Les arbres, enracinés au sol, adaptent leur développement à leur environnement fluctuant et en particulier aux conditions mécaniques imposées en permanence par le vent. Les tempêtes des dernières décennies ont mis en lumière le rôle majeur, pour la survie à long-terme des arbres, du processus d’acclimatation aux contraintes mécaniques dues au vent. Au premier ordre, le vent exerce principalement des efforts de flexion répétés sur les branches et tiges des arbres qui tendent à osciller pendant les épisodes venteux. Ces flexions entrainent une modification de la croissance des arbres en hauteur, en diamètre ou au niveau du système racinaire mais également la formation d’un bois à l’anatomie et aux propriétés particulières ; lequel est désigné par le terme « bois de flexion ». Au cours de ces travaux de thèse, nous avons développé des dispositifs expérimentaux originaux nous permettant, d’une part, d’appliquer des traitements de flexions unidirectionnelles répétées sur des tiges de jeunes peupliers, en contrôlant l’amplitude des déformations appliquées à la tige, et d’autre part, de contrôler différents niveaux de stress hydrique. Les suivis de croissance pendant une saison de végétation complète ont montré que l’acclimatation mécanique des arbres est un processus qui, bien que très couteux en matière de construction de biomasse, s’avère primordial et se met en place même lors de conditions de stress hydrique sévère. De plus, nous avons pu démontrer que l’augmentation de biomasse liée à cette acclimatation s’effectue principalement dans les zones où les déformations tissulaires sont les plus fortes ; conduisant ainsi à des géométries de sections particulières qui accroissent considérablement la rigidité de flexion des tiges. Une modélisation mécanique par éléments finis a aussi permis de révéler que ces configurations issues de l’acclimatation conduisent à une meilleure répartition des contraintes mécaniques, en abaissant en particulier l’intensité des contraintes maximales de compression subies par le bois. Afin de descendre dans les échelles spatiales, nous avons développé des outils et des méthodes de caractérisation originaux qui ont permis de mesurer, au niveau tissulaire, l’impact des différents types de sollicitations (compression et/ou traction répétées), engendrées au cours d’une flexion de tige, sur les propriétés hydrauliques et mécaniques du bois vert ; tant du point de vue des propriétés usuelles (comportement élastique, conduction hydraulique) que du point de vue des fonctions de sécurité (rupture, sensibilité à la cavitation, …). Nous avons alors pu mettre en évidence le comportement singulier du bois formé sous sollicitations de compressions répétées qui montre en particulier une nette augmentation de sa capacité à subir des déformations importantes avec un endommagement très réduit. L’ensemble des résultats expérimentaux et de modélisation aux échelles tissulaires comme de l’organe entier, indique que les acclimatations de la croissance secondaire et des propriétés intrinsèques du matériau bois procurent un bénéfice mécanique pour la pérennité de l’arbre dans son environnement venteux fluctuant. / Trees, anchored in the ground, adjust their development to their fluctuating environment and particularly to the mechanical conditions daily imposed by wind. Storms of last decades enlightened that acclimation of trees to mechanical stresses due to wind is a vital requirement for their long-term survival. Wind mainly leads to repeated bending of the branches and stems of trees that swing during windy events. These bending cause a modulation of trees growth in height, in diameter or at the level of the root system but also imply the formation of a wood with specific anatomy and properties; this wood was defined as “Flexure wood”. In our work, we developed original experimental setups in order to apply a treatment of repeated unidirectional bending to the stem of young poplars, while controlling the magnitude of the strains applied to the stem. The setup controlled different levels of hydric stress too. The monitoring of the growth during a growing season showed that the mechanical acclimation is a process that, although costly in terms of biomass construction, turns out to be essential and takes place even under severe hydric stress. Besides, we demonstrated that the increase of biomass, linked to this acclimation, is mainly provided in the areas where tissue strains are the highest; leading to specific cross-section geometries that considerably increase the stem bending rigidity. A mechanical modelling using Finite Elements also enlightens that these configurations, due to acclimation, lead to a better distribution of mechanical stresses, especially by decreasing the intensity of maximal compressive stresses endured by wood. In order to investigate the microscopic scale, original experimental devices were developed at the tissue scale to measure the effect of different mechanical stresses (repeated compression and/or tension), applied by stem bending, on the hydraulic and mechanical properties of green wood; as much in terms of usual properties (elastic behaviour, hydraulic conduction) as in terms of security functions (rupture, sensibility to cavitation, …). Thus, we enlighten the specific behaviour of wood formed under repeated compressive stresses that shows a significant increase of its ability to withstand high levels of strains with a very reduced damaging. The whole experimental and modelling results, as much at the tissue scale as at the tree scale, points out that the acclimations of secondary growth and wood properties provide a mechanical benefit for tree sustainability in its fluctuating windy environment.

