In adult mammals, the gastrointestinal (GI) epithelium exhibits the highest turnover rate among the endodermal tissues. The harsh luminal environment of the GI tract necessitates replenishment of epithelial cells to maintain organ structure and function during routine turnover and injury repair. This delicate balance between gain and loss of cells is called tissue homeostasis, and multipotent tissue specific adult stem cells serve as the continuous source of self-renewal. Due to their important contribution to homeostatic maintenance the proliferative capacity of the stem cells needs to be tightly controlled, as an imbalance can result in diverse pathologies such as cancer or insufficient injury repair. Despite the crucial role for regulatory processes the molecular mechanisms and the genes governing these processes remain poorly understood. Rnf43 and its paralogue Znrf3 (RZ) act as tumour suppressors in the intestine, but their role in the gastric epithelium has not been previously investigated. Using a novel unpublished stomach specific CreERT2 expressing mouse line I found that simultaneous knockout of RZ (RZ DKO) result in gastric hyperplasia of the corpus epithelium. Gastric RZ DKO organoids show independence from the essential growth factor Rspondin-1 but require exogenous Wnt. A similar exogenous Wnt dependence was identified in a human gastric cancer cell line harbouring homozygous Rnf43 inactivating mutations. Thus, Wnt secretion inhibition might provide a new treatment paradigm for a subset of patients carrying Rnf43 mutations. The prominent role of the E3s Rnf43 and Znrf3 in the intestinal and gastric epithelial led to the question of whether other E3s either closely related to RZ or specifically expressed in stem or niche cells could play a role in homeostatic regulation, specifically in the small intestine. Using a retroviral overexpression screen I identified Rnf24 and Rnf122, two E3s that rendered intestinal organoids insensitive to withdrawal of the BMP inhibitor Noggin. Moreover, potential substrate candidates located at the cell surface membrane were identified and the generation of in vivo models initiated to provide a basis for further studies investigating the role of these E3s. In trying to address the function of the abovementioned genes using in vitro functional genetics I identified gaps in the current technology for organoid genetic engineering. I therefore developed two gene editing methods; a gRNA concatemer system allowing simultaneous knockout of multiple genes and CRISPR-FLIP enabling generation of conditional gene knockouts In summary, this thesis describes the first stomach specific knockout of Rnf43 and Znrf3 in the gastric epithelium, showing that it results in gastric hyperplasia located to the corpus epithelium. The dependence of the Rnf43 and Znrf3 knockout epithelium on exogenous Wnt signalling provides a potential treatment strategy for a subset of patients harbouring Rnf43 mutations. Next, it identifies Rnf24 and Rnf122 as E3 ubiquitin ligases involved in intestinal stem cell regulation and provide preliminary data and a basis for future studies. Finally, it describes the establishment of two advanced genetic engineering approaches which can be applied to various in vitro culture systems such as 3D organoids, mouse embryonic stem cells and conventional cell lines. Collectively this work and the developed methods will contribute to our understanding of the mechanisms regulating adult stem cell homeostasis.

