published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

Submitted in partial fulfillment for the Degree of Master of Applied Sciences in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Prebiotics are defined as non-digestible oligosaccharides (NDOs) or polysaccharides (NDPs), which promote the growth of beneficial lactic acid bacteria in the colon. Algae are rich in polysaccharides and can be exploited as prebiotics for functional food ingredients to improve human and animal health. Currently, inulin is the most widely used ingredient in the prebiotics market, which is produced from live plants and requires expensive production processing. There is a vast repository of marine life with algae as a major source of nutrients. Therefore, this study provides an alternative source for prebiotic production and examines marine and freshwater algae that promote the growth of two strains of Lactobacillus delbrueckii subs. (Lactobacillus lactis and Lactobacillus bulgaricus) and one strain of Bifidobacterium spp. (Bifidobacterium longum). Monosaccharides of the oligosaccharide fraction of marine and freshwater algal extracts were investigated with the use of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) after acidic hydrolysis of cell matrix polysaccharides. A total of fifty-five marine and freshwater aqueous algal extracts were assessed for their effect on the growth of L. lactis, B. longum and L. bulgaricus over a 96 hour period. Relative to the negative control, 34.5% algal extracts showed improved growth on one or more probiotic bacteria. The optimum time for maximum bacterial growth was noted at 48 h for all the tested aqueous algal extracts. Five marine and freshwater algal cultures (Spirulina platensis, Chlorococcum spp., Dunaliella salina, Scenedesmus magnus, Chlorella spp. and algal extract no. 48) from various aquatic environments in Kwa-Zulu Natal showed the best growth dynamics and demonstrated the greatest potential as sources of biomass for prebiotic production. These algal extracts were able to significantly increase the growth of at least one of the three probiotic bacteria (p < 0.05). Aqueous algal extract from S. platensis was regarded as the best algal source for prebiotics as it demonstrated a greater stimulatory effect on the growth of all three probiotic bacteria (L. lactis, B. longum and L. bulgaricus) compared to tested aqueous algal extracts and the inulin used as a positive control. The results obtained by HPLC for characterization confirmed TLC data, as xylose and galactose were detected by both chromatograms. These data indicated that xylose and galactose were present in aqueous algal extracts from S. magnus and S. platensis and galactose in aqueous algal extract no. 48. Xylose was most abundant in aqueous algal extracts from S. platensis (3mg/ml) and S. magnus (2.3mg/ml). In conclusion aqueous algal extracts from S. platensis, Chlorococcum, D. salina, S. magnus, Chlorella and algal extract no. 48 are potential sources for prebiotic production. Spirulina platensis extract was regarded as the best algal source. Xyose and galactose characterized by HPLC in algal extracts make up oligosaccharides that function as prebiotic compounds for stimulation of probiotic bacteria. There is a great scope for successful production of prebiotics from algal sources in South Africa. / M

