Submitted by Adriana Martinez (amartinez@unoeste.br) on 2017-09-22T18:22:04Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Adriano Falvo.pdf: 401706 bytes, checksum: 62cf14b1237c1f73ed1dcb3fc033dd57 (MD5) / Made available in DSpace on 2017-09-22T18:22:04Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Adriano Falvo.pdf: 401706 bytes, checksum: 62cf14b1237c1f73ed1dcb3fc033dd57 (MD5) Previous issue date: 2017-06-28 / According to the World Health Organization (WHO), cigarette smoking accounts for 5 million annual deaths. Cigarette smoke causes malevolent effects in many other systems, including the male reproductive system and functional foods that may mitigate the toxic effects of cigarette smoking. The objective of the present study was to investigate the possible attenuating effects of prebiotic, probiotic and symbiotic oral supplementation on the testis and epididymis of growing rats exposed to cigarette smoke. To perform the present study, 72 male rats were randomly divided into eight groups: control; probiotic; Prebiotic (MOS); Symbiotic; Smoking Control; Prebiotic Tobacco; Probiotic Smoking; And Smoking Symbiotic. The animals in the smoking groups were exposed to cigarette smoke for one hour daily, divided into two 30-minute periods, five days a week. After the experimental period, the animals were anesthetized, sacrificed, the testes and epididymides were removed. Microscopic lesions were analyzed using the chi-square test, Fisher's exact test or G test at 5% significance. The results showed that the smoking groups had a higher prevalence of lesions in the testes and epididymis when compared to the control groups. Functional, prebiotic, probiotic, and symbiotic foods attenuated the harmful effects of chronic exposure to cigarette smoke in the testis and epididymis. The results allow us to state that functional, probiotic, prebiotic and symbiotic foods can attenuate the chronic harmful effects of passive smoking on the testis and epididymis of rats during the growth phase. / Segundo a Organização Mundial da Saúde (OMS), o cigarro é responsável por 5 milhões de mortes anuais. A fumaça do cigarro causa efeitos maléficos em muitos outros sistemas, incluindo entre eles o sistema reprodutor masculino e alimentos funcionais podem atenuar os efeitos tóxicos do tabagismo. O objetivo do presente estudo foi o investigar os possíveis efeitos atenuadores da suplementação oral do prebiótico, probiótico e simbiótico sobre o testículo e epidídimo de ratos em fase de crescimento expostos à fumaça de cigarro. Para realização do presente estudo, foram utilizados 72 ratos machos, divididos aleatoriamente em oito grupos: controle; probiótico; prebiótico (MOS); Simbiótico; Controle Tabagista; Prebiótico Tabagista; Probiótico Tabagista; e Simbiótico Tabagista. Os animais dos grupos tabagistas foram expostos a fumaça de cigarro por uma hora diária, divididos em dois períodos de 30 minutos, cinco dias por semana. Após o período experimental, os animais foram anestesiados, sacrificados, os testículos e epidídimos foram retirados. As lesões microscópicas foram analisadas por meio do teste do qui-quadrado, teste exato de Fisher ou teste G a 5% de significância. Os resultados revelaram que os grupos tabagistas apresentaram uma maior prevalência de lesões nos testículos e epidídimo, quando comparados com os grupos controle. Os alimentos funcionais, prebiótico, probiótico e simbiótico, atenuaram os efeitos nocivos da exposição crônica a fumaça do cigarro no testículo e epidídimo. Os resultados obtidos nos permitem afirmar que os alimentos funcionais, probiótico, prebiótico e simbiótico, podem atenuar os efeitos crônicos nocivos do tabagismo passivo no testículo e epidídimo de ratos na fase de crescimento.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22484号 / 農博第2388号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5264(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The intestinal microflora is an extensive ecosystem of microorganisms that consists of symbiotic and pathogenic species. The microflora is responsible for many important functions in the human body. An unquestionable function is that it affects the health state of the host. The higher the biodiversity, the greater the benefit for the host. However, it is necessary to point out that this should not include a high diversity of pathogenic bacterial species. There are many "beneficial" species, especially from the Bifidobacterium and Lactobacillus families. In recent decades, the popularity of supplementing these "beneficial" species with various supplementary diets (e.g. probiotics) has been growing a lot. The presented diploma thesis deals with pilot studies of liquid commercial probiotic preparations from American companies Ascended Health (not available on the Czech market) and their effects on the human microbiome. The study involved 9 volunteers who provided 70 fecal samples before, during, and after use of the studied products. Two methods were used to determine the biodiversity of intestinal bacterial species. Both are based on the identification by bacterial DNA that encodes gene 16S rRNA. The first method uses PCR-DGGE technique and then identification by Sanger sequencing. The second method...

