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In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and <i>Aspergillus ficuum</i>. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of <i>Lactobacillus plantarum</i> as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-Ò-hydroxamate (DL-ASP), Ò -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of <i>A. ficuum</i>, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in <i>A. ficuum</i> compared to five other nitrogen sources tested.

In the animal feed industries, there is a global need for adding certain nutritional ingredients to augment deficits usually associated with plant-based materials. As a result, the industrial practices require direct addition of ingredients such as amino acids and vitamins. One of the key ingredients in this context is lysine. Alternately, the same goal can be achieved indirectly through in situ co-culturing of microorgan-isms. The focus of this thesis was genetic improvement of bacterial and /or fungal mutants, which could over-produce lysine. The accumulation of free lysine during microbial growth serves this end based on de-regulation of the lysine biosynthetic pathway. Microorganisms used in this thesis were nine species of lactobacilli and <i>Aspergillus ficuum</i>. Having in mind the highly complex nutritional requirements of lacto-bacilli, the assessment of possible lysine auxotrophy was performed. No lysine auxotrophs were found and the choice of <i>Lactobacillus plantarum</i> as the working species among nine others was based on its higher growth rate in minimal medium. Selection of mutants that overproduced lysine was carried out in the minimal medium supplemented with the following lysine analogs: S-aminoethyl-L-cysteine (AEC), DL-aspartic acid-Ò-hydroxamate (DL-ASP), Ò -fluoropyruvic-acid (FPA), L-lysine hydroxamate (LHX) and diaminopimelic acid (DAP). In L. plantarum, LHX was shown to be the most potent inhibitor; although, the bacterium demonstrated high resistance to all the analogs tested. The inhibition by LHX was obtained only after significant alteration of the minimal medium M3. Furthermore, the mutant # 34, resistant to 2 mM of LHX, secreted only 4.52 £gM of lysine in M3. To address the question of low lysine yield obtained by L. plantarum, thorough study of the regulation of aspartokinase (AK) was performed. It was found that AK exists as four isozymes, threonine sensitive, methionine sensitive and two lysine sensitive isozymes. Activity differed with respect to the growth stage of L. plantarum. Beside lysine, threonine and methionine have influenced the repression of AK isozymes, which suggested that effective lysine over-production could be obtained only if AK is simultaneously resistant to threonine and methionine analogs. In the case of <i>A. ficuum</i>, mutant #5-10 secreted 29.25 £gM of lysine in the minimal medium, which was approximately 30 % higher than that of the wild type. DL-ASP was found as the most potent inhibitor only after the conidia were soaked for 8 h in 0.03 % Tween 80. Ammonium phosphate as a nitrogen source enhanced lysine secretion in <i>A. ficuum</i> compared to five other nitrogen sources tested.

No description available.

Background: Approximately 40% of global human immunodeficiency virus-1 (HIV) transmission occurs in the female genital tract (FGT). Epithelial cells lining the FGT comprise the first barrier to HIV-1 entry. The functions of these cells are influenced by female sex hormones and the mucosal microbiota. Studies have suggested that hormonal environment and a dysbiosis of the FGT microbiota may lead to inflammation in the genital mucosa and enhance HIV acquisition. A Lactobacillus dominant microenvironment in the FGT is considered to have protective functions against sexually transmitted pathogens, however the interaction between sex hormones and lactobacilli and their effect on epithelial cell functions remains to be determined. Methods of Study: For these studies, primary genital epithelial cells (GECs) were isolated from hysterectomy tissues obtained following patient consent. GEC cultures were grown to confluence on cell culture inserts in the presence or absence of the female sex hormones estrogen (E2), progesterone (P4), or medroxyprogesterone acetate (MPA). Polarized monolayers were exposed to two probiotic strains of Lactobacillus: L. reuteri (RC-14) or L. rhamnosus (GR-1), or the most common strain of bacteria found in the FGT, L. crispatus in the presence or absence of HIV-1. Cell viability, barrier integrity, and innate