<p> An electronic circuit based on a modified version of the four branch Hodgkin-Huxley electrical equivalent circuit (Roy, 1972) has been proposed and implemented to simulate the pattern of the electrical activities present in the muscle cells of the mammalian small intestine. </p> <p> The analog's implementation comprises two main circuits to simulate these activities. One of them is concerned with generating sub threshold oscillations, while the other is basically a spike-generator circuit. Additional circuitry is included to interface them. Furthermore, the analog provides a parameter set by means of which its performance may be varied. Such settings may alter the intrinsic frequency, the magnitude of the depolarizing phase of the control potential for the response activity to occur, and also the frequency of the electrical response activity. </p> <p> Four such electronic oscillators, having different intrinsic frequencies, were coupled together in a chain structure with passive elements to simulate "frequency pulling" and "entrainment" . The model qualitatively reproduced the observed pattern of electrical activities in the small intestine. </p> / Thesis / Master of Engineering (MEngr)

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Lee, Ying. / On t.p. "beta" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 171-196). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.i / Acknowledgement --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of Tables --- p.vii / List of Figures --- p.x / List of Abbreviations --- p.xvii / Content --- p.xviii / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Probiotics and Prebiotics --- p.1 / Chapter 1.1.1 --- Definitions --- p.1 / Chapter 1.1.2 --- Previous studies --- p.2 / Chapter 1.1.3 --- Properties of enhanced prebiotics --- p.6 / Chapter 1.1.4 --- Synbiotics --- p.7 / Chapter 1.2 --- Colonic fermentation --- p.10 / Chapter 1.2.1 --- Major substrates and metabolites of colonic fermentation --- p.10 / Chapter 1.2.2 --- Health-related effects of Short-Chain Fatty Acids (SCFAs) --- p.12 / Chapter 1.3 --- Bifidogenic effect --- p.14 / Chapter 1.3.1 --- Definition of bifidogenic factor and its health benefits --- p.14 / Chapter 1.3.2 --- Carbohydrate metabolism by related enzymes of bifidobacteria --- p.16 / Chapter 1.3.3 --- Previous studies on bifidogenic effects of carbohydrates --- p.18 / Chapter 1.4 --- Barley β-glucan --- p.18 / Chapter 1.4.1 --- Cereal fibres as prebiotics --- p.18 / Chapter 1.4.2 --- Chemical and physical properties and related health impacts of barley β-glucan --- p.19 / Chapter 1.4.3 --- Impacts on intestinal microecology --- p.21 / Chapter 1.4.4 --- Previous studies on bifidogenic effects of barley β-glucan --- p.21 / Chapter 1.5 --- Methodology for evaluating prebiotic and bifidogenic effect --- p.22 / Chapter 1.5.1 --- In vivo animal models --- p.23 / Chapter 1.5.2 --- Human clinical study --- p.23 / Chapter 1.5.3 --- In vitro fermentation study --- p.24 / Chapter 1.5.3.1 --- Pure culture --- p.24 / Chapter 1.5.3.2 --- Mixed culture bacterial fermenters --- p.25 / Chapter 1.5.3.3 --- Continuous culture systems as in vitro gut models --- p.25 / Chapter 1.5.4 --- Advanced molecular techniques in quantifying intestinal bacteria --- p.26 / Chapter 1.6 --- Factors affecting bifidogenic effect --- p.30 / Chapter 1.6.1 --- Molecular weight --- p.30 / Chapter 1.6.2 --- Species difference --- p.31 / Chapter 1.7 --- Enzymatic activities involved in fermentation of β-glucan --- p.32 / Chapter 1.7.1 --- "Endo-1,3-1,4-(3-glucanase (Lichenase)" --- p.32 / Chapter 1.7.2 --- "Endo-l,4-β-Glucanase (Cellulase)" --- p.33 / Chapter 1.7.3 --- Enzymatic assays --- p.33 / Chapter 1.8 --- Project objectives --- p.36 / Chapter Chapter 2. --- Materials and Methods --- p.37 / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- "Trehalose, chitin