Typescript. Includes vita and abstract. Bibliography: Includes bibliographical references (leaves 172-183). / Cancer magister, the Dungeness crab, occurs in different habitats during its life cycle, habitats which vary widely in the magnitude of salinity and temperature changes. Cancer magister hemocyanin also changes in structure and oxygenation properties during development. The following question was considered in this thesis: what are the effects of environmental salinity and temperature on metabolic rates, ionic and osmotic regulation and hemocyanin oxygen affinity in Q. magister during development. Metabolic rates and hemolYmph ionic and osmotic concentrations were measured in the megalopa, 1st juvenile, 5th juvenile and adult crab eight hours after acute exposure to 100% seawater (=32 ppt), 75% seawater and 50% seawater at both 10°C and 20°C. The oxygen binding properties of the whole hemolymph from these stages in 100% seawater at 10°C was determined. The effects of calcium and magnesium on the oxygen affinity of purified hemocyanin from different stages were also determined. In 100% seawater, routine metabolic rates of the four stages scale with body mass over the size range, 0.05 gm to 500 gm. The Q10 (10°C to 20°C) for the megalopa is higher in 75% seawater and 50% seawater than in 100% seawater. For the 1st juvenile, 5th juvenile and adult the Q10 values (10°C to 20°C) are independent of salinity. The megalopa, 1st juvenile and 5th juvenile are weaker regulators of hemolymph chloride, sodium and osmotic concentrations than the adult. The megalopa and adult, unlike the 1st juvenile and 5th juvenile, strongly regulate hemolymph calcium in reduced salinity. In 100% seawater hemolymph magnesium is significantly higher in the megalopa, 1st juvenile and 5th juvenile than in the adult. The oxygen affinities of whole hemolymph from the four stages are indistinguishable when adjusted for endogenous L-lactate concentrations; the Bohr coefficients are not significantly different among stages. The effect of magnesium on oxygen affinity of purified adult hemocyanin is influenced by proton concentration; the effect of calcium is independent of proton concentration. In 100% seawater, endogenous inorganic ion concentrations in the whole hemolymph of the various stages reduce the intrinsic stage specific differences in hemocyanin oxygen affinity.

Afin de repeupler la mer Caspienne, la propagation artificielle de l’esturgeon Acipenser persicus est maintenant une pratique courante avec rejets annuels de juvéniles directement en mer. Cependant, une forte mortalité est régulièrement observée suite à la libération directe en mer de juvéniles pesant 2-3 g. L'objectif a donc été d'analyser les capacités d’acclimatation de juvéniles de l’eau douce à l’eau saumâtre et d'identifier un moyen possible d’améliorer les taux de survie d’esturgeons de moins de 3 g.Dans une première partie, l'effet de transferts abruptes ou progressifs sur 5 jours de l'eau douce (ED) à une salinité de 11‰ (salinité de la mer Caspienne, CSW) a été étudié chez des juvéniles de 1 à 2 g, 2 à 3 g et de 3 à 5 g. Ont été mesurés les taux de mortalité, l'osmolalité plasmatique, l’expression et la localisation (branchies, reins, valvule spiralée, caecum pylorique) des principales protéines de transport : la Na+, K+-ATPase (NKA), la H+-ATPase vacuolaire (VHA), le Na+, K+, 2Cl- (NKCC) et la ‘Cystic Fibrosis Transmembrane conductance Regulator’ (CFTR). Une étude des changements d'expression génique et protéique de la NKA au niveau des branchies a également été effectuée. Enfin, une étude ultrastructurale (TEM et SEM) des cellules à chlorure branchiales a été effectuée.Dans une deuxième partie, un traitement hormonal avec le cortisol a été effectué afin d’améliorer la capacité d'acclimatation. Des juvéniles de moins de 2g ont été traités pendant 24h en ED avec des bains de cortisol à 3 concentrations différentes : 3, 5 et 7 mg.l-1. Les poissons ont été ensuite directement transférés de l'eau douce à la CSW et échantillonnés après 1, 4 et 9 jours après transfert en CSW. Les taux de mortalité, l'osmolalité plasmatique, le nombre et l’aire des cellules à chlorure des branchies ont été évalués.Ainsi, les poissons de plus de 3 g sont capables de survivre et s’acclimatent à la CSW. Malgré une augmentation initiale de l'osmolalité plasmatique après transfert de salinité, les juvéniles réduisent ensuite leur osmolalité plasmatique jusqu'à la pression osmotique de la mer Caspienne. Cependant, beaucoup de poissons de moins de 3 g ne peuvent survivre à l’augmentation brutale de salinité et la pression osmotique du sang des poissons survivants reste élevée. L’expression de la NKA et du NKCC, la taille et le nombre des cellules à chlorure des branchies sont également plus élevés. Les juvéniles pesants plus de 2 g augmentent aussi fortement leur activité de NKA branchiale après transfert de salinité. L'étude ultrastucturale a révélé des surfaces apicales similaires pour les cellules à chlorure branchiales des poissons en ED et acclimatés à la CSW. Le cytoplasme de ces cellules dans les poissons pesant plus de 2 g apparait plus dense et gonflé par rapport aux poissons pesant moins de 2 g. Cela pourrait être dû à l'allongement des replis de la membrane