Nous nous sommes intéressés à la compréhension des mécanismes régissant la symbiose Rhizobium-Acacia dans les conditions de stress salin. Les lipopolysaccharides jouent un rôle important dans les étapes de cette symbiose. Le but était de mettre en évidence les modifications pariétales de la bactérie en réponse au stress salin par l’étude de la structure des lipopolysaccharides des souches isolées du désert marocain tolérant NaCl 7%. Ainsi, une nouvelle méthode d’hydrolyse des lipopolysaccharides sensible, non destructive et compatible avec la spectrométrie de masse a été développée. En présence de stress salin, nous avons montré que la membrane externe devenait plus hydrophobe en augmentant l’acylation de la région lipidique ainsi qu’en réduisant la présence des molécules de LPSs à longues chaînes de sucres.Des essais d’évaluation de l’efficience et de l’infectivité des Rhizobia étudiés ont été mis en œuvre pour déterminer l’impact de ces modifications des LPSs sur la symbiose sous stress salin. / We were interested in the understanding of the mechanisms governing Rhizobium-Acacia symbiosis in salt stress conditions. Lipopolysaccharides play an important role in the stages of this symbiosis. The aim of this work was to highlight the changes occurring in the bacterial membrane in response to salt stress by studying the structure of the lipopolysaccharides isolated from Moroccan desert strains tolerating 7% NaCl. Thus, a new method of hydrolysis of the lipopolysaccharide - sensitive, non-destructive and compatible with mass spectrometry- was developed. We studied the LPSs strains grown with or without salt stress and we showed that in salt stress conditions, the outer membrane becomes more hydrophobic by increasing acylation of the lipid region and reducing the number of long sugar chains in LPSs. Tests for evaluating the efficiency and infectivity of the studied rhizobia were carried out to determine the impact of these LPS modifications on symbiosis under salt stress.

Os manguezais são ambientes ricos em biodiversidade, cuja funcionalidade reside na ciclagem dos nutrientes e seu principal representante vegetal é Rhizophora mangle. Este estudo objetivou conhecer a diversidade bacteriana endoglicolítica e a tolerância à salinidade de rizobactérias associadas à R. mangle. Das amostras de plantas do manguezal de Bertioga (contaminado com petróleo) e Cananéia (não impactado) isolou-se 129 bactérias, das quais 30 apresentaram atividade endoglicolítica, com Bacillus subtilis isolado 39a como melhor produtor. A presença do gene EglA foi confirmada por amplificação com primers específicos. As linhagens testadas para salinidade mostraram-se halotolerantes, com destaque para o 39a, que cresceu em NaCl 20%. A microscopia eletrônica pós-cultivo em diferentes salinidades mostrou produção de biofilme em concentrações altas. Os resultados indicam que a preservação do ecossistema cria um ambiente bacteriano mais diverso e mostra Bacillus spp. como principal produtor de endoglicanase, além de responder ao stress salino formando biofilme. / Mangroves are environments so rich in biodiversity which functionality made by nutrient cycling. The main vegetable specie is Rhizophora mangle. This study objected to know bacterial endoglucolytic diversity and tolerance saline of rhizobacteria associated to R. mangle. Plants from Bertioga (oil contaminated) and Cananéia (not polluted) were sampled. From both sites, 129 bacteria were isolated, which most diversity observed from Cananéia. These isolates, 30 presented endoglucolytic activity and Bacillus subtilis (strain 39a) was characterized as top producer. The presence of EglA gene was confirmed using specific primers. The salinity test showed halotolerance, mainly strain 39a that growth untill about 20% NaCl. The scan electron microscopy of strains allowed biofilm production at elevated salinity that suggest the biofilm as tolerance mechanism to saline environment. The results indicated that ecosystem preservation makes a most diversity bacterial environment and that Bacillus spp. are main endoglucanase producer and response to saline stress producing biofilm.

