Realizou-se um estudo do processo descontínuo alimentado de fermentação, buscando encontrar as melhores condições de produção do ácido graxo gama-linolênico. Empregou-se como fonte de nitrogênio no meio de fermentação a uréia, que foi adicionada de forma exponencialmente crescente e como inóculo usou-se uma cepa de Spirulina platensis. Utilizou-se planejamento experimental e aplicou-se a metodologia de superfície de resposta, o qual permitiu a obtenção de equações que relacionaram as variáveis dependentes, como produtividade em células, conversão de nitrogênio em células, concentração máxima atingida de biomassa, porcentagem relativa de produção de ácido graxo gama-linolênico. com as variáveis independentes tempo de alimentação (Ta) e massa total de uréia adicionada (MUT). Os melhores resultados obtidos foram os seguintes: Px=117,26 mg/L.dia; Yx/n= 4,9; Xmax= 1759 mg/L e porcentagem de ácido graxo γ-linolênico na fração lipídica de 23,5 %, com 14 dias (Ta) e 1750 mg (MUT). / A Study on the fed-batch fermentation process was made seeking for the best Gamma-linolenic production condition. Mineral media was employed with urea as nitrogen source that was add with exponentially increasing feeding rates and Spirulina platensis as inoculum. Experimental design and a Response Surface Methodology were applied. It was composed by an experimental design which allowed a reduced number of the experiments and by the analysis of the results using equations which combined the interest variables dependent as: Productivity, best Gamma-linolenic production condition, Nitrogen convertion in Biomass, Highest Concentration and the study variables independent (Ta-feeding time and add urea-MUT). The best results were with (Ta-feeding time=14 days; add urea-MUT=1750 mg). In this conditions we had: Productivity=117,26 mg/L.day Best Gamma-linolenic production condition=23,5 per cent Nitrogen convertion in Biomass=4,9 Biggest Concentration.=1759 mg/L.

Os objetivos deste estudo foram avaliar o desempenho produtivo, reprodutivo e parâmetros sanguíneos de ovelhas em lactação alimentadas com teores de grão de soja (GS), e a utilização de teores de bagaço de cana-de-açúcar in natura (BC) na ração de ovelhas secas e gestantes como fonte única de volumoso. Nos experimentos I e II, 56 ovelhas da raça Santa Inês foram alimentadas com rações contendo 0, 7, 14 e 21% de GS na matéria seca total da ração. No experimento I, houve efeito quadrático (P<0,05) no consumo de matéria seca e produção de leite total. Contudo não houve alteração (P>0,05) na produção de leite corrigida para gordura ou para gordura e proteína, ou mesmo na eficiência alimentar. Os teores de gordura, proteína, lactose e sólidos totais do leite foram similares (P>0,05) entre os tratamentos. Houve redução dos ácidos graxos de cadeia curta (C4-C10) e média (C11-C16), enquanto os de cadeia longa (C18) aumentaram linearmente (P<0,05). As proporções dos ácidos graxos C18:2 c9 t11 e o C18:2 t10 c12 aumentaram linearmente (P0,01) e o índice de aterogenicidade reduziu linearmente (P<0,05) com a inclusão crescente de GS na ração. A concentração (g/100g) dos ácidos graxos saturados, insaturados, monoinsaturados, poliinsaturados e a relação insaturados:saturados não foi alterada pelos tratamentos (P>0.05). Não houve diferença no desempenho das crias entre os tratamentos nas fases pré ou pós-desmame (P>0,05). No experimento II, os dias necessários para o retorno à atividade ovariana, a porcentagem de animais que ovularam, o ECC, as concentrações de AGNE e LDL foram semelhantes (P>0,05) entre os tratamentos. Efeito linear crescente (P<0,05) foi observado nas concentrações de HDL e colesterol total, com a inclusão crescente de GS na ração das ovelhas em lactação. Houve efeito (P<0,05) do contraste GS vs. C para as concentrações de HDL e colesterol total, as quais foram maiores