Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Aqüicultura, Instituto de Oceanografia, 2008. / Submitted by Cristiane Silva (cristiane_gomides@hotmail.com) on 2012-07-10T01:17:13Z No. of bitstreams: 1 cristina.pdf: 271225 bytes, checksum: 628c0fc9b0dce68e19ce8e7d60297c5e (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-11-06T17:04:55Z (GMT) No. of bitstreams: 1 cristina.pdf: 271225 bytes, checksum: 628c0fc9b0dce68e19ce8e7d60297c5e (MD5) / Made available in DSpace on 2012-11-06T17:04:55Z (GMT). No. of bitstreams: 1 cristina.pdf: 271225 bytes, checksum: 628c0fc9b0dce68e19ce8e7d60297c5e (MD5) Previous issue date: 2008 / A alimentação é um dos principais custos da piscicultura, sendo importante desenvolver estudos que busquem uma maior eficiência alimentar para o aumento do sucesso da atividade e também para a redução do impacto da emissão de nutritientes ao meio ambiente. Levando em conta o potencial de criação da tainha Mugil platanus na região Sudeste e Sul do Brasil e a carência de informações sobre suas exigências nutricionais, o presente trabalho foi realizado com o objetivo de determinar a exigência protéica para seus juvenis. As tainhas foram alimentadas com cinco dietas com três repetições cada, sendo cada unidade composta por um tanque de 50L com 50 juvenis com peso inicial 1,17 ± 0,02 g e 4,34 ± 0,03 cm (média ± EP). As cinco dietas isocalóricas foram formuladas para conter níveis crescentes de proteína bruta (PB) de 30% , 35%, 40%, 45% e 49% e 18,7 MJ/Kg de dieta (energia metabolizável). As dietas foram oferecidas até a saciedade 5 vezes ao dia durante 35 dias. As dietas não apresentaram diferenças significativas (P>0,05) para sobrevivência, eficiência alimentar e composição corporal.Os resultados indicaram que o nível de 35% PB foi statisticamente superior (P > 0,05) com relação ao ganho em peso, ingestão de alimento e taxa de crescimento específico do que de tainhas alimentas com o maior nível protéico. A necessidade de proteína para os juvenis de tainha foi estimada em 35,8% PB. / Feed is one of the main costs for fish culture. Studies looking for higher feed efficiency are important to increase the success of aquaculture and reduce impacts of nutrient emission into the environment. Considering the potential of the mullet Mugil platanus for aquaculture, as well as the lack of information on its nutritional demands, the main goal of the present work was to determine the dietary protein requirement of juvenile mullets. Five isocaloric diets were formulated in order to contain increasing levels (30, 35, 40, 45,and 50%) of crude protein (CP) corresponding to 18.7 MJ metabolizable energy/Kg. All diets were tested in triplicate. Each experimental unit was composed of a 50 L tank with 50 juveniles (mean ± SE initial weight and length equal to 1.17 ± 0.02 g and 4.34 ± 0.03cm, respectively). Diets were offered five times a day until apparent satiation for 35 days. No significant difference (P > 0.05) was observed in survival rate, feed efficiency and body composition between treatments. However, weight increase, feed ingestion and specific growth rate was higher in fish fed the 35% CP level than those fed the highest protein content diet (50% CP). The amount of postprandial ammonia excreted by mullet was linearly related to protein intake. Intestinal tryptic activity was inversely proportional to the percentage of dietary CP. The dietary protein requirement of juvenile mullet was estimated as 34.28% CP with a P:E ratio of 18.7 g/MJ.

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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.

Glutamine synthetase (GS) is a key enzyme in nitrogen (N) assimilation, especially during seed development. This thesis has identified three cytosolic GS isoforms (HvGS1) in barley (Hordeum vulgare L. cv Golden Promise). The quantitation of gene expression, isoform localisation and response to N supply has revealed that each gene plays a non-redundant role in different tissues throughout seedling development. The localisation of HvGS1_1 in vascular cells of different tissues, combined with its abundance in the stem and its response to changes in N supply, indicate that HvGS1_1 is important in N transport and remobilisation. HvGS1_1 is located on chromosome 6H at 72.54 cM, close to the marker HVM074 which is associated with a major quantitative trait locus (QTL) for grain protein content (GPC). HvGS1_1 may be a potential candidate gene to manipulate barley GPC. HvGS1_2 mRNA was localised to the leaf mesophyll cells, in both the cortex and the pericycle of roots and was the dominant HvGS1 isoform in these tissues. HvGS1_2 expression increased in the leaves with an increasing supply of N, suggesting that its role is in the primary assimilation of N. HvGS1_3 was specifically and predominantly localised in the grain, being highly expressed throughout grain development. HvGS1_3 expression increased specifically in the roots of plants grown on high NH₄⁺ suggesting that it has a primary role in grain N assimilation and also in the protection from ammonium toxicity in roots. The expression of the HvGS1 genes is directly correlated with both protein and enzymatic activity, indicating that transcriptional regulation is of prime importance in the control of GS activity in barley. Analysis of 15 different barley cultivars found no correlation between HvGS expression and various desirable attributes. Transgenics which over-express and silence individual HvGS1 isoforms have been produced and confirmed, to analyse for changes in beneficial traits.