Hui Fong Fong Liza. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 130-182). / Abstracts in English and Chinese. / Chapter I --- Title page --- p.I / Chapter II --- Thesis committee --- p.II / Chapter III --- Acknowledgements --- p.III / Chapter IV --- Abstract (Chinese version) --- p.IV / Chapter V --- Abstract (English version) --- p.VII / Chapter VI --- Table of contents --- p.X / Chapter VII --- List of figures --- p.XIV / Chapter VIII --- List of table --- p.XVIII / Chapter Chapter 1: --- General introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.5 / Chapter 2.1. --- Gill --- p.5 / Chapter 2.2. --- Chloride cells (Mitochondria-rich cells) --- p.6 / Chapter 2.2.1. --- Ion extrusion by fish in seawater --- p.9 / Chapter 2.2.2. --- Ion uptake by fish in hypo-osmotic condition --- p.12 / Chapter 2.3. --- Sparus sarba (Silver seabream) --- p.14 / Chapter 2.4. --- Sodium-potassium adenosinetriphosphatase (Na+-K+-ATPase) --- p.15 / Chapter 2.4.1. --- Na+-K+-ATPase α-subunit --- p.17 / Chapter 2.4.2. --- Na+-K+-ATPase β-subunit --- p.18 / Chapter 2.4.3. --- Regulation of Na+-K+-ATPase --- p.20 / Chapter 2.5. --- Hormones --- p.21 / Chapter 2.5.1. --- Growth hormone-prolactin family --- p.21 / Chapter 2.5.2. --- Structure of hormones --- p.22 / Chapter 2.5.2.1. --- Structure of growth hormone and prolactin in fish --- p.22 / Chapter 2.5.2.2. --- Structure of insulin-like growth factors in fish --- p.26 / Chapter 2.5.2.3. --- Structure of Cortisol in fish --- p.27 / Chapter 2.5.3. --- Regulation of hormones --- p.28 / Chapter 2.5.3.1. --- Regulation of growth hormone in fish --- p.28 / Chapter 2.5.3.2. --- Regulation of prolactin in fish --- p.32 / Chapter 2.5.3.3. --- Regulation of insulin-like growth factor-I in fish --- p.33 / Chapter 2.5.3.4. --- Regulation of Cortisol in fish --- p.33 / Chapter 2.5.4. --- Functions of hormones --- p.33 / Chapter 2.5.4.1. --- Functions of growth hormone in fish --- p.33 / Chapter 2.5.4.2. --- Functions of prolactin in fish --- p.39 / Chapter 2.5.4.3. --- Functions of insulin-like growth factor-I in fish --- p.44 / Chapter 2.5.4.4. --- Functions of Cortisol in fish --- p.45 / Chapter 2.5.4.5. --- "Combined effects of GH, IGF-I, PRL and Cortisol" --- p.49 / Chapter 2.6. --- Salinity effects on Na+-K+-ATPase expression --- p.52 / Chapter Chapter 3: --- In vitro effect of hormones on branchial Na+-K+- ATPase expression in marine teleost Sparus sarba --- p.58 / Chapter 3.1. --- Abstract --- p.58 / Chapter 3.2. --- Introduction --- p.60 / Chapter 3.3. --- Materials and methods --- p.62 / Chapter 3.3.1. --- Overall experimental design --- p.62 / Chapter 3.3.2. --- Fish preparation --- p.62 / Chapter 3.3.3. --- Tissue sampling --- p.62 / Chapter 3.3.4. --- RNA extraction and dot blot analysis --- p.63 / Chapter 3.3.5. --- Protein extraction --- p.65 / Chapter 3.3.6. --- Protein quantification --- p.65 / Chapter 3.3.7. --- Na+-K+-ATPase activity --- p.65 / Chapter 3.3.8. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.66 / Chapter 3.3.9. --- Statistical analysis --- p.67 / Chapter 3.4. --- Results --- p.69 / Chapter 3.4.1. --- Dot blot analysis of Na+-K+-ATPase mRNA subunits --- p.69 / Chapter 3.4.2. --- Analysis of Na+-K+-ATPase protein α-subunit --- p.81 / Chapter 3.4.3. --- Analysis of Na+-K+-ATPase activity --- p.87 / Chapter 3.5. --- Discussion --- p.92 / Chapter 3.5.1. --- Effects of rbGH and rbIGF-I on Na+-K+-ATPase expression --- p.92 / Chapter 3.5.2. --- Effects of oPRL on Na+-K+-ATPase expression --- p.102 / Chapter 3.5.3 --- Effects of Cortisol on Na+-K+-ATPase expression --- p.104 / Chapter 3.6. --- Conclusion --- p.108 / Chapter Chapter 4: --- In vivo effect of salinity on branchial Na+-K+-ATPase expression in marine teleost Sparus sarba --- p.109 / Chapter 4.1. --- Abstract --- p.109 / Chapter 4.2. --- Introduction --- p.110 / Chapter 4.3. --- Materials and methods --- p.112 / Chapter 4.3.1. --- Overall experimental design --- p.112 / Chapter 4.3.2. --- Fish preparation --- p.112 / Chapter 4.3.3. --- Tissue sampling --- p.113 / Chapter 4.3.4. --- "RNA extraction, dot blot analysis, protein extraction, quantification, Na+-K+-ATPase activity, protein gel electrophoresis and immunoblotting (Western blotting)" --- p.113 / Chapter 4.3.5. --- Statistical analysis --- p.114 / Chapter 4.4. --- Results --- p.114 / Chapter 4.4.1. --- Dot blot analysis of Na+-K+-ATPase mRNA subunits --- p.114 / Chapter 4.4.2. --- Analysis of Na+-K+-ATPase protein a-subunit --- p.114 / Chapter 4.4.3. --- Analysis of Na+-K+-ATPase activity --- p.115 / Chapter 4.5. --- Discussion --- p.120 / Chapter 4.6. --- Conclusion --- p.125 / Chapter Chapter 5: --- General discussion and conclusion --- p.126 / References --- p.130

