Dois experimentos foram conduzidos com o objetivo de avaliar o efeito da inclusão de butirato de sódio, monensina sódica ou propionato de cálcio no concentrado inicial, sobre o desempenho, parâmetros sanguíneos e desenvolvimento ruminal de bezerros leiteiros. No primeiro experimento, 24 bezerras recém-nascidas da raça Holandesa foram alojadas em abrigos individuais até a 10a semana de vida, com livre acesso à água, sendo alimentadas com 4 litros de leite/dia e concentrado ad libitum, enquanto feno de capim-coast-cross foi fornecido após o desaleitamento. Os animais foram distribuídos em blocos de acordo com peso ao nascer e data de nascimento e alocados em um dos tratamentos, de acordo com o aditivo no concentrado: 1)Butirato de sódio (0,15%); 2)Monensina sódica (30 ppm); e 3)Propionato de cálcio (0,15%). Os animais foram pesados e avaliados quanto à altura na cernelha, largura do traseiro e perímetro torácico semanalmente. A partir da 4a semana foram realizadas colheitas semanais de amostras de sangue para determinação de glicose, ácidos graxos livres (AGL) e ?-hidroxibutirato (BHBA). Não foram observadas diferenças significativas entre os tratamentos para o consumo de concentrado ou de feno e para o peso e ganho de peso dos animais (P>0,05). As avaliações quanto à altura na cernelha e perímetro torácico também não apresentaram diferenças entre os tratamentos (P>0,05), entretanto as medidas de largura de traseiro foram menores para os animais do tratamento com adição de propionato de cálcio (P<0,05). As concentrações plasmáticas de glicose, AGL e BHBA não foram afetadas pelos tratamentos (P>0,05). Houve efeito significativo da idade (P<0,0001) para a concentração plasmática de glicose, sendo esta reduzida com a idade dos animais. No segundo experimento, 15 bezerros recémnascidos da raça Holandesa, recebendo o mesmo manejo nutricional, foram fistulados no rúmen e alojados em baias individuais até a 10ª semana de vida. A partir da 4ª semana, foram realizadas colheitas semanais de fluído ruminal para determinação de pH, ácidos graxos de cadeia curta (AGCC) e N-amoniacal; e de sangue para determinação de glicose. Ao completar dez semanas os animais foram abatidos para avaliação do desenvolvimento do trato digestório superior e de papilas ruminais. Não foram observadas diferenças significativas entre tratamentos para o consumo de concentrado e para o desempenho dos animais (P>0,05). Houve efeito significativo (P<0,05) de tratamento e horário de colheita para o pH ruminal. As concentrações de AGCC totais, bem como de cada ácido, não foram afetadas pelos tratamentos. As concentrações plasmáticas de glicose foram afetadas pelos tratamentos (P<0,05). O peso total do trato digestório superior, os pesos médios de cada compartimento e a capacidade máxima do retículo-rúmen não foram afetados pelos tratamentos, assim como os parâmetros de desenvolvimento do epitélio ruminal. Os aditivos incluídos no concentrado inicial se mostraram igualmente eficazes no que diz respeito aos seus efeitos no desempenho e desenvolvimento ruminal de bezerros em aleitamento. / Two trials were conducted in order to evaluate the effects of the addition of sodium butyrate, sodium monensin or calcium propionate in the starter feed on the performance, blood parameters and ruminal development of dairy calves. In the first experiment, 24 newborn Holstein calves were housed in individual hutches during ten weeks of life, with free access to water, being fed 4 liters of milk per day and starter ad libitum, with coast-cross hay offered only after weaning. The animals were blocked according to weight and date of birth and allocated in one of the treatments, according to the additive included in the starter feed: 1) sodium butyrate (0.15%); 2) sodium monensin (30 ppm); and 3) calcium propionate (0.15%). Animals were weighed and evaluated for whiter height, hearth girth and hip width weekly. From the fourth week of age blood samples were taken for glucose, free fatty acids and ?-hydroxybutyrate concentration determination. No significant differences were observed among treatments for starter or hay intake, and weight gain or live weight (P>0.05). Measurements of whiter height and hearth girth were also not affected by treatments (P> 0.05); however, measures of hip width of the animals were smaller for treatment with addition of calcium propionate (P<0.05). Plasma concentrations of glucose, free fatty acids and ?-hydroxybutyrate were not affected by treatment (P>0.05). There was significant effect of age (P<0.0001) for the plasma concentration of glucose, with reduction as animals aged. In the second experiment, 15 male newborns Holstein calves, receiving the same nutritional management, were ruminally fistulated and housed in individual pens during ten weeks of life. From the fourth week of age ruminal samples were taken weekly for the determination of pH, short-chain fatty acids (SCFA) and ammonia-N concentration. Blood samples were also taken weekly for glucose determination. By completing ten weeks of age, animals were slaughtered for forestomach growth and papillae development evaluation. No significant differences were observed among treatments for starter intake as well as for animal performance (P>0.05). The ruminal pH was significantly affected (P<0.05) by treatments and by sampling time. Concentrations of total SCFA and individual SCFA were not affected by treatments (P<0.05). Plasma concentrations of glucose were affected by treatments (P<0.05). The total forestomach weight, the average weight of each compartment and the maximum capacity of reticulum-rumen were not affected by treatments, as well as the parameters for ruminal epithelium development. The additives included in the starter feed were equally effective as regard to its effects on animal performance and rumen development of milk-fed dairy calves.