Increasing atmospheric CO2 and other greenhouse gasses coupled to the accelerated rate of global warming puts plants and ecosystems under the strain of a rapidly changing abiotic environment. Understanding the impacts of changing global climate is a strong focus of plant science and the establishment of more resilient crop variants is an important goal for breeding programs. Our understanding of plant responses and acclimation to abiotic conditions has improved substantially over the last decades but the combination of a complex abiotic environment and high biological diversity, both on molecular as well as on species level, leaves us still with a lot of uncertainties. The aim of this doctoral thesis was to establish a link between plant thermal responses and the carbon-nitrogen balance of plants. The work in this thesis focused on ecologically significant species of the boreal region: Picea abies, Pinus sylvestris and Betula pendula; and Betula utilis, which is one of the prominent tree species in the high altitudes of the Himalayas. The results presented demonstrate that sub-optimal temperatures combined with other abiotic factors can have additive effects that are not easily deducible from the effect of the two factors separately. Low nitrogen availability enhanced the negative effect of low temperature, while elevated CO2 enhanced plant growth under moderate increases in temperatures but under a more extreme temperature increase it exacerbated the negative effect of heat. I also show evidence that species, despite being grouped into the same functional group or inhabiting the same biome can have different thresholds to temperature and to shifts in the C/N balance of their environment and that these differences can, to some extent, be explained by their differential growth strategies. Furthermore, I demonstrate results supporting the hypothesis that the C-N fluxes between mycorrhizal fungi and tree are strongly dependent on the C and N in the environment, highlighting the significance of the tree-mycorrhiza associations in the C sequestration capacity of the boreal region. In this thesis I also present a generalised empirically based mathematical model that can describe the respiration-temperature response of plant functional types or biomes with high precision, giving a more accurate estimate of NPP when implemented in global climate models, and has the potential to incorporate the thermal acclimation of respiration, further increasing the precision of estimating carbon fluxes under future warming temperatures. My results provide novel insights into the interactive temperature-carbon-nitrogen responses of plants, taking a step towards better understanding the response of plants and forests to future climates. / Ökande atmosfäriskt CO2 och andra växthusgaser kopplade till den accelererande globala uppvärmningen utsätter växter och ekosystem för stressen av en snabbt förändrande abiotisk miljö. Att förstå påverkan av ett globalt klimat i förändring står i fokus inom växtforskning och utvecklandet av mer motståndskraftiga grödor är ett viktigt mål inom programmen för växtförädling. Vår förståelse av växters responser och acklimatisering till abiotiska förhållanden har förbättrats avsevärt under de senaste decennierna, men på grund av kombinationen av en komplex abiotisk miljö och stor biologisk mångfald, både på molekylär nivå såväl som på art-nivå, kvarstår en del frågetecken. Syftet med denna avhandling var att upprätta ett samband mellan växters responser på temperaturförändringar och kol-kvävebalansen hos växter. Arbetet i denna avhandling inriktades på ekologiskt betydande arter i den boreala regionen, Picea abies, Pinus sylvestris and Betula pendula; samt Betula utilis som är en av de framträdande trädarterna på höga höjder i Himalaya. Resultaten som presenteras visar att suboptimala temperaturer i kombination med andra abiotiska faktorer kan ha additiva effekter som inte enkelt kan härledas från effekten av de två faktorerna var för sig. Låg kvävetillgänglighet ökade den negativa effekten av låg temperatur, medan förhöjd CO2-halt förbättrade planttillväxt under måttliga temperaturökningar, men under en mer extrem temperaturökning förvärrades dock den negativa effekten av värme. Jag framför även bevis på att arter, trots att de grupperas i samma funktionella grupp eller finns inom samma biom, kan ha olika tröskelvärden beträffande temperatur och förskjutningar i C/N-balansen i sin miljö och att dessa skillnader, i viss utsträckning, kan förklaras av deras olika tillväxtstrategier. Vidare visar jag resultat som stöder hypotesen att C-N - flöden mellan mykorrhiza och träd är starkt beroende av C och N i miljön. Detta belyser i sin tur betydelsen av samarbetet mellan träd och mykorrhiza gällande kolbindningskapaciteten i den boreala regionen. I denna avhandling presenterar jag även en generaliserad empiriskt baserad matematisk modell som med hög precision kan beskriva respiration-temperatur svar av växtfunktionella typer eller biom, vilken ger en mer exakt uppskattning av NPP i globala klimatmodeller. Mina resultat åstadkommer nya insikter i de interaktiva temperatur-kol-kväve-responserna hos växter, och tar ett steg mot bättre förståelse för växters och skogars reaktion på framtida klimat.