Fung Chun Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 117-139). / Abstracts in English and Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of figures --- p.vi / List of tables --- p.viii / List of abbreviations --- p.ix / Chapter Chapter I --- General introduction --- p.1 / Chapter 1.1 --- Different fish habitats with various salinities --- p.1 / Chapter 1.2 --- Osmotic challenges faced by teleosts --- p.2 / Chapter 1.3 --- High ionic strength results in DNA damage and excess water gain --- p.3 / Chapter 1.4 --- Osmoregulatory organs and mechanisms for osmotic balance --- p.4 / Chapter 1.5 --- Different tolerance to salinity changes --- p.8 / Chapter 1.6 --- Effective communication among osmoregulatory organs --- p.9 / Chapter 1.7 --- Introduction to GH and PRL --- p.9 / Chapter 1.8 --- Structure of the GHR and PRLR --- p.10 / Chapter 1.9 --- Hypoosmoregulatory action of GH/IGF-I axis in teleosts --- p.11 / Chapter 1.10 --- Hyperosmoregulatory action of PRL in teleosts --- p.11 / Chapter Chapter II --- Research rationale --- p.13 / Chapter 2.1 --- Physiological importance of osmoregulation in fish --- p.13 / Chapter 2.1.1 --- Energy metabolism --- p.13 / Chapter 2.1.2 --- Growth --- p.14 / Chapter 2.1.3 --- Immunity --- p.14 / Chapter 2.1.4 --- Reproduction --- p.15 / Chapter 2.2 --- Aquaculture importance --- p.15 / Chapter 2.3 --- Unknown molecular regulatory mechanism of hormones during salinity changes in fish --- p.16 / Chapter 2.4 --- Animal model --- p.17 / Chapter Chapter III --- In vivo studies of sbGHR and sbPRLR expression in osmoregulatory organs in response to salinity changes --- p.18 / Chapter 3.1 --- Introduction --- p.18 / Chapter 3.1.1 --- Dynamic change of GH level during salinity changes --- p.18 / Chapter 3.1.2 --- Dynamic change of PRL level during salinity changes --- p.19 / Chapter 3.1.3 --- In vitro studies of GH and PRL release from teleost pituitary in response to extracellular osmolality changes --- p.20 / Chapter 3.1.4 --- Biological actions of GH and PRL through the GHR and PRLR --- p.21 / Chapter 3.2 --- Materials and methods --- p.23 / Chapter 3.3 --- Results --- p.28 / Chapter 3.4 --- Discussion --- p.36 / Chapter 3.4.1 --- Plasma osmolality change during salinity changes --- p.36 / Chapter 3.4.2 --- Gene expression after HSW exposure --- p.38 / Chapter 3.4.3 --- Ionic mediators of the gene expression --- p.43 / Chapter 3.4.4 --- Gene expression after BW exposure --- p.44 / Chapter 3.4.5 --- Dynamic changes of the GHR and PRLR in response to salinity changes --- p.45 / Chapter 3.4.6 --- Regulation of the gene expression in response to salinity changes --- p.46 / Chapter Chapter IV --- Gene expression of sbGHR in liver during salinity changes --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.1.1 --- Responses of the somatotropic axis to salinity changes in fish --- p.49 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.3 --- Results --- p.56 / Chapter 4.4 --- Discussion --- p.60 / Chapter 4.4.1 --- Inhibition of GHR and IGF-I gene expression in liver during HSW exposure --- p.60 / Chapter 4.4.2 --- Downregulation of GHR gene expression by hyperosmotic stress --- p.62 / Chapter 4.4.3 --- Growth retardation of fish during hyperosmotic environment --- p.64 / Chapter Chapter V --- Gene expression studies of sbPRLR in gill organ culture --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.1.1 --- Functions of gill in fish osmoregulation --- p.68 / Chapter 5.1.2 --- Gill culture as a model for osmoregulation studies --- p.69 / Chapter 5.2 --- Materials and methods --- p.70 / Chapter 5.3 --- Results --- p.71 / Chapter 5.4 --- Discussion --- p.73 / Chapter Chapter VI --- Regulation of gene expression of sbGHR in liver during hyperosmotic stress: promoter studies --- p.75 / Chapter 6.1 --- Introduction --- p.75 / Chapter 6.1.1 --- What is a promoter? --- p.75 / Chapter 6.1.2 --- Promoter studies of GHR gene --- p.76 / Chapter 6.2 --- Materials and methods --- p.78 / Chapter 6.3 --- Results --- p.85 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter VII --- General discussion and future perspectives --- p.111 / References --- p.117

All these findings can help us to elucidate the mechanisms for the fish to detect changing osmotic conditions and transform signals to osmoregulatory responses. / In the first part of the study, PRL and PRL-releasing peptide (PrRP) cDNAs have been isolated from euryhaline silver sea bream. The PRL cDNA consists of 1360 bp encoding 212 amino acids whereas the PrRP cDNA contains 631 bp encoding prepro-PrRP with 122 amino acids. PRL mRNA was uniquely expressed in sea bream pituitary but PrRP mRNA was expressed in a variety of tissues. Expression levels of both PRL and PrRP mRNA have been examined in sea bream adapted to different salinities (0, 6, 12, 33 and 50 ppt). In pituitary, both PRL and PrRP mRNA were synchronized in their expression, being significantly higher in fish adapted to low salinities (0 and 6 ppt), but the expression profile of hypothalamic PrRP in different salinities was different. These data suggested that PrRP may possibly act as a local modulator in pituitary rather than a hypothalamic factor for regulating pituitary PRL expression in silver sea bream. / In the second part of the study, silver sea bream abruptly transferred from 33 to 6 ppt exhibited a remarkable pituitary PRL secretion following closely with the temporal changes in serum osmolality and ion levels. In order to investigate the direct effect of extracellular osmolality to pituitary PRL secretion, sea bream pituitary cells were dispersed and exposed to a medium with reduced ion levels and osmolality. PRL released from pituitary cells was found to be significantly elevated. In hyposmotic exposed anterior pituitary cells, cell volume exhibited a 20% increase when exposed to a medium with a 20% decrease in osmolality. These enlarged pituitary cells did not shrink until the surrounding hyposmotic medium was replaced, a phenomenon suggesting an osmosensing ability of silver sea bream PRL cells for PRL secretion in response to a change in extracellular osmotic pressure. / In the third part, olfactory rosette in the nasal cavity was surgically removed from silver sea bream adapted to 6 ppt and 33 ppt and mRNA expression of PRL and PrRP in silver sea bream were measured. The elevated pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish were abolished by this olfactory lamellectomy. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change but those in 33 ppt-adapted fish increase significantly after olfactory lamellectomy. These data suggest a possible osmosensing role of the olfactory system for regulation of PRL expression during hypo-osmotic acclimation of the fish. Besides, calcium-sensing receptor (CaSR) was cloned and its mRNA expression in olfactory system, as shown in other fish species previously, was investigated. However, no CaSR expression could be detected in olfactory rosette and nerve but its expression was demonstrated in osmoregulatory tissues and brain. There was no significant difference in CaSR mRNA expression in pituitary, kidney and anterior intestine of fish adapted to different salinities. These studies could not provide conclusive evidence to correlate CaSR with osmosensing in silver sea bream. / The present study used silver sea bream (Sparus sarba ) as a euryhaline fish model to investigate the regulation of prolactin (PRL) expression and secretion in fish adapted to different salinities. / Kwong, Ka Yee. / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3248. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 154-184). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Branchial carbonic anhydrase was purified from silver seabream (Sparus sarba) and antibody against the enzyme was obtained by immunization in rabbits. An assay for quantifying the activity of carbonic anhydrase was developed. Using enzymatic and immunological techniques, the activity, expression and distribution of branchial carbonic anhydrase of silver seabream acclimated to different salinities were studied. Fish gill is one of the most important organs involved in various homeostatic processes. The ability of euryhaline fish to maintain constant internal ionic balance is crucial for the survival of the fish upon change in salinity. The presence of carbonic anhydrase in the chloride cells was suggested to be an important enzyme involved in ion regulation of fish. / In the present study, branchial carbonic anhydrase and erythrocyte carbonic anhydrase were purified from the gill cells of silver seabream with p-aminomethylbenzenesulfonamide-agarose affinity column. They were predominantly cytosolic with a molecular size of 26.6 k Da for branchial carbonic anhydrase and 28.6 k Da for erythrocyte carbonic anhydrase. Investigation of kinetic properties towards the inhibitor acetazolamide has helped determine the inhibition constants (Ki of branchial carbonic anhydrase: 0.54 x 10-9; Ki of erythrocyte carbonic anhydrase: 0.22 x 10-9). The difference in molecular size and inhibition constant towards acetazolamide supported the view that branchial carbonic anhydrase and erythrocyte carbonic anhydrase were two different isozymes. Polyclonal antibody specific to seabream branchial carbonic anhydrase was obtained by immunization in rabbit. The distribution of branchial carbonic anhydrase in the gill of seabream acclimated to different salinities was studied with immunohistochemical method. The enzyme was mainly located at the interlamellar region. The effect of salinity (0, 6, 12, 33, 50 and 70 ‰) acclimation on the expression and activities of branchial carbonic anhydrase has shown a U-shape pattern from freshwater to double-strength seawater on the quantity of seabream branchial carbonic anhydrase. Higher amount of branchial carbonic anhydrase in freshwater was consistent with the current view that the enzyme was actively involved in the ion uptake process through the hydration of carbon dioxide to produce bicarbonate ion and proton for the exchange of chloride and sodium ions, respectively. An interesting finding was obtained with elevated amount of branchial carbonic anhydrase in seabream acclimated to double-strength seawater and the possible role of the enzyme in such extreme environment was discussed. / This study has provided useful information on the properties, localizations and activities of branchial carbonic anhydrase in silver seabream for the understanding of the involvement of the enzyme in salinity adaptation of silver seabream. / Ma, Wing Chi Joyce. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3250. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 127-151). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In the second part of our study, cDNA of AQP-1 and pro-vasotocin were cloned from the silver sea bream. An AQP-1 full clone was isolated from kidney and intestine and it consists of 904 bp with an open reading frame of 774 bp. The deduced amino sequence of sea bream AQP-1 shares highest identity with AQP-1a of gilthead sea bream (97.7%) and AQP-1a of other fish species (83.6% to 95.8%), however, considerably low identity was found between the silver sea bream AQP-1 and AQP-1b of gilthead sea bream (56%). The silver sea bream AQP-1 possesses basic features of a functional aquaporin and AQP-1, which includes two channel-forming asparagine-proline-alanine (NPA) signature motifs, six transmembrane domains, residues of the pore-forming region and a potential mercurial inhibiting site (Cys-178). The water channel was ubiquitously expressed in gills, liver, intestine, rectum, kidney, heart, urinary bladder and blood cells. A partial fragment of pro-vasotocin was isolated from hypothalamus of silver sea bream and consists of 184 bp, including encoding regions for the processing and amidation signal, vasotocin hormone and part of the neurophysin. / Lastly, single doses of cortisol (50 microg/g tissue) or vasotocin (1 microg/g tissue) were administered to seawater-acclimated sea bream with further three-day stabilizing period in seawater followed by an abrupt 6‰ exposure or administered to seawater transfer controls for three days. Cortisol markedly stimulated intestinal expression of AQP-1 in both the seawater transfer control and abrupt 6‰ transfer groups. Vasotocin treatment did not significantly modify AQP-1 expression in all tested organs. Hypothalamic pro-vasotocin expression levels were similar among different treatment groups. / Semi-quantitative RT-PCR analysis was used for studying the effect of salinity and hormones on expression of AQP-1 and pro-vasotocin. In the long-term salinity acclimation experiment, the sea bream were acclimated to six different salinity regimes (0‰, 6‰, 12‰, 33‰, 50‰, 70‰) for four weeks. The abundance of AQP-1 transcript was the highest in intestine of 70‰-acclimated fish among different salinity groups and there was also a statistically significant increase in 12‰-acclimated fish. Branchial AQP-1 expression was significantly upregulated in sea bream acclimated to freshwater. In contrast, the hypothalamic pro-vasotocin expression was significantly downregulated during freshwater acclimation. In addition, the sea bream were also subjected to an abrupt 6%o transfer at different time intervals (2, 6, 12, 72 and 168 hours). RT-PCR analysis revealed there was a transient decrease in branchial AQP-1 expression two hours after abrupt hypo-osmotic exposure and the expression levels subsequently returned to the seawater control levels. The expression levels of hypothalamic pro-vasotocin were not significantly altered by the abrupt exposure treatment. / The present experiments investigated the effects of salinity and hormones on the relative expression of hypothalamic pro-vasotocin, and aquaporin-1 (AQP-1) in intestine, gills and kidney of the silver sea bream Sparus sarba. With the use of immunohistochemical techniques, immunoreactivity of AQP-1 was detected at the basal side of enterocytes and gill chloride cells, and at the apical brush border of kidney tubules whereas AQP-3 was only localized in similar positions in the gills and intestines. AQP-1 was relatively more ubiquitous than AQP-3 and was localized with same cell types as the electrogenic Na+-K+-ATPase in gills and kidney. / The present study had demonstrated the responsiveness of intestinal and branchial AQP-1 expressions of the silver sea bream to environmental salinity perturbations. Further to this, cortisol was observed to upregulate the transcription of AQP-1 in the intestine. Pro-vasotocin expression was altered by long-term salinity adaptation, however, the linkage of this alteration to AQP-1 functioning in different osmoregulatory organs is yet to be elucidated. / Luk, Chun Yin. / Adviser: Norman Y. S. Woo. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 200-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Wong Kwok-Shing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 89-107). / Abstracts in English and Chinese. / Title --- p.i / Abstract (English) --- p.ii / Abstract (Chinese) --- p.v / Content --- p.vii / Acknowledgement --- p.x / Abbreviation --- p.xii / Lists of tables and figures --- p.xiii / Chapter Chapter 1 --- General introduction --- p.1 / Chapter Chapter 2 --- "Effects of salinity on the cardiovascular responses and dipsogenic behaviors and silver seabream, Sparus sarba." / Chapter 2.1 --- Literature review / Chapter 2.1.1 --- Teleost euryhalinity --- p.5 / Chapter 2.1.2 --- Salinity and blood respiratory properties --- p.7 / Chapter 2.1.3 --- Salinity and blood volume --- p.8 / Chapter 2.1.4 --- Salinity and blood pressure --- p.10 / Chapter 2.1.5 --- Intestine physiology --- p.12 / Chapter 2.1.6 --- Summary --- p.14 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Experimental animals --- p.19 / Chapter 2.2.2 --- Salinity adaptation --- p.19 / Chapter 2.2.3 --- Drinking rate measurement --- p.19 / Chapter 2.2.4 --- Respiratory characteristics --- p.20 / Chapter 2.2.5 --- Blood volume measurement --- p.21 / Chapter 2.2.6 --- Blood pressure experiment --- p.23 / Chapter 2.2.7 --- Statistical analysis --- p.23 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Drinking rate --- p.25 / Chapter 2.3.2 --- Oxygen dissociation curves --- p.27 / Chapter 2.3.3 --- Blood volume --- p.29 / Chapter 2.3.4 --- Blood pressure --- p.31 / Chapter 2.4 --- Discussion / Chapter 2.4.1 --- Drinking rate --- p.36 / Chapter 2.4.2 --- Oxygen dissociation curves --- p.37 / Chapter 2.4.3 --- Blood volume --- p.38 / Chapter 2.4.4 --- Blood pressure --- p.40 / Chapter Chapter 3 --- "Manipulation of renin-angiotensin system in relation to the cardiovascular responses and dipsogenic behaviors of silver seabream, Sparus sarba." / Chapter 3.1 --- Literature review / Chapter 3.1.1 --- Renin angiotensin system (RAS) --- p.41 / Chapter 3.1.2 --- RAS