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-09-04T17:22:48Z No. of bitstreams: 2 Luis Antonio - Dissertação mestrado - versão biblioteca.pdf: 706561 bytes, checksum: ba004353ceb320d9ea4f9c0ef0b105bd (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-04T17:22:48Z (GMT). No. of bitstreams: 2 Luis Antonio - Dissertação mestrado - versão biblioteca.pdf: 706561 bytes, checksum: ba004353ceb320d9ea4f9c0ef0b105bd (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-03-18 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Clobetasol propionate is one of the most potent topical corticosteroids usually available and has been established the use in treatment of various skin pathology. However its systemic absorption may cause serious side effects. The use of lipids nanocarriers and application of iontophoretic current shown potential to further controlled release of clobetasol propionate and increase their retention in the stratum corneum, thus making its management more effective and safe. In this study an analytical method was developed and validated for quantification of clobetasol propionate in nanocarriers and pig skin, used as model membranes in vitro permeation studies. Nanostructured lipid carriers (NLC) were obtained by dilution of microemulsion and chitosan-coated NLC (NLC-Ch) by the dripping of NLC in chitosan solution. The developed nanocarriers were characterized to mean diameter, polydispersity index, zeta potential, drug loading, recovery and encapsulation efficiency. The in vitro release and permeation profile of clobetasol free and nanoencapsulated was evaluated using Franz cells. The nanocariers obtained of 125 nm and uniform size distribution, whit PdI 0,25. The increase in size of NLC-Ch and the inverting the value of zeta potential, suggest that there was a coating of the CLN by chitosan, though interaction of charges. The NLC and NLC-Ch showed recovery values greater than 80% and encapsulation efficiency than 90%. The release profile of clobetasol from the developed formulations showed a controlled release with 35% in 24 hours of study. The nanoenapsulation of clobetasol in the NLC-Ch and NLC resulted in retention increased in the stratum corneum of 9.2 an 7.8 fold, respectively, compared to free drug. The retention of the clobetasol in the stratum corneum was 4 and 2 times higher when electric current was applied to the formulation CLNQ, compared to the free drug and encapsulated in NLC, respectively. The accumulation of the drug in the stratum corneum, promoted by lipid carriers, shows that these systems are effective for potential topical delivery to minimize the permeation of the drug into the bloodstream and reduces the side effects. / O propionato de clobetasol é um dos mais potentes corticosteroides tópicos disponíveis atualmente e tem uso consagrado no tratamento de diferentes patologias cutâneas. Contudo, a sua absorção sistêmica pode causar graves efeitos adversos. O uso de nanocarreadores lipídicos e a aplicação de corrente iontoforética apresentam potencial para promover liberação controlada do propionato de clobetasol e aumentar a sua retenção no estrato córneo, tornando assim sua administração mais segura e efetiva. No presente trabalho, um método cromatográfico foi desenvolvido e validado para quantificação do propionato de clobetasol nos nanocarreadores e na pele de porco, utilizada como modelo de membrana nos estudos de permeação in vitro. Carreadores lipídicos nanoestruturados (CLN) foram obtidos pelo método da diluição de microemulsão e CLN revestidos com quitosana (CLN-Q) foram obtidos pelo gotejamento dos CLN em solução de quitosana. Os nanocarreadores desenvolvidos foram caracterizados quanto ao diâmetro médio, índice de polidispersividade, potencial zeta, carga de fármaco, recuperação e eficiência de encapsulação. O perfil de liberação e de permeação in vitro do clobetasol livre e nanoencapsulado foi avaliado utilizando células de Franz. Os CLN obtidos apresentaram tamanho de 125 nm e distribuição de tamanho uniforme, com PdI de 0,25. O aumento no tamanho médio dos CLN-Q, para 257 nm, e a inversão no valor de potencial zeta sugerem que houve revestimento dos CLN pela quitosana, por meio de interação de cargas. Os CLN e CLN-Q apresentaram valores de recuperação maiores que 80% e de eficiência de encapsulação superiores a 90%. O perfil de liberação do clobetasol a partir das formulações desenvolvidas mostrou uma liberação controlada, com cerca de 35% em 24 horas de estudo. A nanoencapsulação do clobetasol nos CLN-Q e CLN resultou em retenção aumentada do fármaco no estrato córneo de 9,2 e 7,8 vezes, respectivamente, comparado ao fármaco livre. A retenção do clobetasol no estrato córneo foi de 4 e 2 vezes maior quando corrente elétrica foi aplicada na formulação CLNQ, comparado ao fármaco livre e encapsulado nos CLN, respectivamente. O acúmulo do fármaco no estrato córneo, promovido pelos carreadores lipídicos, mostrou que esses sistemas são efetivos para entrega tópica deste fármaco com potencial para minimizar sua absorção percutânea, e dessa forma, diminuir os seus efeitos adversos.

It is currently accepted that the most appropriate diet in the treatment of non-insulin-dependent diabetes mellitus &quoteNIDDM&quote is high in carbohydrates, high in fibre and low in fat. Dietary fibre reduces the rate of carbohydrate absorption, which may have a beneficial effect on insulin action. Furthermore, high fibre diets also increase the amount of carbohydrates which are not absorbed from the small intestine. These malabsorbed carbohydrates are fermented by the bacterial population in the large intestine, producing short chain fatty acids &quoteSCFA&quote, including propionate, which has been shown to alter liver carbohydrate metabolism. This thesis investigated the actions of slowed carbohydrate absorption and carbohydrate malabsorption in streptozotocin-induced &quoteSTZ&quote diabetic rats. High carbohydrate diet supplemented with guar gum, a soluble dietary fibre, fed to STZ diabetic rats improved insulin sensitivity. investigation of the alterations in the stomach and small intestine demonstrated that guar increased the viscosity of the meal in the intestine. The action of increased fermentation, producing more propionate, was investigated by supplementing propionate into the diets of STZ diabetic rats or when perfused into isolated rat livers. No changes in insulin action or liver glucose metabolism were measured. in addition, it was shown that guar gum reduces food intake in STZ diabetic rats. Mild reductions in food intake in STZ diabetic rats were shown to increase insulin action. In summary, STZ diabetic rats fed high carbohydrate, high fibre diets reductions in food consumption and slowed carbohydrate absorption are important factors which may lower blood glucose concentrations and increase insulin action. increased SCFA production is unlikely to contribute significantly to the improvements in insulin action.

