<p>Colorectal cancer is one of the leading causes of cancer deaths in the United States and multiple modifiable factors contribute to colorectal carcinogenesis. Gut microbiota are believed to play key roles in colon cancer development. Dietary factors may modulate gut microbiota composition, which may potentially have impact on carcinogenesis. Thus, it is reasonable to develop dietary interventions to effectively prevent colorectal cancer development through alteration of gut microbiota. In this thesis, the first objective is to evaluate the effect of vitamin E forms and metabolites, i.e., δ-tocotrienol (δTE), γ-tocotrienol (γTE) and δTE-13’-COOH (δTE-13’), respectively, on gut microbiota in mice. Healthy male balb/c mice were supplemented with a δTE/γTE mixture or δTE-13’ by gavage for two weeks, while control mice received soybean oil. We isolated DNAs from fecal samples and used 16S rRNA gene sequencing to evaluate the impact of these compounds on gut microbiota compositions. Further, we also examined the effect on short chain fatty acids (SCFAs). We observed that supplementation of δTE-13’ increased microbial richness using the Faith index. On the other hand, supplementation did not separate the microbial communities from the control group. But, these compounds managed to alter the relative abundances of several taxa that might present chemopreventive activities against colon cancer. Specifically, <i>Desulfovibrio</i>, a sulfur-reducing bacterium, was decreased after δTE/γTE supplementation. <i>Eubacterium coprostanoligenes</i> group, a group of microbes that can reduce circulating cholesterol, was increased after δTE/γTE supplementation. In addition, several members from the <i>Lachnospiraceae</i> family were elevated under δTE/γTE and δTE-13’ supplementation, and these microbes are known to produce SCFAs and maintain colonic health. However, the measurement of SCFAs showed that supplementation of δTE/γTE and δTE-13’ did not change SCFAs compared with controls. In the second project, I investigated anti-proliferative effects of combining δTE or δTE-13’ with sodium butyrate (NaBu) on human colorectal carcinoma HCT116 cells. Our data showed promising additive effects against cell growth. Collectively, these results indicate that δTE/γTE and δTE-13’ can modulate gut microbiota under healthy conditions, which provides insights into potential chemopreventive activities of these vitamin E forms. Our cell-based studies also showed additive anticancer effects of combining δTE or δTE-13’ with NaBu, which provides rationale to further develop combination of butyrate producers with vitamin E forms for cancer prevention.</p>

Time-restricted eating is a fasting diet where the food intake is restricted to a short, typically eight-hour, window each day. It is associated with health benefits such as weight loss, improved sleep, protection against cognitive disorders, and improved cognitive function. The cognitive effects of time-restricted eating have primarily been explained by the production of ketogenesis – an alternative energy source produced when calories are restricted – and anti-inflammatory cytokines. The gut microbiota is the trillions of microorganisms inhabiting the intestinal tract and has also been associated with improved mental health through communication via the gut-brain axis. This review aims to investigate whether changes in the microbiota may mediate the effect of time-restricted eating on cognitive function. Studies investigating the effect of time-restricted eating on the microbiota were systematically reviewed. The results indicate that time-restricted eating may alter the microbiome composition and increase butyrate-producing bacteria. Butyrate is a short-chain fatty acid associated with the expression of genes involved in neural development and the reduction of neuroinflammation. Limited by the few studies reviewed, the results may indicate a possible link between time-restricted eating and cognitive function via the microbiota, although more research is needed.