The gut microbiome is an integral component of the human body that plays a role in many physiological processes. Dysbiosis, an imbalance of the microbiome, has been associated with disease states including inflammatory bowel disease, type II diabetes, and obesity, and moreover, contributes to the pathogenesis of these states. Understanding the functional mechanisms governing microbial ecology and microbe-host interactions is essential to understanding the microbiome’s role in health and disease. However, at present, functional genetic studies of diverse natural mammalian gut microbiomes remain challenging, due to a lack of genetic tools for bacteria outside of a handful of well-studied model organisms. Altering the metagenome of a complex microbial community requires novel platform technologies for genetic engineering which can operate in a generalized fashion across many different host organisms. In this thesis, I present two novel genetic tools designed for genetic modification of bacterial communities. The first, the Cas-Transposon platform, is a host-independent targeted genome editing tool that utilizes programmable, targeted transposases to mediate site-specific gene insertions into user-defined loci. The Himar1 transposase naturally inserts transposases into random TA dinucleotides in a genome, but when fused to the dCas9 RNA-guided, DNA-binding protein, the fusion protein Himar1-dCas9 targets transposon insertions to a single TA site. The activity of Himar1-dCas9 was characterized using in vitro experiments, demonstrating that site-specific transposition is dependent on guide RNA (gRNA) orientation relative to the target site and the sequence surrounding the target site, but robust to variations in DNA and protein concentration, presence of background DNA, and temperature. We additionally showed that the Cas-Transposon platform is capable of performing site-specific transposition into a plasmid in vivo in E. coli, although further optimization of the system may be necessary to effect site-specific transposition into a genomic locus. The Himar1-dCas9 protein is the first example of a transposase that inserts transposons into locations programmable by an RNA, making it a novel tool for gene insertion and knockout in potentially any organism, without relying on DNA repair by a host cell. Metagenomic Alteration of Gut microbiome by In situ Conjugation (MAGIC) is an approach to directly modify gut bacteria in their native habitat by harnessing naturally occurring horizontal gene transfer activity to deliver engineered DNA. Because many gut bacteria are difficult to cultivate and thus difficult to genetically manipulate in the laboratory, MAGIC uses donor bacteria, delivered directly into the gut environment, to conjugate mobile vectors bearing engineered genetic payloads. Using payloads with selectable markers, we identified organisms across 4 major phyla of gut bacteria that were amenable to genetic modification with libraries of conjugative vectors we created. Using a lab-adapted E. coli strain as a donor, we achieved transient expression of the engineered payload in the microbiome. We also demonstrated that engineered native gut bacteria containing conjugative vectors could be deployed back into the gut to stably recolonize and mediate secondary transfer of the payload into other microbes, potentially enabling long-term infiltration of the payload into the metagenome. The results from this study suggest that both short-term and long-term genetic alteration of the metagenome are possible by choosing different donors, and that the MAGIC platform could enable development of more diverse microbial chasses for synthetic biology applications. MAGIC could also be used to create personalized engineered probiotics for diagnostic or therapeutic applications. In Chapter 4 of this thesis, we explored the targeted use of MAGIC to genetically modify Segmented Filamentous Bacteria, a gut commensal that is important for immune regulation but recalcitrant to in vitro cultivation. The Cas-Transposon and MAGIC technologies expand our capabilities in the areas of targeted genome editing and gene delivery into bacteria, respectively. Together, they form a suite of complementary approaches to genetically engineer undomesticated gut commensal bacteria and probe the functional genetic networks in the gut microbiome, which will enhance our understanding of microbiome ecology and host-microbiome interactions. In addition, the expanded range of genetic manipulations made possible by these tools may enable production of more diverse, perhaps personalized, probiotics containing engineered functions, such as sensing disease markers or drug delivery.