No description available.

Vitamin B₁₂ deficiency has been difficult to diagnose, mainly due to the vitamin's lack of biological significance in serum in which it is usually assayed. This research has investigated the marker of vitamin B₁₂/cobalt (Co) deficiency in sheep, methylmalonic acid (MMA), in comparison with serum and liver vitamin B₁₂ concentrations in farm situations where vitamin B₁₂ deficiency is expected in order to establish more accurate reference ranges for serum and liver vitamin B₁₂, and MMA. In addition, an attempt was made to ascertain the vitamin B₁₂ requirements of preruminant (PR) lambs, and to determine whether metabolic demand for vitamin B₁₂ influences tissue concentrations. Furthermore, since the vitamin is active in biological tissues in form of its coenzymes, 5’ -deoxyadenosylcobalamin and methylcobalamin, a preliminary assessment of variation in the distribution of these coenzymes in liver in different situations has been sought. The first trial was set up to find out if the addition of propionate to the PR lamb's diet stimulated the uptake and/or storage of vitamin B₁₂ in the liver as a reflection of the need to deal with the incoming propionate. Sixteen ten day old lambs (Dorset Down/Coopworth cross-bred) were housed indoors soon after birth and fed on milk replacer. For half of the lambs 7.5 % (w:w) of the milk powder was replaced by propionate. Within each group, four lambs were treated with 250 µg vitamin B₁₂ twice weekly. Supplementation with vitamin B₁₂ increased liver concentrations from ~250 to ~900 nmol/kg fresh tissue, but there was no effect of propionate. Propionate addition did, however, result in increased plasma vitamin B₁₂ concentrations in vitamin B₁₂ supplemented groups, values being 3323 and 2355 pmol/l in propionate supplemented and control groups, respectively. This suggested that diet could influence plasma vitamin B₁₂ concentrations. An attempt was made to quantify the PR lamb's ability to absorb vitamin B₁₂ from the alimentary tract by comparing the ability of intra-muscular (IM) and oral vitamin B₁₂ to raise plasma and liver vitamin B₁₂ concentrations. Twenty-seven three to four day old lambs from a farm with marginal Co status were housed indoors and fed on milk replacer. They were divided into three groups: control (n=3), IM treatment (n=12) and oral treatment (n=12). The two treatment groups were further subdivided into five sub-groups. These received, respectively, 0.2 (n=3), 0.4 (n=2), 0.8 (n=2), 1.6 (n=2) and 3.2 µg OH-cbl/d (n=3). The oral groups received tenfold the amount of the comparable IM groups, on the assumption that if oral absorption of the vitamin is about 10 % both groups would show similar increases in plasma and liver vitamin B₁₂ concentration. None of the IM groups showed any significant change in plasma or liver vitamin B₁₂. In the oral groups only the group on the highest dose of vitamin B₁₂, viz 32 µg/d, showed increases in plasma and liver concentrations. It was concluded that either absorption of vitamin B₁₂ was greater than 10 % or that the vitamin was retained better when administered orally. The amount retained in the livers of the lambs in the highest oral group was calculated to represent ~ 7.5 % of the dose. In a follow-up 24 h trial, 14 of the above lambs were divided into three groups: Control (n=3), oral (n=6) and IM (n=5) treatment. The IM group received 3.2 µg OH-cbl and the oral group tenfold the amount as single doses at 0800 h. Blood samples were taken at regular intervals throughout the 24 h period and assayed for vitamin B₁₂, Vitamin B₁₂ concentrations in the IM group rose steeply within the first hour after injection to a concentration that was calculated to reflect 100 % uptake of the vitamin. It rose more slowly over about 8 h in the oral group. From the area under the curve absorption of the oral dose was estimated to be ~ 7 %. The next experiment involved a farm where Co deficiency had been reported previously. In the first year, 50 pregnant two-tooth Half-bred ewes were divided randomly into two groups of 25. One group received a Co bullet plus 1000 µg OH-cb1 IM, the other group remained unsupplemented. In the following year the trial was repeated. Ewes from the previous year's trial (by then four-tooths) were augmented by a new cohort of pregnant two-tooths to make up numbers to 75. After lambing the lambs were divided into four groups: first by their dams' vitamin B₁₂ treatment, then half of each group received injections of vitamin B₁₂ at approximately three weekly intervals while the other half remained untreated. The trials lasted about five months, from mid-pregnancy until weaning. Pasture Co was at its lowest at lambing in both years, 0.09 and 0.10µg/g DM, respectively. In the first year, vitamin B₁₂ concentrations in the untreated ewes rose from 340 to 950 pmol/l in plasma and decreased in liver from 330 to 170 nmol/kg fresh tissue. In the Co treated group, vitamin B₁₂ concentrations in plasma rose from 500 to 1550 pmol/l and in liver from 310 to 560 nmol/kg fresh tissue. In the second year, vitamin B₁₂ concentrations in serum in the unsupplemented groups fell from 500 to 260 pmol/l around lambing before rising again to starting values at weaning, and liver vitamin B₁₂ concentrations fell from 450 at the start to 230 nmol/kg fresh tissue at the end of the trial. Serum vitamin B₁₂ concentrations in the two-tooth supplemented group rose from < 500 to > 3000 pmol/l whereas in the four-tooth supplemented group serum vitamin B₁₂ levels started at ~2800 and rose to nearly 5000 pmol/l. The supplemented four-tooths maintained higher liver vitamin B₁₂ concentrations throughout compared to the supplemented two-tooths, viz 680 compared to below 400 at the start and 900 versus 650 nmol/kg fresh tissue at weaning, respectively. MMA in the untreated groups rose to 15 and to 8 µmol/l during early lactation in the first and second years, respectively, whereas MMA in the treated groups stayed below 3 µmol/l in the first season and below 1.5 µmol/l in the second season. There was a live weight response to treatment in the ewes as the unsupplemented groups showed a significantly lower weight gain during the trials than the supplemented groups, viz 10.0 versus 13.6 kg in the first year, and 10.6 versus 13.3 kg in the four-tooths and 9.9 versus 12.1 kg in the two-tooths in the second year. There was also a significant difference in faecal egg count (FEC) in the first year. FEC in the untreated group was higher during lactation than in the treated group, viz 590 versus 170 eggs per gram wet faeces (epg), respectively. In