Thermoplastic blends of several biodegradable polymers with lignin (L) and lignin esters were prepared by solvent casting and melt processing. Among the biodegradable thermoplastics were cellulose acetate butyrate (CAB), poly-hydroxybutyrate (PHB), poly-hydroxybutyrate-co-valerate (PHBV), and a starch-caprolactone blend (SCL). Lignin esters included acetate (LA), butyrate (LB), hexanoate (LH), and laurate (LL). Blend characteristics were analyzed in terms of thermal and mechanical properties. The results indicate widely different levels of interaction between two polymer constituents. Melt blended samples of CAB/LA and CAB/LB were compatible on a 15-30 nm scale when probed by dynamic mechanical thermal analysis, and the glass transition temperatures of the blends followed Fox equation, whereas those of CAB/LH and CAB/LL showed distinct broad transitions on the same scale. Melt blending produced well dispersed phases whereas large phase separation evolved out of solvent castings. Crystallinity and melting points of PHB and PHBV were affected by the incorporation of lignin component, revealing some interaction between the blend constituents. Blends of SCL with L and LB revealed significant effect on crystallinity and melting temperatures of poly-caprolactone component, revealing polymer-polymer interaction between SCL and lignin components. An increased degree of crystallinity was observed in the case of higher-Tg L compared to lower Tg LB. Improvememt in modulus (and in some cases strength also) was observed in almost all blends types due to the glassy reinforcing behavior of lignin. / Master of Science

Biodegradable thermoplastic composites were prepared using high modulus lyocell fibers and cellulose acetate butyrate (CAB). Two reinforcement fiber types: fabric and continuous fiber tow were used. Fabric had advantages of uniform alignment and easier processing, but lacked the use as a unidirectional reinforcement and a continuous method of matrix application. Three different matrix application methods were screened for both fiber types. Matrix application by suspension of particles in water was not feasible because of particle sizes > 15 &micro m. The other disadvantages were high moisture absorption during matrix application and void formation during consolidation. Melt processing technique using alternating sandwich structure of fabrics and CAB films produced composites with impressive appearance, low void contents and low moisture absorption. However, SEM results revealed interfacial failure and extensive fiber pull out. Relatively larger fiber and matrix regions were present on the scale of 10^-3m. Solution prepregging technique using methyl ethyl ketone (MEK) as a solvent for CAB and continuous fibers as reinforcement produced composites with uniform matrix distribution, high tensile strengths and high modulus, and even wetting of fibers by CAB. A maximum tensile modulus of 21.5 GPa and a maximum strength of 251.7 MPa were achieved for a continuous fiber reinforced composites at a volume fraction of 66.5% compared to 0.8 GPa and 76 MPa for the matrix, respectively. Void contents and water absorption were relatively high compared to comparable carbon fiber composites. / Master of Science

A disturbance in the symbiotic mutualism between the intestinal microbiome and the human host’s organism (syn. dysbiosis) accompanies the development of a variety of inflammatory and metabolic diseases that comprise the Metabolic Syndrome, chronic inflammatory gut diseases like Crohn’s disease, Non-alcoholic fatty liver disease (NAFLD) and cardiovascular diseases, among others. The changed uptake and effectiveness of short chain fatty acids (SCFAs) as well as an increase of the intestinal permeability are common, interdependent disease elements in this regard. Short chain fatty acids are end-products of intestinal bacterial fermentation and affect the mucosal barrier integrity via numerous molecular mechanisms. There is evidence to suggest, that SCFAs have a modulating influence on Signal transducer and activator of transcription 3 (STAT3) in intestinal epithelial cells. STAT3 is a central gene-transcription factor in signaling pathways of proliferation and inflammation. It can be activated by growth factors and other intercellular signaling molecules like the cytokine Oncostatin M (OSM). The mode of STAT3’s activation exhibits, finally, a decisive influence on the immunological balance at the intestinal mucosa. Therefore, the posttranslational modification of STAT3 under the influence of SCFAs is likely to be a very important factor within the development and -progression of dysbiosis-associated diseases. In this study, a clear positive in vitro-effect of the short chain fatty acid butyrate on the