basale (réseau tubulo-vésiculaire) et/ou à une densité plus élevée des mitochondries. L’expression branchiale du gène NKA des poissons acclimatés à la CSW est apparue d'abord sur- puis sous-exprimée 4 jours après le transfert de salinité pour atteindre le niveau des poissons en ED. Au niveau intestinal et des reins, aucune différence n'a pu être détectée entre les différents groupes de poids suite au transfert de salinité. Malgré une osmolarité plasmatique réduite, le nombre et la taille des cellules à chlorure des poissons traités avec le cortisol montrent 9 jours après transfert, les mêmes tendances que celles observées pour les poissons non traités.Ainsi, cette étude a révélé que seuls les esturgeons juvéniles de plus de 3 g peuvent être directement rejetés en mer Caspienne. Pour les poissons de 2-3 g, un protocole spécial pendant le transfert de salinité doit être considéré alors que les poissons pesant moins de 2 g ne peuvent tolérer une augmentation de salinité même après un traitement au cortisol. / For restocking purposes, artificial propagation of Persian sturgeon (Acipenser persicus) and annual releases into the Caspian Sea are now common practice. However, high mortality is regularly observed following the direct release of 2-3 g juveniles into the Caspian Sea. Therefore, the aim of the study was to analyse the acclamatory capacities of juvenile Persian sturgeons to elevated salinity and to identify a possible way to improve survival rates of juveniles weighting less than 3 grams.In the first part of this study, the effect of abrupt and 5-day gradual salinity transfers from freshwater (FW) to 11‰ Caspian Sea water (CSW) were investigated in juvenile Persian sturgeons with three different weight groups: 1-2 g (1.62 ± 0.27 g), 2-3 g (2.55 ± 0.41 g) and 3-5 g (4.28 ± 0.76 g). Daily mortality rates, plasma osmolality, immunofluorescence localization of the main ion transporter proteins such as Na+, K+-ATPase (NKA), Vacuolar H+-ATPase (VHA), Na+,K+,2Cl–(NKCC) and Cystic Fibrosis Transmembrane conductance Regulator (CFTR) at the gill, kidney, pyloric caecum and intestinal spiral valve levels, ultrastructural studies (TEM and SEM) of the gills for chloride cell changes and changes of branchial NKA gene and protein expression were measured 4 and 10 days after abrupt transfer to CSW and 9 and 15 days after the initial gradual transfer (i.e. 4 and 10 days after reaching CSW), respectively.In the second part of study, hormonal treatment with cortisol was used to enhance the acclimation capability of fish weighing less than 2g. Fish were treated with a cortisol bathing method using three different concentrations; 3, 5 and 7 mg.l-1 for 24 hours in FW. Fish were then directly transferred from FW to CSW (11‰) and sampled after 1, 4 and 9 days post-transfer to CSW. Daily mortality rate, plasma osmolality and branchial chloride cell number and area were evaluated.The obtained results showed that fish weighing more than 3g are able to survive and could successfully acclimate to CSW. Despite initial plasma osmolality increase after salinity transfer, juveniles could reduce their plasma osmolality down to the CSW osmotic pressure after 15 days of acclimation in CSW. However, fish under 3 g could not survive abrupt salinity increase, and blood osmotic pressure of the remaining surviving fish increased and remained elevated during the whole experimental period. At the gill level, higher chloride cell size and number, with also higher NKA and NKCC content were observed allowing juveniles weighing more than 2 g to sharply increase their NKA activity after salinity transfer. The electron microscopic study revealed similar apical surfaces for branchial chloride cells in FW and CSW-acclimated fish. It also indicated that the cytoplasm of these cells in fish weighing more than 2 g were denser and inflated compared to fish weighing less than 2 g. This could be due to the elongation of the basal membrane infoldings (tubulo-vesicular network) and/or a higher density of mitochondria. Because sequences of NKA and NKCC1 genes were not reported for sturgeon fish, two partial sequences of NKA (632 bp) and NKCC1 (538 bp) were obtained from this present study and were registered in Genbank. Branchial NKA gene of CSW-acclimated fish was firstly upregulated and then downregulated to the level for FW fish after 4 days following salinity transfer. At the kidney and intestinal level, no difference could be detected between the different weight groups during salinity transfer. Despite lower plasma osmolality, number and size of the chloride cells in treated fish with cortisol show the same trends as the untreated control fish after 9-days post transfer. Consequently, this study revealed that only Persian sturgeon juveniles weighing more than 3 g can directly be released into the Caspian Sea. Fish weighting 2-3 g need special care during salinity transfer and fish weighing less than 2 g cannot tolerate Caspian Sea salinity even after cortisol treatment.