Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Orientador: Elisabeth Criscuolo Urbinati / Coorientador: Fábio Sabbadin Zanuzzo / Banca: Marco Antonio de Andrade belo / Banca: Janessa Sampaio de Abreu / Resumo: Manejos inerentes da piscicultura intensiva, como o transporte, desencadeiam resposta de estresse nos animais, podendo causar perdas na produtividade. Como alternativa, o β-glucano, um polissacarídeo derivado da parede celular de cereais, bactérias e fungos, vem sendo muito utilizado na aquicultura pelo seu efeito imunoestimulante. Há evidências de que o β-glucano minimiza os efeitos negativos do estresse por atuar no sistema imune, porém é pouco investigado o seu efeito direto sobre a resposta clássica de estresse. Neste contexto, o presente estudo avaliou, em juvenis de pacu, o uso oral de 0.1% de duas gerações de β-glucano, de diferentes graus de pureza e processamentos, na resposta de estresse e no sistema imune inato, após transporte e inoculação com Aeromonas hydrophila. Avaliamos a concentração de cortisol e glicose plasmáticos como indicadores da resposta de estresse, a atividade respiratória de leucócitos, a atividade hemolítica do sistema complemento, a atividade de lisozima e contagem total e diferencial de leucócitos, como indicadores do sistema imune inato, e o hematócrito, número de eritrócitos, concentração de hemoglobina e volume corpuscular médio de eritrócitos como indicadores hematológicos. As duas gerações de β-glucano utilizadas modularam os níveis de cortisol circulantes, mantendo os níveis elevados até 24 horas após o transporte, sem alteração após o desafio bacteriano. O β-glucano aumentou a atividade hemolítica do sistema complemento e de lisozima apó... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Inherent handling in intensive fish farming, such as transport, trigger stress response in animals, and may cause losses in productivity. As an alternative, the β-glucan, a polysaccharide derived from the cell walls of cereals, bacteria and fungi, has been widely used in aquaculture due their immunostimulatory effect. The β-glucan minimizes the negative effects of stress by acting on the immune system, but it is little investigated its direct effect on the classical stress response. In this context, the present study evaluated in pacu, the oral administration of 0.1% of two generations of β-glucan, with different degrees of purity and processing methods, on the stress and innate immune system responses, after transport and inoculation with Aeromonas hydrophila. We evaluated the plasma concentration of cortisol and glucose, as stress response indicators, the respiratory activity of leukocytes, the hemolytic activity of the complement system, lysozyme activity and total and differential counts of leukocytes as innate immune system indicators, and hematocrit, number of erythrocytes, hemoglobin concentration, and mean corpuscular volume of erythrocytes as hematological indicators. The two generations of β-glucan modulated the circulating levels of cortisol, keeping the high levels 24 hours after transportation, without changes after the bacterial challenge. The β-glucan increased the hemolytic activity of the complement system and lysozyme after handling and after bacterial inoculation, and kept the population of circulating leukocytes after recovery of leukopenia, demonstrating the immunostimulatory effect. The general results of this study suggest the strengthening of the immune response of pacu juveniles fed for 15 with feed containing 0,1% β-glucan derived from yeast cell wall (Sacaromyces cerevisiae) days before stressful ... (Complete abstract click electronic access below) / Mestre