This diploma thesis deals with encapsulation of natural extracts. In the theoretical part the methods of encapsulation, materials for particle preparation, as well as application of encapsulation techniques in food industry were described. In experimental part selected natural extracts of propolis, green barley and probiotics were characterized. There substances were encapsulated into alginate and chitosan. In the total of 25 types of prepared particles long-term stability in some model physiological conditions as well as in four different model foods was evaluated. Additionally, stability of selected particles in several real milk-based products was followed too. The stability of particles was determined spectroptohometrically. In natural extract a content of polyphenols, proteins, chlorophylls, as well as total antioxidant activity were analysed. To analysis of probiotics optical and fluorescence microscopy were used. In propolis and green barley antimicrobial activity was tested too. Moreover, in the sample of propolis also cytotoxic assay was applied. Agar-chitosan was chosen as the best shell material for propolis due to its optimal stability in model physiological conditions as well as model foods. Liposomes were evaluated as unstable and were not recommended for further application. As the suitable shell material for powdered green barley starch-alginate (rate 1:4) and agar-chitosan were proposed, while the second one showed better stability for released proteins. Agar-chitosan shell material was usable for fresh green barley too. For probiotics encapsulation alginate or alginate-starch were chosen because of their porosity and possibility of nutrients diffusion. In real foods the best results were reached with application of probiotic particles into milk. Coencapsulation of powdered barley and probiotics did not confirm inhibition of culture growth. Neither the antimicrobial effect of propolis and barley nor the cytotoxic effect of propolis were confirmed.

Probiotics products are an integral part of the current market. Products containing probiotics cultures are also cosmetic products. The first part of the study focuses on testing of bacterial survival abilities in the environment of preservatives presented in cosmetic products. Collection strains of genus Lactobacillus were used for these tests. Another part of the study focuses on isolation of bacterial DNA from probiotic cosmetic products Ryor, Yoghurt of Bulgaria, FeminaMed and Lactovit Activit in PCR-ready quality. DNA was isolated by fenol extraction and with magnetic particles. Presence of bacteria was proved by genus and species specific PCRs Lactobacillus. Species specific PCR for identification of Lactobacillus pentosus was optimalized. Species identification was in accord with data declared by producers.