com a inclusão de GS. O efeito de semana (P<0,05) foi observado no CMS, AGNE, HDL, LDL e colesterol total, e houve interação tratamento x semana (P<0,05) nas concentrações de HDL e colesterol total. No experimento III 110 ovelhas secas e 64 borregas, ambas em gestação e da raça Santa Inês, foram alimentadas em grupos com rações contendo as relações de 30:70 e 40:60 de volumoso:concentrado (com base na matéria seca), os quais foram compostos por 30 e 40% de bagaço de cana-de-açúcar in natura, respectivamente, como fonte única de volumoso. Houve maiores consumos de MS, MO e GMD nas ovelhas alimentadas com a ração com 30% BC (P<0,05). Por outro lado, os consumos de PB, FDN e FDA não diferiram (P>0,05) entre os tratamentos. A utilização do bagaço de cana-de-açúcar in natura em até 40% na matéria seca supriu as exigências de manutenção e gestação e pode ser utilizado como fonte única de volumoso na ração total de ovelhas secas em gestação. A soja grão pode ser incluída em até 14% da matéria seca da ração total de ovelhas (40:60 de volumoso:concentrado) sem comprometer o desempenho das ovelhas em lactação. / The objectives of this trial were to evaluate blood parameters, productive and reproductive performance of lactating ewes fed increasing levels of soybean (GS), and nutrients intake and body weight variation in dry and gestating Santa Ines ewes fed in natura sugar cane bagasse (BC) levels as the sole roughage source. In experiments I and II 56 Santa Ines ewes were fed total mixed rations (TMR) composed of 0, 7, 14 and 21% of soybean (DM basis). In experiment I, dry matter intake and milk yield showed a quadratic effect (P<0.05). However, milk yield corrected for fat or for fat and protein and feed efficiency were not different (P>0.05). Milk fat, protein, lactose and total solids were similar (P>0.05) among treatments. Short (C4-C10) and medium (C11-C16) chain fatty acids were decreased while long chain (C18) fatty acids were increased linearlly (P<0.05). C18:2 c9 t11 and C18:2 t10 c12 (g/100g) increased linearly (P0,01) and the atherogenicity index decreased linearly (P<0.05) with increasing inclusion of ground soybean in the ration. Unsaturated:saturated ratio and saturated, unsaturated, monounsaturated and polyunsaturated fatty acids concentrations (g/100g) were similar among treatments (P>0.05). There was no difference (P>0.05) in lambs performance in pre or pos weaning period among treatments. In experiment II, BCS, NEFA and LDL concentrations were similar (P>0.05) among treatments. Days to return of the ovarian activity and ovulating animals percentage until the end of the trial were similar (P>0.05). Increasing linear (P<0.05) effect was observed in HDL and total cholesterol concentrations with the increasing GS inclusion in the ewes diet. There was effect (P<0.05) of the contrast GS vs. C for HDL and total cholesterol concentrations, which were higher with the inclusion of GS. Week effect (P<0.05) was observed in DMI, NEFA, HDL, LDL and total cholesterol, and interaction of treatment x week (P<0.05) was also detected in HDL and total cholesterol concentrations. In experiment III, 110 dry and gestating ewes and 64 gestating Santa Ines ewe lambs were fed in group a 30:70 or 40:60 (roughage:concentrate ratio), with the inclusion of 30 and 40% of sugar cane bagasse in the TMR dry matter, respectivelly. Dry and organic matter intake and average daily gain were higher (P<0.05) for the ewes fed 30% BC ration. However, CP, NDF and ADF intakes were similar (P>0.05) among treatments. In natura sugar cane bagasse inclusion up to 40% of the TMR dry matter as the sole roughage supplied the requirements of dry and gestating ewes. Raw soybean may be included in the ewesdiet (40:60 roughage:concentrate) up to 14% (DM basis) with no negative effect on performance and postpartum ovarian activity return.

