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

研究目的：結腸癌是現今最常見的惡性腫瘤之一。在西方國家， 其發病率甚至居惡性腫瘤的第二位，在我國發病率居第四位，並且發病率在不斷攀升。在長江中下游，江淅地區，福建，香港等較為發達地區發病率較高，研究提示可能與經濟發達、生活習慣以及飲食習慣有密切關係。隨著醫療技術的進步，西醫針對結腸癌已有多種治療手段。其中，于術治療、放化療綜合治療是主要的治療手段。但在一定程度上對患者機體有一定的毒副作用，嚴重影響生活品質，並且復發和轉移率較高。研究者發現一些中藥對結腸癌細胞有抑制細胞生長、影響腫瘤細胞相關資訊表達、抑制腫瘤細胞血管生成、誘導腫瘤細胞凋亡、對信號轉導通路的影響等作用。中草藥毒副作用小，成本低廉，提高患者生活品質，降低復發和轉移率。因此，中藥對結腸癌的抑制作用有較高的研究價值。 研究方法：選取人結腸癌HCTl 16 細胞用McCoys 5A 的細胞培養液放置於37 ℃、C02 體積分數為5%的培養箱中靜置培養。研究藥物選擇首先對結腸癌進行中醫辨證，分析每種證型用藥特點。其次在中國期刊全文資料庫進行關於結腸癌文獻檢索，在檢索結果中找出辨證分析中藥物出現頻率較高的。最後，查找相關碩士、博士論文以及有關結腸癌書籍資料篩選出以下幾種進行考證對結腸癌細胞是否有抑制作用的中草藥。（土茯苓、白花蛇古草、馬齒莧、藤梨根、敗醬草、白頭翁） 。分別用DCM 以及MEOH 提取土茯苓、白花蛇古草、馬齒莧、藤梨根、敗醬草、白頭翁，將培養好的細胞種植在96 孔板上，分別加入上述藥物提取物。用SRB 法檢測細胞的增殖情況。 研究結果： 根據實驗所得數據發現， 其中茯夜苓、和敗醬草的DCM 提取液均對HCT116細胞有較強的抑制作用，白花蛇古草對此種結腸癌細胞雖有抑制細胞增殖的作用，但結果並不明顯，實驗所得結果還有待反復實驗，以及進一步探究作用機制。然而藤梨根、馬齒莧、白頭翁的DCM 提取液以及用MEOH 提取的上述藥物均對細胞HCT116 無明顯抑制作用， 考慮實驗僅對單一細胞進行，檢測藥物提取物作用於HCT116 細胞的抑制性 。因此， 本實驗僅為六種中草藥提純物對HCT l 16 細胞的抑制作用初步探究，還有待進一步深入研究。