Nanoflagellates were found to be abundant in a coastal area of the northern Bothnian Sea. The maximum concentration of nanoflagellates, approximately 8000 cells ml-1, was observed in July, coinciding with a decrease in the abundance of cyanobacteria. Pigmented and non-pigmented nanoflagellates were approximately equally distributed throughout the year. Most of the identified genera are known as being phagotrophic, independent if autotrophic or not. A non-cyst-forming pigmented flagellate, Ochromonas sp., was isolated and nutritionally characterized. This chrysophycean flagellate was shown to be a mainly heterotrophic organism: Photosynthesis was too poor to support multiplication of the cells, whereas when feeding on bacteria, high growth rates were obtained. The biological function of the photosynthetic apparatus is suggested to be a survival mechanism during poor bacterial conditions. The flagellate grazed bacteria selectively, preferring cyanobacteria and large cells of heterotrophic bacteria, presumably depending on size-selective grazing. Despite higher growth rates of the bacteria in the sea during summer (July) than spring (May), heterotrophic bacteria in the sea was observed to be smaller in the summer. Nanoflagellates showed a maximum in July, and by selective grazing of large bacteria they might have caused the decrease in the average size of the bacteria and the decrease in the abundance of cyanobacteria. During the consumption of bacteria the flagellate was shown to remineralize nutrients at high rates and excrete dissolved free amino acids. Assuming the existence of a protozoan predator-prey chain of several trophic levels, it seems likely that a significant part of the nutrients fixed by primary producers is remineralized in the euphotic zone. Furthermore, data from this work indicate that flagellate activity may be a significant source of dissolved free amino acids, utilizable for the heterotrophic bacteria. / digitalisering@umu

In contrast to most teleost fish that are ammoniotelic, the gulf toadfish (Opsanus beta) is both facultatively ureogenic and ureotelic. In vivo pharmacological manipulations were used to show that lowering circulating cortisol levels or blocking glucocorticoid receptors (GR) enhanced both urea excretion and urea pulse size. These findings demonstrated that changes in pulsatile urea excretion in the toadfish are mediated by the permissive action of cortisol through GRs. Measurement of urea transport across isolated basolateral gill membranes revealed a cortisol-sensitive carrier mechanism. Cortisol infusion in vivo significantly reduced urea transport capacity, suggesting that cortisol inhibits the recruitment of urea transport proteins (UT) to the basolateral membrane to ultimately decrease the size of the urea pulse in toadfish. A 1.2 kb fragment of the upstream transcription start site for the toadfish urea transporter (tUT) gene was isolated and in silico analysis revealed the presence of several putative glucocorticoid response element (GRE) half sites. Toadfish provided with this regulatory sequence in a reporter gene construct showed increased reporter gene transcription driven by cortisol. The data indicated that cortisol-mediated upregulation of tUT mRNA by GREs may be necessary to maintain tUT activity. Four Rhesus (Rh) glycoproteins (Rhag, Rhbg, Rhcg1, Rhcg2) were isolated from toadfish; these sequences grouped with those of other vertebrates coding for membrane channels that transport ammonia. In vivo increases in circulating cortisol reduced branchial Rh glycoprotein expression and decreased ammonia excretion. These changes were accompanied by cortisol-induced increases in glutamine synthetase activity, an enzyme that captures ammonia for urea synthesis. Taken together, the data indicated that cortisol reduces the loss by branchial excretion of ammonia, instead favouring biochemical pathways that convert ammonia to urea. This thesis confirms that nitrogen excretion in toadfish is controlled and regulated in fashions unlike those in other teleosts. The results demonstrate the importance of the GR signaling pathway in mediating changes in both urea and ammonia transport through molecular mechanisms. As a whole, the data provide a new understanding of branchial nitrogen excretion in the gulf toadfish and enhance our evolutionary perspective of the integrated biological systems involved in nitrogen excretion in fish.

The purpose of this study is to determine the amount of crude fiber in the diet and the laxation rate of college women consuming self-selected diets.

Five normal healthy young university women while on self-chosen diets and living under normal conditions served as experimental subjects in this study. Urinary pregnanediol was measured for 10 days of the menstrual cycle. Meal frequency ( 3 meals vs. 2 meals with no breakfast) was also included in the study. A modification of the method of Eberlein and Bongiovanni on thin layer chromatography was used to determine pregnanediol in the urine sample. Urine pregnanediol excretion was in the pattern as investigated by other workers. The values rose in the latter half of the menstrual cycle, and fell prior to the onset of the next period. No relationship was found between pregnanediol level and meal frequency. A larger number of subjects are recommended in the further work of this problem.

(1→3)-β-D-Glucans are carbohydrate polymers that are present in the cell wall of various fungi and bacteria; they are pathogen associated molecular patterns that circulate during infection and modulate immunity. Our laboratory has previously established the pharmacokinetics of intravenously and orally administered glucans; the present studies investigated the renal excretion of (1→3)-β-D-glucans following intravenous and oral administration. Three fluorescently-labeled glucans were administered to adult male rats in the presence or absence of toxic challenge. Urine specimens were collected and analyzed by fluorescence spectroscopy, size-exclusion chromatography and GPC/MALLS. 71 ± 3% of fluorescence remained in the >5K MWCO fraction; this fraction showed a minor peak with a molecular mass (171 ± 11K) corresponding to injected glucan (~150K). Most excreted glucans were of lower molecular mass (13 ± 8.5K), indicating most (1→3)-β-D-glucans are excreted by the kidneys as smaller polysaccharides. The presence of urinary glucans may be an important indicator of fungal infection.