Thesis (M. CM)--Hong Kong Baptist University, 2006. / Dissertation submitted to the School of Chinese Medicine. Includes bibliographical references (leaves 25-28).

Synthetic biology consists of the design and construction of customized cell-based systems, and metabolic engineering is its co-discipline that aims to engineer these cells into biological factories for the production of drugs, chemical commodities and fuels. Together, these two disciplines continue to provide various innovative solutions to current problems of humanity in the areas of medicine, agriculture and energy. In this dissertation, we use synthetic biology and metabolic engineering approaches to explore the potential of engineered live yeasts as therapeutic platforms for treating inflammatory bowel disease (IBD). The vast majority of microbial-based therapeutics at the moment have focused on bacteria instead of yeasts, and all of these engineered live bacterial platforms use either proteins or peptides as therapeutic agents of choice. This dissertation seeks to enhance yeast’s beneficial properties to humans by genetically engineering them to produce TAN-1612, a small molecule tetracycline with therapeutic potential. We choose tetracyclines as our small molecule therapeutic agent because these compounds are one of the most impactful natural products that humanity has benefited from due to its significant antimicrobial and anti-inflammatory properties. We genetically engineer strains of baker’s yeast Saccharomyces cerevisiae and the probiotic yeast Saccharomyces cerevisiae var boulardii to produce in situ the fungal tetracycline TAN-1612, a natural product with anti-inflammatory properties (instead of anti-microbial so as to not disturb the gut microbiome), and to study the molecular mechanisms involved in their potential beneficial effects for IBD. Our engineered live yeast therapeutics would provide an effective, safe, and cheap alternative to treating IBD and other gastrointestinal tract disorders compared to the currently available but costly and laborious therapies. In Chapter 1, we review key milestones in the fields of synthetic biology and metabolic engineering that have enabled and inspired the generation of both engineered live microbial-based systems and small molecules as the therapeutic agents for the potential treatment of a wide array of human diseases such IBD, cancer, and pathogenic infections. In Chapter 2, we develop synthetic biology and metabolic engineering approaches for designing, building, and testing of the biosynthetic pathway of TAN-1612 in genetically engineered yeasts such as S. cerevisiae and S. boulardii. These approaches enable the production of TAN-1612 in yeasts with titers as high as ~61 mg/L which represent a 100-fold improvement from previous reported yeast strains. These engineering approaches hold great potential to advance the heterologous biosynthesis of other small molecule therapeutics in yeasts. In Chapter 3, we explore the role of TAN-1612 as an anti-inflammatory agent, inhibitor of tetracycline inactivating enzymes, and inducer of gene expression with the goal of identifying its best therapeutic or biological application that can be leveraged for the development of engineered live yeast-based systems for the in situ treatment of IBD. Advances in DNA synthesis and sequencing technologies have spurred the high-throughput construction of microbial strains for numerous applications in synthetic biology and metabolic engineering. Breakthrough technologies in our abilities to screen and select target molecule biosynthesis, however, are needed in order to realize the potential of both of these disciplines for drug discovery and production. Current state-of-the-art methods such as liquid/gas chromatography – mass spectrometry (LC/GC – MS) are applicable to screen or select a variety of target molecules but their throughput remains low (~102 samples/day). Other screening or selection methods available are highly dependent on the molecule of interest and generally inapplicable to other compounds. Therefore, in Chapter 4 we propose that the Fluorescence Polarization (FP) assay can be readily adapted as a general, medium-throughput (~104 samples/day) screen for synthetic biology and metabolic engineering applications. As a proof-of-principle, we develop an FP assay to detect the immunosuppressant polyketide FK506, use this assay to detect FK506 biosynthesized from Streptomyces tsukubaensis cultures in microtiter plates, and finally apply this FP assay to generate S. tsukubaensis strains with increased production of FK506. Lastly, we outline the experimental steps necessary to adapt the FP to screen different classes of natural products beyond polyketides such as FK506 or tetracyclines. The fungal polyketide TAN-1612 is an attractive chemical scaffold for the generation of novel tetracycline-based therapeutics using metabolic engineering approaches. Libraries of > 108 are usually required to generate chemical compound diversity to identify drugs with significant therapeutic activities. Compared to screening methods, genetic selections allow the detection of target molecules at a much higher throughput (> 108 samples/day) because the population of cells can be cultured and assayed in one-pot manner. Thus, in Chapter 5 we establish the yeast three hybrid (Y3H) assay as a general, high-throughput selection technology to detect tetracycline derivatives. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating producer and non-producer yeast strains of TAN-1612. The Y3H assay can be used for the heterologous biosynthesis of tetracycline analogues in yeasts especially because the Y3H would enable the production and detection of these derivatives in the same yeast cell.