In an irritable bowel syndrome (IBS) rat model, S. chinensis reversed the exaggerated visceral nocicptive responses (judged by abdominal withdrawal reflex and electromyographic measurement) to colorectal distension induced by neonatal maternal separation. Relief of visceral hypersensitivity by S. chinensis could be related to the decrease of elevated 5-HT level and the reduction in 5-HT3 receptor expression in colon. / In summary, given the modulatory effects on intestinal motility and visceral sensation, Schisandra chinensis would be potentially useful for the treatment of relieving diarrhea and visceral pain symptoms in IBS patients. Schisandra lignans, the major absorbable components, can be regarded as the active ingredients in S. chinensis for the potential treatment of IBS. / Schisandra chinensis, which is named "Wu-Wei-Zi" in Chinese Pin Yin, is widely used in Chinese medicine as an astringent, tonic and sedative agent. Dibenzo[a,c]-cyclooctadiene lignans are the major components of this herb. In the present study, the chemical constituents of S. chinensis were first characterized. A HPLC-DAD method was developed and validated for quantitative analysis of four major Schisandra lignans, namely, schisandrin (SCH-1), gomisin A (SCH-2), deoxyschisandrin (SCH-3) and gamma-schisandrin (SCH-4), in the aqueous and ethanolic extracts of S. chinensis. The ethanolic extract contains higher amount of lignan components than aqueous extract. The HPLC method has also been employed to obtain chromatographic fingerprintings to distinguish S. chinensis from a related species, S. sphenanthera. / The modulatory effects of both S. chinensis extracts and four major lignans on intestinal motility were evaluated using in vitro intestinal motility assays. The tested compounds induced relaxation on guinea pig ileum contracted by acetylcholine, serotonin and electrical field stimulation, as well as on rat colon with spontaneous contractility. While SCH-3 was most potent in inhibiting sensorimotor response in guinea pig ileum, SCH-1 displayed the highest potency of inhibition on spontaneous contraction of rat colon. / The relaxant effect on rat colon induced by SCH-1 has been demonstrated to involve two or more non-adrenergic non-cholinergic mediators. Nitric oxide was likely to be one of the inhibitory transmitters that involved cGMP-dependent pathways, whereas the non-nitrergic component was apamin-sensitive, but probably excluded vasoactive intestinal peptide (VIP) and adenosine. / With the aid of HPLC-DAD-MS for qualitative and quantitative analyses, the absorption of S. chinensis in the rat everted gut sac and human Caco-2 monolayer in vitro models have been profiled. Fifteen Schisandra lignans were identified as the major absorbable components of S. chinensis in these models. Transport study on SCH-1 has shown a passive diffusion pathway with high permeability. In an in vivo study, metabolites of Schisandra lignans could be found in rat plasma after a single oral administration of S. chinensis extract. The plasma pharmacokinetics of S. chinensis in rats was further evaluated using simultaneous quantification of four representative Schisandra lignans (SCH-1, SCH-2, SCH-3 and SCH-4). / Yang, Jiaming. / Adviser: Chun-Tao Che. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 262-283). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In this thesis, we develop and apply top-down, quantitative approaches to gain novel insights into various complex biological systems. Beginning at the multicellular level, we study human gut microbiome dynamics from an ecological perspective. We develop computational frameworks to enable a global understanding of the spatiotemporal variability of gut bacterial abundances. We demonstrate the utility of our frameworks to elucidate the ecological processes governing abundance changes of gut microbiota. We then shift our focus to the intracellular level by investigating the metabolic limitations of cancer cell growth. We use coarse-grained mathematical modeling to identify a major growth limitation of cancer cells associated with electron acceptor deficiency, which we then experimentally validate. Collectively, these set of approaches help to decipher the organizing principles of complex biological systems at both the individual and multicellular levels.