Submitted by Celso Magalhaes (celsomagalhaes@ufrrj.br) on 2017-09-11T11:16:38Z No. of bitstreams: 1 2016 - Th?r?sse Camille Nascimento Holmstr?m.pdf: 1094101 bytes, checksum: a6473fdeeb8988cb28568635f529a54f (MD5) / Made available in DSpace on 2017-09-11T11:16:38Z (GMT). No. of bitstreams: 1 2016 - Th?r?sse Camille Nascimento Holmstr?m.pdf: 1094101 bytes, checksum: a6473fdeeb8988cb28568635f529a54f (MD5) Previous issue date: 2016-07-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / This study was developed in order to benchmark the performance between organic and conventional milk properties. For this, we analyzed values of livestock and economic performance of organic dairy farm in the state of S?o Paulo with values collected from a database of an industry that had 3259 dairy farms in the same region of the organic property. Data were compared using a benchmark tool. The independent variables identified in all the properties were size in hectares of effective area for milk production, number of lactating cows in the herd and daily milk production. The dependent variables were the zootechnical and economic performances featured in each property. The results of the zootechnical performance of organic property which has 25 hectares area for effective production of milk, dairy cows were 53,60, 2,14 cows per hectare, 1220 liters of daily milk production, 48,80 liter production of milk per area, 83,90% of lactating cows and 22,76 liters of milk per cow in milk, showing better results than conventional properties with similarity in the independent variables. Already the results for the economic margins of organic property were R$ 0,20 and R$ 89,060.00 for gross margins for daily and annual liter, respectively, R$ 0,21, R$ 93,513.00 and R$ 10,47 for banks net per liter per year per hectare, respectively. The economic results of organic property were R$1,00 average cost per liter of milk, -R$ 0,34 return on invested capital, R$ 1,15 Total revenue per liter of milk, R$ 191,989.14 and R$ 639,96 for cash flow in real and actual per hectare per month, respectively. The organic property does not sell milk in its natural form but in derivatives, which have added value and sales value higher than the milk based on CEPEA, which was the analysis parameter. This work also examined the cholesterol and fatty acids in organic milk property, with the results 9.64 ? 0,31mg / 100g cholesterol and the concentration of higher fatty acids in organic milk was palmitic (C16: 0) followed by elaidic (C18: 1 cis 9w9) and stearic (C18: 0). The results of the content of fatty acids in organic milk were 67.06 ? 0.93 mg/100 g for saturated fatty acids (SFA), 26.15 ? 1.10 mg/100g for monounsaturated fatty acids (MUFA) and 2.14 ? 0,15 mg /100g for polyunsaturated fatty acid (PUFA) on a dry matter basis. The values of the quantities of fatty acids represented by ?3 and ?6 were higher in organic milk 0.38 ? 0.04 mg/100h and 2.67 ? 0,41 mg/100 g, respectively. However, the ratio of omega was ?3 / ?6 was 0.19 and ?6/?3 was 22.97. The values of the ratios between the linoleic and linolenic fatty acids were C18: 2 ?6 cis/C18: 3 ?3 were 1.59 and C18: 3 ?3 / C18: 2 cis ?6 was 0.63. It was concluded that, from a technical and economic point of view, the organic system can have values of growth performance and economic compatible or even superior to conventional systems, whatever comparison or benchmark indicators employees. Given the above, it can be concluded that the production of milk in the organic model is sustainable, both from a technical and economic point of view. / O presente trabalho foi desenvolvido com o objetivo de avaliar comparativamente o desempenho entre propriedades de leite org?nica e convencionais. Para isso, foi analisado valores de desempenho zoot?cnico e econ?mico de uma fazenda de leite org?nico localizada no estado de S?o Paulo com valores coletados de um banco de dados de uma ind?stria que possu?a 3259 propriedades leiteiras na mesma regi?o da propriedade org?nica. Os dados foram comparados utilizando a ferramenta benchmark. As vari?veis independentes identificadas em todas as propriedades foram tamanho em hectare da ?rea efetiva destinada ? produ??o leiteira, n?mero de vacas lactantes do rebanho e