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2017-03-30T11:58:43Z No. of bitstreams: 1 PDF - Jeneilson Alves da Silva.pdf: 20934775 bytes, checksum: 51881a33e60d3cdb9a1a77bc362d1e55 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2017-07-20T12:16:36Z (GMT) No. of bitstreams: 1 PDF - Jeneilson Alves da Silva.pdf: 20934775 bytes, checksum: 51881a33e60d3cdb9a1a77bc362d1e55 (MD5) / Made available in DSpace on 2017-07-20T12:16:36Z (GMT). No. of bitstreams: 1 PDF - Jeneilson Alves da Silva.pdf: 20934775 bytes, checksum: 51881a33e60d3cdb9a1a77bc362d1e55 (MD5) Previous issue date: 2017-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Brazil is one of the world's largest producers of papaya (Carica papaya L.). In the Northeast region, more than half of the national production is concentrated. Nevertheless, one of the main limiting factors of the productivity of this culture in this region is the irregularity of the rains. In this context, the objective of this study was to evaluate the brassinosteroid action under the growth characteristics and osmotic mechanisms in the papaya cultivated under irrigation slides. The experiment was conducted in an experimental field at the State University of Paraíba, Catolé do Rocha-PB municipality, using seeds of the cultivar Havaii. Were tested five irrigation slides (60, 80, 100, 120 and 140% of reference evapotranspiration) and three brassinosteroid concentrations (0, 0.5 and 1.0 mg L-1) using a randomized block design, In the 5 x 3 factorial scheme, with four replicates. At 120 days after emergence, the growth and biochemical characteristics were analyzed, being analyzed by F test until (p ≤ 0.05) and for the q uantitative factors polynomial regression analyzes were performed. It was observed that the brassinosteroid influenced the growth of papaya, mainly in the secondary meristem. It was observed that the brassinosteroid influenced the growth of papaya, mainly in the secondary meristem. The content of free amino acids was reduced and there was an increase in soluble protein content with increased irrigation and application of the hormone. Thus, papaya can be irrigated during the vegetative phase, adopting 80% of the reference evapotranspiration when treated with the dose of 0.5 mg L-1 of BR, a fact verified mainly by the analysis of the growth characteristics of stem diameter, leaf area, dry stem mass and root volume. As well as the increase of non-reducing carbohydrates, soluble proteins and the chlorophyll content. / O Brasil é um dos maiores produtores mundiais de mamão (Carica papaya L.). Na região Nordeste concentram-se mais da metade da produção nacional. Todavia, um dos principais fatores limitantes da produtividade dessa cultura nessa região é a irregularidade das chuvas. Nesse contexto, objetivou-se com esse estudo avaliar a ação do brassinosteróide diante das características de crescimento e mecanismos osmóticos no mamoeiro cultivado sob lâminas de irrigação. O experimento foi conduzido em campo experimental na Universidade Estadual da Paraíba, município de Catolé do Rocha-PB, utilizando sementes da cultivar Havaii. Foram testadas cinco lâminas de irrigação (60; 80; 100; 120 e 140% da evapotranspiração de referência) e três concentrações de brassinosteróide (0; 0,5 e 1,0 mg L-1), utilizando delineamento em blocos ao acaso, no esquema fatorial 5 x 3, com quatro repetições. Aos 120 dias após emergência foram analisadas as características de crescimento e bioquímicos, sendo submetidos à análise de variância pelo teste F, até (p ≤ 0,05) e para os fatores quantitativos foram realizadas análises de regressão polinomial. Foi constatado que o brassinosteróide influenciou no crescimento do mamoeiro, principalmente, nos meristemas secundários. O conteúdo de aminoácidos livres foi reduzido e houve um aumento no teor de proteínas solúveis com o incremento da irrigação e aplicação do hormônio. Assim, o mamoeiro pode ser irrigado durante a fase vegetativa, adotando 80% da evapotranspiração de referência, quando tratado com a dose de 0,5 mg L-1 de BR, fato esse verificado principalmente pela análise das características de crescimento do diâmetro de caule, área foliar, massa seca do caule e volume de raiz. Como também pelo aumento de carboidratos não-redutores, proteínas solúveis e o teor de clorofilas.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2017-11-09T17:40:18Z No. of bitstreams: 1 PDF - Edilene Daniel de Araújo.pdf: 24128593 bytes, checksum: d5e6c19239a3ef7fd6272bd5e58ab15b (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2017-11-17T18:19:59Z (GMT) No. of bitstreams: 1 PDF - Edilene Daniel de Araújo.pdf: 24128593 bytes, checksum: d5e6c19239a3ef7fd6272bd5e58ab15b (MD5) / Made available in DSpace on 2017-11-17T18:19:59Z (GMT). No. of bitstreams: 1 PDF - Edilene Daniel de Araújo.pdf: 24128593 bytes, checksum: d5e6c19239a3ef7fd6272bd5e58ab15b (MD5) Previous issue date: 2017-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Cowpea (Vigna unguiculata (L.) Walp.) is one of the most important food crops in the Northeastern semi-arid region for its high nutritional value. However, in the Northeast region, water scarcity is one of the factors that reduce its yield, with various consequences for its development and yield. This study aimed to evaluate the application of silicon (Si) as inductor of tolerance to water restriction in cowpea genotypes. The experiment consisted of a 5 x 2 x 3 factorial scheme, corresponding to five cowpea genotypes (‘BRS Marataoã’, ‘BR 17 Gurgueia’, ‘BRS Rouxinol’, ‘Paulistinha’ and ‘Costela de Vaca’) subjected to two water conditions (E 0- without water deficit/W-100% of field capacity and E1- with water deficit/W-50% of field capacity) and three Si doses (0; 49 and 70 mg L^-1 of Si), resulting in 30 treatments, arranged in randomized blocks, with five replicates. Agronomic (growth and production), physiological (leaf water potential and relative water content) and biochemical (superoxide dismutase, catalase, ascorbate peroxidase, free proline, total soluble proteins and total soluble carbohydrates) evaluations were performed. The obtained data were subjected to univariate and multivariate analyses, using the software Statistica 7.0. The evaluated genotypes showed increase in growth and development, when cultivated under water limitation of 50% of available water and treated with Si. Si reduced the leakage of electrolytes in ‘Paulistinha’ and ‘BRS Marataoã’. The antioxidant activity of the enzymes SOD, CAT and APX were potentiated by Si leaf application. The genotypes ‘BRS Marataoã’ withstood the water restriction better through the application of Si. / O feijão caupi (Vigna unguiculata (L.) Walp.) é uma das culturas alimentares mais importantes do semiárido nordestino por possuir alto valor nutritivo. Todavia, na região Nordeste, a escassez de água é um dos fatores que reduz a produtividade dessa cultura com diversas consequências para o seu desenvolvimento e produtividade. Objetivou-se avaliar a aplicação de silício (Si) como indutor de tolerância à restrição hídrica em genótipos de feijão-caupi. O experimento constou de um fatorial 5 x 2x 3, sendo cinco genótipos de feijão-caupi (BRS Marataoã, BR 17 Gurgueia, BRS Rouxinol, Paulistinha e Costela de Vaca) submetidas a duas condições hídricas (E0- sem déficit hídrico/W-100% da capacidade de campo e E1- com déficit hídrico/W-50% da capacidade de campo) e três aplicações de Si (0; 49 e 70 mg L^-1 de Si), resultando em 30 tratamentos, arranjados no delineamento em blocos casualizados, com cinco repetições. Realizou-se avaliações agronômicas (crescimento e produção), fisiológicas (potencial hídrico foliar e conteúdo relativo de água) e bioquímicas (superóxido dismutase, catalase, ascorbato peroxidase, prolina livre, proteínas solúveis totais e carboidratos solúveis totais). Os dados obtidos foram submetidos à análise univariada e multivariada, utilizando-se o software Statistica 7.0. Os genótipos avaliados expressaram aumento no crescimento e desenvolvimento, quando cultivados na limitação hídrica de 50% de água disponível e tratados com Si. O Si reduziu o extravasamento de eletrólitos em ‘Paulistinha’ e ‘BRS Marataoã’. A atividade antioxidante das enzimas SOD, CAT e APX foram potencializadas pela aplicação foliar de Si. O genótipo BRS Marataoã suportou melhor à restrição hídrica por meio da aplicação de Si.

A (Na+,K+)-ATPase presente no tecido branquial dos crustáceos osmorreguladores é um componente essencial de seu sistema de regulação iônica e osmótica. Esta enzima também apresenta um papel relevante no processo de excreção ativa de NH4+ através do tecido branquial dos crustáceos. Uma fração microsomal rica em (Na+, K+)ATPase foi preparada por centrifugação diferencial a partir de um homogeneizado do tecido branquial de Callinectes ornatus. A centrifugação em gradiente de sacarose revelou a presença de um unico pico de proteina com atividade ATPase, mas a eletroforese em gel de poliacrilamida em condições desnaturantes revelou a presença de várias bandas protéicas. O uso do anticorpo monoclonal 5 contra a subunidade da (Na+, K+) ATPase, revelou a presença de uma única banda proteica de 110 kDa com atividade (Na+, K+)?ATPase. A (Na+, K+) ATPase hidrolisou o PNPP (V= 52,0 ± 2,0 U/mg e K0,5 = 1,1 ± 0,1 mM) através de interações sítio-sítio (nH= 1,6). A modulação da enzima pelos íons magnésio (V= 52,3 ± 1,3 U/mg e K0,5 = 1,1 ± 0,05 mM), potássio (V= 51,4 ± 1,5 U/mg e K0,5 = 2,3 ± 0,1 mM) e amônio (V= 56,7 ± 2,6 U/mg e K0,5 = 9,8 ± 0,4 mM) ocorreu através de interações sítio-sítio. Os íons sódio atuaram como inibidores da atividade K+-fosfatase da enzima (Ki= 1,7 ± 0,1 mM) e a ouabaína inibiu cerca de 80% a atividade PNPPase independentemente da presença de íons amônio. A (Na+, K+) ATPase hidrolisou o ATP de acordo com cinética de Michelis-Menten, com KM= 0,16 0,01 mM e V= 116,3 5,6 U/mg, enquanto a modulação da atividade da enzima pelos íons magnésio (V= 111,0 ± 5,4 U/mg e K0,5= 0,54 ± 0,03 mM), sódio (V= 110,6 ± 5,3 U/mg e K0,5= 6,3 ± 0,3 mM), potássio (V= 116,0 ± 5,5 U/mg e K0,5= 1,5 ± 0,1 mM) e amônio (V= 173,3 ± 5,4 U/mg e K0,5= 5,4 ± 0,3 mM) ocorreram através de interações sítio-sítio. Também foi observado que na presença de concentrações crescentes de ions amonio, a estimulação da atividade (Na+,K+)-ATPase pelo íons potássio acarretou um aumento de 50% na atividade específica da enzima. A ouabaína inibiu cerca de 86% a atividade (Na+,K+)-ATPase com Ki= 74,5 ?M, sugerindo a presença de 14% de fosfatases e/ou outras ATPase contaminantes. Este é o primeiro trabalho onde se observa uma estimulação sinergística da atividade K-fosfatase da (Na+,K+)-ATPase de crustáceo pelos íons potássio e amônio. Os resultados cinéticos obtidos para a (Na+,K+)-ATPase branquial de Callinectes ornatus, analisados em conjunto com os já