inflammatory factors were among the primary measures performed. Results: In our system, cell viability was unaltered in the presence of Lactobacillus species and/or female sex hormones. All three strains of bacteria (L. crispatus and probiotic lactobacilli GR-1 and RC-14) significantly increased GEC barrier integrity, as measured by transepithelial electrical resistance (TER). Both GR-1 and RC-14 significantly reduced GEC barrier permeability as measured by a dextran dye leakage assay, whereas L. crispatus did not. Conversely, hormones did not alter barrier integrity nor barrier permeability. However, hormones did alter secretion of cytokines and chemokins by GECs. GECs grown in the presence of estrogen decreased TNF-α, IL-1α, IL-1β and IL-8 secretion in comparison to no hormone treatment, while GECs grown in the presence of MPA significantly decreased MIP-1α and TNF-α secretion. In the presence of HIV both GR-1 and RC-14 were able to confer an increase in barrier integrity similar to that observed with GR-1 and RC-14 treatment alone. Addionally, GECs grown in the presence of E2 and MPA displayed a less inflammatory (TNF-α, IL-1α, and IL-1β) environment when exposed to HIV compared to no hormone and P4. Interstingly, the decrease in inflammation was not observed when measuring chemokines such as IL-8 and RANTES. Furthermore, probiotic bacteria were able to significantly reduce HIV mediated increases in TNF-α when grown in the presence of no hormone, P4, and MPA. A similar trend was observed for GECs grown in the presence of E2 however, given that E2 reduced the TNF-α response mediated by HIV, results were not significant. Overall, probiotic lactobacilii GR-1 and RC-14 enhanced GEC barrier functions while E2 and MPA appeared to exert an anti-inflammatory effect on epithelial cell innate responses in both the presence and absence of HIV. Conclusions: In our system, probiotic lactobacilli enhanced GEC barrier functions and estrogen appeared to exert an anti-inflammatory effect on epithelial innate responses. Enhanced barrier function and decreased inflammation correlate with decreased in HIV acquisition and replication. These studies provide an insight into how factors in the genital microenvironment can affect HIV acquisition in the FGT, and will subsequently assist in the development of prophylactic strategies to reduce HIV transmission. / Thesis / Master of Science (MSc) / Approximately 40% of global HIV transmission occurs in the female genital tract. Although women make up more than 50% of infected individuals worldwide, the details regarding how HIV infection starts in the female genital tract remains poorly understood. The cells that line the genital tract are the first barrier against HIV entry. These cells are influenced by common factors within the genital tract microenvironment such as female sex hormones and natural bacterial populations. Previous studies have suggested that certain hormonal contraceptives or a build-up of pathogenic bacteria within the genital tract, leads to an inflammatory microenvironment and may enhance HIV acquisition. Comparatively, ‘good bacteria’ within the microenvironment have been shown to have protective effects against sexually transmitted infections. For this study, we were interested in understanding how different hormones (estrogen, progesterone and progesterone based hormonal contraceptives) and ‘good bacteria’ (specifically probiotic strains of lactobacilli), affect the cells that line the genital tract and local inflammation in the presence and absence of HIV. Therefore, we obtained cells that line the genital tract (epithelial cells) from women undergoing hysterectomies. The cells were grown in the presence or absence of hormones, exposed to ‘good bacteria’ and then challenged with HIV. In our system, probiotic lactobacilli enhanced genital epithelial cell barrier functions and estrogen appeared to exert an anti-inflammatory effect on epithelial cells. Furthermore, when genital epithelial cells were pre-treated with lactobacilli and exposed to HIV, lactobacilli treatment was able to protect against HIV mediated barrier disruption. Lactobacilli treated genital epithelial cells also reduced inflammatory markers in the presence HIV. Enhanced barrier function and decreased inflammation correlate with decrease in HIV infection and replication. This study provides insight into how factors in the genital microenvironment can affect HIV infection in the female genital tract and suggests potential prophylactic strategies to reduce HIV infection.