posttranslational serine727-phosphorylation of STAT3 and its total protein amount in the human adenocarcinoma cell line CACO2 is verified. Moreover, an increased gene expression of the OSM-receptor subunit OSMRβ can be observed after butyrate incubation. Histone deacetylase inhibition is shown to have a predominant role in these effects. Furthermore, a subsequent p38 MAPK-activation by Butyrate is found to be a key molecular mechanism regarding the STAT3-phosphorylation at serine727-residues. To consider the portion of butyrate receptor signaling in this context in future assays, a CACO-2 cell 3D-culture model is introduced in which an improvement of the GPR109A-receptor expression in CACO-2 cells is accomplished. / Eine Störung der symbiotischen Wechselbeziehung zwischen dem Darmmikrobiom und dem Wirtsorganismus (syn. Dysbiose) begleitet die Entwicklung vieler verschiedener entzündlicher und metabolischer Erkrankungen. Zu ihnen zählen unter anderem das Metabolische Syndrom, chronisch entzündliche Darmerkrankungen wie M. Crohn, die Nicht-alkoholische Fettlebererkrankung (NAFLD) und kardiovaskuläre Erkrankungen. Eine veränderte Aufnahme und Wirkung von kurzkettigen Fettsäuren (Short-chain fatty acids = SCFAs) und eine Schwächung der Darmbarriere bedingen sich in diesem Zusammenhang gegenseitig. Kurzkettige Fettsäuren sind Endprodukte des bakteriellen Stoffwechsels und beeinflussen die Integrität der Darmbarriere über eine Vielzahl molekularer Mechanismen. Es gibt Hinweise darauf, dass kurzkettige Fettsäuren einen modulierenden Einfluss auf „Signal transducer and activator of transcription 3“ (STAT3) in intestinalen epithelialen Zellen ausüben. STAT3 ist hier ein zentraler Gentranskriptionsfaktor in proliferations- und entzündungsregulierenden Zellsignalwegen. Er kann durch Wachstumsfaktoren und andere interzelluläre Botenstoffe, wie beispielsweise das Zytokin Oncostatin M (OSM), aktiviert werden. Die Art der Aktivierung von STAT3 wirkt sich nicht zuletzt entscheidend auf die immunologische Balance an der Darmbarriere aus. Die Modifikation von STAT3 durch kurzkettige Fettsäuren ist aufgrund dessen mit hoher Wahrscheinlichkeit ein sehr wichtiger Faktor im Hinblick auf Entstehung und Progression der im Zusammenhang mit einer Dysbiose stehenden Erkrankungen. In dieser Arbeit kann eine klare Steigerung der posttranslationalen Phosphorylierung von STAT3 an den Serin-Molekülendungen der Position 727 sowie eine Steigerung der Gesamtproteinmenge von STAT3 in der humanen Karzinomzellline CACO2 in vitro durch Butyrat-Inkubation gezeigt werden. Weiterhin wird unter Butyrat-Einfluss auch die Genexpression der OSM-Rezeptor-Untereinheit OSMRβ gesteigert. Diese Effekte können größtenteils auf den Mechanismus der Histondeacetylase-Hemmung durch Butyrat zurückgeführt werden. Die in der Folge erhöhte P38 MAPK-Phosphorylierung durch Butyrat ist in Bezug auf die Serin727-Phosphorylierung an STAT3 entscheidend beteiligt. Um in künftigen Assays auch den möglichen Einfluss der Butyrat-Rezeptor-Wirkung in diesem Zusammenhang besser zu erfassen, wird ein 3D-Zellkultur Ansatz getestet und in diesem eine verbesserte Expression des Butyrat-Rezeptors GPR109A in CACO-2-Zellen erreicht.

Syntrophic relationships between fatty acid degrading bacteria and hydrogenotrophic methanogens are important for a well-functioning anaerobic degradation process during biogas production from organic waste material. This is particularly important in biogas processes fed protein-rich material as their degradation gives rise to high levels of ammonia, which inhibits many microorganisms. A common problem arising in the high-ammonia biogas process is the accumulation of volatile fatty acids, such as acetate, propionate or butyrate, which negatively affects the methane production. The aim of this study was to isolate and identify the syntrophic butyrate-oxidizing bacteria present in the enriched syntrophic communities originating from thermophilic continuously fed laboratory-scale reactors. Butyrate was added to batch assays containing the enrichment cultures. Sequencing of a 464 bp region within the 16S rRNA gene was conducted to study the change in microbial community structure over time during the butyrate degradation. The enrichment culture was also used as an inoculum source during the isolation attempts using agar cultivation, colony transfer and 16S rRNA gene sequencing of the obtained isolates. From the Illumina sequencing data, it could be concluded that the novel species of interest belonged to the genus Syntrophothermus. Two species were isolated, however neither appeared to be the butyrate-degrading bacterium. One of the species was Defluviitoga tunisiensis and the other was a novel species related to the mesophilic bacterium Schnuerera ultunensis.