Les maladies inflammatoires chroniques de l’intestin (MICI), qui comprennent les deux principales formes, la maladie de Crohn (MC) et la rectocolite hémorragique (RCH), sont caractérisées par une inflammation chronique et récurrente de la muqueuse intestinale, ayant un impact considérable sur la qualité de vie. À l'heure actuelle, la prise en charge thérapeutique de la MC n'est pas curative et un tiers des patients ne réagissent pas aux traitements biologiques et aux immunosuppresseurs. Par conséquent, de nouvelles stratégies pour traiter cette maladie sont fortement attendues. La dérégulation de l'interaction entre d'une part les facteurs génétiques et le système immunitaire de l'hôte, et d'autre part le microbiote intestinal et les facteurs environnementaux, est impliquée dans le développement des MICI. Cette perturbation entraîne effectivement une augmentation de la perméabilité intestinale et une inflammation persistante. Restaurer le microbiote «dysbiotique» et les fonctions intestinales altérées représentent donc une thérapeutique alternative intéressante. De ce fait, les probiotiques sont une option intéressante et ont été utilisés avec succès chez des patients souffrant de pouchite et de RCH. Cependant, leur effet protecteur est clairement souche-dépendant et plusieurs souches probiotiques bien connues n’ont pu conduire à un résultat clinique probant, en particulier chez les patients souffrant de MC. Le décryptage des mécanismes moléculaires sera donc la clé pour permettre une recommandation efficace des probiotiques dans le traitement ou la prévention des MICI. La sélection de souches basée sur des critères de sélection bien définis et en utilisant des modèles bien maitrisés est indispensable à ce processus. L'objectif principal de cette thèse était de sélectionner des lactobacilles et des bifidobactéries parmi une collection de souches françaises et libanaises, capables de présenter des propriétés protectrices contre les MICI, en se concentrant sur leurs capacités immuno-régulatrices et leurs capacités à renforcer la barrière épithéliale. Des approches in vitro ont été utilisées pour sélectionner des souches ayant une activité anti-inflammatoire et également capables d'améliorer la fonction de la barrière intestinale. Cinq souches ont été identifiées présentant des caractéristiques différentes, mais avec un potentiel thérapeutique élevé. Deux souches se sont révélées hautement protectrices dans deux modèles différents de colite aiguë et de colite de bas grade. Nos résultats ont confirmé en outre l'hypothèse selon laquelle la capacité des souches à atténuer l'inflammation est en partie due à l'amélioration de la barrière intestinale et à la restauration des protéines de jonction serrés.Un nombre croissant d’études génétiques ont prouvé que l’autophagie peut affecter plusieurs aspects de la réponse immunitaire des muqueuses, notamment via l’élimination de bactéries intracellulaires, la sécrétion de peptides antimicrobiens, la production de cytokines pro-inflammatoires et la présentation des antigènes. Par conséquent, l'autophagie peut être considérée comme un mécanisme de régulation clé impliqué dans la physiopathologie de la MC. Nous avons donc évalué la capacité des souches à activer cette voie et montré que les souches sélectionnées étaient en effet capables d’induire une activation de l’autophagie dans des cellules dendritiques murines. Nous avons démontré in vitro que le blocage de l'autophagie pouvait diminuer la capacité des souches à induire la sécrétion d'IL-10, cytokine anti-inflammatoire et, inversement, à exacerber la sécrétion d'IL-1β, cytokine pro-inflammatoire. Nous avons pu confirmer, à l'aide d'un modèle murin de colite, que la capacité protectrice d’une souche impliquait la machinerie autophagique, et nous avons pu mettre en évidence le rôle des cellules dendritiques dans ce processus [...] / Inflammatory bowel disease (IBD), including the two main types, Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing inflammation of the gut mucosa with considerable impact on the quality of life. At present, the therapeutic management of CD is not curative and one third of patients fails to respond to current biologicals and immunosuppressive drugs. Therefore new strategies for treating this disease are imperative.The deregulation of the normal interplay between the genetics and immune system of the host on the one hand, and the gut microbiota and environmental factors on the other hand, is known to be associated with the development of IBD, as this disturbance is leading to increased intestinal permeability and persistent inflammation. Restoring the “dysbiotic” microbiota and the impaired intestinal functions represent an attractive therapeutic alternative. Probiotics represent therefore an interesting option and have been used quite successfully in patients suffering from pouchitis and UC. However, their protective effect is clearly strain-dependent and several well-known probiotic strains failed to fulfill the expected clinical outcome, especially when applied in CD. Deciphering the molecular mechanisms will be the key to the recommendation of probiotics for the treatment or prevention of IBD. Selecting strains on well-defined selection criteria and using well-studied models is indispensable to this process.The main objective of this thesis was first to select lactobacilli and bifidobacteria from a collection of French and Lebanese strains that exhibited protective properties against IBD, focusing on their immunoregulatory capacities and their capacities to strengthen the epithelial barrier.In vitro approaches were used to select strains with anti-inflammatory activity and also able to enhance intestinal barrier function. Five strains were identified with different characteristics, but entailing a high potential for the management of IBD. Two strains, e.g. were found to be highly protective in two different models of acute and low grade colitis. Our results furthermore support the hypothesis that the capacity of the strains to alleviate inflammation is in part mediated by the improvement of the intestinal barrier and the restoration of tight junction proteins.A growing number of genetic studies provided strong evidence that autophagy machinery can affect several aspects of the mucosal immune response, including intracellular bacterial killing, antimicrobial peptide secretion, pro-inflammatory cytokine production and antigen presentation. Therefore, autophagy can be considered as a key regulator mechanism most likely involved in the physio-pathogenesis of CD.We therefore evaluated the capacity of the strains to activate this pathway and showed that the selected strains were indeed able to induce autophagy activation in dendritic cells. We demonstrated in vitro that blocking the autophagy machinery can abolish the capacity to induce the secretion of the anti-inflammatory cytokine IL-10 after immune cell stimulation, while exacerbating the secretion of the pro-inflammatory cytokine IL-1β. We could confirm, using a murine model of colitis, that the protective capacity of the selected strains indeed involves autophagy mechanisms, and we could highlight the role of dendritic cells in this process. We therefore propose here that autophagy is a novel mechanism through which probiotics can exhibit their immunoregulatory capacities.