Orientador: Elisabeth Criscuolo Urbinati / Banca: Sérgio Fonseca Zaiden / Banca: Marco Antonio de Andrade Belo / Banca: João Batista Kochenborger Fernandes / Banca: Maria José Tavares Ranzani de Paiva / Resumo: Este trabalho avaliou o efeito da administração oral de β-glucano no crescimento, no aproveitamento do alimento, na dinâmica metabólica e nas respostas imunes inatas de juvenis de pacu (Piaractus mesopotamicus) submetidos a ciclos curtos de restrição alimentar e realimentação, e a ação imunoestimulante de β-glucano oferecido por 15 dias na fase final dos ciclos. Um total de 210 peixes foi distribuído em 21 tanques e submetido a diferentes estratégias de alimentação durante sete semanas: GC - Alimentação todo dia com ração comercial (RC); G 6/1- Alimentação com RC por 6 dias e jejum por 1 dia (6/1); G 2/1 - Alimentação com RC por 2 dias e jejum por 1 dia (2/1); GCS - Alimentação todo dia com RC, e com suplementação de 0,5% de β-glucano (RCS), nos últimos 15 dias; GS 6/1 - Alimentação com RC por 6 dias e 1 dia de jejum, e RCS nos últimos 15 dias; GS 2/1 - Alimentação com RC, por 2 dias de alimentação e 1 dia de jejum, e RCS nos últimos 15 dias. Um sétimo grupo (GC-PBS) foi alimentado todos os dias para controle do efeito da manipulação e do veículo da inoculação bacteriana. No dia 50, nove peixes de cada tratamento foram anestesiados com benzocaína (1,0g/15L de água) e pesados antes da obtenção de amostras de sangue, fígado, intestino anterior e músculo branco. Foram determinadas a taxa de crescimento especifico (TCE), a conversão alimentar (CA), a morfologia da mucosa intestinal, a concentração de metabólitos circulantes e teciduais, a atividade respiratória dos leucócitos (AR... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of this study was to evaluate the effect of oral administration of β-glucan on growth, feed utilization, metabolic dynamics and the innate immune responses of pacu (Piaractus mesopotamicus) subjected to short-term starvation and refeeding and the immunostimulatory action of β-glucan offered for 15 days in the final phase of the cycle. A total of 210 fish was distributed in 21 tanks and submitted to different feeding strategies for seven weeks: GC - feeding daily with commercial feed (RC); G 6/1- 6-day feeding with RC and 1 restraint day (6/1); G 2/1 - 2-day feeding with RC and 1 restraint day (2/1); GCS - Feeding daily with RC and supplemented with 0.5% β-glucan (RCS) in the last 15 days; GS 6/1 - 6-day feeding with RC and 1 restraint day, and RCS in the last 15 days; GS 2/1 - 2-day feeding with RC and 1 restraint day, and RCS in the last 15 days. A seventh group (GC-PBS) was fed every day to control the effect of the handling and bacterial inoculation (injected PBS control). On day 50, nine fish of each treatment were sampled, weighed and anaesthetized with benzocaine (1.0g / 15L water) for blood, liver, white muscle and foregut sampling, to determine the specific growth rate (SGR), feed conversion (CA), morphology of intestinal mucosa, concentration of circulating and tissue metabolites, leukocyte respiratory burst, serum lysozyme concentrations, serum hemolytic activity of complement system (alternative pathway), total leukocyte differential count and intest... (Complete abstract click electronic access below) / Doutor

Um papel importante foi atribuído ao receptor toll-like 4 (TLR4) no desenvolvimento da placa aterosclerótica. O TLR4 foi primeiramente descrito como um receptor para bactérias gram-negativas; posteriormente foi demonstrado que sua expressão está aumentada em placas ateroscleróticas e que pacientes que possuem um polimorfismo disfuncional do TLR4 são menos suscetíveis ao desenvolvimento dessa doença. Portanto, o objetivo desse estudo foi o de investigar, em um modelo experimental de aterosclerose, a influência da deleção do TLR4 na formação e morfologia da placa aterosclerótica, no perfil lipídico e em marcadores inflamatórios. Camundongos duplo knockout (DKO), deficientes no receptor de LDL e TLR4, foram gerados cruzando-se camundongos deficientes para o receptor de LDL (LDLrKO) com camundongos deficientes para o TLR4 (TLR4KO). Todos os grupos receberam dieta rica em gordura e colesterol por 12 semanas. As concentrações plasmáticas de colesterol e triglicérides foram medidas por ensaio colorimétrico. Cortes seriados da raiz aórtica foram corados com Oil red O e as áreas de lesão quantificadas por analisador de imagens. O colágeno foi medido por coloração de picrossirius. A formação de nitrotirosina e expressão de CD40L, MMP9 e iNOS nas placas foram feitas por imunohistoquímica. As comparações foram feitas por ANOVA com pós teste de Student Newman-Keuls. Os dados foram expressos como média ± EPM. Camundongos DKO desenvolveram placas menores que camundongos LDLrKO (117.6 ±1.4 vs 198.8 ± 3.3 104m2). Camundongos TLR4KO não formaram placa. As placas dos camundongos DKO apresentaram menor núcleo lipídico que as dos LDLrKO (76.2± 13.2 vs 161.7 ± 2.9 104m2). O colágeno ao redor do núcleo lipídico é maior nos camundongos DKO do que nos LDLrKO (24.9 ± 1.8 vs 16.5 ± 2.5 % da placa). A distribuição do colágeno nos camundongos DKO ocorre principalmente ao redor da placa, de forma mais organizada, enquanto que nos LDLrKO onde sua distribuição é mais difusa. As placas dos camundongos DKO apresentaram menor expressão de CD40L e iNOS do que as dos LDLrKO (13.1 ± 0.7 vs 18.5 ± 2.5 AU e 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectivamente). A expressão de MMP9 foi menor nas placas dos camundongos DKO do que as dos LDLrKO (2.99 ± 0.3 vs 1.99 ± 0.2 AU). A marcação para nitrotirosina foi maior nos camundongos LDLrKO quando comparada com as dos grupos DKO e TLR4KO (142.89 ± 208.5, 77.16 ± 227.7 e 71.73 ± 95.9 10m2, respectivamente). Todos esses resultados sugerem que o processo inflamatório é menor na ausência do TLR4. As concentrações plasmáticas de colesterol não foram diferentes entre os grupos LDLrKO e DKO mas os camundongos LDLrKO apresentaram concentrações plasmáticas de triglicérides maiores do que os camundongos DKO após a dieta (265.2 ± 27.6 vs 150.5 ± 8.8 mg/dL). O receptor toll-like 4 influencia na estrutura e formação da placa aterosclerótica independentemente dos níveis séricos de colesterol / A crucial role has been suggested for toll-like receptor 4 (TLR4) in atherosclerotic plaque formation and development. TLR4 was described primarily, as a receptor for gram-negative bacteria lipopolisacharide; later it was showed that its expression is increased in atherosclerotic plaques and patients that carries a TLR4 dysfunctional polymorphism are less susceptible to development of this disease. Therefore, the aim of this study was to investigate, in an experimental model of atherosclerosis, the influence of TLR4 deletion in atherosclerotic plaque formation and morphology, cholesterol profile and inflammatory markers. Double knockout mice (DKO), deficient in LDL receptor and TLR4, were generated by breeding LDL receptor knockout mice (LDLrKO) with TLR4 knockout mice (TLR4KO). All three experimental groups, LDLrKO, TLR4KO and DKO were fed a high fat-cholesterol diet for 12 weeks. Plasma cholesterol and triacylglicerol concentrations were measured by colorimetric assay. Cross sections of aortic sinus were stained with Oil red O and lesion areas were quantified by an image analyzer. Collagen content was measured by picrossirius staining. We also