Made available in DSpace on 2014-06-11T19:30:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-29Bitstream added on 2014-06-13T18:41:13Z : No. of bitstreams: 1 stelzer_m_dr_botib.pdf: 918862 bytes, checksum: bc6a3bc2154c49c9ab02546521651045 (MD5) / O estudo da atividade do sistema digestório apresenta vários desafios, pois para termos medidas eficientes do seu funcionamentos as técnicas atualmente aplicadas ou são invasivas ou apresentam o uso de radiação ionizante, o que pode causar desconforto ou outros problemas ao individuo. ABiosusceptometria de Corrente Alternada (BAC), tem sido utilizada com sucesso no estudo da motilidade do trato gastrintestinal, sem a necessidade de radiação ionizante e sem ser invasiva.Essatese foi desenvolvida em três partes, com cada parte representando um trabalho, no primeiro foi estudado aOscilação Tônica no Estomago Proximal, no segundo foi feita a validação da BAC in vitro e in vivo como um método biomagnético para registrar contrações gástricas e finalmente no terceiro foi iniciado o desenvolvimento de uma nova instrumentação de modo a permitir a obtenção de imagens tomográficas utilizando a BAC. Desta forma, este trabalho apresenta estudos de validações da BAC para análise das atividades de contração tônica e fásica do estômago e de emprego como técnica tomográfica / The evaluation of gastric-intestinal activity presents a lot of challenges, because, the techniques to obtain efficient measures or is an invasive technique or it makes use of ionizing radiation, what could cause some discomfort or another problems related to the radiation. The Alternated Current Biosusceptometry (ACB) has been utilized with success, in the gastricintestinal activity studies, without the necessity of ionizing radiation and it isn´t a invasive method. This thesis has been developed in three parts, in the first part was studied the stomach proximal tonic oscillation, in the second was studied the validation of theACB method in vitro and in vivo as a biomagnetic method to register the gastric contractions, and finally the third, it was started the development of a new instrumentation capable of obtain tomography images by means of BAC

Orientador: José Ricardo de Arruda Miranda / Banca: Andrés Vercik / Banca: Sérgio Paulo Amaral Souto / Banca: José Roberto Corrêa Saglietti / Banca: Eder Rezende Moraes / Resumo: O estudo da atividade do sistema digestório apresenta vários desafios, pois para termos medidas eficientes do seu funcionamentos as técnicas atualmente aplicadas ou são invasivas ou apresentam o uso de radiação ionizante, o que pode causar desconforto ou outros problemas ao individuo. ABiosusceptometria de Corrente Alternada (BAC), tem sido utilizada com sucesso no estudo da motilidade do trato gastrintestinal, sem a necessidade de radiação ionizante e sem ser invasiva.Essatese foi desenvolvida em três partes, com cada parte representando um trabalho, no primeiro foi estudado aOscilação Tônica no Estomago Proximal, no segundo foi feita a validação da BAC in vitro e in vivo como um método biomagnético para registrar contrações gástricas e finalmente no terceiro foi iniciado o desenvolvimento de uma nova instrumentação de modo a permitir a obtenção de imagens tomográficas utilizando a BAC. Desta forma, este trabalho apresenta estudos de validações da BAC para análise das atividades de contração tônica e fásica do estômago e de emprego como técnica tomográfica / Abstract: The evaluation of gastric-intestinal activity presents a lot of challenges, because, the techniques to obtain efficient measures or is an invasive technique or it makes use of ionizing radiation, what could cause some discomfort or another problems related to the radiation. The Alternated Current Biosusceptometry (ACB) has been utilized with success, in the gastricintestinal activity studies, without the necessity of ionizing radiation and it isn't a invasive method. This thesis has been developed in three parts, in the first part was studied the stomach proximal tonic oscillation, in the second was studied the validation of theACB method in vitro and in vivo as a biomagnetic method to register the gastric contractions, and finally the third, it was started the development of a new instrumentation capable of obtain tomography images by means of BAC / Doutor