M.A. / Irritable Bowel Syndrome (IBS) can be described as a bodily idiom - a nonverbal language which may have its roots in unspeakable dilemmas (Griffiths & Griffiths, 1994). The splitting of languages and silencing of the body may be the soil in which such symptoms grow. Unutterable conflicts lead to the symptoms being trapped within the body until the body itself begins to "speak" (Griffiths & Griffiths, 1994). In essence, this study seeks to evaluate the effects of attaching language, feelings and awareness to these symptoms and communicating this with other IBS subjects within the group context. Psychiatric illness is often found in IBS health care seekers (Drossman & Thompson, 1992). The specific aim of this study was to ascertain the effects of a holistic short-term group intervention in the treatment of IBS with comorbid depression and anxiety. The sample consisted of 24 South African women who had been positively diagnosed with severe IBS by either a gastroenterologist or a general practitioner. Furthermore, each subject had to have associated moderate to severe depression and anxiety. Four questionnaires were utilised, namely the Biographical Questionnaire, the Irritable Bowel Syndrome Client Questionnaire, the Personality Assessment Inventory (PAI) and the Functional Bowel Disorder Severity Index (FBDSI). The Biographical Questionnaire mainly requested personal details and sought a family history of psychological disorders. The Irritable Bowel Syndrome Client Questionnaire, based on the standardised Rome Criteria (Drossman, 1994; Drossman, Zhiming, Toner, Creed, Thompson, Read et al., 1995; Talley, Phillips, Melton, Mulvihill, Wiltgen & Zinsmeister, 1989), verified a positive IBS diagnosis, while the Functional Bowel Disorder Severity Index rated the severity of the subject’s IBS. Lastly, the depression score was rated on the depression scale of the Personality Assessment Inventory (PAI) and the anxiety score was rated on the anxiety scale of the PAI. The subjects were divided into two groups of twelve members each - Group 1 was the experimental group and Group 2 was the control group. The group design was a pre-test, post-test control group design where subjects in Group 1 (the experimental group) received group intervention and subjects in Group 2 (the control group) were placed on a waiting list and received no intervention. The subjects in the control group were offered individual therapy once the post-tests were completed. All the subjects completed the IBS Severity Index Questionnaire and the Depression and Anxiety subscales of the Personality Assessment Inventory before commencement of group therapy for Group 1 and again one month after completion of this intervention. The effect of the intervention was determined utilising comparative statistics with reference to the pre-test versus post-test scores. The t-test for the equality of means for between group variance was utilised for two analyses. Firstly, it was used to determine the variance regarding the pre-test scores between Group 1 (the experimental group – who received intervention) versus Group 2 (the control group – who received no intervention) (Hypothesis 1). Secondly, it was utilised to determine the between group variance in terms of the post-test scores for Group 1 (the experimental group) versus Group 2 (the control group) (Hypothesis 2). The paired samples t-test was also used for two analyses. Firstly, it was used to determine the within group variance regarding the pre-intervention test scores versus the post-intervention test scores for Group 1 (the experimental group)(Hypothesis 3). Secondly, the paired samples t-test was also utilised to determine if there were statistically significant differences in terms of the pre-test scores versus the post-test scores of Group 2 (the control group) who did not receive the intervention (Hypothesis 4). A short-term holistic group therapy model was applied based on the work of Broom (1997), Crafford (1985), Pretorius (1996) and Yalom (1970). The results of the study showed that there was a statistically significant improvement in the anxiety scores of Group 1 (the experimental group) after completion of the intervention when compared with Group 2 (the control group) who received no intervention. The within group depression and anxiety scores in the experimental group also revealed a statistically significant improvement after the intervention. However, the IBS symptom severity remained unchanged. Thus, it is concluded that holistic short-term group therapy is indicated in the treatment of severe IBS with comorbid depression and anxiety even if the IBS symptoms are unaltered. It is recommended that further research be conducted to ascertain whether holistic group therapy of a moderate duration (approximately eight to ten weeks) has a greater impact on the IBS symptom severity.