Žmogaus organizmas yra linkęs aklimuotis ir prie besikartojančių šilumos ir prie besikartojančių šalčio stimulų (Michael, 2001). Tai sukelia skirtingus fiziologinius atsakus organizme. Žmogaus organizmas aklimuodamasis šilumai siekia sušvelninti šilumos sukeltą fiziologinį stresą, ir pagerinti šiluminį komfortą bei treniravimosi galimybes. Aklimacija yra sukeliama pakartotinių šilumos stimulų, kurie sukelia pakankamą stresą, galintį pakelti odos ir vidines temperatūras, padidinti prakaitavimą. Dažniausiai aklimacija kasdien pasikartojantiems šilumos stimulams atsiranda per pirmas 4 dienas ir galutinai susiformuoja per 3 savaites. Šilumos aklimacijos sukelti požymiai – žemesnė vidinė temperatūra, padidėjęs prakaitavimas ir odos kraujotaka, sumažėjusi metabolinė medžiagų apykaita, sumažėjęs širdies ir kraujagyslių sistemai sukeliamas krūvis, pagerėjęs skysčių balansas ir padidėjusi šiluminė tolerancija (Michael ir kt., 2001). Šiais laikais profesinė ir rekreacinė veikla šaltomis oro sąlygomis yra gana dažna. Šalčio poveikis sukelia termoreguliacijos atsakus, kurie pasireiškia elgesiu arba fiziologiniais pokyčiais, kad išlaikytų šiluminę pusiausvyrą- padidėjusia metabolinė šilumos gamyba drebant arba vazokonstrikcija- sumažėjusiu šilumos atidavimu. Šie fiziologiniai atsakai priklauso nuo asmens individualių savybių- amžiaus, kūno sudėjimo, bendro prisitaikymo šalčiui. Stiprus šalčio stimulas gali sukelti medicinines traumas ar net mirtį (Launay, 2009). Aklimacija šalčiui yra... [toliau žr. visą tekstą] / Human body tends to acclimate to repeated heat or cold stimulus (Michael, 2001). This leads to different physiological responses in the body. During acclimation to heat the body seeks to soften heat-induced physiological stress, to improve thermal comfort and increase training opportunities. Acclimation is induced by repeated heat stimuli that cause sufficient stress, that can raise skin and core temperature, increase sweating. Usually the acclimation to daily repeated heat stimuli occurs within the first 4 days and finally forms during 3 weeks. Heat acclimation causes symptoms such as lower core temperature, increased sweating and skin blood circulation, decreased metabolism, decreased stress for cardiovascular system, improved fluid balance and increased thermal tolerance (Michael et al., 2001). Nowadays, professional and recreational activities in cold weather conditions are quite common. Cold exposures cause thermoregulatory responses that affects behaviour or physiological changes to maintain thermal balance- increased metabolic heat production while shivering, or vasoconstriction- reduced heating. These physiological responses depend on the person’s individual characteristics- age, body composition and general adaptation to cold. A strong cold stimulus can cause medical injuries or even death (Launey, 2009). Acclimation to cold is classified into several categories- metabolic, insulation, hypothermic and intermediate versions (Bittel, 1992). Acclimation to cold may... [to full text]

Biological processes in living organisms consist of a vast number of different molecular networks and interactions, which are complex and often hidden from our understanding. This work is focused on recovery of such details for two quite distant examples: acclimation to extreme freezing tolerance in Siberian spruce (Picea obovata) and detection of proteins associated with prostate cancer. The first biological system in the study, upon P. obovata, is interesting by this species ability to adapt and sustain extremely low temperatures, such as -60⁰C or below. Despite decades of investigations, the essential features and mechanisms of the amazing ability of this species still remains unclear. To enhance knowledge about extreme freezing tolerance, the metabolome and proteome of P. obovata’s needles were collected during the tree’s acclimation period, ranging from mid August to January, and have been analyzed. The second system within this study is the plasma proteome analysis of high risk prostate cancer (PCa) patients, with and without bone metastases. PCa is one of the most common cancers among Swedish men, which can abruptly develop into an aggressive, lethal disease. The diagnostic tools, including PSA-tests, are insufficient in predicting the disease’s aggressiveness and novel prognostic markers are urgently required. Both biological systems have been analyzed following similar steps: by two-dimensional difference gel electrophoresis (2D-DIGE) techniques, followed by protein identification using mass spectrometry (MS) analysis and multivariate methods. Data processing has been utilized for searching for proteins that serve as unique indicators for characterizing the status of the systems. In addition, the gas chromatography-mass spectrometry (GC-MS) study of the metabolic content of P.obovata’s needles, from the extended observation period, has been performed. The studies of both systems, combined with thorough statistical analysis of experimental outcomes, have resulted in novel insights and features for both P. obovata and prostate cancer. In particular, it has been shown that dehydrins, Hsp70s, AAA+ ATPases, lipocalin and several proteins involved in cellular metabolism etc., can be uniquely associated with acclimation to extreme freezing in conifers. Metabolomic analysis of P. obovata needles has revealed systematic metabolic changes in carbohydrate and lipid metabolism. Substantial increase of raffinose, accumulation of desaturated fatty acids, sugar acids, sugar alcohols, amino acids and polyamines that may act as compatible solutes or cryoprotectants have all been observed during the acclimation process. Relevant proteins for prostate cancer progression and aggressiveness have been identified in the plasma proteome study, for patients with and without bone metastasis. Proteins associated with lipid transport, coagulation, inflammation and immune response have been found among them.