Two commercial carboxymethylcellulose acetate butyrate (CMCAB) polymers, high and low molecular weight (MW) forms, were analyzed in this study. High-solids water-borne dispersions of these polymers were studied as renewable wood adhesives. Neat polymer analyses revealed that the apart from MW, the CMCAB systems had different acid values, and that the high MW system was compromised with gel particle contaminants. Formulation of the polymer into water-dispersions was optimized for this study, and proved the "direct method", in which all formulation components were mixed at once in a sealed vessel, was the most efficient preparation technique. Applying this method, 4 high-solids water dispersions were prepared and evaluated with viscometry, differential scanning calorimetry, dynamic mechanical analysis, light and fluorescence microscopy, and mode I fracture testing. Thermal analyses showed that the polymer glass transition temperature significantly increased when bonded to wood. CMCAB dispersions produced fairly brittle adhesive-joints; however, it is believed toughness can likely be improved with further formulation optimization. Lastly, dispersion viscosity, film formation, adhesive penetration and joint-performance were all dependent on the formulation solvents, and moreover, these properties appeared to correlate with each other. / Master of Science

O carcinoma hepatocelular (HCC) é uma neoplasia primária com mau prognóstico e alta taxa de recorrência. Estudos recentes demostram que o HCC pode ser classificado em três subtipos segundo o perfil molecular. Destes subtipos, o HCC pouco diferenciado apresenta pior prognostico. Neste sentido, torna-se de particular interesse o estudo de compostos com efeitos diferenciadores e citotóxicos nas células destas neoplasias pouco diferenciadas. O butirato, um ácido graxo de cadeia curta produzido pela fermentação microbiana da fibra alimentar no intestino, tem demonstrado atividade anti-neoplásica e capacidade moduladora da diferenciação celular em diversos tipos celulares, incluindo linhagens de HCC humano e células progenitoras hepáticas. Assim, objetivou-se neste estudo, caracterizar o efeito do butirato de sódio (NaBu) em duas linhagens de células neoplásicas de rato: uma pouco diferenciada (GP7TB) e a outra, uma linhagem derivada de um HCC diferenciado (JM-1). A linhagem GP7TB mostrou maior resistência ao NaBu (ED50= 7,7 mM) do que as células JM-1 (ED50= 5,2 mM). A redução na viabilidade celular após 72 h de tratamento com NaBu esteve relacionada com a diminuição na proliferação celular e no caso das células GP7TB, de um aumento na apoptose. O tratamento com NaBu induziu alterações morfológicas nas duas linhagens celulares, porém apenas nas células do tipo GP7TB, essas alterações sugerem um processo de diferenciação/transdiferenciação celular. O aumento na expressão de genes envolvidos no controle da pluripotência de células tronco, assim como de alguns marcadores de células tronco, sugere que o NaBu induziu uma reprogramação profunda das células GP7TB. Por outro lado, a redução na expressão de genes relacionados com migração e plasticidade celular assim como de proliferação celular apontam que estas células diminuíram seu potencial invasivo e a capacidade de autorenovação. Embora sejam necessárias análises adicionais para confirmar o efeito observado nos perfis de expressão gênica, os resultados deste estudo sugerem que o NaBu apresenta efeito antineoplásico por meio da redução da proliferação, aumento da apoptose e modulação da expressão de genes associados com a transição epitéliomesenquimal em células com características tronco tumorais. / Hepatocellular carcinoma (HCC) is a primary neoplasia with poor prognosis and high recurrence rate. Recent evidence suggests that HCC can be classified in three different subtypes based on their molecular