measured nitrotyrosine formation, CD40L, MMP9 and iNOS expression by immunohistochemistry. Comparisons were made by ANOVA followed by Student-Newman-Keuls post- test. Data are mean ± SEM. DKO mice developed smaller plaques than LDLrKO mice (117.6 ±1.4 vs 198.8 ± 3.3 104m2). TLR4KO mice developed no plaque. Plaques from DKO mice have also a smaller lipid core than the ones from LDLrKO mice (76.2± 13.2 vs 161.7 ± 2.9 104m2). Collagen content around the lipid core is higher in DKO mice compared to LDLrKO mice (24.9 ± 1.8 vs 16.5 ± 2.5 % of the whole plaque). Interestingly, collagen distribution in DKO mice seems to occur mainly on the plaque periphery, in a more organized manner, while in LDLrKO mice it is fuzzier, being present also inside the plaque. Plaques from DKO present lower expression of CD40L and iNOS than LDLrKO mice (13.1 ± 0.7 vs 18.5 ± 2.5 AU and 7.7 ± 0.9 vs 10.2 ± 0.4 AU, respectively). MMP9 expression is lower in DKO mice as compared to LDLrKO mice (2.99 ± 0.3 vs 1.99 ± 0.2 AU). Nitrotyrosine staining was higher in LDLrKO mice as compared to DKO and TLR4KO groups (142. 89 ± 208.5, 77.16 ± 227.7 and 71.73 ± 95.9 10m2, respectively). All together, these findings suggest that inflammatory process is milder in the absence of TLR4. Serum cholesterol were not different between LDLrKO and DKO mice but LDLrKO presented higher triacylglicerol serum levels after 12 weeks on high fat high cholesterol diet as compared to DKO mice (265.2 ± 27.6 vs 150.5 ± 8.8). Toll like receptor 4 influences atherosclerotic plaque formation and structure independently from serum cholesterol levels

Neisseria lactamica está envolvida na aquisição da imunidade natural contra Neisseria meningitidis, causadora da doença meningocócica. Vesículas de membrana externa (OMV) de N. lactamica são antígenos potenciais contra N. meningitidis. Analisou-se a cinética de biomassa, de produção de OMV, da fonte de carbono (lactato), e dos metabólitos, para maximizar a produção de OMV. Realizaram-se ensaios em biorreator, em batelada simples, batelada alimentada com lactato, com ou sem pulsos de aminoácidos e extrato de levedura (YE). Utilizou-se o meio de Catlin (MC) como meio mínimo, e analisaram-se efeitos das concentrações do lactato, aminoácidos e YE. Lactato foi consumido e citrato e acetato produzidos. Os melhores resultados obtidos foram no meio com adição de 2 g/L de YE e concentrações dobradas de lactato e 5 aminoácidos constitutivos do MC, em batelada alimentada com pulsos. O lactato apresentou efeito positivo sobre o rendimento de OMV e o YE sobre a biomassa. Os 5 aminoácidos constitutivos do MC foram necessários para obtenção de biomassa e rendimento de OMV. / Neisseria lactamica is involved with the acquisition of natural immunity to Neisseria meningitidis. N. lactamica outer membrane vesicles (OMV) are potential antigens against N. meningitidis, the pathogen of meningococcal disease. The objective of this work was to analyze the kinetics of bacterial growth, OMV production, the carbon source (lactate), and products of metabolism, to improve growing conditions and OMV production. Groups were studied in batch process, fed-batch process with lactate, fed-batch process with pulses of amino acids and YE. MC was considered as minimal medium and it was analyzed the effect of lactate, amino acids and YE. Lactate was consumed and citrate and acetate increased in the medium. The best results were in fed-batch with pulses, in MC with the double concentrations of lactate and amino acids, added with 2 g/L of YE. The lactate had a positive effect over OMV yield and YE had a positive effect over biomass. 5 amino acids of MC were necessary to obtain biomass and OMV yield.