and lactulose" --- p.37 / Chapter 2.1.2 --- Barley β-glucan --- p.37 / Chapter 2.1.3 --- Pure Bifidobacterium species of human origin --- p.39 / Chapter 2.2 --- Static batch culture fermentation using fecal inoculums --- p.39 / Chapter 2.2.1 --- Substrate preparation --- p.39 / Chapter 2.2.2 --- Human fecal inoculum preparation --- p.41 / Chapter 2.2.3 --- Inoculation of human fecal inoculums --- p.41 / Chapter 2.3 --- Static batch culture fermentation using pure culture of bifidobacteria --- p.42 / Chapter 2.3.1 --- Substrate preparation --- p.42 / Chapter 2.3.2 --- Cultivation of pure bifidobacterium cultures --- p.43 / Chapter 2.3.3 --- Inoculation of bifidobacterium culture --- p.44 / Chapter 2.3.4 --- Growth curve of Bifidobacterium species --- p.44 / Chapter 2.4 --- Dry matter and organic matter disappearance in batch fermentation --- p.47 / Chapter 2.5 --- Gas chromatographic (GC) determination of short-chain fatty acids (SCFAs) --- p.48 / Chapter 2.6 --- MTT assay --- p.51 / Chapter 2.7 --- Microbial identification and enumeration --- p.53 / Chapter 2.7.1 --- Fluorescent in situ hybridization --- p.53 / Chapter 2.7.1.1 --- Oligonucleotide probes for fluorescent in situ hybridization --- p.53 / Chapter 2.7.1.2 --- Cell fixation --- p.54 / Chapter 2.7.1.3 --- In situ hybridization --- p.55 / Chapter 2.7.1.4 --- Automated image analysis --- p.55 / Chapter 2.7.1.5 --- Quantification of bacteria --- p.57 / Chapter 2.7.2 --- Optical Density (OD) measurement --- p.58 / Chapter 2.7.3 --- Direct microscopic count --- p.59 / Chapter 2.8 --- Enzyme assays --- p.60 / Chapter 2.8.1 --- Enzyme extraction --- p.60 / Chapter 2.8.2 --- "Endo-1, 3:1, 4-β-glucanase (Lichenase)" --- p.61 / Chapter 2.8.2.1 --- Principle --- p.61 / Chapter 2.8.2.2 --- Preparation of substrate and assay solutions --- p.63 / Chapter 2.8.2.3 --- Enzyme assay procedures --- p.64 / Chapter 2.8.3 --- "Endo-l,4-β-Glucanase (Cellulase)" --- p.65 / Chapter 2.8.3.1 --- Principle --- p.65 / Chapter 2.8.3.2 --- Dissolution of substrate and preparation of assay solutions --- p.65 / Chapter 2.8.3.3 --- Enzyme assay procedures --- p.66 / Chapter 2.8.4 --- API@ ZYM kit --- p.67 / Chapter 2.8.4.1 --- Principle --- p.67 / Chapter 2.8.4.2 --- Specimen preparation --- p.68 / Chapter 2.8.4.3 --- "Preparation, inoculation and reading of the strips" --- p.70 / Chapter 2.9 --- Statistical analysis --- p.71 / Chapter Chapter 3 --- Results and Discussions --- p.72 / Chapter 3.1 --- Growth curves of Bifidobacterium species --- p.72 / Chapter 3.2 --- Batch in vitro fermentation using human fecal inoculum --- p.79 / Chapter 3.2.1 --- Dry matter and organic matter disappearance --- p.79 / Chapter 3.2.2 --- Colonic bacterial profile evaluated by FISH with CellC software --- p.81 / Chapter 3.2.2.1 --- Total colonic bacteria --- p.81 / Chapter 3.2.2.2 --- Bifidobacterial growth --- p.82 / Chapter 3.2.3 --- SCFA production --- p.86 / Chapter 3.2.3.1 --- Acetate --- p.88 / Chapter 3.2.3.2 --- Propionate --- p.89 / Chapter 3.2.3.3 --- Butyrate --- p.89 / Chapter 3.2.3.4 --- Total SCFA production --- p.90 / Chapter 3.2.3.5 --- Molar ratio of SCFAs --- p.92 / Chapter 3.3 --- In vitro fermentation of barley β-glucans with different molecular weight using pure culture of Bifidobacterium species --- p.95 / Chapter 3.3.1 --- Dry matter and organic matter disappearance --- p.96 / Chapter 3.3.2 --- Evaluation of bifidobacterial growth by optical density (OD) --- p.100 / Chapter 3.3.3 --- Time course study of SCFAs production --- p.109 / Chapter 3.3.3.1 --- "Total and individual SCFAs (Acetate, Propionate and Butyrate) production" --- p.109 / Chapter 3.3.4 --- Correlation between various parameters related to fermentation --- p.124 / Chapter 3.4 --- Enzymatic activities in 2 selected Bifidobacterium species during