El estudio tuvo por objetivo estimar la prevalencia de helmintos gastrointestinales en alpacas de dos comunidades del distrito de Macusani, Provincia Carabaya-Puno, durante la época de seca. Así como determinar la prevalencia de las variables: sexo, edad y procedencia; establecer el promedio de carga parasitaria e identificar los géneros de helmintos presentes. Se colectaron muestras de heces de 1319 alpacas durante agosto a octubre del 2010. Las muestras fueron procesadas en el Laboratorio de Microbiología y Parasitología – Sección Parasitología; empleándose las técnicas coproparasitológicas de flotación con solución Willis y sedimentación espontánea; así mismo para la estimación de la carga e identificación de larvas de nematodos se utilizó el método Mcmaster modificado y Baermann respectivamente. Obteniéndose una prevalencia de helmintos de 63.9 ± 2.6% en alpacas y observándose mayor porcentaje en machos (73.9%); así como en el grupo etario de 5 meses a 1 año (77.7%). Con respecto a la comunidad Hatun Phinaya y Queracucho se halló prevalencias de 60.7 y 66.6% respectivamente. La mayoría de la carga parasitaria por nematodos no superó los 100 hpg. Los géneros de helmintos identificados fueron: Nematodirus, Trichuris, Moniezia, Cooperia, Oesophagostomum, Trichostrongylus, Ostertagia, Bunostomum, Haemonchus, Capillaria y Lamanema. Donde Nematodirus presento prevalencia del 52.8% seguido de Trichuris (10.8%) y Moniezia (9.6%). La edad constituyo un factor de riesgo para la presencia de helmintos; donde, animales de 5 meses a 1 año y animales de 1 a 3 años presentaron riesgo de 2.93 y 1.98 veces (p<0.05) respecto a la población mayor a 3 años. Palabras clave: Alpacas, parásitos gastrointestinales, Puno, época seca / The study aimed to estimate the prevalence of gastrointestinal helminth communities alpacas two Macusani District, Province Carabaya-Puno, during the dry season. And to determine the prevalence of the variables: sex, age and origin. Set the load average and identify parasitic helminths genres present. Stool samples were collected in 1319 alpacas during August to October 2010. Samples were processed in the Laboratory of Microbiology and Parasitology - Section Parasitology; techniques being used coproparasitologic Willis flotation and sedimentation spontaneous solution, likewise for load estimation and identification of nematode larvae Mcmaster method was used and modified Baermann respectively. With a prevalence of helminths of 63.9 ± 2.6% in alpacas and highest percentage observed in males (73.9%), and in the age group of 5 months to 1 year (77.7%). With respect to the community and Queracucho Phinaya Hatun was found prevalences of 60.7 and 66.6% respectively. There was significant difference (p <0.05) between parasitism against variables such as sex, age and origin. Most nematode parasite load not exceeded 100 epg. Helminth genera were identified: Nematodirus, Trichuris, Moniezia, Cooperia, Oesophagostomum, Trichostrongylus, Ostertagia, Bunostomum, Haemonchus, Capillaria and Lamanema.Where present Nematodirus prevalence 52.8% followed by Trichuris (10.8%) and Moniezia (9.6%). The age constituted a risk factor for the presence of helminths, where animals from 5 months to 1 year and animals 1-3 years had risk of 2.93 and 1.98 times (p <0.05) compared to the population over three years. Key Word: Alpacas, gastrointestinal parasites, Puno, dry season. / Tesis

The microflora inhabiting the human gastrointestinal tract can be considered an essential 'metabolic organ', in a symbiotic relationship with its host. Due to the low cultivability and inappropriate sampling methodology the microflora is poorly explored and ill-defined. Preliminary, molecular-based research at the University of Waikato revealed the presence of 16S rRNA gene sequences originating from novel Mollicute-like species inhabiting the human GI tract. A ~830bp 'consensus' sequence representing these novel Mollicute-like sequences was classified within the Mollicute Genus Anaeroplasma the type species of which is Anaeroplasma abactoclasticum. It also displayed near exact matches with 16S rRNA sequences obtained from the human GI tract and matches of high similarity to those from the mouse GI tract in the NCBI database. This thesis describes an attempt to design and create primers that would amplify and characterize full-length versions of these Mollicute-like sequences from samples obtained from the mucosal surface of the human gastrointestinal tract. Primers sets targeted extended 5' and 3' versions of these novel 'known' sequences and were designed from sequence matches found in the preliminary work and other related sequences from the NCBI database. The attempt to amplify a full-length version of these novel Mollicute-like sequences was proven to be unsuccessful. No sequences were classified within the Genus Anaeroplasma, although 81% of amplicons from the 5' extending primer sets were classified within the same division as the Mollicutes, the Firmicutes, only 6% of the sequenced amplicons from the 3' extending primer set belonged to this division. Phylograms containing these 'relevant' sequences and the 'consensus' sequence grouped the 'consensus' sequence separately, indicating a lower relatedness than would have been seen if any of the amplicons contained the 'consensus' sequence.