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The objectives of this study were to quantify the meteorological elements in agroforestry system (AFS) and full sun, to analyze the evaluate the leaf area, percentage of leaves and stems, quantify the production of dry matter (DM) and crude protein (CP) of annual ryegrass grown in the understory of canafístula and eucalyptus. In addition to evaluating the anatomic changes completely expanded ryegrass leaves resulting from acclimatization of this species to shading of angico-vermelho and canafístula. The experiment was conducted in an area that has an agroforestry system (AFS). Forest species are distributed in range and line, where the rows are separated on 12 and 6 meters respectively. The experiment was conducted in a randomized block design with three replications in split splot scheme. The ryegrass was sown in the understory of the forest species and full sun on 05/06/2012. The weather data were measured on AFS and in full sun for the purpose of characterization of production environments. For measurements of leaf area, relating the percentage of leaves and stems, quantify the production of DM and determine CP content of six cuts with box 0.25 m2 were performed in each of the treatments. To evaluate the effect of shading on the anatomy of leaves of annual ryegrass, samples of fully expanded leaf blades were collected from all treatments. In AFS s, range and line, there was no significant difference in the percentage photosynthetically active radiation transmited (PARt) and transmission with only relative to full sun. Among species, the PARt was reduced by approximately 30%. The air temperature (T°) showed variation between the AFS compared to full sun during the ryegrass development cycle. Shading caused by the presence of forest species, eucalyptus and canafístula in AFS, range and line, did not limit the growth and development of ryegrass grown in shaded understory. Independent of the systems used, the DM production of ryegrass and CP content, reached satisfactory levels for the use of this species as alternative forage in AFS. The leaf anatomy of Lolium multiflorum Lam cv. Common, is influenced by the environmental conditions in which it develops. Annual ryegrass can be used to compose Agroforestry Systems. / Os objetivos deste trabalho foram quantificar os elementos meteorológicos em sistemas agroflorestais (SAFs) e pleno sol, avaliar a área foliar e relação folha/colmo, quantificar a produção de matéria seca (MS), e o teor de proteína bruta (PB) de azevém cultivado em sub-bosque de canafístula e eucalipto, além de avaliar as alterações anatômicas em folhas completamente expandidas de azevém decorrentes da aclimatação desta espécie ao sombreamento de angico-vermelho e canafístula. O experimento foi realizado em uma área que possui um SAFs. As espécies florestais estão distribuídas em faixa e em linha, onde os renques se separam em 12 e 6 metros, respectivamente, e conduzido no delineamento experimental blocos ao acaso com três repetições no esquema split splot. O azevém foi semeado no sub-bosque das espécies florestais e pleno sol no dia 05/06/2012. Os elementos meteorológicos foram medidos nos SAFs e em pleno sol para fins de caracterização dos ambientes de produção. Para as medições de área foliar, relacionar o percentual de folhas e colmos, quantificar a produção de MS e determinar o teor de PB foram realizados seis cortes com quadro de 0,25 m2, em cada um dos tratamentos. Para avaliar o efeito do sombreamento sobre a anatomia de folhas de azevém anual, foram coletadas amostras de lâminas foliares totalmente expandidas em todos os tratamentos. Nos SAFs, faixa e linha, não houve diferença significativa para a RFAt e percentual de transmissão havendo apenas em relação ao pleno sol. Entre as espécies florestais, a RFAt foi reduzida em aproximadamente 30%. A temperatura do ar apresentou variação entre os SAFs em relação ao pleno sol durante o ciclo de desenvolvimento do azevém. O sombreamento provocado pela presença das espécies florestais, canafístula e eucalipto nos SAFs, faixa e linha, não limitaram o crescimento e desenvolvimento do azevém cultivado no sub-bosque sombreado. Independente dos sistemas utilizados, a produção de MS de azevém e o teor de PB, atingiram níveis satisfatórios para a utilização desta espécie como alternativa forrageira em SAFs. A anatomia foliar do azevém é influenciada pelas condições do meio em que se desenvolve. O azevém pode ser usado para compor Sistemas Agroflorestais.