Background: Type 2 diabetes mellitus is a metabolic disorder characterized by the rise of fasting plasma glucose from its normal range (≥125mg/dl). It is marked by insufficient production of insulin from pancreatic β-cells as a result of failed compensation due to insulin resistance. Several treatments are available for the disorder, which mainly focus on improving the sensitivity of insulin in different body tissues. Recently, probiotics were suggested as candidate treatments for type 2 diabetes and for extending lifespan as well. This experiment aims to investigate such claims using Drosophila melanogaster as a disease model.   Results: Other than the observed low average weights in treated larva samples, probiotics did not show any other significant results in affecting the length, glucose, glycogen, and trehalose levels (One-Way ANOVA and Kruskal-Wallis, p>0.05). Real-time PCR was only carried out once. Thus, no statistical tests were reliable enough to analyse the data obtained. The longevity study, on the other hand, did show significance (Log-rank (Mantel-Cox) test and Gehan-Breslow-Wilcoxon test, p<0.0001), as the probiotic Bifidobacterium lactis extended the lifespan of adult flies feeding on a high sugar diet significantly when compared to the control ones feeding on only high sugar diet without probiotics.   Conclusion: Except for weight measurements, none of the other results was reliable enough to make a concrete conclusion on whether the treatments indeed worked in reversing type 2 diabetes symptoms or not. Real-time PCR results did show some effects of some of the treatments at different developmental stages. However, unless Real-time PCR is repeated at least once using the same protocol, no deduction can be made. Additionally, the data obtained hint that the dosage used (0.025 g) was too high for larvae and adult flies and might have caused malnutrition by blocking their midgut and decreasing food absorption. Hence, false significant or non-significant results were acquired instead.   Further studies are required using a much lower probiotic dosage if Drosophila is used as a disease model. Although, other models such as mice or rats are recommended in this case, in order to reach a solid conclusion about the effectiveness of probiotics in treating type 2 diabetes mellitus. Baring these thoughts in mind and based on the results of this experiment, the null hypothesis indicating that there is no significant relationship between the use of probiotics and reversing type 2 diabetes mellitus symptoms is therefore accepted.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) prevention, screening, and treatment strategies. With CRC incidence on the rise in younger populations, there is an increased need to engineer technologies that enhance patient access to diagnostic exams and disease management. This dissertation presents the development of an orally-delivered probiotic to screen for and treat early CRC lesions with a particular emphasis on translatability factors including: safety of probiotic use, exploration of oral delivery, and testing in clinically relevant models. At the interface of immunology, synthetic biology, and the microbiome fields is the overarching concept that microbes play a critical role in the tumor microenvironment (TME). The innate ability of bacteria to seek out tumor-specific signatures and proliferate within their necrotic cores due to reduced immune surveillance enables the precise immunoengineering of the local TME. Here, we will design, characterize, and test a probiotic encoded with a lysis mechanism to aid in biocontainment and maximize the release of recombinantly-produced diagnostic and immunotherapeutic cargo. In this lysis circuit, bacteria grow to a critical density within tumors and synchronously lyse, locally releasing their payload. A small fraction of bacteria remains to reseed the population and the cycle continues, resulting in repeated and sustained drug delivery. Drawing from advancements in immunology, we engineered bacteria to produce immune checkpoint