produ??o leiteira di?ria. As vari?veis dependentes foram os desempenhos zoot?cnicos e econ?micos caracterizados em cada propriedade. Os resultados dos desempenhos zoot?cnicos da propriedade org?nica, que possui 25 hectares de ?rea para produ??o efetiva de leite, foram 53,60 vacas em lacta??o, 2,14 vacas por hectare, 1220 litros de produ??o di?ria de leite, 48,80 litros de produ??o de leite por ?rea, 83,90% de vacas em lacta??o e 22,76 litros de leite por vaca em lacta??o, mostrando resultados superiores aos das propriedades convencionais com similaridade nas vari?veis independentes. J? os resultados para as margens econ?micas da propriedade org?nica foram R$ 0,20 e R$ 89.060,00 para margens bruta por litro di?rio e anual, respectivamente, -R$ 0,21, -R$ 93.513,00 e -R$ 10,47 para margens l?quidas por litro, por ano e por hectare, respectivamente. Os resultados econ?micos da propriedade org?nica foram R$ 1,00 de custo m?dio por litro de leite, -R$ 0,34 de retorno sobre capital investido, R$ 1,15 de receita total por litro de leite, -R$ 191.989,14 e -R$ 639,96 para fluxo de caixa em reais e em reais por hectare por m?s, respectivamente. A propriedade org?nica n?o comercializa leite em sua forma natural e sim em derivados, que possuem valor agregado e valores de venda superiores ao do leite com base no CEPEA, o qual foi o par?metro de an?lise. Neste trabalho tamb?m foi analisado o colesterol e ?cidos graxos presentes no leite da propriedade org?nica, tendo como resultados 9,64 ? 0,31mg/100g de colesterol e a concentra??o de maior ?cido graxo no leite org?nico foi de palm?tico (C16:0), seguido pelo ela?dico (C18:1 cis 9 ? 9) e este?rico (C18:0). Os resultados do conte?do dos ?cidos graxos em leite org?nico foram 67,06 ? 0,93 mg/100g para ?cidos graxos saturados (AGS), 26,15 ? 1,10mg/100g para ?cidos graxos monoinsaturados (AGMI) e 2,14 ? 0,15mg/100g para ?cido graxo poliinsaturado (AGPI) em base de mat?ria ?mida. Os valores das quantidades dos ?cidos graxos representados pelos ?3 e ?6 foram altos no leite org?nico 0,38 ? 0,04mg/100g e 2,67 ? 0,41mg/100g, respectivamente. No entanto, a raz?o entre os ?megas foi ?3/?6 foi 0,19 e ?6/?3 foi de 22,97. Os valores entre as raz?es entre os ?cidos graxos linoleico e linol?nico foram C18:2 ?6 cis / C18:3 ?3 foram 1,59 e C18:3 ?3 / C18:2 ?6 cis foram de 0,63. ? poss?vel concluir que, do ponto de vista t?cnico e econ?mico, o sistema org?nico pode ter valores de desempenho zoot?cnico e econ?micos compat?veis ou at? superiores aos sistemas convencionais, quaisquer que sejam os indicadores de compara??o ou benchmark empregados. Diante do exposto, pode-se concluir que a produ??o de leite no modelo org?nico ? sustent?vel, tanto do ponto de vista t?cnico quanto econ?mico

Ho, Tik Shun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 88-102). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Table --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiii / Chapter CHAPTER 1 --- General Introduction --- p.1 / Chapter 1.1 --- Fatty Acid Synthase (FAS) - 7-domain multifunctional enzyme --- p.1 / Chapter 1.1.1 --- Functions --- p.1 / Chapter 1.1.2 --- Structure --- p.2 / Chapter 1.2 --- Fatty Acid biosynthesis reactions --- p.4 / Chapter 1.3 --- Malonyl Coenzyme A - An important mediator in lipogenesis --- p.7 / Chapter 1.4 --- FAS expression in different histotypes --- p.8 / Chapter 1.4.1 --- FAS in normal cells --- p.8 / Chapter 1.4.2 --- FAS in pathological cells --- p.8 / Chapter 1.4.3 --- Tumor-associated FAS (Oncogenic antigen-519) in cancer cells --- p.9 / Chapter 1.5 --- FAS signaling models in breast and prostate cancers --- p.12 / Chapter 1.5.1 --- Association between FAS and PI3K／Akt pathway --- p.12 / Chapter 1.5.2 --- Hypothetical model of FAS hyperactivity in breast and prostate cancer cells --- p.13 / Chapter 1.6 --- FAS inhibition to tackle cancer cell growth --- p.15 / Chapter 1.6.1 --- FAS inhibitors --- p.15 / Chapter 1.6.1.1 --- Cerulenin --- p.16 / Chapter 1.6.1.2 --- C75 --- p.17 / Chapter 1.6.2 --- Small interfering RNA --- p.17 / Chapter 1.7 --- FAS inhibition to enhance chemoresistant cancer cells sensitivity to drugs --- p.19 / Chapter 1.8 --- Hypothesis --- p.20 / Chapter CHAPTER 2 --- Methods and Materials --- p.21 / Chapter 2.1 --- Chemicals and antibodies --- p.21 / Chapter 2.2 --- Cell cultures --- p.21 / Chapter 2.3 --- MTT assay --- p.22 / Chapter 2.4 --- 5-Bromo-2'-deoxyuridine (BrdU)-labeling cell proliferation assay --- p.22 / Chapter 2.5 --- Cytotoxicity detection assay of LDH release --- p.23 / Chapter 2.6 --- DNA flow cytometry --- p.23 / Chapter 2.7 --- Confocal micocropy --- p.24 / Chapter 2.8 --- Immunoblot analysis --- p.24 / Chapter 2.8.1 --- Preparation of protein lysates --- p.24 / Chapter 2.8.2 --- Immunoblotting --- p.25 / Chapter 2.9 --- Caspase inhibitor studies --- p.26 / Chapter 2.10 --- Analysis of mitochondrial membrane potential --- p.26 / Chapter 2.11 --- Determination of caspase activities --- p.27 / Chapter 2.12 --- siRNA transfection --- p.27 / Chapter 2.13 --- Statistical analysis --- p.28 / Chapter CHAPTER 3 --- Results --- p.29 / Chapter 3.1 --- Cytostatic & cytotoxic studies of FAS inhibitors on human cancer cells --- p.29 / Chapter 3.1.1 --- Cerulenin and C75 suppress cell growth of different cancer histotypes --- p.29 / Chapter 3.1.2 --- Cerulenin and C75 suppress cell growth of A-375 dose- and time-dependently --- p.32 / Chapter 3.1.3 --- Cerulenin and C75 exert cytotoxic effect on A-375 but not normal skin HS68 cells --- p.36 / Chapter 3.1.4 --- Cerulenin and C75 arrest cell cycle progression and induce apoptosis with DNA Fragmentation --- p.39 / Chapter 3.2 --- Mechanistic studies of FAS inhibitors in A-375 cells --- p.46 / Chapter 3.2.1 --- Cerulenin and C75 induce caspase-dependent apoptosis --- p.46 / Chapter 3.2.2 --- Cerulenin- and C75-induced apoptosis involve extrinsic death receptor pathway --- p.52 / Chapter 3.2.3 --- Cerulenin- and C75-induced apoptosis involve intrinsic mitochondrial pathway --- p.57 / Chapter 3.2.4 --- Extrinsic death receptor pathway serves as a pioneer and links with intrinsic mitochondrial pathway in cerulenin- and C75-induced apoptosis --- p.65 / Chapter 3.3 --- Small interfering RNA on Fatty Acid Synthase (FAS siRNA) --- p.68 / Chapter 3.3.1 --- FAS siRNA induces PARP cleavage --- p.68 / Chapter 3.3.2 --- FAS siRNA triggers caspase-dependent apoptosis as FAS inhibitors --- p.70 / Chapter CHAPTER 4 --- Discussion --- p.72 / Chapter CHAPTER 5 --- Future Prospect --- p.85 / Chapter CHAPTER 6 --- References --- p.88

Realizou-se um estudo do processo descontínuo alimentado de fermentação, buscando encontrar as melhores condições de produção do ácido graxo gama-linolênico. Empregou-se como fonte de nitrogênio no meio de fermentação a uréia, que foi adicionada de forma exponencialmente crescente e como inóculo usou-se uma cepa de Spirulina platensis. Utilizou-se planejamento experimental e aplicou-se a metodologia de superfície de resposta, o qual permitiu a obtenção de equações que relacionaram as variáveis dependentes, como produtividade em células, conversão de nitrogênio em células, concentração máxima atingida de biomassa, porcentagem relativa de produção de ácido graxo gama-linolênico. com as variáveis independentes tempo de alimentação (Ta) e massa total de uréia adicionada (MUT). Os melhores resultados obtidos foram os seguintes: Px=117,26 mg/L.dia; Yx/n= 4,9; Xmax= 1759 mg/L e porcentagem de ácido graxo &#947;-linolênico na fração lipídica de 23,5 %, com 14 dias (Ta) e 1750 mg (MUT). / A Study on the fed-batch fermentation process was made seeking for the best Gamma-linolenic production condition. Mineral media was employed with urea as nitrogen source that was add with exponentially increasing feeding rates and Spirulina platensis as inoculum. Experimental design and a Response Surface Methodology were applied. It was composed by an experimental design which allowed a reduced number of the experiments and by the analysis of the results using equations which combined the interest variables dependent as: Productivity, best Gamma-linolenic production condition, Nitrogen convertion in Biomass, Highest Concentration and the study variables independent (Ta-feeding time and add urea-MUT). The best results were with (Ta-feeding time=14 days; add urea-MUT=1750 mg). In this conditions we had: Productivity=117,26 mg/L.day Best Gamma-linolenic production condition=23,5 per cent Nitrogen convertion in Biomass=4,9 Biggest Concentration.=1759 mg/L.