profile. Among these subtypes, the poorlydifferentiated HCC has the worst prognosis. Therefore, the study of compounds with pro-differentiating and cytotoxic effects on poorly-differentiated neoplastic cells represents a matter of primary concern. Butyrate which is a short-chain fatty acid produced by microbial fermentation in the intestine, has demonstrated anti-neoplastic activity and pro-differentiating potential in several cell types, including, human HCC cell lines and liver progenitor cells. In this study, we aimed to characterize the effect of sodium butyrate (NaBu) on two neoplastic cell lines derived from rats: a poorlydifferentiated cell line (GP7TB) and, a cell line derived from a well-differentiated HCC. GP7TB showed increased resistance to NaBu treatment (ED50= 7.7 mM) compared to JM-1 (ED50= 5.2 mM). The reduction in cell viability observed after 72 h of treatment was explained by a reduction in cell proliferation and, in the case of GP7TB, by increased levels of apoptosis. The NaBu treatment induced morphological alterations in both cell lines. However, only in the case of GP7TB cells, the alterations suggested a differentiation/transdifferentiation process. The up-regulation of genes involved in pluripotency and genes expressing stem cell markers indicated that NaBu triggered a deep reprogramming of GP7TB cells. Besides, a down-regulation in the expression of genes related with cell migration and plasticity suggested that these cells reduced their invasive potential and their self-renewal capacity. Additional analyses are necessary to confirm the observed effect on gene expression profiles. However, the results of this study suggest that NaBu exert anti-neoplastic effects through apoptosis, reduction of cell proliferation and downregulation of genes associated with epithelial-mesenchymal transition of cancer stem-like cells.

As limitações ao uso de antibióticos promotores de crescimento vêm aumentando a procura por aditivos substitutos para manter a performance animal, a saúde intestinal e a resposta imune de frangos de corte. Este experimento foi conduzido para comparar o efeito e a dose resposta do produto Ava Cid P (composto por substância húmica, butirato de sódio 30% protegido e uma pequena porção de acidificantes) suplementado na dieta, sobre a performance, resposta imune e saúde intestinal de frangos de corte. O experimento seguiu um design inteiramente casualizado, envolvendo um arranjo fatorial 2 x 5 (2 sexos e 5 tratamentos) com 7 repetições de 15 aves por tratamento. Os tratamentos seguiram a suplementação em diferentes fases de 1 a 49 dias: 1) Controle: dieta basal sem nenhuma suplementação; 2) AVA1-21: aves receberam 0,91 kg/t de Ava Cid P de 1 a 21 d; 3) AVA1-35: 0,91 kg/t de Ava Cid P de 1-21 d e 0,45 kg/t de 22-35 d; 4) AVA1-42: 0,91 kg/t de Ava Cid P de 1 a 21 d e 0,45 kg/t de 22- 42 d; 5) AVA1-49: 0,91 kg/t de Ava Cid P de 1 a 21 d, 0,45 kg/t de 22-35 d e 0,23 kg/t de 36-49 d. A suplementação com Ava Cid P não influenciou a performance de machos e fêmeas, nem mesmo a densidade de células caliciformes (P > 0,05). No entanto, o Ava Cid P foi capaz de modificar a morfometria intestinal, aumentando a altura de vilosidades aos 9 e 35 d (P < 0,05). A área superficial aparente dos vilos e altura de vilos, nas aves que receberam Ava Cid P durante todas as fases experimentais, foi superior à das aves suplementadas somente na fase inicial. No íleo, a área superficial aparente dos vilos também foi superior nas aves suplementadas aos 9 d. Além disso, a expressão do gene para mucina 2 (MUC2) e para o fator de necrose tumoral (TNF-α) diminuiu nas aves recebendo Ava Cid P aos 21 d (P < 0,05), mas não foram observadas diferenças estatísticas para interleucina-1 beta e interleucina-10. Os resultados sugerem que Ava Cid P pode alterar a expressão de mRNA de algumas citocinas e MUC2 