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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 Using a novel unpublished stomach specific CreERT2 expressing mouse line I found that simultaneous knockout of RZ (RZ DKO) result in gastric hyperplasia of the corpus epithelium.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 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.

Previous studies examining the canine intestinal microflora have focused on cultivation of bacteria from intestinal content. Recently, it has been recognized that the majority of bacteria cannot be identified using standard culture techniques. The aim of this study was to describe the composition and dynamics of the canine intestinal microflora using molecular methods based on identification of the 16S ribosomal DNA (16S rDNA) and to evaluate the clinical use of a 13C-glycocholic acid blood test (13CGCBT) as a serum marker for small intestinal bacterial biomass. Intestinal content was obtained from healthy dogs and the microflora was characterized in different compartments of each dog by denaturing gradient gel electrophoresis (DGGE) and comparative 16S rDNA analysis. A 13C-glycocholic acid blood test (13C-GCBT) was developed as a marker for small intestinal bacterial biomass and the influence of tylosin administration on the 13C-GCBT, serum concentrations of cobalamin, folate, and unconjugated cholic acid (SUCA) was evaluated. There was marked variation in DGGE profiles between individual dogs and also between different intestinal compartments within dogs. DGGE profiles from duodenal juice samples collected endoscopically at different time-points varied within individuals, possibly due to variations over time or a slight variation in sampling location. Direct sequencing revealed 106 individual 16S rDNA sequences. Forty-two sequences showed less than 98% similarity to described sequences in public databases and may constitute previously uncharacterized bacterial species. Serum folate concentrations, SUCA, and the cumulative percent dose/min of 13C administered as 13C-glycocholic acid (CUMPCD) increased significantly following tylosin administration (p<0.01). The results indicate that dogs have a complex intestinal microflora with marked differences between individual dogs. Different intestinal compartments appear to host a unique microflora and the assessment of a fecal sample does not yield accurate information about the composition of the microflora in proximal compartments of the gut. The intestine harbors many previously uncharacterized bacterial species. The clinical significance of these uncharacterized intestinal bacterial species needs to be further investigated in dogs with gastrointestinal disease. Increased serum folate, SUCA, and CUMPCD in the 13C-GCBT suggest that, in the dogs described here, tylosin administration increased the biomass of organisms carrying out these metabolic functions.

Cell-cell communication between bacteria, belonging to the same species or to different species (Intra-kingdom signaling), or communication between bacteria and their animal host (Inter-kingdom signaling) is mediated through different chemical signals that are synthesized and secreted by bacteria or the host and is crucial for the survival of bacteria inside their host. The overall goal of this work was to understand the role of inter- and intra-kingdom signaling in phenotypes such as chemotaxis, colonization and biofilm formation, and virulence that are associated with infections caused by the human gastrointestinal (GI) tract pathogens. A part of our work also aimed at developing microfluidics-based models to study inter- and intra-kingdom signaling in biofilm formation, inhibition, and dispersal. We showed that norepinephrine (NE), an important host signal produced during stress, increases human opportunistic pathogen Pseudomonas aeruginosa growth, motility, attachment, and virulence, and also showed that the actions of NE are mediated primarily through the LasR, and not the RhlR QS system. We investigated the molecular mechanism underlying the chemo-sensing of the intra-kingdom signal autoinducer-2 (AI-2) by pathogens Escherichia coli and Salmonella typhimurium by performing different chemotaxis assays (capillary, microPlug and microFlow assays), and discovered that AI-2 is a potent attractant for E. coli and S. typhimurium, and that the Tsr chemoreceptor and periplasmic AI-2 binding protein LsrB are necessary for sensing AI-2, although uptake of AI-2 into the cytoplasm is not required. We concluded that LsrB, when bound to AI-2, interacts directly with the periplasmic domain of Tsr primarily at the Thr-61 and Asp-63 residues of LsrB, making LsrB the first known periplasmic-protein partner for Tsr. We fabricated a simple user-friendly microfluidic flow cell (microBF) device that can precisely measure the effect of a wide range of concentrations of single or combinations of two or more soluble signals on bacterial biofilm formation and development. We also constructed a synthetic biofilm circuit that utilizes the Hha and BdcA dispersal proteins of E. coli along with a quorum sensing (QS) switch that works based on the accumulation of the signal N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-o-C12HSL) and implemented it in an upgraded �BF device. We showed that a QS system may be utilized with biofilm dispersal proteins to control consortial biofilm formation by removing an existing biofilm and then removing the biofilm that displaced the first one. These types of synthetic QS circuits may be used to pattern biofilms by facilitating the re-use of platforms and to create sophisticated reactor systems that will be used to form bio-refineries.