Red blood cells (RBC) have been shown to mediate plaque development seen in coronary artery disease (CAD). This study determined whether differences in RBC fatty acid (FA) composition were related to CAD risk. FAs were extracted from RBCs of 38 individuals who have undergone cardiac catheterization, 9 of whom had obstructive CAD, and analyzed via gas chromatography. Ferric reducing ability of plasma (FRAP) assay was used to determine oxidative stress. Food frequency questionnaires were used to correlate RBC omega-3 FA to daily intake of omega-3 FA. No correlation was found between RBC content and intake of omega-3 FA. FRAP values and RBC FA composition did not differ between the 2 groups with exception of the saturated FA, palmitic acid (p=0.018). These results suggest that RBC FA composition may differ between individuals with or at risk for CAD. Additional research is needed to validate this biomarker as a predictor of CAD.

N-acylethanolamines (NAEs) including anandamide are lipid derivative molecules, which play vital roles in physiological and developmental processes in plants and animals and mediate stress responses. In mammals, NAEs are synthesized from hydrolysis of their precursor molecule N-acylphosphatidylethanolamine (NAPE) by NAPE-specific phospholipaseD (NAPE-PLD). All NAEs including anandamide (NAE20:4) are hydrolyzed by fatty acid amide hydrolase (FAAH) into free fatty acid and ethanolamine. To date, different NAEs including anandamide have been identified in Physcomitrella patens but its metabolic pathway remains undiscovered. It is hypothesized that NAE metabolic pathway in P. patens is conserved and is similar to that of other eukaryotic systems. To this extent, putative PpNAPE-PLD and PpFAAH were identified and cloned for heterologous expression and characterization. Expression of PpFAAH was further verified by Western blot analysis. Future studies will involve biochemical characterization of putative PpNAPE-PLD and PpFAAH, to establish the evolutionarily conserved nature of NAE functions in early land plants.

N-acylethanolamines (NAEs) with C12-C18 acyl chain are ubiquitous in seed plants and play a role in mediating abscisic acid (ABA)-dependent or -independent responses to stress. In moss Physcomitrella patens, using selective lipidomics approach, we recently identified the occurrence of anandamide or N-arachidonylethanolamide (NAE 20:4) and its precursors that were previously not reported in plants. Occurrence of anandamide in moss provides us with a unique opportunity to address if early land plants retained NAE-mediated signaling mechanism that is akin to animals but not to vascular plants. It is hypothesized that a distinctive NAE profile and metabolic pathway occurs in P. patens. To this extent, putative genes that might be responsible for anandamide metabolic pathway were identified and their expression levels were determined for three developmental stages of moss. The NAE metabolite levels and transcript levels for putative genes were higher in protonema stage and anandamide showed higher growth inhibitory effects, chlorophyll reduction, and putative gene induction than NAE 12:0, compared to ABA, when applied exogenously.