descritos para outras espécies de crustáceos poderão abrir novas perspectivas em relação ao papel dessa enzima na adaptação fisiológica-bioquímica, bem como para a sobrevivência desses animais em diferentes ambientes. / (Na+, K+)-ATPase present on branchial tissue osmoregulatory crustaceans is an essential component of their osmotic and ionic regulation system. Apparently, this enzyme also have a relevant role in the active excretion de NH4+ through the branchial crustacean tissue. A (Na+, K+) ATPase-rich microsomal fraction was prepared by differential centrifugation from Callinectes ornatus homogenized branchial tissue. The sucrose gradient sucrose centrifugation showed the presence of a single protein peak with ATPase activity, and SDS-PAGE revealed the presence of several proteins bands. The use of the 5 monoclonal antibody, against the ? subunit, revealed the presence of a unique protein band of 110 kDa corresponding to the (Na+, K+) ATPase. (Na+, K+) ATPase hydrolyzed the PNPP (V= 52.0 2.0 U/mg and K0.5= 1.1 0.1 mM) through the site-site interactions (nH= 1.6). The modulation of (Na+, K+) ATPase by magnesium (V= 52.3 1.3 U/mg and K0.5= 1.1 ? 0.05 mM), potassium (V= 51.4 1.5 U/mg and K0.5= 2.3 0.1 mM) and ammonia ions (V= 56.7 2.7 U/mg and K0.5= 9.8 ? 0.4 mM) followed cooperative kinetics. However, sodium ions inhibited PNPPase activity of (Na+, K+)?ATPase with Ki= 1.7 0.1 mM. Ouabain also inhibited up to 80% the total activity PNPPase independent of the presence of ammonium ions. The hydrolysis of ATP by (Na+, K+) ATPase followed Michaelis-Menten kinetics with Km= 0.16 0.01 mM and V= 116.3 5.6 U/mg, while enzyme modulation by magnesium (V= 111.0 5.4 U/mg and K0.5= 0.54 0.03 mM), sodium (V= 110.6 5.3 U/mg and K0.5= 6.3 0.3 mM), potassium (V= 116.0 5.5 U/mg and K0.5= 1.5 ? 0.1 mM) and ammonium ions (V= 173.3 . Interestingly, the stimulation of (Na+, K+)-ATPase activity by potassium ions in the presence of increasing concentration of ammonium ions to K+ resulted in a 50% higher specific activity. Ouabain inhibited approximately 86% the activity (Na+, K+) ATPase with (Ki= 74.5 M), suggesting the presence of about 14% of phosphatases and/or other ATPases. This is the first work showing synergistic stimulation of crustacean (Na+, K+) ATPase by potassium and ammonium ions when PNPP is used a substrate. The results reported herein for Callinectes ornatus branchial (Na+, K+) ATPase might open new perspectives concerning the physiological adaption and the survival of these animals in different environmental.

Les probiotiques sont des microorganismes vivants qui, ingérés en quantité suffisante, exercent des effets positifs sur la santé. La lyophilisation est aujourd’hui questionnée quant à sa consommation d’énergie et son caractère discontinu. S’il offre une alternative pour produire massivement des poudres probiotiques à faible coût, le séchage par atomisation induit quant à lui des stress thermiques et oxydatifs conduisant à des pertes de viabilité rédhibitoires.Dans ce travail, un procédé innovant de séchage par atomisation est proposé. Du lactosérum doux concentré (jusqu’à 30% p/p) est utilisé à la fois comme support de culture et de séchage de P. freudenreichii et L. casei. Ce procédé élimine les étapes intermédiaires à risque de contamination élevé, accroît la biomasse et améliore la viabilité des bactériesLes mécanismes sous-jacents ont été explorés au plan de la résistance bactérienne et des conditions de séchage. Le milieu concentré induit une osmoadaptation des bactéries par expression de protéines de stress et accumulation de solutés compatibles, conduisant à une tolérance accrue des probiotiques à différents stress. La présence d’agrégats et la concentration en Mg2+ du milieu concentré pourraient également être impliquées.Le scale-up du procédé a été étudié : un schéma technologique semi-industriel impliquant séchage par atomisation, sur bande et en lit fluidisé a permis d’atteindre une viabilité de 100% (> 109 CFU g-1). Par ailleurs, la fonctionnalité des poudres probiotiques a été évaluée in vitro and in vivo sur modèle porcelet. Ce travail ouvre de nouvell / Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Freeze drying, the reference drying method, is currently challenged because of its low energy-efficiency and productivity. Therefore, spray drying is expected to be an alternative and sustainable method for producing probiotic powders. The issue remains in the considerable inactivation of probiotics caused by high temperature and dehydration during the process. In this work, a novel spray-drying process for continuous production of probiotics was challenged. Concentrated sweet whey (up to 30% w/w dry matter) was used to both culture and spray dry P. freudenreichii ITG P20 and L. casei BL23. This process cut down the steps between culturing and drying (e.g. harvesting, washing), increases the cell population after growth and improves spray drying productivity and probiotic viability. The mechanisms were explored from bacterial physiology and drying process conditions. The hypertonic stress led to overexpression of key stress proteins and accumulation of intracellular compatible solutes, which enhanced multistress tolerance. The presence of protein aggregates and optimal concentration of Mg2+ in matrix may also be involved.The feasibility of scaling up this process was validated. A multi-stage semi industrial drying process, coupling spray-drying with belt drying and fluid-bed drying, was applied to further improve the probiotic viability to 100% (> 109 CFU g-1). Moreover, the functionality of these probiotic powders was investigated in vitro and in viv