Thesis (Ph. D.)--University of Nevada, Reno, 1998. / Includes bibliographical references. Online version available on the World Wide Web.

The normal sonographic appearance of the adult canine gastrointestinal tract has been well described. Interpretation of ultrasonographic findings in puppies presented for gastrointestinal evaluation is difficult due to the lack of information on normal ultrasonographic findings. The gastrointestinal tract, jejunal lymph node size and appearance as well as the presence or absence of peritoneal fluid were prospectively investigated in a group of 23 normal, 7 – 12 week old Beagle puppies. The duodenal wall thickness was significantly greater than other parts of the gastrointestinal tract (mean 3.8 mm). The mean stomach wall thickness was 2.7 mm, mean jejunal wall thickness 2.5 mm and mean colonic wall thickness 1.3 mm. In addition, the mean thickness of the duodenal mucosal layer (2.7 mm) was significantly thicker than that of the jejunal mucosal layer (1.5 mm). The mucosa was isoechoic to the muscularis layer and had a crisp luminal-mucosal interface in all puppies. There were no intestinal corrugations observed and wall layering was distinct in all gastrointestinal segments. The homogenous, hypoechoic jejunal lymph nodes were easily found and their mean thickness measured 7.1 mm (± SD 2.2 mm). A mild amount of anechoic free peritoneal fluid was seen in all puppies. Conclusions drawn from this study were that prominent jejunal lymph nodes and a mild amount of anechoic free peritoneal fluid can be considered normal findings in puppies. Information from the above study was utilised to interpret findings of a prospective clinical study on the ultrasonographic appearance of the gastrointestinal tract of puppies suffering from parvoviral enteritis. Forty puppies between six and 24 weeks of age were examined ultrasonographically within 24 hours of admission for canine parvoviral enteritis confirmed on faecal transmission electron microscopy. A clinical score (assessing habitus, appetite, vomiting, faecal consistency, mucous membranes, abdominal palpation and borborygmi) was attributed to each puppy prior to the ultrasonographic examination. Sonographic findings included fluid filled small intestines in 92.5% of cases, and stomach and colon in 80% and 62.5% of cases respectively. Generalised atony was seen in 30 cases and weak peristaltic contractions indicative of functional ileus observed in the remaining 10 cases. The duodenal and jejunal mucosal layer thicknesses were significantly reduced when compared to values obtained in the normal Beagle puppies with mean duodenal mucosal layer measuring 1.7 mm and jejunal mucosal layer 1.0 mm. Additionally, a mucosal layer with diffuse hyperechoic speckles was seen in the duodenum (15% of cases) and the jejunum (50% of cases). The luminal surface of the duodenal mucosa was irregular in 22.5% of cases and the jejunal mucosa in 42.5% of cases. In all of these puppies, changes were accompanied by generalised indistinct wall layering. Small intestinal corrugations were seen within the duodenum in 35% of cases and within the jejunum in 7.5%. A mild amount of anechoic free peritoneal fluid was observed in 26 cases and was considered within normal limits for puppies and a moderate amount of anechoic free peritoneal fluid was observed in six cases. The jejunal lymph node size was within normal limits for puppies and thus parvoviral enteritis does not appear to be associated with ultrasonographic evidence of regional lymphadenopathy. There was a tendency for animals with the most dramatic ultrasonographic changes to be in poor condition clinically i.e. they had a low clinical score. Each of the above described changes cannot be considered pathognomonic for canine parvoviral enteritis but in combination, are suggestive of the disease. It is hoped that information from this study may alert the clinician as to the possibility of underlying parvoviral enteritis in puppies presented for abdominal ultrasound for investigation of gastrointestinal disease. Further studies are needed to document the ultrasonographic appearance of other paediatric gastrointestinal diseases such as severe verminosis, giardiasis, coccidiosis and distemper etc. before further conclusions can be drawn from this study. Daily ultrasonographic examinations of puppies suffering from canine parvoviral enteritis are needed to further understand the progression of this disease over time as well as the possible ultrasonographic indicators of clinical improvement or deterioration. / Dissertation (MMedVet)--University of Pretoria, 2009. / Companion Animal Clinical Studies / unrestricted