Probiotic bacteria such as Lactobacillus and Bifidobacterium species are normal inhabitants of healthy gastrointestinal (GI) tract, which may promote beneficial effects on host through limiting the growth of undesirable micro-organisms and modulating the immune system. In the present study, Lactobacillus and Bifidobacterium strains were screened for their in vitro acid and bile tolerance, autoaggregation, coaggregation and hydrophobic abilities to identify potential probiotic bacteria. Lactobacillus acidophilus LAFTI L10 and Lactobacillus paracasei LAFTI L26 were selected based on their overall tolerance to in vitro acidic conditions to further investigate their influence on various immune functions and gene expression in mice. Immunofluorescent analysis of small intestine in mice fed with L. acidophilus or L. paracasei demonstrated an increase of immunoglobulin (Ig)-A, interleukin (IL)-10 and interferon (IFN)- producing cells compared to control mice. In summary, L. acidophilus and L. paracasei showed tolerance to various gastric conditions and bile salts. Lactobacillus acidophilus and L. paracasei enhanced gut and systemic immune functions, particularly non-specific and specific immune responses in normal and CT mice. Moreover, L. acidophilus regulated the genes involved in various biological functions in small bowel of normal and CT mice, which provided a basis in understanding the pathways through which these bacteria are beneficial to the host. / Doctor of Philosophy (PhD)

Mucositis is the damage that occurs to the alimentary canal from anti - cancer therapies. It is caused by chemotherapy, radiotherapy and combination therapy and affects a large proportion of patients. Despite its prevalence, an effective anti - mucositis agent has yet to be developed that protects the whole tube, although the use of keratinocyte growth factor ( Amgen ' s Palifermin ) has recently been approved for the prevention of oral mucositis. It is important to understand mechanisms controlling mucositis so that treatment can be targeted appropriately. This thesis has investigated some of the key components identified as being involved in mucositis as well as identifying new genes which contribute to chemotherapy - induced intestinal injury. The research chapters investigated : 1 ) Gene expression of the apoptosis - regulating Bcl - 2 family, p53 and caspase - 3, and the changes which occur in the intestine following chemotherapy treatment for cancer. 2 ) The effect of different chemotherapeutic agents on intestinal cells in vitro and the role p53 plays. 3 ) The mucositis caused by single dose irinotecan in the rat with breast cancer and the role of p53 in induction of intestinal damage. 4 ) The early gene changes that occur in the small intestine of the rat with breast cancer following irinotecan treatment. Firstly, to investigate the difference in susceptibility to damage between the small and large intestine, the protein expression of 8 members of the Bcl - 2 family ( 4 pro - apoptotic ; Bax, Bak, Bid, Bim and 4 anti - apoptotic ; Bcl - 2, Bcl - xL, Bcl - w, Mcl - 1 ) was quantified in jejunal and colonic sections taken from rats inoculated with breast cancer. It was found that there was significantly higher expression of the pro - apoptotic proteins, Bax, Bak, Bim and Bid, in the crypts of the jejunum compared to the colon. Furthermore, expression of the anti - apoptotic proteins, Bcl - 2, Bcl - xL and Bcl - w, was significantly lower in jejunal crypts compared to colonic crypts. Mcl - 1 expression was similar in both regions. Thus, the small intestine is an environment balanced to favour apoptosis through specific Bcl - 2 family protein expression profiles. The Bcl - 2 family regulates apoptosis in response to a variety of chemotherapy agents. However, it is unknown how Bcl - 2 family gene expression changes along with other apoptogenic factors following cytotoxic therapy in the normal intestine. To investigate this, sections of rat jejunum treated with methotrexate and duodenal biopsies from chemotherapy patients treated with various regimens for cancer were subjected to quantitative immunohistochemistry to detect Bcl - 2 family proteins, p53 and caspase - 3. Treatment caused expression of p53 and caspase - 3 to increase within the crypts and follow a similar pattern to apoptosis levels. Pro - apoptotic Bcl - 2 family members, Bax and Bak, were increased, while the anti - apoptotic protein, Mcl - 1, was significantly reduced. A significant increase in mRNA expression for Bax and Bak was noticed at 6 h, without a concurrent decrease in Mcl - 1. Thus, Bcl - 2 family genes were altered in the small intestine in both humans and rats, and this was irrespective of chemotherapy agent or regimen used. The best characterised changes which occur during chemotherapy - induced damage in the intestine are in the epithelial layer, although it is thought that pan #45 mucosal alterations are involved. Two intestinal cell lines were chosen to investigate changes in apoptosis, proliferation and protein expression following cytotoxic treatment with various chemotherapeutic agents. These were the rat IEC - 6 and human