Doctor en Ciencias Farmacéuticas / Autor no autoriza el acceso a texto completo de su documento / En el área farmacéutica, la investigación en nanotecnología ha tenido un progreso sostenido en la última década. El desarrollo de este tipo de sistema, ha permitido incrementar la solubilidad, la biodisponibilidad, la estabilidad y la llegada al blanco farmacológico de moléculas convencionales, mejorando su perfil terapéutico. Por lo tanto, se presenta como una alternativa al tratamiento común de patologías crónicas, donde existen medicamentos con limitada efectividad. Este es el caso de la enfermedad inflamatoria intestinal (EII), cuya remisión se logra con el uso de fármacos anti-inflamatorios como corticoides, a menudo con indeseables efectos sistémicos. Así, el objetivo de este trabajo, fue preparar nanopartículas (NPs) para encapsular Prednisolona (Prd) e Inulina (Inu), destinadas a la entrega local en el colon. El procedimiento de pulverización y congelamiento sobre nitrógeno líquido (PCN), usando Quitosano (nQPCN) o Alginato (nAPCN), fue propuesto como una alternativa a la clásica plataforma Quitosano-Tripolifosfato (nQTPP). Las NPs fueron completamente caracterizadas y evaluadas de acuerdo a la carga (CA), rendimiento de partículas (RP), eficiencia de encapsulación (EE) y liberación de activos en buffer fosfato y Krebs. Además, su sensibilidad a la degradación por bacterias, enzimas y en un ensayo de fermentación anaeróbica con una suspensión fecal humana. En todas las formulaciones, Prd estuvo dispersa en estado molecular en la matriz del vehículo, de acuerdo a la difracción de rayos X, espectrocopía Raman y análisis térmico. No se detectaron por espectroscopía IR, uniones covalentes entre los activos y los polímeros, pero si interacciones débiles como formación de puentes de hidrógeno y de tipo hidrofóbicas. Adicionalmente, enlaces iónicos entre los grupos aminos protonados de Quitosano y aniones tripolifosfatos, explicarían la estructura más condensada del producto nQTPP. En relación a la CA de las formulaciones, esta superó el 5,0%, con valores entre el 12,8% al 14,5% para Prd, que son adecuados para la fabricación de un producto farmacéutico conteniendo la dosis recomendada de este corticoide. Para Inu la CA estuvo entre el 6,6% al 13,9%. Otras propiedades de las NPs de Quitosano, como carga, tamaño, estructura y comportamiento viscoelástico fueron similares, pero nQPCN mostró una mayor liberación promedio de los agentes activos en los buffer. Al mismo tiempo, fue menos degradada que nQTPP en la presencia de Lisozima y E.coli, sin embargo, fue lábil a β-glucosidasa y B.thetaiotaomicron, pero no a la suspensión fecal. Por otro lado, los resultados obtenidos con nAPCN fueron promisorios, debido a que sus NPs mostraron buenos parámetros de encapsulación para ambos activos (Ingredientes hidrofóbico e hidrofílico) y degradabilidad biológica por E.coli y la suspensión fecal, no obstante, será necesario usar derivados de Alginato de menor solubilidad para mejorar el comportamiento mecánico. De acuerdo a los resultados obtenidos, la formulación nQTPP fue elegida para el escalamiento y la forma farmacéutica final. En consecuencia, la geometría del proceso fue mantenida e incrementada de 100 mL a 400 mL (Factor = 4), utilizando dos bombas de infusión. El producto obtenido (tres lotes) fue cualitativamente similar al inicial, medido mediante espectroscopía infrarroja, carga y análisis de tamaño, no obstante, la magnitud del potencial zeta fue significativamente mayor. Con respecto al promedio de CA, esta fue menor para Prd (12,7% versus 14,5%) e Inu (6,1% versus 9,7%), pero no inferior al valor recomendado (5,0%). Además, la liberación de los activos desde el vehículo inicial y escalados, fueron similares con valores de F2 (factor de similitud) mayores a 50. Finalmente, el producto solido fue introducido en cápsulas de gelatina dura que fueron recubiertas con Eudragit® S100 (Polimero entérico). No hubo desintegración de los vehículos en fluido gástrico simulado y en buffer fosfato pH 6,8, demostrando la consistencia de la película con una concentración del polímero mayor que la recomendada por el proveedor. En el buffer colónico simulado (fosfato pH 7,4), no hubo diferencias en la liberación de los activos de las cápsulas con y sin recubrimiento. En conclusión, las NPs de nQTPP son adecuadas para la entrega de Prd e Inu en el colon y nuevos estudios son necesarios para mejorar el producto nAPCN / In the pharmaceutical field, research in nanotechnology has had a sustained progress in the past decade. The development of this type of system has permitted to enhance solubility, bioavailability, stability and pharmacological targeting of conventional molecules improving their therapeutic profile. Therefore, is presented as alternative to regular treatment of chronic diseases where there are medicines with limited effectivity. This is the case of inflammatory bowel disease (IBD), which remission is achieved using anti-inflammatory drugs such as corticoids, often with undesirable systemic effects. Thus, the objective of this work was to prepare nanoparticles (NPs) for encapsulating Prednisolone (Prd) and Inulin (Inu), intended for local colonic delivery. Spray freezing over liquid nitrogen (SFL), using chitosan (nSFLC) or alginate (nSFLA) was proposed as an alternative to classic chitosan-tripolyphosphate platform (nCTPP). NPs were fully characterized and assessed for loading capacity (LC), yield of particles (YP), efficiency of encapsulation (EE), and release of actives in phosphate and Krebs buffers. Moreover, its sensitivity to degradation by bacteria, enzyme and in anaerobic fermentation assay with human faecal slurry. In all formulations, Prd was dispersed in the carrier matrix at molecular level, according to X ray diffraction, Raman spectroscopy and Thermal analysis. Were not detected covalent linkages by infrared spectroscopy between actives and polymers, instead of it, weak interactions like hydrogen bridges and hydrophobic. Furthermore, ionic bonds between protonated amine groups of chitosan and tripolyphosphate anions could to explain more condense particles present in nCTPP product. In relation to LC of the formulations, it was over 5,0%, with values from 12,8% to 14,5% for Prd, that are suitable for manufacturing of pharmaceutical product containing recommended dose of this corticoid. For Inu CA were from 6,6% to 13,9% Another property of chitosan NPs such as charge, size, structure and viscoelastic behavior were similar, but nSFDC showed higher mean release of both active ingredients in buffers. At the same time, this was lesser degraded than nCTPP in the presence of lysozyme and E.coli, however, it was labile to β-glucosidase and B.thetaiotaomicron, but not for faecal slurry. On the other hand, the results obtained with nSFDA were promising because its NPs shows good encapsulation parameters for both actives (hydrophobic and hydrophilic ingredients) and biological degradability by E.coli and faecal slurry, but it will be necessary to use alginate derivatives with lesser solubility for improving its mechanical behavior. In according to performance obtained, nCTPP formulation was chosen for scale up and final pharmaceutical dosage. Accordingly, the geometry of the process was maintained and it was increasing from 100 mL to 400 mL (Factor = 4), utilizing two delivery pumps. The product obtained (three batch) was qualitatively similar to Initial measured by infrared spectroscopy, charge and size analysis, but the magnitude of zeta potential was significantly higher. Regarding the mean LC, it was inferior for Prd (12,7% versus 14,5%) and Inu (6,1% versus 9,7%), but no lower than recommended value (5,0%). Moreover, the release of the actives from initial carrier and scale up were similar, with F2 (similarity factor) values over 50. Finally, the solid product was introducing in gelatin capsules that were coating with Eudragit® S100 (Enteric polymer). There weren’t disintegration of the carriers in simulated gastric fluid and phosphate buffer pH 6,8, demonstrating the suitability of the film with a higher concentration of polymer than recommended by the supplier. In simulated colonic buffer (phosphate pH 7,4), there weren’t difference in the release of actives from coated and uncoated capsules. In conclusion, NPs of nCTPP are adequate for delivery of Prd and Inu to the colon and new studies are necessary to improve nSFDA product / Fondecyt

Cartas al editor

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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21446号 / 医博第4413号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 萩原 正敏, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

The electrical activity recorded from the human colon could play an important role in analyzing the pattern of contractions under different physiologic or experimental states. In general, the frequency of the electrical activity is extremely irregular and time-varying. Its analysis requires a technique that considers variations in both time and frequency domains. The research undertaken was to analyze time-frequency variations of the human colonic electrical activity, to implement positive time- frequency distribution techniques in a computer system and to analyze theoretical signals using this technique to characterize a kernel function. Our results show that the uncertainty coefficient together with the marginal conditions and the average of the conditional PTFD in time and also in frequency can be applied to determine which kernel function and c-value were appropriate for calculating the PTFD of a sinusoidal signal. The selected kernel function constituted a comparative template of signals with similar characteristics. We found that those results were useful in analyzing the time- frequency variations of the electrical activity recorded in the human colon. The comparison of relative contributions of frequency bands showed that the band with the highest values during the pre- and interprandial period was 30-40 cpm suggesting an important role in the generation of bursts of these signals. The numerical results suggested that a meal can induce changes in the relative importance of frequencies below 10 cpm and a significant change in the 30-40 cpm band. In addition, a computer program of the Chakravarti method was implemented to calculate the Fourier transform of nonperiodical signals. This program was part of the computer program system developed to compute the PTFD of theoretical and experimental signals. / Thesis / Master of Engineering (MEngr)

No description available.

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.