fermentation --- p.125 / Chapter 3.4.1 --- Dry matter and organic matter disappearance --- p.126 / Chapter 3.4.2 --- Bifidobacterial growth evaluated by direct microscopic count --- p.128 / Chapter 3.4.3 --- Time course study of SCFAs production --- p.131 / Chapter 3.4.3.1 --- "Total and individual SCFAs production (Acetate, Propionate and Butyrate)" --- p.131 / Chapter 3.4.3.2 --- MTT assay --- p.137 / Chapter 3.4.3.2.1 --- Effect of metabolites in the fermentation medium on the proliferation ofSW620 --- p.137 / Chapter 3.4.3.2.2 --- Effect of metabolites in the fermentation medium on the proliferation of Caco-2 --- p.145 / Chapter 3.4.4 --- Enzyme assays using commercial kits --- p.153 / Chapter 3.4.4.1 --- API @ZYM assay --- p.153 / Chapter 3.4.4.2 --- Efficiency of intra-cellular enzyme extraction using labiase --- p.156 / Chapter 3.4.5 --- Time course enzyme assays --- p.157 / Chapter 3.4.5.1 --- Lichenase activity assay --- p.157 / Chapter 3.4.5.2 --- Cellulase activity assay --- p.160 / Chapter Chapter 4. --- Conclusions and Future work --- p.168 / References --- p.171

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Call number: LD2668 .T4 1984 F46 / Master of Science

Thesis (PhD)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: Research on probiotics has increased over the past years, which led to commercialization of a number of probiotic supplements and functional foods. In vitro assays such as tolerance to acid and bile, adhesion to mucus and epithelial cells, antimicrobial activity and antibiotic resistance tests are performed to screen lactic acid bacteria for probiotic properties. Enterococcus mundtii ST4SA produces an antimicrobial peptide (peptide ST4SA) with activity against Gram-positive and Gram-negative bacteria. Lactobacillus plantarum 423 produces plantaricin 423, a typical class II bacteriocin, active against a number of Gram-positive bacteria. A gastro-intestinal model (GIM) simulating the gastro-intestinal tract (GIT) of infants, was developed to study the survival of E. mundtii ST4SA and L. plantarum 423 and evaluate them as possible probiotics. Growth of the two strains in the GIM was compared to the growth of commercially available probiotics. Infant milk formulations were used as growth medium. Changes in pH, the addition of bile salt and pancreatic juice, and intestinal flow rates were controlled by peristaltic pumps linked to a computer with specifically designed software. Strain ST4SA was sensitive to low pH and high concentrations of bile salts. Growth of strain ST4SA was repressed in the first part of the GIM, however, the cells recovered in the ileum. Strain 423 was also sensitive to acidic conditions. However, the cells withstood the presence of bile and pancreatin in the first part of the GIT. Neither of the two strains displayed bile salt hydrolase (BSH) activity. Both strains were resistant to amoxicillin, ampicillin, chloramphenicol, cefadroxil, roxithromycin, meloxicam, doxycycline, erythromycin, novobiocin, rifampicin, tetracyclin, bacitracin, oflaxacin and cephazolin, anti-inflammatory drugs Na+- diklofenak and ibuprofen, and painkillers codeine terprim hydrate aminobenzoic acid, metamizole aspirin and paracetamol. Strain 423 was resistant to ciprofloxacin. Genes encoding cytolysin, non-cytolysin β-hemolysin and cell aggregation substances were detected on the genome of strain ST4SA but they were not expressed. L. plantarum 423 does not contain genes encoding gelatinase, cell aggregation, enterococcus surface protein, hemolysin, non-cytolysin β- hemolysin and enterococcus endocarditis antigen. Both strains inhibited the growth of Listeria monocytogenes ScottA in the GIM. Survival of the strains improved when used in combination and compared well with the survival of commercially available probiotics. Adhesion to epithelial