the second year, the two-tooths had a higher FEC than the four-tooths, viz 120 versus 40 epg during the same time span, respectively. While there was a trend for treatment having an effect on FEC similar to that in the first year it was not significant. Supplementation of ewes in the first year increased mean milk vitamin B₁₂ concentrations at lambing from 800 to 1400 pmol/l and at weaning from 1750 to 4000 pmol/l. In the second year, Co bullet treatment increased milk vitamin B₁₂ concentrations in the four-tooths and two-tooths from 1500 and 2300 to 4000 and 2900 pmol/l at lambing, and from 1800 and 1400 to 6200 and 4500 pmol/l at weaning, respectively. Treatment of ewes increased vitamin B₁₂ concentrations in the lambs which were not themselves supplemented. Plasma values in the first year increased from 160 to 325 pmol/l soon after birth and from 650 to 900 pmol/l at weaning, and liver values from 75 to 140 nmol/kg fresh tissue soon after birth and from 150 to 240 nmol/kg fresh tissue at weaning. In the second year, plasma vitamin B₁₂ concentrations increased from 160 to 380 pmol/l soon after birth and from 500 to 700 pmol/l at weaning, and in liver from 130 to 260 nmol/kg fresh tissue soon after birth and from 220 to 340 nmol/kg fresh tissue at weaning. There was also a significant effect of ewe supplementation on lamb MMA in 1997/1998 when values decreased from 19 to 8 µmol/l around the time of rumen development. MMA in the second year stayed below 3 µmol/l throughout in all groups of lambs. There was no difference in LWG between any groups of lambs. FEC was lowest in the group where both ewes and lambs were supplemented and highest in the group where neither ewes nor lambs were treated. Further investigations were conducted on farms in Southland with lambs post-weaning in order to compare changes in serum and liver vitamin B₁₂ with serum MMA and LWG to determine the critical time and level of deficiency. In the first year, three farms with 50 lambs each participated. Lambs from each farm were allocated to five groups of 10 animals each. The first group received a Co bullet at weaning, and each month another group was treated with a Co bullet. The lambs were weighed monthly, and blood and liver samples were taken prior to treatment and each subsequent month from five lambs of the first supplemented group. The trial lasted about four months. Serum vitamin B₁₂ concentrations in lambs at weaning were between 500 and 1000 pmol/l. Although supplementation increased serum levels for the first month this was followed by a drop to near or below starting concentrations. An exception was Farm 3 where serum vitamin B₁₂ concentrations rose again at the end of the trial. Liver vitamin B₁₂ concentrations also showed an overall decline from starting levels (200 to 300 nmol/kg fresh tissue) to the end of the trial (100 to 200 nmol/kg fresh tissue). MMA started around 2 µmol/l and reached between 6 and 7 µmol/l in the untreated lambs on Farms 1 and 3 two months after weaning before decreasing to around 3 µmol/l at the end of the trial, whereas the treated lambs maintained MMA concentrations around 2 µmol/l. On Farm 2 MMA started just below 5 µmol/l, decreased to around 1 µmol/l for treated and untreated lambs one month later and rose again to between 2.5 and 4 µmol/l, respectively, at the end of the trial. LWG was below average for all lambs (between 0.20 and 0.04 kg/d except for Farm I in the first month after weaning) but no significant differences were noted between treated and untreated lambs on any of the farms. Another trial was conducted on one of these farms in the following year. One hundred lambs were divided into two groups of 50 each at weaning and sampled monthly for about six months. One group was treated with two Co bullets, the other group remained untreated. Pasture Co was between 0.04 and 0.07 µg/g DM, yet serum levels for the untreated group stayed ~500 pmol/l throughout the trial. Serum vitamin B₁₂ concentrations for the treated group started at ~500 pmol/l, rose to ~2500 pmol/l before falling back to ~2000 pmol/l. Liver vitamin B₁₂ concentrations for the untreated and treated groups were 529 and 427 nmol/kg fresh tissue at weaning, respectively. This decreased for both groups to ~350 nmol/kg fresh tissue one month after weaning. In the untreated lambs liver values decreased further to ~290 nmol/kg fresh tissue whereas they increased to ~450 nmol/kg fresh tissue for the treated group at the end of the trial. MMA concentrations started between 2 and 3 µmol/l for both groups and increased to 4.5 µmol/l for the untreated group one month later before falling back to 3.2 µmol/l. In the treated group MMA decreased to ~1µmol/l and stayed at that level throughout the trial. There was no difference in weight gain. In order to obtain an understanding of the distribution of corrinoids in biological tissues a High Performance Liquid Chromatography method was developed. The sensitivity of the analytical method meant that it was only practical to assay mainly liver samples because of the higher vitamin B₁₂ concentrations than in other tissues. The general finding was that the coenzyme 5’ –deoxyadenosylcobalamin (ado-cbl) constituted the highest proportion of corrinoids in liver (45 %), followed by analogues (28 %), OH-cbl (24 %) and lastly methy1cobalamin (3 %). Ado-cbl did tend to be proportionately higher in supplemented than in unsupplemented animals (56 and 42 %, respectively), whereas biologically non-active analogues tended to be higher in untreated than in treated sheep (29 and 21 %, respectively). It was concluded that in the farm trials Co deficiency was only mild or not present although deficiency would have been predicted from the low vitamin B₁₂ concentrations in serum and liver and from raised MMA values. Therefore, currently used thresholds in New Zealand appear to be too high for vitamin B₁₂, and overseas values for MMA do not seem to be appropriate. Revised marginal ranges of 100 to 200 pmol/l for serum, 100 to 200 nmol/kg fresh tissue for liver and 10 to 20 µmol/l for MMA are suggested. Further, this work shows that Co bullets were effective in elevating blood and liver vitamin B₁₂concentrations for longer than one year. In the trials with preruminant lambs it was found that maintenance requirements were met by the vitamin B₁₂ content of milk replacer. There is evidence from indoor and farm trials that vitamin B₁₂ from milk was much more readily absorbed than vitamin B₁₂ from supplements. It was estimated that suckling lambs probably require between 1200 and 4000 pmol vitamin B₁₂/d, depending on age.