Les crevettes Palaemonidae sont issues d'un clade ancestral marin, qui a montré une tendance évolutive remarquable à s'adapter à des conditions non-marines, envahissant avec succès les milieux estuariens et limniques. Adulte, Macrobrachium amazonicum (A) est une espèce d'eau douce (ED) avec une stratégie d'exportation vers les estuaires des larves qui ont besoin d'eau salée pour se développer. Des populations se sont trouvées au cours du temps isolées en ED ; elles ont récemment été décrites comme une nouvelle espèce, M. pantanalense (P), qui a acquis au cours de son évolution la capacité d'effectuer tout son cycle en ED, grâce à l'acquisition de l'hyper-osmorégulation en ED dès l'éclosion, et en perdant l'hypo-osmorégulation en eau salée. Ces deux espèces représentent un bon modèle pour la reconstruction des transitions évolutives des crevettes de l'eau salée à l'ED. L'objectif de ce travail est de comprendre les différences liées à l'adaptation physiologique et moléculaire à l'ED et donc à l'osmorégulation entre les deux espèces au cours de l'ontogénèse. Pour cela nous avons étudié l'ontogenèse comparative des organes osmorégulateurs, en particulier de la cavité branchiale, et la localisation et expression de différents transporteurs ioniques. Au niveau structural, aux stades larvaires, P a un développement branchial plus précoce que A. La Na+/K+ ATPase (NKA) a été essentiellement immunolocalisée au niveau des branchies chez P et au niveau des branchiostégites chez A aux mêmes stades larvaires. Ceci suggère que la forte capacité d'hypo-osmorégulation durant l'ontogenèse de A est liée aux transports ioniques dans les branchiostegites, alors que les lamelles branchiales ne sont pas complètement développées. Sur le plan ultrastructural, les lamelles branchiales des deux espèces comportent deux types de cellules associées, les cellules septales et les cellules piliers qui toutes deux présentent des caractéristiques d'ionocytes. Une différentiation ultrastructurale a été observée au niveau des cellules piliers et de l'épithélium interne des branchiostégites suite à une acclimatation en ED. Ces cellules présentent des microvillosités apicales, profondes et nombreuses, ce qui semble être une adaptation aux faibles salinités permettant une absorption efficace d'ions. Les transporteurs ioniques impliqués dans l'osmorégulation ont été étudiés. La V-H+ ATPase (VHA) a été détectée au niveau des cellules piliers et de l'épithélium interne des branchiostégites. La NKA et l'échangeur Na+/H+ (NHE-3) ont été localisés au niveau des cellules septales. Des différences d'expressions géniques de la VHA, du NHE-3 et de la NKA ont été mesurées en comparant les 2 espèces à certains stades de développement. La distribution différentielle de ces transporteurs entre les cellules piliers et septales suggère que ces deux cellules pourraient fonctionner comme un complexe cellulaire pour absorber ou sécréter des ions. Chez P, la capacité de tous les stades à hyper-réguler en ED peut provenir du développement précoce des branchies fonctionnelles, et la perte de l'hypo-régulation peut être liée au manque de transports ioniques au niveau des branchiostégites. Enfin, les glandes excrétrices antennaires produisent de l'urine hypotonique chez les juvéniles et adultes des deux espèces en ED, ce qui diminue les pertes ioniques. Ces résultats illustrent des adaptations évolutives (perte et gain de fonctions) qui ont permis l'invasion des habitats d'ED. / Palaemonid shrimps originate from an ancestral marine clade showing a remarkable evolutionary ability to adapt to non-marine conditions, successfully invading estuarine and limnic habitats. Macrobrachium amazonicum (A) is a freshwater (FW) species as an adult with an export strategy toward estuaries of larvae requiring salt water for their development. Over time, some populations ended up isolated in FW; recently, they have been described as a new species, M. pantanalense (P), which during its evolution has become able to complete its entire life cycle in FW, thanks to the acquisition of hyper-osmoregulation in FW from hatching, while loosing hypo-osmoregulation in salt water. The two species offer a valuable model to reconstruct the evolutionary transitions of shrimps from salt water to FW. The objective of this study was to decipher the differences in physiological and molecular adaptations to FW, thus in osmoregulation, between both species during ontogeny. We studied the comparative ontogeny of osmoregulatory organs, particularly the branchial chamber, and the localization and expression of ion transporters. During the larval phase, we found that the gill development starts earlier in P than in A. Na+/K+ ATPase (NKA) was mainly localized in gills of P and in branchiostegites of A at the same larval stages. This suggests that the high capacity to hypo-osmoregulate during the ontogeny of A originates