cells is an important prerequisite for bacterial colonization in the GIT. The adhesion of E. mundtii ST4SA and L. plantarum 423 was studied using Caco-2 (human colon carcinoma epithelial) cells. Both strains revealed good adhesion compared to other probiotic strains. No correlation was found between hydrophobicity, auto-aggregation and adhesion to Caco-2 cells. Antibiotics and anti-inflammatory medicaments had a negative effect on adhesion. Different combinations of proteins were involved in the adhesion of E. mundtii ST4SA and L. plantarum 423 to Caco-2 cells. E. mundtii ST4SA, L. plantarum 423 and L. monocytogenes ScottA were stained with fluorescent dyes to visualize adhesion to Caco-2 cells. Adhesion of L. monocytogenes ScottA to Caco-2 cells was not reduced in the presence of strains ST4SA and 423. Cell-free culture supernatants of both strains inhibited the invasion of L. monocytogenes ScottA. The cell structure of Caco-2 cells changed in the presence of L. monocytogenes ScottA. Strains ST4SA and 423 protected Caco-2 cells from deforming. / AFRIKAANSE OPSOMMING: Navorsing op probiotika het die afgelope tyd drasties toegeneem en aanleiding gegee tot die kommersialisering van ‘n groot hoeveelheid probiotiese supplemente en funksionele voedselsoorte. In vitro studies, soos bv. weerstand teen suur en gal, vashegting aan mukus en epiteelselle, antimikrobiese aktiwiteit en weerstand teen antibiotika word uitgevoer om te bepaal of melksuurbakteriëe aan probiotiese standaarde voldoen. Enterococcus mundtii ST4SA produseer ’n peptied met antimikrobiese werking teen Grampositiewe en Gram-negatiewe bakteriëe. Lactobacillus plantarum 423 produseer ‘n tipiese klas II bakteriosien, plantarisien 423, met aktiwiteit teen sekere Gram-positiewe bakteriëe. ’n Gastro-intestinale model (GIM) wat die spysverteringskanaal (SVK) van babas simuleer, is ontwikkel om die oorlewing van E. mundtii ST4SA en L. plantarum 423 te bepaal en hul eienskappe met dié van kommersiële probiotiese stamme te vergelyk. Babamelk formules is as groeimedium gebruik. Verandering in pH, byvoeging van galsoute en pankreassappe, en intestinale vloei is met behulp van peristaltiese pompe gereguleer wat seine vanaf ‘n spesiaal ontwikkelde rekenaarprogram ontvang. E. mundtii ST4SA was sensitief vir lae pH en hoë galsoutkonsentrasies en groei is in die eerste deel van die GIM onderdruk. Selgetalle het wel in die ileum herstel. Stam 423 was ook sensitief vir lae pH, maar het die galsout- en pankreatienvlakke in die laer deel van die SVK weerstaan. Geen galsout-hidrolase aktiwiteit is by enige van die twee stamme gevind nie. Beide stamme het weerstand getoon teen amoksillien, ampisillien, chloramfenikol, cefadroksiel, roksitromisien, meloksikam, doksisiklien, eritromisien, novobiosien, rifampisien, tetrasiklien, basitrasien, oflaksasien, kefazolien, die anti-inflammatoriese medikamente Na+-diklofenak en ibuprofen, en die pynstillers kodeïenterprimhidraataminobensoësuur, metamisoolaspirien en parasetamol. L. plantarum 423 was bestand teen ciprofloksasien. Gene wat kodeer vir sitolisien, nie-sitolisien β-hemolisien III en sel-aggregasie is op die genoom van E. mundtii ST4SA gevind, maar word nie uitgedruk nie. L. plantarum 423 besit nie die gene wat vir gelatinase, selaggregasie substansies, enterokokkus selwandproteïen, hemolise, nie-sitolisien β-hemolisien en enterokokkus endokarditis antigeen kodeer nie. Albei stamme inhibeer die groei van Listeria monocytogenes ScottA in die GIM. Die twee stamme in kombinasie het tot beter oorlewing in die GIM gelei. Stamme ST4SA en 423 vergelyk goed met kommersieël beskikbare probiotika. Vashegting van probiotiese stamme aan epiteelselle is belangrik vir kolonisering in die SVK. Vashegting van E. mundtii ST4SA en L. plantarum 423 is bestudeer deur van Caco-2 (kolon epiteel) selle van die mens