e a morfometria intestinal de frangos de corte, aumentando a superfície aparente e a altura dos vilos, o que demonstra a potencialidade do produto como alternativa aos antibióticos promotores de crescimento. / The limitations of the antibiotic growth promoter’s (AGP) usage have been increasing the search for new products to improve poultry performance, gut healthy and immune response. This experiment was conducted to compare the effect and dose response of Ava Cid P (consisted of a humic substance, coated sodium butyrate 30% and a small acidifier portion) diet supplementation on performance, immune response and gut health of broilers. Five dietary regimens were used: 1) birds didn’t receive Ava Cid P in any phase (Control), 2) birds received 0.91 kg/t of Ava Cid P from 1 to 21 d (AVA1-21), 3) 0.91 kg/t of Ava Cid P from 1 to 21 d and 0.45 kg/t from 22 to 35 d (AVA1-35), 4) 0.91 kg/t of Ava Cid P from 1 to 21 d and 0.45 kg/t from 22 to 42 d (AVA1-42), 5) 0.91 kg/t of Ava Cid P from 1 to 21 d, 0.45 kg/t from 22 to 35 d and 0.23 kg/t from 36 to 49 d (AVA1-49). They were applied in a completely randomized design, involving a 2 × 5 factorial arrangement with 2 sex and 5 levels of inclusion, and 7 replications with 15 birds each. The supplementation with Ava Cid P showed no influence on males and females growth performance and goblet cell density (P > 0.05). However, it modified the gut morphometry, increasing jejunum villi height at 9 and 35 days (P < 0.05). The apparent villus surface area and villi height on birds fed with Ava Cid P during all phases also increased in relation to those who received only in the early phase. The expression of mucin 2 (MUC2) and tumor necrosis factor-alpha (TNF-α) decreased on birds that received Ava Cid P at 21 days (P < 0.05), but no differences were seen for interleukin-1beta (IL-1β) and interleukin-10 (IL-10). The results suggest that Ava Cid P can alter the mRNA expression of some inter-leukins, MUC2 and intestinal morphometry in broilers, increasing apparent villus surface area and villi height, which demonstrates the product potential as an alternative growth promoter.

Sodium butyrate (NaBt) is a four-carbon short chain fatty acid, produced naturally in colon as the end product of the bacterial anaerobic metabolism on dietary fibers. It was previously shown that NaBt can induce differentiation and may inhibit proliferation. The objective of this study was to investigate the effect of NaBt-induced differentation on inflammatory pathways in HT29 colon cancer cells. For this purpose, first, cells were treated with varying concentrations of NaBt from 1-5 mM and amount required to induce differentiation was determined as 3 mM. To understand the effect of NaBt on inflammation, the NF-kappaB pathway (p50 and p65) was investigated. Immunofluorescent staining showed increased nuclear translocation of p50 subunit with no remarkable change in subcellular localization of p65 / moreover a synergistic effect was observed when cells were co-treated with NaBt and an NF-kappaB repressor, Bay 11-7085 / implying the formation of repressive p50 homodimers in the nucleus. Our preliminary chromatin immunoprecipitation results showed that p65 recruitment v to the promoters of ICAM-1 was reduced, whereas p50 recruitment was increased. However, analysis of NF-kappaB target genes showed that cells treated with 3 mM NaBt have higher expression of the cytokines IL1-&beta / and TNF-&alpha / , adhesion molecules ICAM-1 and VCAM-1 but not COX-2. These results suggest that NaBt-induced differentiation could cause the emergence of an inflammatory signal in HT29 cells as an anti-tumor mechanism, independent from the NFkappaB activity. This work will be important in understanding the role of SCFAs in the colon microenvironment and may provide alternative therapeutic options in colorectal cancer.