Neste trabalho foi avaliada a permeação/penetração cutânea in vitro (pele suína) de propionato de clobetasol nanoencapsulado incorporado em um semissólido, empregando células de difusão de Franz. A nanoencapsulação foi capaz de reduzir a quantidade de fármaco que penetra nas camadas da pele (estrato córneo, epiderme e derme) sem alterar a forma (distribuição percentual) como o propionato de clobetasol se distribui. A adequabilidade de diferentes membranas sintéticas (acetato de celulose, policarbonato e membrana de diálise) para a avaliação da liberação in vitro, empregando células de difusão de Franz, a partir desta formulação foi também estudada. A partir da combinação de diferentes técnicas analíticas (espalhamento de luz dinâmica, microscopias eletrônicas de transmissão e varredura) foi observado que a membrana de menor tamanho de poro (membrana de diálise, 12 kDa de cut off) é a mais adequada para a condução deste tipo de avaliação, pois é a única capaz de evitar a passagem de nanocápsulas íntegras da formulação para o meio receptor das células de difusão, em detrimento das membranas de policarbonato e acetato de celulose (0,05 μm e 0,45 μm de tamanho de poro, respectivamente). Além disso, uma nova estratégia para a avaliação da liberação in vitro de fármacos associados a nanocápsulas de núcleo lipídico, combinando fluxo contínuo de meio de liberação e sacos de diálise foi proposta neste trabalho. A técnica mostrou-se adequada para a obtenção do perfil de liberação in vitro a partir de suspensões de nanocápsulas contendo diferentes fármacos modelo (prednisolona e propionato de clobetasol), possibilitando a diferenciação destes sistemas de soluções contendo os fármacos livres, graficamente e pelos valores de fluxo calculados. Adicionalmente, esta estratégia mostrou-se apropriada para a manutenção da concentração de fármaco no meio de liberação afastada da saturação, contribuindo para o atendimento da condição sink. Ainda, classificamos o sistema como um protótipo semi-automatizado para a avaliação da liberação in vitro de fármacos, capaz de gerar resultados com maior precisão em relação à diálise convencional. / The in vitro cutaneous permeation/penetration (porcine skin) of clobetasol propionate-loaded lipid-core nanocapsules incorporated into a semisolid dosage form was evaluated, using the Franz diffusion cells technique. It was shown that the nanoencapsulation was able to reduce the drug amount penetration into skin layers (stratum corneum, epidermis and dermis) without changing the way (percentual distribution) that it was distributed. The suitability of different synthetic membranes (cellulose acetate, polycarbonate, and dialysis membrane) to assess the in vitro drug release using Franz diffusion cells from this formulation was also studied. It was ascertained by combining different analytical techniques (dynamic light scattering, scanning and transmition electron microscopy) that the membrane with smaller pore size (dialysis membrane, 12 kDa cut off) is the most appropriate for conducting this kind of study, because it is the only one able of preventing the passage of intact nanocapsules from formulation to Franz diffusion cells receptor media, instead of polycarbonate and cellulose acetate membranes (0.05 and 0.45 pore size, respectively). In addition, a new strategy to assess in vitro drug release drug-loaded lipid-core nanocapsules was proposed, associating continuous flow of release media and dialysis sac. The proposed system was adequate to assess the in vitro drug release profiles from nanocapsule suspensions containing different model drugs (prednisolone and clobetasol propionate), enabling the differentiation of these systems from drug solutions, graphically and by the calculated flux values. Furthermore, this strategy was suitable to maintain the drug concentration into release media far away from saturation, contributing to the sink condition. Also, the proposed system was described as a semi-automated prototype for in vitro drug release evaluation, able to produce results with greater accuracy than conventional dialysis technique.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fluticasone propionate (FP) is a synthetic glucocorticoid with potent anti-inflammatory activity that has been effectively used for the treatment of seasonal and allergic perennial rhinitis, minimizing systemic activity. In the present study, the methods were developed and validaded for assessment of FP in pharmaceutical products. The analysis by reversed-phase liquid chromatography were performed using Shim-pack CLC-ODS column (150 x 4.6 mm), maintained at 35 °C. The mobile phase was consisted of acetonitrile/ methanol/ 0.01M phosphate buffer pH 4 (35:35:30), run at flow rate of 1 mL/min and using photodiode array detection at 240 nm. The chromatographic separation was obtained within 8 minutes and it was linear in the concentration range of 0.05-150 μg/mL (r2 = 0,9999). The method was successfully applied for the determination of FP in creams and nasal sprays pharmaceutical formulations. The micellar electrokinetic chromatography was also developed and validaded. The analyses were performed on a fused-silica capillary (50 μm i.d.; effective length, 40 cm) and background electrolyte consisted of 25 mM borate and 25mM SDS solution at pH 9. The capillary temperature was maintained at 35 °C and the applied voltage was 20 kV. The injection was performed using the hydrodynamic mode at 50 mbar for 6 s, with detection at 238 nm. The electrophoretic separation was obtained with migration time of 5.6 and 5.1 minutes for the FP and prednisolone acetate (internal standard), respectively, and with run time of 7 minutes. The method was linear in the concentration range of 2-80 μg/mL (r2 = 0.9956). The procedures were validated evaluating parameters such as the specificity, linearity, precision, accuracy, and robustness, limit of detection and limit of quantitation, whose results have met the requirements recommended. The proposed methods were used for the analysis of pharmaceutical products, demonstrating nonsignificative difference of the results (P > 0.05). Then, the