gebruik te maak. Die aanhegting van beide stamme aan Caco-2 selle het goed vergelyk met kommersieël beskikbare probiotiese stamme. Geen korrelasie is gevind tussen hidrofobisiteit, aggregasie en vashegting aan Caco-2 selle nie. Antibiotika en antiinflammatoriese medikamente het ‘n negatiewe effek op vashegting gehad. Verskillende kombinasies van proteïene is betrokke in die vashegting van E. mundtii ST4SA en L. plantarum 423 aan Caco-2 selle. E. mundtii ST4SA, L. plantarum 423 en L. monocytogenes ScottA is met fluoreserende kleurstowwe gemerk om vashegting aan Caco-2 selle te monitor. Vashegting van L. monocytogenes ScottA aan Caco-2 selle is nie deur die teenwoordigheid van stamme ST4SA en 423 beïnvloed nie. Sel-vrye kultuursupernatante van beide stamme het die binnedring van L. monocytogenes ScottA verhoed. Die selstruktuur van Caco-2 selle het in die teenwoordigheid van L. monocytogenes ScottA van vorm verander. E. mundtii ST4SA en L. plantarum 423 het die Caco-2 selle teen vervorming beskerm.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

The intestine is constantly exposed to a wide variety of dietary antigens, commensal bacteria and pathogens, toward which it has evolved complex immune responses to protect the host. The intestinal immune system relies on innate immune cells, such as mononuclear phagocytes (MNPs), that include dendritic cells (DCs), monocytes (Mo) and macrophages (Mfs), to sense and respond to luminal and mucosal challenges. MNPs are essential players as they instruct adaptive immune cells, in particular T cells, to discriminate between innocuous and harmful antigens. Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by epithelium-associated commensal segmented filamentous bacteria (SFB). Several reports suggest that the cytokine environment induced by gut bacteria is sufficient to drive LP Th17 cell differentiation. In this context, intestinal DCs are proposed to facilitate the conversion of naïve CD4 T cells to Th17 cells within gut-draining lymph nodes. Whether such mechanisms control commensal-mediated Th17 cell differentiation has not been examined. In this work, I explore the mechanisms of induction of Th17 cells by SFB, with a particular focus on the role of antigen-presenting cells in this process. Initiation of CD4 T cell responses requires both major histocompatibility II (MHCII)-mediated antigen presentation and cytokine stimulation, which can be provided by the same or different subsets of intestinal MNPs. To test the requirement for either function in the induction of Th17 cells by SFB, we analyzed the role of SFB-induced cytokine environment in driving Th17 cell differentiation of non-SFB transgenic CD4 T cells. We find that although the cytokine environment is important, it is not sufficient to promote Th17 cell differentiation of activated CD4 T cells. In fact, we show that MHCII-dependent antigen presentation of SFB antigens by intestinal MNPs is crucial for Th17 cell induction. Expression of MHCII on CD11c+ cells was necessary and sufficient for SFB-induced Th17 cell differentiation. We also show that most SFB-induced Th17 cells respond to SFB antigens, which stressed that they carry T cell receptors that recognize SFB moieties. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. Our results outline the complex role of MNPs in the regulation of intestinal Th17 cell homeostasis, and we investigated the contribution of individual subsets to SFB-specific Th17 cell differentiation. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. To this end, I examined the role of mucosal DCs and Mfs in Th17 induction by SFB in vivo. Employing DC and Mf subset-specific depletion and gain-of-function mouse models, I show that Mfs, and not conventional CD103+ DCs, are essential for generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.