Isovaleric Acid (IVA) is a 5-carbon branched chain fatty acid present in fermented foods and produced by the fermentation of leucine by colonic bacteria. IVA activates G-protein coupled receptors such as FFAR2, FFAR3, and OR51E1 known to be expressed on enteric neurons and enteroendocrine cells. We previously reported that the shorter, straight chain fatty acids acetate, propionate and butyrate, differentially affect colonic propulsion; however, the effect of branched chain fatty acids on gastrointestinal motility is unknown. We hypothesize that IVA relaxes smooth muscle in a cAMP/PKA dependent manner by direct action on smooth muscle cells. IVA will also decrease peristalsis and encourage retention of luminal contents. This thesis investigates the effect of IVA on smooth muscle tension and peristaltic activity in isolated colon and individual smooth muscle cells. Colon segments from C57BL/6J mice were placed in a longitudinal orientation in organ baths in Krebs buffer and fastened to force transducers. Segments were contracted with 10 μM acetylcholine (ACh) and the effects of IVA at several concentrations were measured in the absence and presence of Nitric Oxide Synthase inhibitor L-N-nitroarginine (L-NNA), neuronal action potential inhibitor tetrodotoxin (TTX), and adenylate cyclase inhibitor SQ22536. To study individual live cells, mouse smooth muscle was isolated from colon, suspended in smooth muscle buffer, and after contraction with ACh were relaxed with micromolar concentrations of IVA. For peristalsis studies, whole colonic segments isolated from C57BL/6J were catheterized and placed horizontally in organ baths with circulating Krebs buffer. The colon was clamped on the anal end, and a solution (5 μL per mm of colon length) of either Krebs buffer or 50 mM IVA was delivered from the oral end to the lumen. Video of the peristalsis was then analyzed for diameter, changes in diameter, velocity of diameter changes along the length of the colon, normalized to the anatomical changes in the proximal region. IVA in concentrations of 10 mM to 50 mM relaxed the ACh-induced contraction in a sigmoidal fashion. In separate studies, L-NNA nor TTX affected the ability of IVA to inhibit relaxation. SQ22536 inhibited IVA induced relaxation in longitudinal colon compared to vehicle control. In isolated cells, SQ22536 and PKA inhibitor H-89 inhibited IVA-induced relaxation. In peristalsis studies, 50 mM IVA in Krebs buffer changed the character of the peristaltic action by increasing proximal diameter, inhibiting contractions in the proximal end of the colon, and decreasing overall velocity of peristaltic contractions in the proximal region. The data indicate that the branched chain fatty acid IVA causes a concentration-dependent relaxation of colonic smooth muscle that is direct to the smooth muscle and independent of neuronal activity. This relaxation is cAMP/PKA dependent. In addition to the direct relaxation of smooth muscle, intraluminal IVA decreased overall colonic propulsive activity and encouraged retention of the luminal contents. We conclude that the ingestion and production of branched chain fatty acids could affect overall GI motility and is an area for study in dietary and therapeutic control of bowel activity.