procedures contribute to improve the quality control, assuring the safety and therapeutic efficacy of pharmaceutical formulations. / O propionato de fluticasona (PF) é um glicocorticóide sintético com potente atividade antiinflamatória utilizado efetivamente no tratamento de rinites alérgicas sazonais e perenes, minimizando a atividade sistêmica. No presente trabalho foram desenvolvidos e validados métodos para avaliação de PF em produtos farmacêuticos. As análises por cromatografia líquida em fase reversa foram realizadas utilizando coluna Shim-pack CLC-ODS (150 x 4,6 mm), mantida à 35ºC. A fase móvel foi composta de acetonitrila/ metanol/ tampão fosfato 0,01M pH 4 (35:35:30), eluída na vazão de 1 mL/min e detecção no ultravioleta a 240 nm. A separação cromatográfica foi obtida no tempo de 8 minutos, sendo linear na faixa de concentração de 0,05-150 μg/mL (r2 = 0,9999). O método foi aplicado para análise de PF em cremes e sprays nasais. Paralelamente, desenvolveu-se e validou-se método por cromatografia eletrocinética micelar. Executaram-se as análises utilizando capilar de sílica (comprimento efetivo de 40 cm e diâmetro de 50 μm) e solução eletrolítica composta de borato 25 mM e SDS 25 mM, pH 9. O capilar foi mantido a temperatura de 35ºC, e aplicada voltagem de 20 kV. O tempo de injeção foi de 6 s com pressão de 50 mbar e detecção no UV a 238 nm. Realizou-se a separação eletroforética com tempo de migração de 5,6 e 5,1 minutos para o PF e acetato de prednisolona (padrão interno), respectivamente, com tempo de corrida de 7 minutos. O método foi linear na faixa de 2-80 μg/mL (r2 = 0,9956). Os procedimentos foram validados, avaliando-se os parâmetros de especificidade, linearidade, precisão, exatidão, robustez, limite de detecção e quantificação, cujos resultados cumpriram os requisitos preconizados. Os métodos propostos foram utilizados para análise de produtos farmacêuticos, demonstrando que não há diferenças significativas dos resultados (P > 0,05). Assim, os procedimentos pesquisados contribuem para aprimorar o controle da qualidade, garantindo a segurança e eficácia terapêutica das formulações farmacêuticas.

Dois experimentos foram conduzidos com o objetivo de avaliar o efeito da inclusão de butirato de sódio, monensina sódica ou propionato de cálcio no concentrado inicial, sobre o desempenho, parâmetros sanguíneos e desenvolvimento ruminal de bezerros leiteiros. No primeiro experimento, 24 bezerras recém-nascidas da raça Holandesa foram alojadas em abrigos individuais até a 10a semana de vida, com livre acesso à água, sendo alimentadas com 4 litros de leite/dia e concentrado ad libitum, enquanto feno de capim-coast-cross foi fornecido após o desaleitamento. Os animais foram distribuídos em blocos de acordo com peso ao nascer e data de nascimento e alocados em um dos tratamentos, de acordo com o aditivo no concentrado: 1)Butirato de sódio (0,15%); 2)Monensina sódica (30 ppm); e 3)Propionato de cálcio (0,15%). Os animais foram pesados e avaliados quanto à altura na cernelha, largura do traseiro e perímetro torácico semanalmente. A partir da 4a semana foram realizadas colheitas semanais de amostras de sangue para determinação de glicose, ácidos graxos livres (AGL) e ?-hidroxibutirato (BHBA). Não foram observadas diferenças significativas entre os tratamentos para o consumo de concentrado ou de feno e para o peso e ganho de peso dos animais (P>0,05). As avaliações quanto à altura na cernelha e perímetro torácico também não apresentaram diferenças entre os tratamentos (P>0,05), entretanto as medidas de largura de traseiro foram menores para os animais do tratamento com adição de propionato de cálcio (P<0,05). As concentrações plasmáticas de glicose, AGL e BHBA não foram afetadas pelos tratamentos (P>0,05). Houve efeito significativo da idade (P<0,0001) para a concentração plasmática de glicose, sendo esta reduzida com a idade dos animais. No segundo experimento, 15 bezerros recémnascidos da raça Holandesa, recebendo o mesmo manejo nutricional, foram fistulados no rúmen e alojados em baias individuais até a 10ª semana de vida. A partir da 4ª semana, foram realizadas colheitas semanais de fluído ruminal para determinação de pH, ácidos graxos de cadeia curta (AGCC) e N-amoniacal; e de sangue para determinação de glicose. Ao completar dez semanas os animais foram abatidos para avaliação do desenvolvimento do trato digestório superior e de papilas ruminais. Não foram observadas diferenças significativas entre tratamentos para o consumo de concentrado e para o desempenho dos animais (P>0,05). Houve efeito significativo (P<0,05) de tratamento e horário de colheita para o pH ruminal. As concentrações de AGCC totais, bem como de cada ácido, não foram afetadas pelos tratamentos. As concentrações plasmáticas de glicose foram afetadas pelos tratamentos (P<0,05). O peso total do trato digestório superior, os pesos médios de cada compartimento e a capacidade máxima do retículo-rúmen não foram afetados pelos tratamentos, assim como os parâmetros de desenvolvimento do epitélio ruminal. Os aditivos incluídos no concentrado inicial se mostraram igualmente eficazes no que diz respeito aos seus efeitos no desempenho e desenvolvimento ruminal de bezerros em aleitamento. / Two trials were conducted in order to evaluate the effects of the addition of sodium butyrate, sodium monensin or calcium propionate in the starter feed on the performance, blood parameters and ruminal development of dairy calves. In the first experiment, 24 newborn Holstein calves were housed in individual hutches during ten weeks of life, with free access to water, being fed 4 liters of milk per day and starter ad libitum, with coast-cross hay offered only after weaning. The animals were blocked according to weight and date of birth and allocated in one of the treatments, according to the additive included in the starter feed: 1) sodium butyrate (0.15%); 2) sodium monensin (30 ppm); and 3) calcium propionate (0.15%). Animals were weighed and evaluated for whiter height, hearth girth and hip width weekly. From the fourth week of age blood samples were taken for glucose, free fatty acids and ?