vr9, and Vr27 are typical of the Mediterranean area according to group (Bahri et al., 2009) and corresponded to the worldwide invasive pathotype described since 2000 (Milus et al., 2009). The Vr8 was not fixed in this population, whereas this virulence is frequent in the Mediterranean genetic group (Bahri et al., 2009).Recently Pst strains have been described for adaptation to warm temperature (Milus et al., 2009; Mboup et al., 2012). The question of temperature adaptation in this study was whether the strains adapted to warm temperature are found in few clones of invasive strains or if they are selected in different pathogen genotypes locally under specific climate conditions. We selected 26 Pst isolates from the Middle East, 13 isolates from warm and 13 isolates from cold areas. We assessed their infection efficiency and latent period under four temperature regimes (high and warm temperature for the spore penetration phase, and high and warm temperature for the latency period). The isolates differed for the thermal aptitude for infection efficiency and latent period, but no clear relationship was established between the climate of the origin location of the isolate and its thermal aptitude. Some isolates were able to infect at high temperature but had long latency at high temperature and vice versa, some isolates had low infection efficiency and short latent period at high temperature, and few isolates were efficient either at high temperature or cold temperature for infection efficiency. Latency period showed pattern of local adaptation. Warm dew temperatures retarded sporulation, but this effect was far less marked for isolates from warm climates when incubated under warm conditions.This study provides details about probable effective yellow rust genes present in different genotypes and the prevalent pathotypes in the region. Moreover, the thermal aptitude for infection efficiency and latent period of some isolates under contrasting temperature will help us to build a better integrated disease management in the highlight of global warming.

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