and blood pressure --- p.47 / Chapter 3.1.3 --- RAS and drinking --- p.53 / Chapter 3.1.4 --- RAS and Cortisol --- p.55 / Chapter 3.1.5 --- RAS and kidney --- p.58 / Chapter 3.1.6 --- Summary --- p.58 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Experimental animals --- p.61 / Chapter 3.2.2 --- Salinity adaptation --- p.61 / Chapter 3.2.3 --- Drinking rate measurement --- p.61 / Chapter 3.2.4 --- Determination of angiotensin converting enzyme (ACE) activity --- p.61 / Chapter 3.2.5 --- Blood pressure experiment --- p.62 / Chapter 3.2.6 --- Statistical analysis --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Drinking rate --- p.64 / Chapter 3.3.2 --- ACE activity --- p.69 / Chapter 3.3.3 --- Blood pressure --- p.71 / Chapter 3.4 --- Discussion / Chapter 3.4.1 --- Drinking rate --- p.77 / Chapter 3.4.2 --- ACE activity --- p.81 / Chapter 3.4.3 --- Blood pressure --- p.83 / Chapter Chapter 4 --- General conclusion --- p.86 / Reference --- p.89

Submitted by (ana.araujo@ufrpe.br) on 2017-02-10T14:18:44Z No. of bitstreams: 1 Gabriela de Morais Guerra Ferraz.pdf: 1062151 bytes, checksum: b0123f5d7101341f981de975826dc25b (MD5) / Made available in DSpace on 2017-02-10T14:18:44Z (GMT). No. of bitstreams: 1 Gabriela de Morais Guerra Ferraz.pdf: 1062151 bytes, checksum: b0123f5d7101341f981de975826dc25b (MD5) Previous issue date: 2008-02-29 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The Northeast of Brazil, major national producer of bananas, presents as a major limiting factor the stretch of saline soil. The urgency in the development of tolerant cultivars to salinity has led to breeding programs with the aimed to differ the bananas cultivars in diploid genotypes tolerant and sensitive, to be considered viable genetic material for pollination of triploid and tetraploid cultivars. Generally, the present study aimed to characterize diploid genotypes belonging to the AA banana genomic group on salinity and identify genes differentially expressed in contrasting genotypes. At first, this research assessed the growth and accumulation of inorganic ions in 19 diploid genotypes (AA) of bananas subjected to salt stress. The genotypes were grown in a greenhouse and submitted to irrigation with no saline water (0 mM NaCl) or with saline water (100 mM NaCl) for a period of 21 days, when the experiment was collected. To the physiological evaluation, were considered the parameters of growth:leaf area, height, number of leaves, diameter of the pseudostem, weight of the tax burden of fresh and dry, while the chemical assessment observed the concentration of sodium ions, potassium, chloride, magnesium and calcium in limbo leaf, and root pseudostem / subroot. The addition of NaCl to the cultivar solution has, in general, reduced the growth expressed by height, formation of new leaves, leaf area, diameter of the pseudostem and production of fresh and dry materials. This reduction in growth probably due to factors such as: the toxic effect of ions that have been absorbed, the low osmotic and water potential of the cells as well as the use of metabolic energy in the process of osmotic adjustment. In assessing chemical factors, it was possible to observe that the ions concentration has been preferentially presented in root tissue, showing that it is a culture moderately tolerant to salinity.The sodium and chlorine ions increased significantly with the salt increase in differentparts of the plant, while potassium suffered reduction in the leaf lamina and the pseudostem probably because it is associated to the competition with the sodium ion leading to the conclusion that the differential accumulation of ions potentially toxic (sodium and chlorine) and the maintenance of potassium, contribute to the tolerance to salinity in banana. From the physiological analysis and by means of the toxicity symptoms caused by NaCl, it was possible to observe that the effects were less intense in Birmania and Khai Nai On genotypes, than in Sowmuk, its indicates the possibility of use of this plants in programmes for cultivars improvement as tolerant and sensitive, respectively. Seven genotypes were selected for characterization by means of molecular markers RAPD, with the aim to link the physiological responses to salt stress with the formation of groups. We tested 16 random primers. The resultsshow a broad molecular genetic variability among seven genotypes studied. The formation of groups, in part is related to the data obtained in the physiological assessment, keeping in the same group the Birmania and Khai Nai On genotypes.These genotypes that showed higher tolerance to salt stress, when compared to the more salt-sensitive genotype (Showmuk), became distant genetically, so, it can be noticed the possibility of use of this molecular marker in the study of genetic diversity for this species. For the genome functional study, or transcriptoma, this study aimed the detection of possible changes in the pattern of genes expressed in the three diploid genotypes (AA) of banana, Khai Nai On, Burma and Sowmuk with contrasting results when in the absence and presence of high salt concentrations. The Differential Display PCR - ddPCR technique was used to identify and compareregions of bands from fragments of cDNA on agarose gel. A total of 43 fragments of differentially expressed cDNA was generated from the combination of four primers anchors and six random