-hydroxybutyrate concentration determination. No significant differences were observed among treatments for starter or hay intake, and weight gain or live weight (P>0.05). Measurements of whiter height and hearth girth were also not affected by treatments (P> 0.05); however, measures of hip width of the animals were smaller for treatment with addition of calcium propionate (P<0.05). Plasma concentrations of glucose, free fatty acids and ?-hydroxybutyrate were not affected by treatment (P>0.05). There was significant effect of age (P<0.0001) for the plasma concentration of glucose, with reduction as animals aged. In the second experiment, 15 male newborns Holstein calves, receiving the same nutritional management, were ruminally fistulated and housed in individual pens during ten weeks of life. From the fourth week of age ruminal samples were taken weekly for the determination of pH, short-chain fatty acids (SCFA) and ammonia-N concentration. Blood samples were also taken weekly for glucose determination. By completing ten weeks of age, animals were slaughtered for forestomach growth and papillae development evaluation. No significant differences were observed among treatments for starter intake as well as for animal performance (P>0.05). The ruminal pH was significantly affected (P<0.05) by treatments and by sampling time. Concentrations of total SCFA and individual SCFA were not affected by treatments (P<0.05). Plasma concentrations of glucose were affected by treatments (P<0.05). The total forestomach weight, the average weight of each compartment and the maximum capacity of reticulum-rumen were not affected by treatments, as well as the parameters for ruminal epithelium development. The additives included in the starter feed were equally effective as regard to its effects on animal performance and rumen development of milk-fed dairy calves.

Made available in DSpace on 2014-12-17T15:36:43Z (GMT). No. of bitstreams: 1 FernandoRFS_TESE.pdf: 469950 bytes, checksum: a8f86cc336421fd74e299ebe8f8d1daf (MD5) Previous issue date: 2014-11-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The use and the demand for substances that enhance masculinity, strength and sexual power are not novel. Over the years, this search has assisted the research directions in this area, leading to the discovery of the primary male sex hormone testosterone in 1935. Since then, numerous testosterone analogue compounds were synthesized, which are generically called Anabolic Androgenic Steroids (AAS). The AAS were produced for therapeutic purposes, but an increase in the use of these compounds for other purposes occurred over time. Initially they were used mainly to improve performance in athletes. However, recent studies have shown that the use of AAS by non-athletes with aesthetical purposes have been increasing as well. The abuse of AAS with non-clinical purposes can promote a number of physiological alterations, such as heart, liver, respiratory and psychological problems such as changes in mood, levels of anxiety and aggression. Exposure to supraphysiological doses of AAS is associated with behavioral changes, however, little is known about the effects of AAS on cognitive functions. In this work, we aimed to mimic the AAS abuse in humans with intramuscular administration of a supraphysiological dose of testosterone propionate (TP) in rats. We investigated the effects of this treatment on different aspects of cognitive function, specifically learning, memory and anxiety. Adult male Wistar rats were tested in the spontaneous alternation, novel object recognition and plus-maze discriminative avoidance tasks. The control group received intramuscular injections of vegetable oil (vehicle), and the TP group received injections of TP (10 mg/kg, i.m.). The injections were administered for 40 days, with intervals of 48 hours (chronic treatment) or in a single injection (acute treatment). In addition to the behavioral assessments, we performed biochemical analyzes as indicators of the endocrine effects of the treatment. Our results show that chronic treatment with a supraphysiological dose of TP caused memory impairments in the novel object recognition and the discriminative avoidance tasks. The spatial working memory (evaluated by spontaneous alternation task) was not affected. Also, we did not observe changes in anxiety levels. Regarding the biochemical parameters, chronic treatment increased serum levels of glutamicpyruvic transaminase, an indicator of hepatic and pancreatic lesions (as those observed after chronic use of these substances in humans). On the other hand, acute treatment with PT did not promote significant changes in any of these parameters when compared to the control group. In summary, we conclude that chronic treatment with a supraphysiological dose of testosterone propionate produces memory deficits in novel object recognition and retrieval of the discriminative avoidance task in adult male rats / A utiliza??o e a busca por subst?ncias que aumentem a masculinidade, a for?a e a pot?ncia sexual n?o ? recente. Com o tempo, essa busca auxiliou no direcionamento de pesquisas na ?rea, levando a descoberta do principal horm?nio masculino a testosterona em meados da d?cada de 30. A partir desse momento, in?meros compostos foram sintetizados com o intuito de mimetizar os efeitos deste horm?nio, aos quais hoje chamamos genericamente de Esteroides Anab?licos Androg?nicos (EAA). A princ?pio, esses EAA foram sendo produzidos com prop?sitos terap?uticos. No entanto, iniciou-se o uso crescente desses compostos com outras finalidades, principalmente para a melhoria de desempenho em atletas. Al?m disso, estudos recentes t?m demonstrado que os EAA est?o sendo cada vez mais utilizados por n?o atletas, por indiv?duos que n?o s?o atletas, mas, buscam um corpo esteticamente perfeito. Paralelamente, o crescente abuso dos EAA com finalidades n?o cl?nicas pode promover uma s?rie de altera??es fisiol?gicas nocivas, tais como problemas card?acos, hep?ticos, respirat?rios e tamb?m psicol?gicos como altera??es de humor, nos n?veis de ansiedade e na agressividade. A exposi??o a doses suprafisiol?gicas de EAA