Abstract Chlamydia pneumoniae is an obligatory intracellular pathogen that causes upper and lower respiratory tract infections. Like other Chlamydial species, also C. pneumoniae has a tendency to cause persistent infections, which have been associated with different cardiovascular, neurological, and respiratory diseases. In addition, a few studies have reported an association between C. pneumoniae seropositivity and an elevated body mass index (BMI), and it has been shown that C. pneumoniae is capable of infecting preadipocytes and adipocytes. The main aims of this study were to study if certain gene polymorphisms regulate the serum levels of innate immunity and inflammation proteins, and if the polymorphisms are associated with markers of C. pneumoniae infection; to compare different methods in detection of C pneumoniae in atherosclerotic tissue; and to study if serum levels of chlamydial LPS (cLPS) are associated with BMI. The serum levels of inflammatory and innate immunity markers, namely interleukin 6 (IL-6), C-reactive protein (CRP), LPS-binding protein (LBP), and soluble CD14, in apparently healthy individuals were found to correlate with each other and possibly be regulated by the polymorphisms of genes important in inflammation and innate immunity. Especially the serum LBP levels may be regulated by the LBP (rs2232618) and toll-like receptor 4 (rs4986790) polymorphisms. The IL-6 (rs1800795) polymorphism was found to be associated with C. pneumoniae antibody positivity. C. pneumoniae DNA and cLPS could be found from atherosclerotic tissue. A new, cLPS enzyme immunoassay method was developed in this study, and it might provide a standardized, commercial method for the detection of chlamydia in tissue samples, if the sensitivity of the method could be increased e.g. by testing multiple pieces of tissue. In situ hybridization method was found to be complicated by technical problems and the repeatability of polymerase chain reaction was poor. C. pneumoniae IgG positivity and elevated serum cLPS and CRP levels were associated with an elevated BMI. There was also a strong association between cLPS levels and inflammation as measured by CRP levels. The lack of association between serum total endotoxin activity and BMI implies that the association between infection and an elevated BMI may be specific to certain pathogens.

Thesis (M. Sc. (Chemistry)) -- University of Limpopo, 2021 / Tuberculosis is one of the top 10 leading causes of death in the world. The development of drug resistant strains of Mycobacterium tuberculosis such as Multidrug resistant (MDR) and Extensively drug resistant (XDR) strains further complicate the TB control. Medicinal plants present a possible source for new potential antitubercular drugs. They have played an important role in drug discovery, with many pharmaceutical products originating from them. Isolation and characterisation of new antitubercular compounds from plant extracts is relevant today because of the development of resistant strains. The aim of the study was to evaluate the antimycobacterial activity of the leave extracts of Croton gratissimus and Leonotis leonorus. The first step was to extract fine powder leaves of the two plant species using four (dichloromethane, acetone, hexane and ethanol/water) different solvent systems. Isolation of the fractions was done using column chromatography and preparative thin layer chromatography. Minimum inhibitory concentrations were determined using the broth dilution method and the values were recorded in μg/mL. All the isolated fractions from both plant species were evaluated for preliminary in-vitro antimycobacterial activity. Some of the isolated fractions showed an increased activity against the pathogen as compared to the crude extracts. All the crude extracts of the two plants had activity with MIC90 values greater than 125 μg/mL. Seven fractions obtained from Croton gratissimus showed potential activity against the pathogen with MIC90 values ranging from 30.61 to 64.88 μg/mL. Leonotis leonurus had three fractions with promising activity with MICs ranging from 1.963 to 62.51 μg/mL. The crude extracts of the two plant species showed that the two plant species have antioxidant properties. The qualitative antioxidant assay showed that DCM crude extracts had more antioxidants than all other extracts because of more clear zones against the purple background colour on the TLC plates. These was confirmed by the qualitative antioxidant assay where DCM crude extracts was able to inhibit the highest percentage of DPPH at different concentrations than all other solvent extracts. The DCM crude extracts of L. leonurus and Croton gratissimus inhibited 87 and 93 % of DPPH respectively at 250 μg/mL. The structures of the compounds within the isolated fractions were elucidated using NMR and confirmed by MS and FTIR spectroscopies. The NMR data showed that the isolated fractions were not pure compounds but mixtures of closely related compounds. The compounds whose structures were elucidated included two labdane diterpenoids (Croton A and Croton B) and a Cembranolide ((5E,10E,13R)-4-isopropyl-7,11-dimethyl-15-oxo-14-oxa-bicyclo [11.2.1] hexadeca-5,10-dien-7-yl acetate) from Croton gratissimus and a phenol (4-(3,3,4,4-tetramethylheptyl) benzene-1,2-diol)) from Leonotis leonurus. / National Research Foundation (NRF) and Sasol Foundation

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