from ionic transports in branchiostegites, while gill lamellae are not fully developed. In both species, the gill lamellae contain two associated cells types, septal and pillar cells, displaying features of ionocytes. After FW acclimation, ultrastructural differences were observed in pillar cells and in the inner epithelium of branchiostegites. These cells possess numerous deep apical microvilli, a possible adaptation to low salinities for efficient ion uptake. Regarding ion transporters involved in osmoregulation, V-H+ ATPase (VHA) was detected in pillar cells and in the inner branchiostegite epithelium. NKA and Na+/H+ exchanger (NHE-3) were localized in septal cells. Differences in VHA, NHE-3 and NKA gene expression were observed by comparing the two species at certain developmental stages. The differential distribution of these transporters between pillar and septal cells suggest that these two cells may function as a cell complex for ion absorption or secretion. In P, the capacity of all stages to hyper-regulate in FW may originate from the early development of functional gills; and the loss of hypo-regulation may originate from an absence of ion transport in branchiostegites. Finally, the excretory antennal glands produce hypotonic urine in juveniles and adults of both species in FW, thus reducing ion loss. These results illustrate evolutionary adaptations (gain and loss of functions) that have permitted the invasion of FW habitats.

Renal function of fresh water acclimated Glaucous-winged Gulls, Larus qlaucescens, was studied during infusion of hypotonic and hypertonic NaCl. Two experimental protocols were followed. In one, the closed urine collection system (CCS), ureteral urine was collected using catheters glued over ureteral openings of a supine, previously anesthetized gull. In the other, the open urine collection system (OCS), ureteral urine was collected through a funnel placed in the urodeum of a standing, unanesthetized bird. In both protocols, there was continuous saline infusion of hypotonic (hydration) and hypertonic (LOAD) saline at 0.286 ml⋅min⁻¹. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), ml(kg⋅min) ⁻¹, were determined by ¹⁴C-polyethylene glycol (PEG) and ³H-para-aminohippuric acid (PAH) clearances. Plasma vasotocin (PAVT, pg⋅ml⁻¹) was measured. At the end of 4 h hydration with 0.02 M NaCl, urine flow was high but matched infusion rate only in CCS birds (CCS, 0.29 ± 0.05; OCS, 0.17 ± 0.03 ml⋅min⁻¹), GFR (CCS, 5.56 ± 0.85; OCS, 5.36 ± 0.77) and ERPF (CCS, 15.80 ± 1.60; OCS, 14.35 ± 1.65) were high; urine sodium (UNa+) concentration was low (CCS, 15.0 ± 7.3; OCS, 36.4 ± 6.0 mEq⋅1⁻¹), UNa+ excretion was low (CCS, 6.38 ± 4.2; OCS, 5.19 uEq⋅min⁻¹) ; urine/plasma PEG ratio (U/PPEG) was high (CCS, 22.4 ± 4.4, OCS, 39.6 ± 8.5); free water clearance (CH₂O) was positive (CCS, 0.143 ± 0.011; OCS, 0.052 ± 0.019 ml⋅min⁻¹) , and PAVT was low (ccs,14.7 ± 7.4; OCS, 16.1 ± 2.4) in both groups. Immediately following infusion of 5 M NaCl, GFR, ERPF and urine flow increased for about 10 mins. Fifteen minutes later, the GFR of CCS gulls fell to 70% of pre-load values (P < 0.05) and in OCS gulls, GFR and ERPF fell to 64% (P < 0.01) and 61% (P < 0.05). Eighty mins after infusion of 5 M NaCl, the GFR and ERPF of CCS gulls returned to pre-LOAD levels, but remained low in OCS gulls. Twenty-five minutes after salt load, urine flow had fallen to 49% (P < 0.05) and remained low. In OCS gulls, urine flow had fallen to 13% (P < 0.001) after 185 mins. In both CCS and OCS gulls, UNa+ concentration and excretion increased significantly. Sixty minutes after salt load, UNa+ excretion returned to pre-LOAD levels but UNa+ concentration remained high in CCS (111.7 ± 57.5) and OCS (132.8 ± 12.5) gulls. U/PPEG attained 134.3 ± 26.5 in CCS and 181.2 ± 32.4 in OCS gulls. CH₂O fell significantly (P < 0.05) in CCS gulls but remained unchanged in OCS gulls. Mean PAVT increased to 122.5 ± 5.5 in CCS and 96.0 ± 12.6 in OCS gulls. In both CCS and OCS gulls, salt gland secretion was initiated but ceased 60 mins after 5M NaCl infusion, although 60% of the load was retained in the gull. / Science, Faculty of / Zoology, Department of / Graduate

Chapter 1: Animals can respond to extreme climate by behaviorally avoiding it, or by physiologically coping with it. We understand behavioral thermoregulation and physiological thermal tolerances, but water balance has largely been neglected. Climate change includes both global warming and changes in precipitation regimes, so improving our understanding of organismal water balance is increasingly urgent. We assessed the hydric physiology of endangered Blunt-nosed Leopard Lizards (Gambelia sila) by measuring cutaneous evaporative water loss (CEWL), plasma osmolality, body mass, and body condition throughout their active season. On average, G. sila had low CEWL that is likely desert-adaptive, and high plasma osmolality that is indicative of dehydration. Given that our study was in a drought year, it is reasonable to believe that every lizard measured was dehydrated to a degree. We hypothesized that throughout the G. sila active season, as their habitat got hotter and drier, G. sila would become increasingly dehydrated and watertight. Instead, CEWL and plasma osmolality