M.A. / The purpose of this study was to ascertain whether patients suffering from irritable bowel syndrome (IBS) differed from non-IBS clients in terms of their coping styles. Gastrointestinal disorders are among the most common of all illnesses; half of the population suffers from acute gastrointestinal illnesses every year (Read, 1985). More than 10% have chronic illnesses, and these illnesses are a major cause of absenteeism from work. In view of this it is surprising that there is such a faucity of psychological and psychophysiological research focusing on gastrointestinal activity. Perhaps one reason for this is that investigators conceptualise the gastrointestinal tract as a system that is unresponsive to psychological intervention. Another reason may be the widespread belief that adequate techniques are not available for studying gastrointestinal psychology and psychophysiology (Haynes & Gannon, 1981). Today there is consensus that IBS is a psychosomatic disorder that accounts for between 40 to 70% of referrals to gastroenterologists. Unfortunately, this is a very misunderstood disorder. Sufferers are often misinformed or poorly educated by their physicians. Misunderstanding and lack of patient education often results in increased anxiety and physical distress. There are cases in which unnecessary surgery, expensive diagnostic procedures and addictive pain killers are mistakenly employed. In addition, IBS patients represent an expensive group because they use up a considerable amount of medical resources in money and time (Moser, 1986).

Doctor of Philosophy / Nineteenth century philosophy and anatomy regarded the nervous system as the only pathway of communication between the brain and body but now, research in the field of psychoneuroimmunology (PNI) has provided evidence to prove the age-old belief that there is a connection between the mind (or mental/emotional states) and the body. Researchers in PNI have now shown that the communication between the nervous and immune systems is bi-directional – i.e. there is a psychological reaction to physical disease and a somatic presentation of psychological disorders - and that the immune system, the autonomic nervous system, the endocrine system and the neuropeptide systems all communicate with each other by means of chemicals called messenger molecules or ligands. This paper outlines research into the treatment of Irritable Bowel Syndrome (IBS) with hypnotherapy, taking into account the mind-body connection and treating both the patient’s physiological and emotional/psychological symptoms rather than treating the physiological symptoms only. In other words, using a more holistic approach to the treatment of IBS. IBS is probably the most common functional gastrointestinal disorder encountered by both gastroenterologists and physicians in primary care. It is estimated that from 10% to 25% of the general population suffer from this condition and that it comprises about 30-50% of the gastroenterologists’ workload, yet the aetiology of IBS is unknown and, so far, there is no cure. Researchers are beginning to view IBS as a multi-faceted disorder in which there appears to be a disturbance in the interaction between the intestines, brain, and autonomic nervous system, resulting in an alteration in the regulation of bowel motility and/or sensory function. Most researchers agree that a subset of IBS sufferers have a visceral hypersensitivity of the gut or, more specifically, an increased perception of sensations in the gut. To date, studies of IBS have proposed previous gastroenteritis, small intestine bacterial overgrowth, psychosocial factors, a genetic contribution, and an imbalance of neurotransmitters as either possible causes or playing a part in the development of IBS. It is generally agreed that a patient’s emotional response to stress can exacerbate the condition. In section 1 of the thesis, the introduction, a detailed description and background appropriate to the study undertaken are provided, including aspects of epidemiology, diagnostic symptom criteria and clinical relevance of the Irritable Bowel Syndrome. Previous studies of various forms of treatment for IBS are discussed with the main emphasis being on treatment with hypnotherapy. All these therapies have concentrated on either mind or body treatments whereas this study demonstrates how hypnotherapy, and the use of imagery, addresses both mind and body. Finally, the rationale for the current study and the specific aims of the thesis are outlined. In section 2, the methodology and assessment instruments used in the clinical trial are discussed, as well as recruitment processes, research plan and timetable, and treatment schedule. Statistical analyses are provided and the main outcomes measures of the clinical trial, its limitations and scientific implications are addressed.