Orientador: Daniela Cardoso Umbelini Cavallini / Orientador no exterior: Bruce A. Vallance / Coorientador: Caetano Martha Antunes / Banca: Bruce Vallance / Banca: Carla Fontana / Banca: Xiaonan Lu / Banca: Ligia Sassaki / Resumo: O trato gastrointestinal abriga a maior população de microrganismos no corpo humano, onde eles desempenham um papel importante na promoção da saúde do hospedeiro. A alteração na composição da microbiota pode levar à disbiose intestinal, que consequentemente desencadeia ou agrava doenças intestinais e extra-intestinais. Microrganismos benéficos também conhecidos como probióticos são constantemente investigados como uma terapia complementar nas doenças relacionadas à disbiose. No entanto, sua eficácia no tratamento de condições severas, como por exemplos as doenças inflamatórias intestinais (DII), é bastante variável e apresenta resultados controversos. Para abordar a importância de uma abordagem probiótica personalizada para tratar a inflamação intestinal, primeiro examinamos o efeito de bactérias personalizadas usando um modelo de colite induzida por produtos químicos. Os animais que receberam comensais isolados de suas próprias fezes foram mais protegidos contra a inflamação, pois mostraram sinais reduzidos de colite, menor dano histológico e menores níveis de marcadores inflamatórios, quando comparados aos ratos que receberam uma cepa probiótica comercial. Em seguida, o papel da mucina intestinal Muc2 e da enzima Core-1 que glicosilam foram explorados usando o modelo de colite infecciosa Citrobacter rodentium. A camada de muco intestinal é a primeira linha de defesa no intestino e é composta em grande parte pela mucina Muc2. Uma vez que quase todas as bactérias entéricas de... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The intestinal tract harbours the largest population of microbes in the human body where they play an important role in promoting the health of their host. If the composition of these microbes is altered, this may lead to dysbiosis that triggers or exacerbates intestinal and extra-intestinal diseases. Probiotics have been investigated as a complementary therapy in dysbiosis-related diseases. However, their effectiveness in treating severe conditions such as Inflammatory Bowel Disease (IBD) is quite variable and have shown controversial results. To address the importance of a personalized probiotic approach to treat intestinal inflammation, we first examined the effect of personalized bacteria using a model of chemical induced colitis. The animals that received commensals isolated from their own feces were more protected against inflammation as they showed reduced signs of colitis, less histological damage and lower levels of inflammatory markers as compared to mice given a commercial probiotic strain. Next, the role of the intestinal mucin Muc2 and the Core-1 enzyme that glycosylates it were explored using the Citrobacter rodentium model of infectious colitis. The intestinal mucus layer is the first line of defense in the intestine and is largely composed of the secreted mucin Muc2. Since almost all enteric bacteria must cross the overlying mucus layer to infect the host, the mucus-enteric bacterial interactions provide fundamental knowledge about infectious diseases as well as inflammatory conditions linked to dysbiosis... (Complete abstract click electronic access below) / Doutor