Les biofilms ont longtemps été décrits comme des organisations évolutives de microorganismes, attachés à une surface et englués dans une matrice contenant, entre autre, des polysaccharides. En partant de ce constat BioFilm Control a souhaité cribler des microorganismes pour la production d’exopolysaccharides, en utilisant le BioFilm Ring Test® (BRT). Le principe repose sur la coincubation de microorganismes avec des particules magnétiques en microplaque. Les particules sont plus ou moins attirées par un aimant en fonction du stade d’organisation du biofilm. En se formant, il piège, dans sa matrice visqueuse, les particules qui perdent leur mobilité. Celle-ci est révélée par une aimantation qui provoque l’apparition d’un spot (pas de biofilm) ou non (biofilm). Une analyse d’images quantifie ce processus et permet de le standardiser. La démarche a consisté dans un premier temps à vérifier le comportement de microorganismes modèles producteurs de polysaccharides (bactéries et microalgues) avec le BRT. L’étude a été étendue au criblage d’une banque de lactobacilles. Les résultats inattendus ont orienté l’étude vers l’analyse du rôle exact des polysaccharides et plus généralement de l’implication des macromolécules dans la structuration du biofilm. Pour cela, la dégradation séquentielle de chaque famille macromoléculaire a été réalisée via des enzymes dépolymérisantes sur les biofilms de Leuconostoc mesenteroides et Bacillus sp. Au regard des résultats obtenus, l’utilisation du BRT a été étendue à la caractérisation qualitative et quantitative d’enzymes de dégradation de polysaccharides. / Biofilms were described for a long time as evolutionary structures elaborated by microorganisms, fixed on a surface and maintained in a polysaccharidic matrix. From this assessment, BioFilm Control chose to screen microorganisms for their capacity to produce exopolysaccharides (EPS), using theBioFilm Ring Test® (BRT). The principle is the co-incubation of magnetic particles with microbial culture on microplates. The mobility of particles depends on the stage of biofilm formation. During this formation, particles are trapped in the matrix and loose their mobility. Revelation is induced by magnet which causes a spot in the absence of biofilm. The pictures analysis quantifies this phenomenon and standardizes different results. This approach was realised, at first step, by the test of EPS-producing bacteria or microalgae with the BRT. The study was extended to the screening of a lactobacilli collection. Unexpected results guided the research toward the understanding of the role of macromolecules in biofilm structuring. To study their implication, sequential enzymatic degradation has been achieved for each macromolecular family of Leuconostoc mesenteroïdes and Bacillus sp. biofilms. Using the results, BRT was then appreciated as a suitable method to detect and quantify polysaccharide degrading enzymes.

Submitted by biblioteca unifenas (biblioteca@unifenas.br) on 2017-05-15T18:46:23Z No. of bitstreams: 1 Thailson Fernando Faustino Dissertacao.pdf: 714335 bytes, checksum: e9b1bc519ff0d96e7bd8cbe84dfe1746 (MD5) / Made available in DSpace on 2017-05-15T18:46:23Z (GMT). No. of bitstreams: 1 Thailson Fernando Faustino Dissertacao.pdf: 714335 bytes, checksum: e9b1bc519ff0d96e7bd8cbe84dfe1746 (MD5) Previous issue date: 2016-09-30 / The objective of this research was to evaluate the nutritional quality of sorghum silage processed and rehydrated with water or whey. The experimental design was a completely randomized 4x2x2 factorial design, with rehydration of the grain in four moisture contents (25, 30, 35 and 40%), rehydration with water or whey and, with or without the addition of bacterial inoculant, totaling 16 treatments with four replicates each. The sorghum grains were ground, rehydrated and conditioned in experimental PVC silos with capacity of 4.5 kg for 90 days, to evaluate the aerobic stability as well as the microbiological and chemical-bromatological characteristics of the silage. The results were submitted to analysis of variance of the statistical program SAS (9.2), and the humidity levels were evaluated by linear and quadratic orthogonal contrasts and, the vehicle of rehydration and inoculant were compared by F test. The use of water compared to the whey in the rehydration decreased the losses by gases, effluents and dry matter of the silages. The increase of moisture in the silages promoted a lower development of fungi and yeasts. The inoculated silages showed higher dry matter contents in relation to the silages without inoculant. The crude protein (CP) concentration in the grain silage of rehydrated sorghum was not altered as a function of the treatments. The