Doctor of Philosophy / Nineteenth century philosophy and anatomy regarded the nervous system as the only pathway of communication between the brain and body but now, research in the field of psychoneuroimmunology (PNI) has provided evidence to prove the age-old belief that there is a connection between the mind (or mental/emotional states) and the body. Researchers in PNI have now shown that the communication between the nervous and immune systems is bi-directional – i.e. there is a psychological reaction to physical disease and a somatic presentation of psychological disorders - and that the immune system, the autonomic nervous system, the endocrine system and the neuropeptide systems all communicate with each other by means of chemicals called messenger molecules or ligands. This paper outlines research into the treatment of Irritable Bowel Syndrome (IBS) with hypnotherapy, taking into account the mind-body connection and treating both the patient’s physiological and emotional/psychological symptoms rather than treating the physiological symptoms only. In other words, using a more holistic approach to the treatment of IBS. IBS is probably the most common functional gastrointestinal disorder encountered by both gastroenterologists and physicians in primary care. It is estimated that from 10% to 25% of the general population suffer from this condition and that it comprises about 30-50% of the gastroenterologists’ workload, yet the aetiology of IBS is unknown and, so far, there is no cure. Researchers are beginning to view IBS as a multi-faceted disorder in which there appears to be a disturbance in the interaction between the intestines, brain, and autonomic nervous system, resulting in an alteration in the regulation of bowel motility and/or sensory function. Most researchers agree that a subset of IBS sufferers have a visceral hypersensitivity of the gut or, more specifically, an increased perception of sensations in the gut. To date, studies of IBS have proposed previous gastroenteritis, small intestine bacterial overgrowth, psychosocial factors, a genetic contribution, and an imbalance of neurotransmitters as either possible causes or playing a part in the development of IBS. It is generally agreed that a patient’s emotional response to stress can exacerbate the condition. In section 1 of the thesis, the introduction, a detailed description and background appropriate to the study undertaken are provided, including aspects of epidemiology, diagnostic symptom criteria and clinical relevance of the Irritable Bowel Syndrome. Previous studies of various forms of treatment for IBS are discussed with the main emphasis being on treatment with hypnotherapy. All these therapies have concentrated on either mind or body treatments whereas this study demonstrates how hypnotherapy, and the use of imagery, addresses both mind and body. Finally, the rationale for the current study and the specific aims of the thesis are outlined. In section 2, the methodology and assessment instruments used in the clinical trial are discussed, as well as recruitment processes, research plan and timetable, and treatment schedule. Statistical analyses are provided and the main outcomes measures of the clinical trial, its limitations and scientific implications are addressed.

M.A. / The purpose of the study was to ascertain whether patients suffering from irritable bowel syndrome (IBS) differed from non- IBS clients in terms of their defense mechanisms. Although irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders encountered by primary care physicians and gastroenterologists, it is one of the least well understood. Part of the reason for this is the lack of real consensus of opinion regarding the nature of the complaint (Read, 1985). Today it is widely agreed that irritable bowel syndrome is a psychosomatic disorder, that is, a disorder of physiological functioning and anatomical structure, which are determined for most part by psychological factors (Lachman, 1972; Moser, 1986). Evidence linking psychological variables to gastrointestinal disorders is surprisingly sparse, and all too often confusing and contradicting. Such conflicting results probably reflect the many methodological weaknesses common to all areas of study (Bennett, .1989). Although evaluation of the impact of psychological interventions on both symptomatic and psychological relief has been pursued, its findings provide tangential support for the importance of psychological disorders. Nevertheless, these studies have shown a consistency of positive results not found in the etiological research (Read, 1985). Using a variety of techniques, most with the therapeutic goal of stress reduction, psychological therapy has been shown to produce. symptomatic relief, increase periods of remission, and to reduce the impact of stress resulting from severe symptomatic flare ups in IBS (Bennett, 1989). In .addition, most IBS patients may not identify their gut symptoms in psychological terms. Therefore, they inappropriately and repeatedly subject themselves to unnecessary, expensive and harmful medical procedures in search of an organic cause. Further research into the psychological factors of IBS, including the defense mechanisms underlying it may lead to a reduction in type of anxiety (Folkman, Lazarus, Gruen & DeLongis, 1986). The ways in which people cope with intense emotions may have a significant effect on their psychological and physical health. StresS factors and the suppression of emotions, for example through defense mechanisms, are thought to be especially relevant in the etiology and exacerbation of psychosomatic illness (Ogden & Von Sturmer, 1984). The role that defense mechanisms play in the development of IBS forms the cornerstone of the present research.

Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 109-120).

Irritable bowel syndrome (IBS), majorly characterized by irregular bowel movements and abdominal pain, is one of the most prevalent functional gastrointestinal disorders (FGIDs) in the world. Disturbance of gut microbiota, closely linking with gut dysfunction, has been regarded as one of important pathogenetic factors for IBS. However, gut microbiota-driven mechanism underlying IBS remains unclear, which leads to inefficient and non-specific effects of current microbiota-oriented therapy. In this thesis, function-based microbiota investigation with combination of metagenomic and metabolomic analyses was separately performed in IBS cohort and model to precisely link pathogenic species with disordered GI motor function. A series of microbiota manipulation studies in rodents were conducted to explore bacteria-driven molecular mechanism. Firstly, a pilot study with 'omics' analyses revealed fecal microbial structure significantly varied in IBS patients with disorder GI motility relative to healthy controls (HC). Such changed IBS enterotype was functionally characterized by disturbed metabolism of bile acids (BAs) that are previously proved to regulate GI motor function. It indicates microbiota-driven GI dysmotility relevant to disturbance of BA metabolism in IBS. Secondly, a systematic review with meta-analysis was performed to comprehensively understand existing findings related to BA metabolism and its linkage with IBS. Results showed that abnormal BA excretion, previously reported in at least one IBS subtype, is associated with dysregulation of BA synthesis, marked with abnormalities of circulating indices 7α-hydroxy-4-cholesten-3-one (C4) and fibroblast growth factor 19 (FGF19). However, what's the role of gut microbiota in abnormal BA excretion is undetermined. Thirdly, to explore possible role of gut microbiota in abnormal BA excretion in IBS, BA metabolites and BA-related microbiome were simultaneously analyzed in stools of recruited subjects. Results found that total BA and microbiota-derived BAs were remarkably elevated in a quarter of IBS-D patients (BA+IBS-D) who exhibited more frequent defecation, higher level of serum C4 but lower level of serum FGF19 than those with normal BA excretion (BA-IBS-D). In line with metabolic results, abundances of BA-metabolizing bacteria, particularly Clostridium scindens (C. scindens) simultaneously expressed hdhA and bais that are responsible for BA 7α oxidation and dehydroxylation, were highly enriched in fecal metagenomes of such particular IBS-D population. These findings suggest the increased BA-metabolizing microbiome is associated with the dysregulated host BA synthesis in the subgroup of BA+IBS-D patients. Fourthly, by analyzing metabolites and bacteria related to BA metabolism, a neonatal maternal separation (NMS)-induced IBS-D rat model characterized by accelerated GI motility and excessive BA excretion were found to largely mimic gut microbial BA metabolism in BA+IBS-D patients. Specifically, intraluminal total and secondary BAs were significantly elevated in the large intestinal lumens (cecum, proximal colon and feces) of NMS rats, together with increased abundances of hdhA- and bais-expressing Clostridium species, including C. scindens. Moreover, quantitative polymerase chain reaction (PCR) analysis showed upregulated mRNA expression of cholesterol 7 α-hydroxylase (CYP7A1) whereas downregulated mRNA expression of small heterodimer partner (SHP) in the liver of NMS rats, indicating enhanced hepatic BA synthetic level. These observations based on such IBS-D model suggest the association of excessive BA-metabolizing microbiome and increased hepatic BA synthesis. Fifthly, to further clarify whether excessive BA-metabolizing bacteria contribute to enhanced hepatic BA synthesis and to explore the underlying molecular mechanism, we performed bacterial intervention in pseudo germ-free (GF) or/and specific pathogen free (SPF) mice by transplantation of human fecal microbiota and the signal strain C. scindens. Compared with GF mouse recipients of HC and BA-IBS-D fecal microbiota, BA+IBS-D fecal microbial recipients displayed shorter GI transit and increased subsistence of