Bacteria in the gastrointestinal tract play an important role in intestinal motility, inflammation, homeostasis, and behavior. Bacteria, through the natural synthesis of neuroactive compounds and secondary metabolites, can modulate the host immune system and communicate with the host along the signaling pathway along the gut-brain axis. Here, we functionally design, develop, test, and characterize a platform for the study of microbial-host interactions using advancements in the field of synthetic biology. First, we describe the engineering of Escherichia coli Nissle to biosynthesize serotonin within the mammalian gut using a native-plasmid optimized approach. Serotonin is crucial for neurotransmission throughout the body and may be playing a role in microbial gut-brain communication. In the gastrointestinal tract, serotonin regulates intestinal motility, cell turnover, intestinal inflammation, and gastrointestinal homeostasis. Upon serial daily oral gavages, our engineered bacterium populates a murine colon to produce serotonin locally in the mucosa layers along the epithelial lining. Changes in host physiology were observed including decreased gastrointestinal motility, increased colonic Muc2 expression, induction of host TPH2, responsible for serotonin biosynthesis in enteric neurons, and upregulation of serotonin receptors HTR3, HTR4, and HTR7 in the colon. Behavioral tests revealed a statistically significant decrease in anxiety and depression in stress-induced environments in mice treated with the engineered bacterium. This work suggests that gut bacteria engineered to modulate host gut-brain axis may have both scientific and clinical uses to study microbial-host interactions and treat gastrointestinal and behavioral mood disorders in humans. Second, we engineered bacteria to produce exogenous butyrate and other SCFAs in the murine gut. Short chain fatty acids (SCFAs) play an important role in intestinal homeostasis, fluid dynamics, inflammation, oxidative stress, and intestinal hypersensitivity and motility. With this development, we characterized the effects of our butyrate-producing bacteria on a high-fat diet and DSS-induced colitis model within the colon. Although energetically burdensome to produce, our strains produced butyrate in the colon at higher density in an actively inflamed colitis model. After 14 days of oral administration, our engineered strain (EcN:B) increased the colon length of normal wild-type mice, in high fat fed mice, and in mice with recovering and actively inflamed DSS-induced colitis. EcN:B increased mucosal barrier thickness, upregulated gene expression of the barrier integrity markers Cldn1, Ocln, Zo1, and altered crypt and villus height during inflammation recovery. Furthermore, as butyrate is known to induce Foxp3+ Regulatory T cells, we saw a 13.01% percent increase in Foxp3+ cells in the colon of mice fed our engineered bacteria. This work suggests that synthetic gut bacteria engineered to produce short chain fatty acids may have future clinical uses to treat patients with inflammatory bowel disease including Crohn’s and Colitis with future potential to serve as a therapeutic for irritable bowel syndrome, idiopathic constipation, obesity, and colorectal cancer. This platform, with the use of synthetic biology to natively engineer Escherichia coli Nissle to produce bioactive compounds in the distal gastrointestinal tract, creates a framework for future characterization of bacterial-host communication and future microbial-based therapeutics.

H. pylori is one of the most significant discoveries in gastroenterology in the past century. It is associated with a wide range of gastroduodenal pathologies and gastric cancer. Antibiotic resistance in H. pylori has emerged as a significant clinical problem. The body of work contained within this dissertation was carried out to investigate an alternative therapy based on observations of the traditional therapy for gastric disease in the Middle East. One of these traditional therapies centres on plants belonging to the Pistacia genus. This study represents the first reported investigation into the composition and biological activity of the trunk bark exudates of Pistacia atlantica Kurdic (P. a. Kurdica), Pistacia atlantica Mutica (P. a. Mutica) and Pistacia atlantica Cabolica (P. a. Cabolica), resinous gums that have been termed here ‘Kurdica Gum’, ‘Mutica Gum ’ and ‘Cabolica Gum ’ respectively. The antimicrobial screening of the trunk exudates of the genus Pistacia led to the characterization of the most active fraction of the Kurdica gum. This fraction was subsequently subjected to sub-fractionation leading to the discovery of fundamentally new information that went beyond H. pylori, expanding the original parameters of the project. The extent of these findings suggests that new classes of antibiotics might have been discovered. Primary studies on their structure and potential mechanism of action has been undertaken. Thirteen novel antimicrobial agents were identified. Based on the characteristics of these isolated fractions, 50 new compounds were modelled; of which 30 hypothetically have an MIC consistent with contemporary antibiotics and could represent viable lead compounds for commercial development. / Doctor of Philosophy

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.

Bibliography: leaves 197-220. / xv, 220 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Analyses some general problems of laparoscopy, particularly in relation to induced physiological disturbances; by development and evaluation of new applications in laparoscopic gastrointestinal surgery ; and by assessing the efficacy of laparoscopic procedures which have already entered rountine practice. / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 1996?

Includes bibliographical references. / 59 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Various articles published by John Robin Warren on the discovery and pathology of Helicobacter pylori. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 2000

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences