Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 4 uppsatser.

M.A. / The disorder known as Irritable Bowel Syndrome (IBS) is a clinical conundrum. Of chronic magnitude, this disorder of the gastrointestinal system affects up to 20% of the population in developed countries. Yet, it remains elusive in terms of its accurate definition and diagnosis, while its origins and etiology are unknown. To date, clinical confirmation of this disorder is complex and uncertain, while medical intervention has proven to be largely unsuccessful. Symptomatology relating to IBS varies widely and is not confined to the gastrointestinal tract. Diagnoses are made on the grounds of the manifestation of certain physical symptoms such as constipation, diarrhoea, abdominal pain and distension and disordered bowel habits. The role of psychological factors in the manifestation of IBS is controversial, although many clinicians postulate IBS to be a psychosomatic disorder due to the presence of many concomitant psychological features such as anxiety and depression. By all appearances, one of the most dramatic psychosocial concomitants of IBS is stress. Research has indicated that factors such as income, social support, life stress, psychological status, coping styles and personality all play a role in terms of whether the disorder develops and how successfully or adaptively the sufferer copes with his/her disorder. In terms of a research project undertaken by the Counselling and Research Centre for Gastroenterology under the auspices of the Department of Counselling Psychology at the Rand Afrikaans University, an exhaustive investigation into the physiological and psychological concomitants of IBS was conducted. As part of this endeavour to bring IBS to the attention of the medical profession, the public at large, and the individuals who suffer from this disorder, a number of individual studies were undertaken by MA (Psychology) students as part of the larger project. These researchers attempted to highlight various essential aspects associated with IBS, the focus of which included psychopathological features, stress, abuse, coping styles and strategies, defence mechanisms, personality factors and eating disorders. A second phase of this research project is currently in operation in order to provide remediation in the form of psychotherapy and stress management to the subjects who participated in the initial research project. The focus of the present treatise aims to provide an all-encompassing integration of the various research studies referred to above. Thereafter, on the basis of the data obtained from these studies, attention turns to the identification of a psychological profile with respect to the typical patient suffering from IBS. In accordance with the scientific demands of psychosocial research, a thorough review of the literature and various theoretical explanations of IBS were conducted as part of the integrative process. A number of theoretical models were considered in terms of their application to IBS, including the Digestive Disease model; the Psychiatric model; the Psychophysiological model; a Behavioural iii model; a Biopsychosocial model and a relatively recent newcomer, the Salutogenic Orientation proposed by Antonovsky, that offers an unusual approach to health and disease. In terms of the psychological profile mentioned above, the data demonstrated interesting findings. It was ascertained that the large majority of IBS patients are women, a finding that has been internationally verified. These women tend to be married with children, well-educated and employed on a full-time basis. The emotional status of the typical IBS patient reflects varying degrees of psychopathological tendencies, while her interpersonal relationships are characteristically unstable. Demonstrating high stress levels, the IBS patient tends to utilize inadequate coping skills, while these women also report using a maladapative defence mechanism to cope with the difficulties of their lives. Furthermore, personality traits such as introversion and neuroticism have been observed in these women. Lastly, it has been noted that the typical IBS patient exhibits certain aberrant eating patterns that are characteristic of women who are diagnosed as anorexic and/or bulimic. The IBS patient experiences her physical symptoms as severely disruptive and debilitating. Living in a very stressful and demanding world, she is lonely and isolated and shows the tendency to somatize her problems in the form of a disorder that is neither life-threatening, nor results in other more serious diseases, but which causes major distress in her life.

Stress-related functional bowel disease is a common, often incapacitating, problem in humans; the symptomatology of stress-related intestinal dysfunction is: (1) impaired small intestinal transit and motility, and (2) increased large intestinal transit and, commonly, diarrhea. The etiology of stress-induced intestinal dysfunction is completely unresolved, and the lack of an appropriate animal model has hindered studies of causality. We compared a number of stressors and their resultant effects on intestinal transit, a measure of the propulsive motor activity of the gut, in the rat. We found that the response of the intestine to stress, and the neural systems activated by stress, were dependent on the type and duration of stress, as well as the animal strain, and gender. We developed a model, acute wrapping restraint stress, to fully characterize the effects of stress on intestinal transit. Wrap restraint stress is a nonulcerogenic model in which rats are subjected to acute restraint by wrapping them in a harness of paper tape to restrict, but not prevent movement of the upper body and forelimbs. Transit was evaluated by the geometric center method, in which a radiomarker (⁵¹Cr) is instilled directly into the proximal duodenum and proximal colon via a surgically placed intestinal cannula, in fasted, adult female Sprague Dawley rats (150-200g). Subjecting animals to 35 min. of wrap restraint stress resulted in (1) inhibition of small intestinal transit, and (2) increased large intestinal transit and increased fecal output. The effects of stress on intestinal transit in rats resembled symptoms associated with stress in humans, suggesting that wrap restraint stress may be suitable as a model of stress-induced intestinal dysfunction. We found a close correlation between stress-induced intestinal dysfunction and stress-activation of endocrine systems. Stress-induced changes in intestinal function was strongly influenced by circadian variations in endocrine levels, suggesting that stress-induced intestinal dysfunction may be hormonally mediated. However, neither pituitary nor adrenal factors mediated the effects of stress on the gut. To evaluate the role of corticotropin-releasing factor (CRF), the major hypothalamic factor released in response to stress, in stress-induced intestinal dysfunction, we studied the effects of exogenous CRF on intestinal transit. CRF resulted in (1) a potent, dose-dependent inhibition of small intestinal transit, (2) a dose-dependent increase in large intestinal transit, and (3) increased fecal excretion. The effects of exogenously administered CRF closely paralleled the effects of stress on intestinal transit and on ACTH secretion in the rat. Blockade of CRF receptors by means of an antagonist, α helical CRF (9-41), prevented the effects of stress on colonic transit and fecal excretion. These data strongly suggest that endogenous CRF may mediate the effects of wrap restraint stress on intestinal motor activity and coordination in the rat.

Dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Chiropractic, Technikon Natal, 1999. / The aim of this placebo-controlled clinical trial was to determine the role of spinal manipulation in the management of irritable bowel syndrome (IBS), in terms of the patients' subjective response to treatment. It was hypothesized that spinal manipulation would have a greater effect than placebo in reducing the intensity of the symptoms of IBS. Thirty subjects diagnosed with IBS were randomly divided into two groups. Each group consisted of 15 subjects, aged between 18 and 50. Patients were treated twice a week for three weeks and once in the fourth week. Thereafter, each patient returned approximately 1 month later to be assessed for any longer-term benefit to treatment. Patients in the experimental group received spinal manipulation directed at areas of spinal fixation, as determined by motion palpation. Patients in the control group were treated using a detuned ultrasound machine over areas of spinal fixation. Treatment was performed with the same degree of enthusiasm in both groups, where possible. / M

Memoria para optar al título profesional de Bioquímico / El síndrome de intestino irritable (SII) es un trastorno funcional digestivo caracterizado por la presencia de dolor abdominal asociado a alteraciones del hábito intestinal. Si bien su fisiopatología no ha sido dilucidada completamente, se reconoce la existencia de un desequilibrio del eje cerebro-intestino, que afecta diversas funciones intestinales, entre ellas el aumento de la permeabilidad paracelular y la activación de células inmunes. La elevada activación de mastocitos se asocia a aumento de la permeabilidad intestinal debido a alteraciones en la organización de la unión estrecha (UE), mecanismo mediado por la activación de PAR-2 (receptor activado por proteasa-2) por triptasa. La proteína inmuno-moduladora Bcl-3 (B-cell leukemia/lymphoma-3) es un regulador transcripcional de genes activados por NF-κB, entre ellos los que regulan la UE. En este trabajo se proponen las siguientes hipótesis: 1) En pacientes con SII, existe una pérdida de la función de barrera intestinal asociada a un aumento en la expresión de Bcl- 3, a una elevada activación de mastocitos y a alteraciones de la unión estrecha epitelial; 2) La expresión de Bcl-3, es inducida por triptasa, mediante la activación de PAR-2, afectando el estado de la unión estrecha en líneas celulares de epitelio intestinal. Para ello, muestras de mucosa ileal y colónica de pacientes con SII y SC (sujetos controles) fueron recolectadas. En ellas se evaluó la expresión de Bcl-3, mediante q-PCR, western blot e inmunofluorescencia indirecta (IFI); el número de mastocitos y su grado de activación, por IFI y microscopía electrónica de transmisión (TEM); y las alteraciones de la UE, mediante IFI y TEM. El efecto de triptasa sobre la expresión de Bcl-3 y las alteraciones en la UE fueron evaluadas in vitro, en líneas celulares DLD-1 y Caco-2, mediante western blot e IFI. Los resultados evidenciaron una elevada expresión de Bcl-3 un aumento en el número y actividad de mastocitos, alteraciones en la distribución y expresión de ZO-1 en mucosa y alteración de la arquitectura de la UE epitelial intestinal en pacientes con SII en comparación con SC. Nuestros resultados in vitro evidenciaron que la expresión de Bcl-3 fue inducida por triptasa, a través de la activación de PAR-2, induciendo este estimulo alteraciones en la distribución de proteínas de la UE. Nuestros hallazgos sugieren que en el SII, la proteína Bcl-3 actuaría como un factor intermediario de las alteraciones de la UE epitelial inducida por la activación de triptasa/PAR-2. Futuros estudios dirigidos estudiar con profundidad este mecanismo permitirán contribuir a la fisiopatología del SII, en miras de un nuevo marcador diagnostico y blanco terapéutico / Irritable bowel syndrome (IBS) is a digestive functional disorder characterized by the presence of abdominal pain associated with alterations of the intestinal habit. Although its pathophysiology has not been fully elucidated, the existence of an imbalance of the brainintestine axis is recognized, which affects various intestinal functions, among them the increase in paracellular permeability and the activation of immune cells. The high activation of mast cells is associated with increased intestinal permeability due to alterations in the organization of the tight junction (TJ), a mechanism mediated by the activation of PAR-2 (receptor activated by protease-2) by tryptase. The Bcl-3 immunomodulating protein (B-cell leukemia / lymphoma-3) is a transcriptional regulator of genes activated by NF-κB, including those that regulate the TJ. In this work, the following hypotheses are proposed: 1) In patients with IBS, there is a loss of intestinal barrier function associated with an increase in the expression of Bcl-3, a high activation of mast cells and alterations of the epithelial tight junction ; 2) The expression of Bcl-3 is induced by tryptase, through the activation of PAR-2, affecting the state of the tight junction in intestinal epithelial cell lines.. To do this, samples of ileal and colonic mucosa from patients with IBS and CS (control subjects) were collected. In them, the expression of Bcl-3 was evaluated by means of q-PCR, western blot and indirect immunofluorescence (IFI); the number of mast cells and their degree of activation, by IFI and transmission electron microscopy (TEM); and the alterations of the TJ, through IFI and TEM. The effect of tryptase on the expression of Bcl-3 and the alterations in the EU were evaluated in vitro, in cell lines DLD-1 and Caco-2, by means of western blot and IFI. The results showed a high expression of Bcl-3 an increase in the number and activity of mast cells, alterations in the distribution and expression of ZO-1 in mucosa and alteration of the architecture of the intestinal epithelial TJ in patients with IBS compared to CS . Our in vitro results showed that the expression of Bcl-3 was induced by tryptase, through the activation of PAR-2, inducing this stimulation alterations in the distribution of proteins of the TJ. Our findings suggest that in IBS, the Bcl-3 protein would act as an intermediary factor of epithelial TJ alterations induced by the activation of tryptase / PAR-2. Future studies aimed at studying in depth this mechanism will allow contributing to the pathophysiology of IBS, in view of a new diagnostic marker and therapeutic target

Thesis (Ph. D.)--UCLA, 2009. / Vita. Description based on print version record. Includes bibliographical references (leaves 244-259).

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).