Immunofluorescence was used to study immunoreactivity (IR) for corticotropin-releasing factor (CRF) in the guinea pig enteric nervous system. CRF-IR was expressed in both the myenteric and the submucosal plexuses of all regions of the large and small intestine and the myenteric plexus of the stomach. CRF-IR nerve fibers were present in the myenteric and submucosal plexuses, in the circular muscle coat, and surrounding submucosal arterioles. Most of the CRF-IR fibers persisted in the myenteric and submucosal plexuses after 7 days in organotypic culture. CRF-IR was not coexpressed with tyrosine hydroxylase-IR or calcitonin gene-related peptide-IR fibers. The proportions of CRF-IR cell bodies in the myenteric plexus increased progressively from the stomach (0.6%) to the distal colon (2.8%). Most of the CRF-IR myenteric neurons (95%) had uniaxonal morphology; the remainder had Dogiel type II multipolar morphology. CRF-IR cell bodies in the myenteric plexus of the ileum expressed IR for choline acetyltransferase (56.9%), substance P (55.0%), and nitric oxide synthase (37.9%). CRF-IR never colocalized with IR for calbindin, calretinin, neuropeptide Y, serotonin, or somatostatin in the myenteric plexus. CRF-IR cell bodies were more abundant in the submucosal plexus (29.9-38.0%) than in the myenteric plexus. All CRF-IR neurons in submucosal ganglia expressed vasoactive intestinal peptide-IR and were likely to be secretomotor/vasodilator neurons. CRF-IR neurons did not express IR for the CRF1 receptor. CRF 1-IR was expressed in neuronal neighbors of those with CRF-IR. Collective evidence suggests that VIPergic secretomotor neurons might provide synaptic input to neighboring cholinergic neurons.

Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/ CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF 2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

Complex medication regimens have major implications on patient therapy. When we consider that these regimen therapies can also be further convoluted by co-morbidity, it is then seen as an essential opportunity to research possible solutions to alleviate such complications. Globally identified conditions such as the Human Immuno-deficiency Virus (HIV) and Tuberculosis (TB) are known to have such complications within their respective regimens. In many cases, the regimental therapies themselves are overbearing with high pill burdens having to be taken in segregated manners throughout the day. Within a standard TB regimen, isoniazid and rifampicin are seen to have a deleterious drug-drug interaction in which the bioavailability is compromised through formation of an insoluble complex. Despite this interaction, the 2 active drugs must be taken concurrently for successful TB therapy. No true solution exists as fixed dose combinations of isoniazid and rifampicin (Rifinah®) are still in production despite the detrimental interaction that impedes successful bioavailability. The once daily multi-unit drug delivery system (ODMUS) has the benefits of superseding the described problems and aiding in therapeutic outcomes. Preliminary studies utilized preliminary testing to ascertain the science surrounding the 2 components of the ODMUS, the memblet and the multiparticulate components. pH-sensitive polymers (Eudragit® L100-55 and E 100) were of critical importance to the success of the system and were individually manipulated for each component to produce a novel memblet and multiparticulate system through a unique salting out approach. Primary studies focused on drug release testing and drug entrapment for the multiparticulate component. Testing of the memblet system addressed dissolution and thermal analysis. Utilizing this data, a series of process variables were used to achieve an optimized formulation through a Box- Behnken statistical design. Optimized formulations used response testing to establish the optimal characteristics of both components. Multiparticulates achieved controlled release for 12 hours with an enhanced 71% drug entrapment efficiency. Memblet release profiles were confirmed over 2 hours with a maximal Tg of 56°C. Molecular modeling corroborated release understanding for both components. Surface area and porosity analysis, surface morphology, fourier transform infrared spectroscopy as well as thermal, rheological and mechanical analysis were additional tests undertaken on the optimized formulations. In vivo analysis was the final testing to verify validity of the ODMUS components and utilized a pig model for the investigation. UPLC blood analysis revealed increase blood levels of INH (CmaxINH= 0.0138ng/mL) and RIF (CmaxRIF= 0.052ng/mL) in relation to conventional dosage forms validating segregated site-specific release and increased bioavailability. Ideally, a segregated means of drug delivery throughout the gastrointestinal tract was achieved such that an enhanced bioavailability, a more controlled release and a simplified medication regimen was produced. This study aimed to achieve said goals through novel technique analysis, innovation and globally approved science to critically assess the success of the ODMUS as a potential means to reduce the complexities of medication regimen therapy.