addition of inoculant decreases the concentration of acid detergent fiber (ADF), the lowest values being in the silage with 40% moisture with water. Silage rehydrated with whey had higher values of mineral matter, compared to silages rehydrated with water. The use of whey in the rehydration of sorghum grains for silage is a good strategy to avoid its disposal in the environment, and its addition is recommended to the point where the sorghum grain reaches 30% moisture. In addition, it is recommended to use the bacterial inoculant, as it ensures less fermentative loss in the process with greater aerobic stability of the silages. / Objetivou-se com esta pesquisa avaliar a qualidade nutricional da silagem de grão de sorgo processada e reidratada com água ou soro de leite. O delineamento experimental utilizado foi o inteiramente casualizado em esquema fatorial 4x2x2, sendo a reidratação do grão em quatro teores de umidade (25, 30, 35 e 40%), a reidratação com água ou soro e com ou sem a adição de inoculante bacteriano, totalizando 16 tratamentos com quatro repetições cada. Os grãos de sorgo foram moídos, reidratados e acondicionados em silos experimentais de PVC com capacidade de 4,5 kg por 90 dias, para então, avaliar a estabilidade aeróbia, bem como as características microbiológicas e químico-bromatológicas da silagem. Os resultados foram submetidos à análises de variância do programa estatístico SAS (9.2), sendo que os níveis de umidade foram avaliados por contrastes ortogonais lineares e quadráticos, e o veículo de reidratação e inoculante foram comparados por teste F. O uso de água comparado com o soro na reidratação diminuiu as perdas por gases, efluentes e matéria seca das silagens. O aumento da umidade nas silagens promoveu menor desenvolvimento de fungos e leveduras. As silagens inoculadas apresentaram teores de matéria seca superiores em relação às silagens sem inoculante. A concentração PB na silagem de grão de sorgo reidratado não foi alterada em função dos tratamentos. A adição de inoculante diminui a concentração de FDA, sendo que os valores mais baixos foram na silagem com 40% de umidade com água. As silagens reidratadas com soro de leite apresentaram maiores valores de matéria mineral, comparadas às silagens reidratadas com água. A utilização de soro de leite na reidratação de grãos de sorgo para ensilagem é uma boa estratégia para evitar seu descarte no meio ambiente, sendo recomendada sua adição até o ponto em que o grão de sorgo atinja 30% de umidade. Além disso, recomenda-se a utilização do inoculante bacteriano, por assegurar menor perda fermentativa no processo com maior estabilidade aeróbia das silagens.

Lactobacillus is a genus of lactic acid bacteria frequently used as healthpromoting probiotics. Using probiotics to treat or prevent infections is a novel experimental approach with vast impact on future therapy. Lactobacillus rhamnosus GR-1 is a probiotic investigated for its ability to reduce urogenital disease including urinary tract infections caused by pathogenic Escherichia coli. L. rhamnosus GR-1 has been shown to modulate immunity, thought to influence its probiotic effect. In this thesis, the aim was to study immunomodulation by L. rhamnosus GR-1 and other lactobacilli, with emphasis on elicited immune responses such as nuclear factor-kappaB (NF-κB) activation and cytokine release from human urothelial cells. Viable, heat-killed, and isolated released products from L. rhamnosus GR-1 augmented NF-κB activation in E. coli-challenged urothelial cells. Blocking of lipopolysaccharide binding to toll-like receptor 4 completely quelled this augmentation. Size-fractionation, urothelial cell challenge, and two-dimensional gel electrophoresis of L. rhamnosus GR-1 released products presented several candidate proteins with NF-κB modulatory actions including chaperonin GroEL, elongation factur Tu, and a protein from the NLP/P60 protein family. While tumor necrosis factor was correspondingly augmented by L. rhamnosus GR-1, the release of two other cytokines, interleukin (IL)-6 and CXCL8, was reduced. Similar effects were observed in macrophage-like cells stimulated with L. rhamnosus GR-1. Many immunomodulatory effects of lactobacilli are believed to be species and strain dependent. Therefore, twelve Lactobacillus strains were used to screen for their effects on CXCL8 release from urothelial cells. A majority of these strains were able to influence CXCL8 release from the cells. Phylogenetic analysis revealed close evolutionary linkage between lactobacilli with similar actions on CXCL8. Increased knowledge on probiotic bacterial products and the mechanism(s) of action could lead to improved future treatments for infections.