FHs 74 cell lines, which represent untransformed epithelial cells. The human breast carcinoma cell line, MCF - 7, was also used as a positive control. Intestinal cells were resistant to the occurrence of methotrexate toxicities within 24 h of treatment, modestly affected by irinotecan and extremely sensitive to doxorubicin. Doxorubicin caused a marked increase in p53 and p21 expression, which for irinotecan was less pronounced. The effect of cytotoxic treatment on Bcl - 2 family expression in intestinal cells varied, however the pro - apoptotic proteins, Bax and Bak, were generally upregulated following doxorubicin. Temporary inhibition of p53 using pifithrin alpha resulted in a significant improvement in cell survival in cancerous cell only and did not alter Bcl - 2 family expression. It was concluded that cultured epithelial cells exhibit varying sensitivities to different chemotherapeutic agents which is dependent on induction of p53 gene expression. The topoisomerase I inhibitor, irinotecan, is a chemotherapeutic agent commonly used in the treatment of colorectal cancer. It often induces severe mucositis with the most common symptom being diarrhoea. Previous research has shown that irinotecan damages the small and large bowel equally, which is unusual. This is characterised by an increase in apoptosis and a reduction in proliferation within epithelial crypts, an increase in inflammatory cell infiltrate in the lamina propria and excess mucin production. These investigations used two sequential doses of irinotecan. The early effect of a single dose of irinotecan on the intestine have yet to be studied. Thus the primary aim of this experiment was to examine in detail the changes caused by irinotecan at 6 and 48 h in the rat. A secondary aim was to investigate the role of p53 on induction of apoptosis and cell cycle arrest within intestinal crypts and the effect of temporary inhibition of the protein. Single dose irinotecan caused a decrease in body and small intestinal weight by 48 h after treatment. This was accompanied by crypt and villous degeneration, increased apoptosis and reduced proliferation within crypt epithelium as well as inflammatory infiltrate throughout lamina propria. An increase in Bax expression was seen at 6 h, however p53 protein levels remained relatively low until 48 h. Rats also treated with pifithrin alpha to inhibit p53 and had a significantly lower peak in apoptosis in the colon at 6 h, however did not show improvements in any other parameters tested. It was concluded that irinotecaninduced damage in the rat intestine is primarily p53 - independent, and that pifithrin alpha acts to inhibit apoptosis in the large intestine via a p53 - independent pathway. A study was designed to investigate the early genome - wide changes which occur following irinotecan treatment in the rat small intestine. Microarray analysis found that regulation of many genes was altered at 6 h following dual dose irinotecan. These genes were involved in apoptosis, cell cycle regulation, immune function, calcium homeostasis and protein turnover. Multiple genes from the MAP kinase pathway were also activated by irinotecan. The cystine protease, caspase - 1 was upregulated and was chosen for further investigations due to its role in apoptosis and inflammation. Real time PCR analysis confirmed the increase in gene expression at 6 h and also showed a return to baseline levels by 24 h which was followed by another modest increase at 48 h. It was concluded that irinotecan induces a wide range of gene changes within the intestine and that apoptosis and inflammatory damage pathways are activated during treatment. This thesis described key molecules in apoptosis and their role in induction of chemotherapy - induced intestinal mucositis. It has provided evidence of the importance of apoptosis in mucosal injury and also highlighted areas requiring further research. Results presented herein show that the Bcl - 2 family is involved in intestinal damage following many chemotherapy agents, whereas p53 is agent - specific. It has also shown that irinotecan causes intestinal damage via a mainly p53 - independent manner in the rat. It can be concluded that gastrointestinal mucositis is complex and activates multiple pathways to induce damage. Findings from this thesis will aid targeting of new anti - mucotoxic agents. / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2006.

Bibliography: leaves 159-192. / viii, 192 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Examines aspects of the control and measurement of pyloric motor function believed to be relevant to the role(s) of the pylorus in the regulation of normal gastric emptying. / Thesis (M.D.)--University of Adelaide, Dept. of Medicine, 1992

Copy of author's previously published article inserted. / Bibliography: leaves 210-234. / xiii, 235 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates the effect of chemotherapy on the mucosa of the small intestine and the prevalence, duration and severity of mucositis, both in humans and in rats. / Thesis (M.D.)--University of Adelaide, Depts. of Gastroenterology and Haematology/Oncology, 1998