primers. Among the transcripts, 30 were once expressed by the genotype Burma and Kha Nai On, and 13 only by Sowmuk genotype. Theregions of bands with greater consistency of formation, is between 4000 bp and 150 bp. By the results of this research, it was possible to identify some fragments potentially involved to the condition of salinity in banana. The isolation, purification and sequencing of these - Expressed sequence tags - ESTs can assist in the development of new varieties of banana, more adapted to salt stress, in addition to enriching the database of public functional sequences of genomes banks. / O Nordeste do Brasil, maior produtor nacional de banana, apresenta como fator limitante a grande extensão de solos salinos. A urgência no desenvolvimento de cultivares tolerantes a salinidade tem levado programas de melhoramento genético da cultura a classificar os genótipos diplóides de bananeira em tolerantes e sensíveis, por serem considerados materiais genéticos viáveis para polinização de cultivares triplóides e tetraplóides. De modo geral, o presente trabalho buscou caracterizar genótipos diplóides pertencentes ao grupo genômico AA de bananeira quanto a salinidade além de identificar genes diferencialmente expressos nos genótipos contrastantes. Na primeira fase desta pesquisa, foi avaliado o crescimento vegetativo e o acúmulo de íons inorgânicos em 19 genótipos diplóides (AA) de bananeiras submetidas a estresse salino. Os genótipos foram cultivados em casa de vegetação e submetidos à irrigação com água não salina (0 mM de NaCl) ou águasalina (100 mM de NaCl) durante um período de 21 dias, quando foi coletado o experimento. Para a avaliação fisiológica, foram considerados os parâmetros de crescimento: área foliar, altura, nº de folhas, diâmetro do pseudocaule, peso da matéria fresca e peso da matéria seca; enquanto que a avaliação química observou a concentração dos íons sódio, potássio, cloro, magnésio e cálcio no limbo foliar, pseudocaule e raiz/rizoma. A adição de NaCl à solução de cultivo, provocou, de modo geral, redução no crescimento expresso pela altura, formação de novas folhas, área foliar, diâmetro do pseudocaule e produção de matéria fresca e seca, provavelmente devido a fatores como: o efeito tóxico dos íons que foram absorvidos; o baixo potencial osmótico e hídrico das células; bem como a utilização de energia metabólica no processo de ajustamento osmótico. Na avaliação química, foi possívelobservar que a concentração dos íons foi preferencialmente no tecido radicular, reafirmando tratar-se de uma cultura moderadamente tolerante a salinidade. Os íonssódio e cloro aumentaram significativamente frente ao incremento salino nas diferentes partes da planta, enquanto o potássio sofreu redução no limbo foliar e no pseudocaule, possivelmente por estar associado a competição com o íon sódio levando a conclusão de que o acúmulo diferencial de íons potencialmente tóxicos (sódio e cloro) e a manutenção do potássio, contribuem para a tolerância à salinidade em bananeira. A partir da análise fisiológica e por meio da sintomatologia de toxidez provocada pelo NaCl, foi possível observar que os efeitos foram menos intensos nos genótipos Birmânia e Khai Nai On, do que no Sowmuk, indicando possíveis plantas a serem utilizadas nos programas de melhoramento da cultura como tolerantes e sensível, respectivamente. Sete genótipos foram selecionados para caracterização por meio de marcadores moleculares RAPD, buscando-se relacionar as respostas fisiológicas ao estresse salino com a formação de grupos.Foram testados 16 primers randômicos. Os resultados moleculares mostram uma ampla variabilidade genética entre os sete genótipos estudados. A formação dos agrupamentos, em parte correspondeu aos dados obtidos na avaliação fisiológica, mantendo em um mesmo grupo os genótipos Birmânia e Khai Nai On. Estes genótipos que apresentaram maior tolerância ao estresse salino, quando comparados com a mais sensível ao sal (Showmuk), mostraram-se distantes geneticamente, o que vem a demonstrar a possibilidade de utilização deste marcador molecular no estudo da diversidade genética para esta espécie. Para o estudo do genoma funcional, ou transcriptoma, o presente trabalho objetivou a detecção das possíveis alterações no padrão de genes expressos nos três genótipos diplóides (AA) de bananeira, Khai Nai On, Birmânia e Sowmuk, com respostascontrastantes quando na ausência e presença de alta concentração salina. A técnica de Differential Display PCR – ddPCR foi utilizada para identificar e comparar regiões de bandas de fragmentos de cDNA em gel de agarose. Um total de 43 fragmentosde cDNA diferencialmente expressos foram gerados a partir da combinação de quatro primers âncoras e seis primers aleatórios. Dentre os transcritos, 30 foram expressos unicamente pelos genótipos Birmânia e Kha Nai On, e 13 apenas pelo genótipo Sowmuk. As regiões de bandas com maior consistência de formação, encontraram-se entre 4000 pb e 150 pb. Pelos resultados deste trabalho, foi possível identificar alguns fragmentos potencialmente envolvidos em respostas à condição de salinidade em bananeira. O isolamento, purificação e sequenciamento destes –Expressed sequence tags - ESTs poderá auxiliar no desenvolvimento de novos cultivares de bananeira, mais adaptados ao estresse salino, além de enriquecer a base de dados de bancos públicos de seqüências de genomas funcionais.