est? associada com altera??es comportamentais, contudo, pouco se sabe sobre os efeitos dos EAAs sobre as fun??es cognitivas. Neste trabalho, mimetizamos o abuso de EAA em humanos atrav?s da administra??o intramuscular de uma dose suprafisiol?gica de propionato de testosterona (PT), em ratos, com o objetivo de investigar os efeitos desse tratamento sobre diferentes aspectos das fun??es cognitivas, especialmente aprendizado, mem?ria e ansiedade. Ratos Wistar machos adultos foram submetidos aos testes de alterna??o espont?nea, reconhecimento de objetos e esquiva discriminativa em labirinto em cruz elevado. O grupo controle recebeu inje??es intramusculares de ?leo vegetal (ve?culo); e o grupo testosterona recebeu inje??es de PT (10 mg/kg, i.m.). As inje??es foram administradas por 40 dias, com intervalos de 48 horas (tratamento cr?nico) ou em uma ?nica inje??o (tratamento agudo). Al?m das avalia??es comportamentais, foram realizadas an?lises bioqu?micas como indicadores dos efeitos end?crinos do tratamento. Nossos resultados mostram que o tratamento cr?nico com uma dose suprafisiol?gica de PT acarretou preju?zos na mem?ria de reconhecimento de objetos novos e na evoca??o da tarefa da esquiva discriminativa. A mem?ria espacial operacional (avaliada pelo teste de alterna??o espont?nea) n?o foi afetada bem como n?o observamos altera??es nos n?veis de ansiedade. Em rela??o aos par?metros bioqu?micos avaliados, o tratamento cr?nico elevou os n?veis s?ricos da transaminase glut?mica pir?vica (TGP), um indicador da presen?a de les?es hep?ticas e pancre?ticas (assim como as observadas ap?s o uso cr?nico dessas subst?ncias em humanos). Por outro lado, o tratamento agudo com PT n?o promoveu altera??o significativa em nenhum dos par?metros avaliados, quando comparados ao grupo controle. Em s?ntese, podemos concluir que o tratamento cr?nico com uma dose suprafisiol?gica de testosterona produz d?ficits de mem?ria de reconhecimento de objetos bem como preju?zos na mem?ria na tarefa da esquiva discriminativa em ratos machos adultos

Neste trabalho foi avaliada a permeação/penetração cutânea in vitro (pele suína) de propionato de clobetasol nanoencapsulado incorporado em um semissólido, empregando células de difusão de Franz. A nanoencapsulação foi capaz de reduzir a quantidade de fármaco que penetra nas camadas da pele (estrato córneo, epiderme e derme) sem alterar a forma (distribuição percentual) como o propionato de clobetasol se distribui. A adequabilidade de diferentes membranas sintéticas (acetato de celulose, policarbonato e membrana de diálise) para a avaliação da liberação in vitro, empregando células de difusão de Franz, a partir desta formulação foi também estudada. A partir da combinação de diferentes técnicas analíticas (espalhamento de luz dinâmica, microscopias eletrônicas de transmissão e varredura) foi observado que a membrana de menor tamanho de poro (membrana de diálise, 12 kDa de cut off) é a mais adequada para a condução deste tipo de avaliação, pois é a única capaz de evitar a passagem de nanocápsulas íntegras da formulação para o meio receptor das células de difusão, em detrimento das membranas de policarbonato e acetato de celulose (0,05 μm e 0,45 μm de tamanho de poro, respectivamente). Além disso, uma nova estratégia para a avaliação da liberação in vitro de fármacos associados a nanocápsulas de núcleo lipídico, combinando fluxo contínuo de meio de liberação e sacos de diálise foi proposta neste trabalho. A técnica mostrou-se adequada para a obtenção do perfil de liberação in vitro a partir de suspensões de nanocápsulas contendo diferentes fármacos modelo (prednisolona e propionato de clobetasol), possibilitando a diferenciação destes sistemas de soluções contendo os fármacos livres, graficamente e pelos valores de fluxo calculados. Adicionalmente, esta estratégia mostrou-se apropriada para a manutenção da concentração de fármaco no meio de liberação afastada da saturação, contribuindo para o atendimento da condição sink. Ainda, classificamos o sistema como um protótipo semi-automatizado para a avaliação da liberação in vitro de fármacos, capaz de gerar resultados com maior precisão em relação à diálise convencional. / The in vitro cutaneous permeation/penetration (porcine skin) of clobetasol propionate-loaded lipid-core nanocapsules incorporated into a semisolid dosage form was evaluated, using the Franz diffusion cells technique. It was shown that the nanoencapsulation was able to reduce the drug amount penetration into skin layers (stratum corneum, epidermis and dermis) without changing the way (percentual distribution) that it was distributed. The suitability of different synthetic membranes (cellulose acetate, polycarbonate, and dialysis membrane) to assess the in vitro drug release using Franz diffusion cells from this formulation was also studied. It was ascertained by combining different analytical techniques (dynamic light scattering, scanning and transmition electron microscopy) that the membrane with smaller pore size (dialysis membrane, 12 kDa cut off) is the most appropriate for conducting this kind of study, because it is the only one able of preventing the passage of intact nanocapsules from formulation to Franz diffusion cells receptor media, instead of polycarbonate and cellulose acetate membranes (0.05 and 0.45 pore size, respectively). In addition, a new strategy to assess in vitro drug release drug-loaded lipid-core nanocapsules was proposed, associating continuous flow of release media and dialysis sac. The proposed system was adequate to assess the in vitro drug release profiles from nanocapsule suspensions containing different model drugs (prednisolone and clobetasol propionate), enabling the differentiation of these systems from drug solutions, graphically and by the calculated flux values. Furthermore, this strategy was suitable to maintain the drug concentration into release media far away from saturation, contributing to the sink condition. Also, the proposed system was described as a semi-automated prototype for in vitro drug release evaluation, able to produce results with greater accuracy than conventional dialysis technique.