inhibitors. Monoclonal antibodies targeting immune checkpoints have revolutionized cancer therapy, but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Due to their preferential colonization of tumors, bacteria are a natural chassis for the localized delivery of such therapeutics. Therefore, we engineered a commercially available probiotic, E.coli Nissle 1917 (EcN), for the controlled production and intratumoral release of nanobodies targeting programmed cell death protein – ligand 1 (PD-L1) and cytotoxic T- lymphocyte-associated protein-4 (CTLA-4) using the described lysing release mechanism. We demonstrate that a single injection of this engineered probiotic enhanced therapeutic response compared to analogous clinically-relevant antibodies, resulting in tumor regression in syngeneic mouse models. In an effort to create a more effective therapeutic for poorly immunogenic cancers, we utilized the modularity of our platform to slow tumor growth in mouse models of established CRC by combining it with a probiotically-produced cytokine, granulocyte-macrophage colony stimulating factor (GM-CSF). We sought to expand upon the relevance of this approach for early-stage CRC screening and treatment, by characterizing the platform in CRC precancerous lesions, or adenomas. When orally-delivered, EcN robustly colonized adenomas in genetically-engineered and orthotopic murine models of CRC, and human CRC patients. Leveraging adenoma-specific colonization, we probed for EcN presence in fecal matter, demonstrating its utility as a non-invasive screen for adenomas. For more accessible testing, we engineered EcN to produce salicylate and showed that it could be detected in the urine of tumor-bearing mice for days after oral delivery of the probiotic. Moreover, we demonstrated that the therapeutic effectiveness of our previously engineered therapeutic strain, producing PD-L1, CTLA-4 and GM-CSF, was maintained when delivered orally, ultimately resulting in significant adenoma reduction. Altogether, this dissertation aims to highlight the potential for engineered EcN to be used as a safe, orally-deliverable screening and therapeutic platform for early-stage CRC disease. While we have chosen to focus on CRC here, we will conclude by discussing efforts to adapt this platform to work in combination with other cellular therapies and therapeutic indications, ultimately engineering a platform to impact a broader patient population.

No description available.

A study was conducted to isolate and identify bacteria from “amabere amaruranu” cultured-milk from Kenya and to evaluate the isolates’ potential to be used as probiotics. Isolates were identified using PCR sequence analysis of the 16S rRNA gene and the API® 50 identification system. Identified isolates included: Acetobacter tropicalis, Bacillus pumilus, Bacillus safensis, Lactobacillus paracasei, Lactobacillus rhamnosus, Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus sciuri. Their potential as probiotics was evaluated using their ability to survive in acid-then-bile conditions, antibacterial activity (against Escherichia coli, Klebsialla pneumonia, Enterococcus faecalis, Pseudomonas aeruginosa and Enterobacter cloacae), mucin degradation activity, and sensitivity to antibiotics (ampicillin, bacitracin, chloramphenicol, erythromycin, kanamycin, penicillin, streptomycin and tetracycline). Lactobacillus isolates were tolerant to acid-then-bile conditions, non-mucinolytic and inhibited growth of indicator strains but only L. rhamnosus was sensitive to all test antibiotics. Bacillus isolates were tolerant to acid-then-bile conditions, non-mucinolytic, lacked antibacterial activity and only B. safensis was sensitive to all test antibiotics. Acetobacter tropicalis isolates were non-mucinolytic but were intolerant to acid-then-bile conditions. In conclusion, both Lactobacillus rhamnosus and Bacillus safensis isolates that showed tolerance to digestive tract conditions, were sensitive to antibiotics and were non-mucinolytic would be recommended for further consideration as candidate probiotics.