showed minimal change for females and nonlinear change for males, which we hypothesize is connected to sex-specific reproductive behaviors and changes in food availability. We also measured thermoregulation and microhabitat use, expecting that more hydrated lizards would have higher body temperature, better thermoregulatory accuracy, and spend more time aboveground. However, we found no effect of CEWL, plasma osmolality, body mass, or body condition on these thermal and behavioral metrics. We posit either that G. sila tolerate dehydration to maintain activity during their brief active season, or that because every lizard was dehydrated due to the drought, they all experienced equally constrained thermoregulation and microhabitat use. Finally, G. sila spend considerable time underground in burrows, and we believe burrows serve as essential hydric, not only thermal, refugia. Our findings suggest that these lizards might benefit from artificial humid refugia and supplemental hydration, especially during drought. Chapter 2: Testing acclimation plasticity informs our understanding of functional biodiversity and applies to conservation management amidst our rapidly changing climate. While there is a wealth of research on the plasticity of thermal and hydric physiology in response to temperature acclimation, there is a comparative gap for research on acclimation to different hydric regimes, as well as the interaction between water and temperature. We sought to fill this gap by acclimating Western Fence Lizards (Sceloporus occidentalis) to experimental climate conditions (crossed design of Hot or Cool, Dry or Humid) for eight days, and measuring cutaneous evaporative water loss (CEWL), plasma osmolality, hematocrit, and body condition before and after acclimation under common conditions. CEWL changed plastically in response to the different climates, with lizards acclimated to Hot Humid conditions experiencing the greatest increase in CEWL. Change in CEWL among individuals was negatively related to treatment vapor pressure deficit. Plasma osmolality, hematocrit, and body condition all showed greater changes in response to temperature than to humidity or vapor pressure deficit. CEWL and plasma osmolality were positively related across treatment groups before acclimation and within treatment groups after acclimation, but the two variables showed different responses to acclimation, suggesting that they are interrelated but governed by different mechanisms. This study is among just a small number of studies that assess more than one metric of hydric physiology and that test the interactive effects of temperature and humidity. Such measurements will be essential for predictive models of activity and survival for animals under climate change.

In seawater, drinking is a fundamental part of the osmoregulatory strategy for teleost fish, and presents a unique challenge. The intestine has an established role in osmoregulation, and its ability to effectively absorb fluid from imbibed seawater is crucial to compensating for water losses to the surrounding hyperosmotic environment. Alongside solute-linked water transport (driven by NaCl cotransport), intestinal bicarbonate (HCO3-) secretion also benefits fluid absorption directly (via apical Cl-/HCO3- exchange), and indirectly through the formation of calcium carbonate (CaCO3) thus removing the osmotic influence of Ca2+ within the gut fluid. For the European flounder (Platichthys flesus), elevated luminal Ca2+ has proven to be a specific, potent stimulator of HCO3- secretion both in vitro and in vivo where these actions are presumably modulated by an extracellular Ca2+-sensing receptor (CaR). The focus of this work was to learn more about how intestinal HCO3- secretion is regulated, the role of Ca2+, and more specifically the CaR. To achieve this, in vitro ‘gut sac’ experiments investigated how luminal Ca2+ influenced HCO3- secretion, and associated ion and fluid transport. Contrary to expectation, increasing Ca2+ from 5 to 20 mM did not stimulate HCO3- secretion. In an attempt to elucidate the role of CaCO3 precipitation in fluid absorption, and further explore the physiological implications of HCO3- secretion, the intestine was perfused in vivo with salines containing varying concentrations of Ca2+ (10, 40 and 90 mM). The production and secretion of HCO3-, in addition to CaCO3 formation increased accordingly with Ca2+, and was associated with a dramatic 25 % rise in the fraction of fluid absorbed by the gut. Additional in vitro experiments, utilising the Ussing chamber, helped establish some of the characteristics of intestinal HCO3- secretion by the euryhaline killifish (Fundulus heteroclitus), but was unresponsive to elevated mucosal Ca2+. Further attempts to potentiate the activity of the CaR, and application of the receptor agonists gadolinium (Gd3+) and neomycin, failed to produce responses consistent with the effect of Ca2+ observed previously, either in vitro or in vivo. With no evidence supporting a direct role for an extracellular, intestinal CaR in HCO3- secretion it was argued that secretion would be principally regulated by two factors, the ability of the epithelia to generate high levels of intracellular HCO3- and the rate of CaCO3 formation.