C. scindens in the cecal contents. In line with higher level of serum C4, taurine-conjugated BA contents and mRNA expressions of BA synthetase CYP7A1 and sterol 12α-hydroxylase (CYP8B1) were significantly elevated in the liver of BA+IBS-D recipients. These findings showed bioactive effects of BA+IBS-D fecal microbiota with enrichment of C. scindens on hepatic BA synthesis. Next, to further confirm the effects of the species C. scindens on host BA synthesis, we individually colonized C. scindens strains (ATCC 37504) to pseudo GF and SPF mice. Results showed both mice models with single strain colonization exhibited accelerated GI transit and higher contents of hepatic total and taurine-conjugated BAs compared with individual vehicles treated with PBS. Combining metabolic changes, the upregulated expressions of hepatic CYP7A1 mRNA in colonized mice indicate that C. scindens substantially promote hepatic BA synthesis in colonized mice. Furthermore, contents of taurine-conjugated BAs, served as natural antagonists of farnesoid X receptor (FXR) that negatively control of new BA synthesis, were elevated in ileal lumens of colonized mice. Expressions of FXR-targeted genes SHP and fibroblast growth factor 15 (FGF15) were consistently reduced in the liver and ileum tissues of colonized mice, respectively. Results suggest that suppression of FXR-mediated feedback signaling is involved in Clostridium-driven hepatic BA oversynthesis, which deserve the further investigation. Collectively, the works of this thesis integrating clinical and animal studies indicate that BA-metabolizing bacteria, particularly C. scindens, enhance hepatic BA synthesis and consequently leads to BA overexcretion. It provides novel bacteria-driven mechanism for enhanced GI motility, and supply a direction in precise microbiota-related pathogenesis and medication for IBS-D population in future.

M.A. / Irritable Bowel Syndrome is defined as a chronic relapsing functional bowel disorder of unknown causes (Weber & McCallum, 1992). IBS is characterized by attacks of abdominal pain and change of bowel habit resulting in diarrhoea, constipation or both, where no structural alteration of the colon is found (Varis, 1987). The symptoms appear to result from a dysfunction of the intestine and are therefore said to be “functional” (Heaton & Thompson, 1999). The prevalence of IBS in the general population of Western countries is 14-24% of women. It is the most common cause of gut symptoms, and the most common reason that people go to their family doctor with a gut complaint. Despite all of this, physicians are still groping to understand the pathogenesis of IBS. The secret of success with IBS is to recognize it quickly and confidently. This is done primarily from the history, as there are no clinical tests that may be done to diagnose IBS. Once the diagnosis has been made it is of utmost importance that the sufferer is told, the syndrome is explained, and a good relationship is established with the health-care giver. Thereafter it becomes important to search for unspoken agendas in the life of the sufferer. According to the literature, stress can exacerbate IBS, and sexual, physical and emotional abuse can pose complex problems that require the assistance of a skilled counsellor. These problems, if left, may lead to the intensified symptoms of IBS. Society is becoming increasingly abusive and women and children often bear the brunt of physical, emotional and sexual abuse. Studies in America of women who present at medical facilities as well as those sampled from the community have found abuse rates that range from 20-76%. There is no reason to believe that these figures would be that different for South Africa. These studies have also found that abused women report a significantly higher number of medical problems and health-care system usage. A number of researchers have also found that there was a significant association between IBS and sexual abuse and physical abuse in childhood and adulthood. For the counselling psychologist the challenge is to unravel the mechanisms behind the symptoms, and to provide a rationale for therapy. The role that abuse may play in the development of IBS forms the cornerstone of the present study.