The constitutively obsereved up-regulation of Hsp70 expression often led to premature conclusions about its critical role as a repair mechanism of cold injury that is, besides, expressed by protein misfolding/denaturation. In this study, we analyze the cold tolerance and the expression of 24 different mRNA transcripts of Hsp complex and other genes, that are associated with the repair of injury caused by cold. We use two strains of D. melanogaster: the wild type and the mutant type Hsp70- null, that lacks all 6 copies of the gene hsp70. We found out, that the larvae of two strains do not differ in their patterns of target genes expression during long term acclimation nor during recovery from chronic cold exposure and acute cold shock, therefore there is no transcriptional compensation of any other Hsp gene for the missing hsp70 in Hsp70- strain. The cold tolerance of Hsp70- strain larvae was impaired only, when they were exposed to strong acute cold shock by temperatures below -8°C. No difference in cold tolerance was observed, when the larvae were exposed to chronic cold exposure in 0°C or to mild acute cold shock by temperatures up to -4°C. Based on our results we assess, that the cold injury caused by strong acute cold schock is of another nature than caused by mild cold conditions and only in the first case Hsp70 expression is critical for the repair of cold injury in Drosophila melanogaster larvae.

Le gel est un des facteurs majeurs limitant l’aire de répartition des végétaux. Il a pour conséquence de limiter l’efficience de la circulation de sève brute par formation d’embolie dans les vaisseaux du xylème. Lorsqu’il y a formation de glace dans les tissus, les cellules vivantes peuvent également être lysées dans les organes lignifiés, et dans les bourgeons. Pour pallier cela, différents mécanismes de résistance sont développés pour limiter la formation d’embolie ou la résorber, pour augmenter transitoirement la tolérance à la formation de glace dans les tissus et pour éviter d’exposer les bourgeons et jeunes feuilles aux gels printaniers tardifs. Ces différents traits ont été caractérisés pour onze espèces communes ayant des limites altitudinales potentielles contrastées. Puis la physiologie de l’acclimatation au gel chez Juglans regia L. a été caractérisée, essentiellement à travers les relations entre humidité pondérale, contenu en sucres solubles, climat et levée de dormance. L’ensemble de ces données a permis d’élaborer un modèle prédictif de la résistance au gel en relation avec la physiologie de l’arbre qui pourrait, couplé avec d’autres modèles simulant la période estivale, permettre d’estimer la conjonction de différents stress sur le potentiel de résistance. / Frost is one of the major limiting constraints for plants causing embolism, living cell lysis and damages on buds during spring. Several processes are observed in trees in order to limit embolism or refill vessels, to increase frost resistance and to avoid freezing period for buds while bursting. Those different processes were monitored in eleven common species according to their potential alitudinal limt. And one was selected (Juglans regia L.) and physiology of acclimation was caracterised through interaction between water content, soluble carbohydrate (which are the best explaining factors for frost resistance), climate and dormancy release. According to those data, a predicting model for frost hardiness was built related to physiology of acclimation. This kind of model would be usefull related to a summer simulating model in order to estimate interaction between different stresses for frost hardiness potential.

As propriedades bioquímicas da (Na+,K+)-ATPase branquial do siri eurialino Callinectes danae aclimatado à salinidade de 15 o/oo foram estudadas. A análise do gradiente de centrifugação em sacarose revelou a presença de um único pico entre 30-35% de sacarose, com uma boa correlação entre as atividades PNFFase a ATPase totais e (Na+,K+)-ATPase. A atividade residual observada na presença de ouabaína 3 mM sugere a presença de outros sistemas de enzimas atuantes. A eletroforese em condições desnaturantes nos microsomas de brânquias de C. danae em animais recém-coletados em salinidade de 33 o/oo (não aclimatados) e de aclimatados a salinidades de 15 e 33 o/oo por um período de 10 dias mostrou a presença de pequenas diferenças nos padrões eletroforéticos das diferentes amostras. A análise por Western blot mostrou um aumento significativo da proporção relativa da subunidade alfa da (Na+,K+)-ATPase em relação à proteína total na fração microsomal do tecido branquial de animais aclimatados à salinidade de 15 o/oo quando comparados aos animais aclimatados a 33 o/oo. Entretanto, proporções similares de subunidade alfa foram observadas para amostras de animais recém-coletados a salinidade de 33 o/oo e aclimatados a 15 o/oo. A estimulação da atividade (Na+,K+)-ATPase pelo ATP ocorreu através de uma curva de saturação monofásica apresentando interações sítio-sítio (nH=1,2), com V= 298,8 ± 16,7 U/mg, com K0,5 de 174,2 ± 9,8 uM. A estimulação da atividade ATPase da (Na+,K+)-ATPase por íons Mg2+ (V= 299,16 ± 14,06 U/mg; K0,5= 767,31 ± 36,06 uM), íons Na+ (V= 309,0 ± 15,8 U/mg; K0,5= 7,8 ± 0,4 mM), íons K+ (V= 300,6 ± 15,3 U/mg; K0,5= 1,63 ± 0,08 mM) e íons NH4+ (V= 345,1 ± 19,0 U/mg; K0,5= 6,0 ± 0,3 mM) ocorreu através de interações sítio-sítio. A atividade da enzima foi modulada sinergisticamente pelos íons K+ com atividade máxima variando de 300,6 ± 15,3 U/mg para 514,6 ± 26,2 U/mg, na ausência e na presença 50 mM de íons NH4+, respectivamente. Além disso, foi observado um significativo aumento na afinidade aparente da enzima pelo íon K+ da ordem de 10 vezes (diminuiu de 1,6 ± 0,08 mM para 0,157 ± 0,008 mM). Similarmente ao observado para os íons K+, o íon NH4+ estimulou sinergisticamente a atividade da enzima na presença de diferentes concentrações de íons K+. A estimulação da atividade da enzima pelo íon NH4+ também ocorreu através de interações cooperativas entre os sítios. Embora tenha sido observado um aumento da atividade específica da enzima de 345,1 ± 19,0 U/mg para 516,8 ± 27,9 U/mg, não foram observadas variações significativas nos valores de nH e K0,5 com o aumento da concentração de íons K+. A ouabaína inibiu cerca de 90% da atividade ATPase total. A inibição pela ouabaína apresentou valor de KI de 45,09 ± 2,51 uM. O ortovanadato também inibiu atividade (Na+,K+)-ATPase na mesma faixa (90%) através de uma curva de inibição monofásica, com valor de KI da ordem de 1,31 ± 0,06 uM. O emprego de bafilomicina A1, tapsigargina e teofilina, juntamente com a ouabaína, na atividade ATPase total descartam a presença de V-ATPase, Ca2+-ATPase ou fosfatase, respectivamente. Apesar da inibição por oligomicina corresponder a menos de 3,7%, esse valor aparentemente sugere a presença de uma F0F1-ATPase. Além disso, a inibição por ácido etacrínico, em conjunto com os experimentos de estimulação por da atividade ATPase da enzima por íons Na+ sugere fortemente a presença de uma K+-ATPase. A (Na+,K+)-ATPase hidrolisou o substrato PNFF obedecendo à cinética Michaeliana com velocidade de V= 102,9 ± 4,3 U/mg e KM= 1,7 ± 0,1 mM. Já a estimulação da atividade K+-fosfatase da enzima por íons Mg2+ (V= 93,7 ± 2,3 U/mg; K0,5= 1,40 ± 0,03 mM), K+ (V= 94,9 ± 3,5 U/mg; K0,5= 2,9 ± 0,1 mM) e NH4+ (V= 106,2 ± 2,2 U/mg; K0,5= 9,8 ± 0,2 mM) seguiu uma cinética cooperativa, sugerindo a presença de múltiplos sítios de ligação. Entretanto, a atividade K+-fosfatase não foi estimulada sinergísticamente na presença de íons K+ mais NH4+. Os íons sódio (KI= 22,7 ± 1,7 mM) e ortovanadato (KI= 28,1 ± 1,4 nM) inibiram completamente a atividade fosfatase total através de uma única curva de inibição. / The biochemical properties of the (Na+,K+)-ATPase from the gill microsomal tissue of the euryhaline, marine, swimming crab Callinectes danae, acclimated to 15 0/00 salinity, were investigated. Sucrose gradient centrifugation analyses revealed a unique peak, between 30-35% sucrose, coincident with the total PNPPase, ATPase, and (Na+,K+)-ATPase activities. The residual activity observed in the presence of 3 mM ouabain suggests the existence of other enzyme systems. Electrophoresis under denaturing conditions, using material from fresh-caught crabs (33 o/oo salinity, not acclimated), and from crabs acclimated to 15 or 33 o/oo salinity, for 10 days, revealed differences in migration pattern. Western blot analyses showed a significant increase in the amount of (Na+,K+)-ATPase alpha-subunit relative to total protein, for crabs acclimated to 15 o/oo compared to those acclimated to 