Submitted by (ana.araujo@ufrpe.br) on 2017-02-21T15:30:56Z No. of bitstreams: 1 Roberta Lane de Oliveira Silva.pdf: 816508 bytes, checksum: 8ee58f1e26cefa223321e756c9e49f07 (MD5) / Made available in DSpace on 2017-02-21T15:30:56Z (GMT). No. of bitstreams: 1 Roberta Lane de Oliveira Silva.pdf: 816508 bytes, checksum: 8ee58f1e26cefa223321e756c9e49f07 (MD5) Previous issue date: 2008-02-21 / The salinity is a common factor of abiotic stress that seriously affects the agricultural production. Currently, over 800 million hectares worldwide are affected by salinity. One of the strategies to promote reincorporation of salinity areas and the productivity increase consists in development and selection of tolerant genotypes, which allows parental identification for crossings. The diploid (AA) of bananas Germplasm Bank's Active of the National Center for Research of Cassava and Fruticulture (CNPMF/Embrapa), sources of interest genes to improvement programmes, still are not characterized for their salinity tolerance. This research aimed to identify the salinity tolerance among nine banana diploid genotypes and characterize them genetically through ISSR and RAPD markers, to obtain cultivars adapted to saline soils. In physiological assessing, the growth variables analyzed were leaf area, plant height, diameterof pseudostem, leaves number, weight of fresh and dry matter. Twenty RAPD primers and twenty ISSR primers were used in molecular evaluation. The consensus dendrogram of similarity genetic analyses grouped the genotypes Monyet with Borneo, Buitenzorg with Tjau Lagada and 0323-03 with 0116-01, in ISSR and RAPD dendrogramas. The 0116-01 genotype showed greater salinity tolerance and could be used in future improvement programs. The 0337-02 genotype, presenting minor reduction in leaf area, number of leaves and fresh/dry biomass of limbo variables, was considered the most tolerant to salinity stress, while the Borneo genotype was the most sensitive on salt presence. / A salinidade é um fator comum de estresse abiótico que afeta a produção agrícola mundial. Atualmente, cerca de 800 milhões de hectares no mundo são afetados pela salinidade. Uma das estratégias para promover a reincorporarão de áreas salinizadas e o aumento da produtividade consiste no desenvolvimento e na seleção de genótipos tolerantes, o que permitirá a identificação de parentais a serem utilizados em cruzamentos. Os diplóides (AA) de bananeiras do Banco Ativo de Germoplasma do Centro Nacional de Pesquisa de Mandioca e Fruticultura – CNPMF/Embrapa, que são fontes de genes de interesse para os programas de melhoramento, não estão caracterizados, ainda, quanto à sua tolerância à salinidade. Esta pesquisa teve como objetivo identificar dentre nove genótipos diplóides de bananeiras,aqueles tolerantes a salinidade e caracterizá-los geneticamente, através de marcadores ISSR e RAPD, visando à obtenção de cultivares adaptados a solos salinos da região nordeste brasileira. Na avaliação fisiológica, as variáveis de crescimento analisadas foram área foliar, altura da planta, diâmetro do pseudocaule, número de folhas, peso da matéria fresca e o peso da matéria seca. Na avaliação molecular foram testados 20 primers RAPD e 20 primers ISSR. O dendograma consenso das análises de similaridades genéticas obtidas a partir dos marcadores ISSR e RAPD agruparam em mesmo subgrupos os genótipos Monyet e Borneo, Buitenzorg e Tjau Lagada e também 0323-03 e 0116-01, os quais se repetiram nos dendogramas de ISSR e RAPD. O genótipo 0116-01 apresentou maior tolerância à salinidade e poderá serutilizado em futuros programas de melhoramento. Considerando o conjunto das variáveis analisadas, o genótipo 0116-01 foi considerado o mais tolerantes dosnove genótipos diplóides AA avaliados no primeiro trabalho. No segundo trabalho, o genótipo 0337-02 por apresentar menor redução nas variáveis área foliar, número de folhas e fitomassa fresca e seca do limbo, foi considerado tolerante ao estresse salino, enquanto o genótipo Borneo foi o mais sensível na presença do sal.