Neisseria meningitidis, Auslöser der Meningokokken-Meningitis und Sepsis, trägt auch heute noch zur hohen Kindersterblichkeit in Entwicklungsländern bei und sorgt, vor allem im afrikanischen Meningitis-Gürtel, immer wieder für Epidemien mit gravierenden Folgen für die Betroffenen. Im Rahmen dieser Arbeit wurden zwei an der Pathogenität von N. meningitidis beteiligte Proteine, der Transkriptionsregulator FarR und der Transportkanal HrpB, näher charakterisiert, um weitere Einblicke in die immer noch nicht vollständig entschlüsselte Pathogenese der Meningokokken-Meningitis zu erhalten. Das Neisseria adhesin A NadA ist Bestandteil der sich aktuell in der Entwicklung befindenden Impfung gegen Meningokokken der Serogruppe B. Im dem bekapselten B-Stamm MC58 wurde gezeigt, dass nadA unter der negativen Kontrolle des Transkriptionsregulators FarR steht (Schielke et al., 2009). In den ebenfalls zur Gattung Neisseria gehörenden Neisseria gonorrhoeae (Ng) wurde bereits 2001 ein FarR-Homolog beschrieben (Shafer et al., 2001). NgFarR ist an der Resistenz gegenüber antimikrobiellen, langkettigen Fettsäuren beteiligt, indem es die Expression des FarABEffluxpumpen-Systems reguliert, welches eingedrungene Fettsäuren wieder nach extrazellulär befördert. Dagegen zeigten Palmitinsäure-Resistenztests, dass FarR nicht an der intrinsischen Fettsäure-Resistenz der Meningokokken beteiligt ist. Die Deletion und die Komplementierung von farR hatten weder in bekapselten noch in unbekapselten Meningokokken Einfluss auf das normale Wachstumsverhalten. Ein Western Blot- Nachweis des FarR-Proteins in der frühen, mittleren und späten exponentiellen Wachstumsphase von Wildtyp, Kapsel-Deletionsmutante und farR-Komplementante zeigte, dass die Menge an FarR im zeitlichen Verlauf kontinuierlich zunimmt und FarR damit Wachstumsphasen-abhängig exprimiert wird. Dabei scheint es einer posttranskriptionalen oder posttranslationalen Regulation zu unterliegen, da auch in dem farRkomplementierten Stamm unabhängig vom farR-Promotor eine entsprechende Hochregulation stattfindet. In Infektionsversuchen wurde die Interaktion zwischen Meningokokken und humanen polymorphkernigen Granulozyten untersucht. In den Infektionsassays wurde die farRDeletionsmutante innerhalb des dreistündigen Versuchsrahmens deutlich stärker durch die Granulozyten abgetötet als der Serogruppe B-Wildtyp. Als Mitglied der in Bakterien und Archaeen weit verbreiteten Familie der MarR-Transkriptionsregulatoren (Multiple antibiotic resistance Regulator, MarR) bindet FarR mit hoher Wahrscheinlichkeit auch als Homodimer an seine Bindesequenz auf der DNA. FarR erkennt eine 16 bp lange, palindromische Sequenz in der Promotorregion von nadA (NMB1994), wodurch die nadA-Expression verhindert wird. Außerdem erkennt FarR eine ähnliche Bindesequenz im Promotorbereich von farAB (NMB0318/0319), wobei es aber keinen regulatorischen Einfluss ausübt. Mit einer aus diesen beiden Bindestellen berechneten minimalen Bindesequenz wurde im Genom von MC58 weitere mögliche Bindepartner detektiert. Eine Auswahl dieser möglichen Bindestellen wurde in Electrophoretic Mobility Shift Assays auf eine direkte Interaktion mit dem FarR-Protein hin untersucht, wobei sich allerdings keine direkte Bindung nachweisen ließ. Diese Ergebnisse darauf hin, dass der Transkriptionsregulator FarR hoch spezifisch bestimmte DNA-Bindesequenzen erkennt und die entsprechenden Gene reguliert. In der Promotorregion des TpsB-Proteins HrpB wurde in den sequenzierten Referenzstämmen Z2491, MC58, FAM18 und α14 eine mit der minimalen FarR-Bindesequenz kompatible Sequenz gefunden. In Electrophoretic Mobility Shift Assays konnte allerdings gezeigt werden, dass FarR nicht direkt daran bindet. Um das Transport-Protein HrpB näher zu charakterisieren, wurde das entsprechende Gen in 22 N. meningitidis-Isolaten sequenziert. Dabei zeigte sich, dass das Transportprotein hrpB in allen untersuchten invasiven und nicht-invasiven Stämmen vorhanden ist. Dieses äußerst konservierte Protein weist nur im seinem C-terminalen Bereich eine relativ variable Region auf, was vermutlich auf Rekombinationsereignisse zurückzuführen ist. Ein Alignment der Aminosäure-Sequenz des Serogruppe C-Stamms FAM18 mit der des homologen Bordetella pertussis TpsB-Proteins FhaC zeigte, dass die dreidimensionale Struktur des HrpB ebenfalls eine α-Helix, eine transmembranöse Domäne und variable extrazelluläre Loops enthält. Zusammengenommen erfüllt HrpB somit wichtige Bedingungen, um als Vakzine-Bestandteil in Betracht gezogen zu werden. / Function of the transcriptional regulator FarR in Neisseria meningitidis