33 o/oo salinity. However, the proportion of alpha-subunit in samples from fresh-caught crabs acclimated to 33 o/oo and those acclimated to 15 o/oo salinity was similar. (Na+,K+)-ATPase activity was stimulated by ATP and showed a single saturation curve, exhibiting site-site interactions (nH=1.2), with V= 298.8 ± 16.7 U/mg, and K0.5= 174.2 ± 9.8 uM. Stimulation of the ATPase activity by Mg2+ (V= 299.16 ± 14.06 U/mg; K0.5= 767.31 ± 36.06 uM), Na+ (V= 309.0 ± 15.8 U/mg; K0.5= 7.8 ± 0.4 mM), K+ (V= 300.6 ± 15.3 U/mg; K0.5= 1.63 ± 0.08 mM) and NH4+ ions (V= 345.1 ± 19.0 U/mg; K0.5= 6.0 ± 0.3 mM) occurred through site-site interactions. (Na+,K+)-ATPase activity was synergistically modulated by K+ ions, maximum activity varying from 300.6 ± 15.3 U/mg to 514.6 ± 26.2 U/mg, in the absence and presence of 50 mM NH4+ ions, respectively. K+ ions induced a 10-fold increase in enzyme apparent affinity (from 1.6 ± 0.08 mM to 0.157 ± 0.008 mM). As for K+ ions, NH4+ synergistically stimulated enzyme activity in the presence of variable K+ concentrations. The stimulation by NH4+ ions exhibited cooperative, site-site interactions. Although an increase in specific activity from 345.1 ± 19.0 U/mg to 516.8 ± 27.9 U/mg was seen, no significant changes in nH and K0.5 were observed. Ouabain inhibited total ATPase activity by about 90%, showing a KI= 45.09 ± 2.51 uM. Orthovanadate also inhibited the (Na+,K+)-ATPase with a KI of 1.31 ± 0.06 uM. Although the inhibitory effect of oligomycin was minimal (3.7%), this inhibition may suggest F0F1-ATPase activity. The inhibition by ethacrynic acid, in association with Na+ ion stimulation of the ATPase activity, suggests the presence of a K+-ATPase. The (Na+,K+)-ATPase hydrolyzed PNPP (K+-phosphatase activity) obeying Michaelian kinetics, with V= 102.9 ± 4.3 U/mg and KM= 1.7 ± 0.1 mM. The stimulation of K+-phosphatase activity by Mg2+ (V= 93.7 ± 2.3 U/mg; K0.5= 1.4 ± 0.03 mM), K+ (V= 94.9 ± 3.5 U/mg; K0.5= 2.9 ± 0.1 mM), and NH4+ ions (V= 106.2 ± 2.2 U/mg; K0.5= 9.8 ± 0.2 mM) following cooperative kinetics, suggests multiple binding sites. K+-phosphatase activity, however, was not synergistically stimulated by K+ and NH4+. Sodium ions (KI= 22.7 ± 1.7 mM), and orthovanadate (KI= 28.1 ± 1.4 nM) totally inhibited the total phosphatase activity.

Le changement climatique entraîne un changement profond des régimes thermiques qui a des conséquences importantes sur la croissance des plantes. Toutefois, les effets des températures extrêmes ont été peu étudiés chez espèces fourragères pérennes, et ne sont pas intégrés dans les modèles actuels de prairie. Or ces modèles ont un rôle central à jouer dans le développement de stratégies d'adaptation au changement climatique. Dans ce contexte, cette thèse a cherché à approfondir la compréhension de la réponse de la luzerne et de la fétuque élevée à une large gamme de températures et à améliorer la prise en compte de ces réponses dans le modèle STICS. Un volet de travail expérimental en conditions contrôlées n'a pas permis de mettre en évidence de différences de réponses entre génotypes d'origine contrastées (tempéré/Méditerranéen) pour différents processus de croissance et de développement des deux espèces. En revanche, il est apparu des différences significatives entre les réponses normalisées de différents processus ; l'allongement des tiges en particulier se distinguant nettement des autres processus chez la luzerne. La réponse de la photosynthèse à la température a montré une forte acclimatation à la température de croissance, aussi bien pour des génotypes d'origine tempérée que méditerranéenne. Ces derniers sont apparus plus sensibles aux températures élevées du fait d'une désactivation de la RUBISCO. La modification des formalismes de réponse à la température de la croissance et du développement dans STICS (fonction non linéaire, pas de temps) a eu un fort impact sur le cumul de temps thermique pour des scénarios climatiques « futurs ». Toutefois, du fait de l'effet majoritaire d'autres stress conjoints durant les périodes de stress thermique et d'une rapide saturation du rayonnement intercepté, l'impact sur les projections de production fourragère s'est avéré limité pour les conditions testées. Au total ces résultats suggèrent que les productions fourragères pourraient être moins exposées au stress thermique que les cultures annuelles. / Due to climate change, the global average temperature is expected to rise with marked impacts on crop growth. However, extreme temperature's impacts on perennial herbaceous species have received much less attention to date than other crops and aren’t currently implemented in grassland models. Yet, grassland models have a major role to play in defining adaptation options to global warming’s impacts on agriculture. In this context, our aims were to characterise the responses to temperature of two major perennial forage species over a large range of growth temperature and to improve the temperature response formalisms in the STICS model. First, experiment conducted in growth chambers indicated that the responses of normalized developmental rates did not differ between genotypes from contrasting thermal areas (Mediterranean/temperate) within each species. On the other hand, the responses to temperature of normalised rates differed significantly between several of the physiological processes studied; in particular stem elongation rate differed from other developmental rates in alfalfa. Irrespective of cultivar origin, a remarkable acclimation of photosynthesis to growth temperature was observed. Mediterranean genotypes displayed a greater sensitivity of the maximum rate of Rubisco carboxylation to elevated temperatures. Modifications of temperature formalism in the STICS crop model (implementation of nonlinear response, hourly/daily step of response) had a significant impact on thermal time accumulation and crop development for future climate scenarios. However, due to larger effects of water stress and to saturation of radiation interception, impacts on projections of grassland dry matter production were limited. This result suggests that grassland production could be less exposed to thermal stress than annual crops.

Algae have been the centre of recent research as a sustainable feedstock for fuel because of their higher oil yield in comparison to other plant sources. However, algae biofuel still performs poorly from an economic and environmental perspective due to the high reliance on freshwater and nutrients for cultivation, among other challenges. The use of wastewater has been suggested as a sustainable way of overcoming these challenges because wastewater can provide a source of water and nutrients for the algae. Moreover, the ability of the algae to remove contaminants from wastewater also enhances the total economic output from the cultivation. However, the success of this strategy still depends greatly on efficient strain selection, cultivation and harvesting. Therefore, this PhD thesis has focussed on strain isolation, characterisation, optimisation and cultivation in open pond systems. Five algae strains were isolated from wastewater treatment tanks at a municipal water treatment plant in North West England. The isolated strains were morphologically and genetically characterised as green single-celled microalgae: Chlamydomonas debaryana, Hindakia tetrachotoma, Chlorella luteoviridis, Parachlorella hussii and Desmodesmus subspicatus. An initial screening of these strains concluded that C. luteoviridis and P. hussii were outstanding in all comparisons and better than some of the strains previously reported in the literature. Further tests carried out to elucidate the underlying tolerance mechanisms possessed by these strains were based on stress tolerance and acclimation hypotheses. In the following experiments, C. luteoviridis and P. hussii were found to have higher anti-oxidant enzyme activity that helps in scavenging reactive oxygen species produced as a result of exposure to wastewater. This result provides a new argument for screening microalgae strains for wastewater cultivation on the basis of anti-oxidant activity. In addition, the two strains could grow heterotrophically and are better adapted to nutrient deficiency stress than the other three isolates. In order to understand the role of microalgae acclimation in wastewater cultivation, strains identical or equivalent to the wastewater treatment tank isolates were obtained from an algae culture collection. These strains had not been previously exposed to wastewater secondary effluent. The initial growth of these strains in wastewater secondary effluent was very poor. However, after two months of acclimation to increasing concentrations of secondary wastewater effluent, it was observed that growth, biomass and lipid productivities of most of the strains were significantly improved, although still not as high as the indigenous strains. Therefore, it was concluded that continuous acclimation is an additional factor to the successful growth of algae in wastewater. Furthermore, addition of 25% activated sludge centrate liquor to the secondary effluent was found to increase algal growth and biomass productivity significantly. Futher tests to examine the continous cultivation of C. luteoviridis and P. hussii in wastewater showed that a biomas productivity of 1.78 and 1.83 g L-1 d-1 can be achieved on a continual basis. Finally, the capability of C. luteoviridis and P. hussii for full seasonal cultivation in a 150 L open pond in a temperate climate was studied, using the optimised secondary wastewater +25% liquor medium. Each strain was capable of growth all year including in autumn and winter but with strongest growth, productivity and remediation characteristics in the summer and spring. They could maintain monoculture growth with no significant contamination or culture crash, demonstrating the robustness of these strains for wastewater cultivation in a northern European climate.