Thesis (MSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The work presented in this thesis deals with the association between cancers of the upper gastro-intestinal tract and the tumor suppressor gene, TP53, and can be divided into three parts: (i) the analysis of the mutational spectrum of TP53 with respect to laryngeal cancer, (ii) the analysis of the mutational spectrum of TP53 with respect to esophageal cancer and (iii) the analysis of TP53 transcriptional levels in esophageal cancer. Laryngeal cancer (LC) is the 6th most common cancer in the world and the 2nd most common respiratory cancer, with approximately 500 000 new cases per annum detected worldwide. Over the last few years, LC has become increasingly prevalent within the Coloured Community of the Western Cape. The mechanisms of tumorigenesis in LC remain unknown, although smoking and alcohol consumption are considered to be major risk factors. Mutations within the gene TP53 have been strongly implicated as playing a role in cancer development, as they are frequently found in several cancer types. We therefore screened exons 5 - 8 of TP53 for mutations in DNA from tumor biopsies (n=44) and blood samples (n=42) from Coloured LC patients, using polymerase chain reaction - single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing. Blood samples from a healthy, matched control group (n=40) were included in the study as controls. Significant correlations were found between the occurrence of LC and age and smoking, whereas daily meat consumption was a possible protective factor. In tumor-derived samples, mutations were found in 3 of the exons under investigation, representing 25% of the samples. The mutations were unique to the tumor biopsies, indicating a somatic origin for mutations. The data confirms that the region between codons 175 and 273 of TP53 is a mutational hotspot for cancers in general. This study reports 6 novel mutations within this same region. Esophageal cancer (EC) has a very high incidence in South Africa, relative to the rest of the world, and is particularly common amongst the Black Transkei population. The goal of this study was to determine whether there are differences in the TP53 mutational pattern observed in the Coloured Western Cape community as compared to that observed in the Black Transkei community. This required the analysis of the molecular structure of TP53, specifically exons 5 - 8, in a group of Coloured EC patients (n=44) treated at Tygerberg Hospital, Cape Town, South Africa. DNA obtained from tumor biopsies and blood (from patients) as well as from apparently healthy surrounding tissue was screened via PCR-SSCP and direct sequencing analysis. Only 4 nucleotide changes were observed from a total of 124 sequences obtained, of which two were novel to esophageal squamous cell carcinoma. These 4 nucleotide alterations were found only within the tumor biopsy sample set, representing 9% of the tumors investigated. This study revealed that the mutational spectrum of TP53 within the Coloured population of the Western Cape greatly differs from that of the Black community of the Transkei. This suggests that a different set of etiological factors are involved in the tumorigenic process for each of these distinct geographical communities, which is the subject of an epidemiological study undertaken by the MRC. The final part of this thesis deals with the quantification and comparison of TP53 transcription levels in esophageal cancer tumor tissue to the TP53 levels in healthy esophageal tissue obtained from patients from a unique geographical and ethnic background. The cohort used in this study consisted of Coloured patients (n=2) treated at Tygerberg Hospital. The LightCycler system was implemented in order to try to accurately quantify TP53 mRNA levels. Unfortunately, the desired results were unattainable due to unforeseen difficulties encountered during the study. These difficulties included the insufficient preservation of samples for RNA based studies. Several recommendations were made concerning future similar studies, including an improved planning strategy as well as the employment of an RNA stabilizing agent. Additionally, a few important contributions were made through this study, including the design and optimization of TP53 primers specifically intended for future RNA studies. These primers would enable the identification of the presence of TP53 RNA species as well as the absence of DNA contamination in a single PCR amplification step. Other contributions include the development of a well-optimized RNA extraction method for the extraction of RNA from tough tissues (such as the human esophageal tissue used in this study). This method makes the extraction of large quantities of RNA from small amounts of tough tissue types possible. In conclusion, this study has made a significant contribution to the field of cancer research, by shedding light on the TP53 mutational spectrum with regards to laryngeal as well as esophageal cancer in a population unique to the Western Cape. The first part of this thesis has been published in Cancer Genetics and Cytogenetics (Barnard, D., K. Lehmann, E.G. Haal, P.O. van Heiden, and l.C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarities to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145:126-132), of which a copy can be found in Appendix I. This work has also been presented (by D. Barnard) at an international conference entitled "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", held in Amsterdam, the Netherlands during the period 13 - 15 December 2002. / AFRIKAANSE OPSOMMING: Die werk wat in hierdie tesis voorgelê word handel oor die assosiasie tussen kankers van die boonste gastrointestinale weg en die tumor suppressor geen, TP53, en kan in 3 dele gedeel word, (i) die analise van die mutasiespektrum van TP53 in laringiale kanker (LK), (ii) die analise van die mutasiespektrum van TP53 in slukderm kanker (SK) en (iii) die analise van die transkripsievlakke van TP53 in SK. Laringeal kanker (LK) is die 6de algemeenste kanker in die wêreld en die 2de algemeenste respiratoriese kanker, met "n benaderde 500 000 nuwe gevalle jaarliks wêreldwyd. Oor die afgelope paar jare het LK "n toenemende probleem geraak, veral in die Kleurling gemeenskap van die Wes Kaap. Die meganismes van die tumorvorming in LK is onbekend, alhoewel rook-en alkoholgebruik vername risiko faktore is. Die voorkoms van mutasies in TP53 is verskeie kere aangetoon in verskillende kanker tipes en daar word vermoed dat dit "n rol speel in tumorvorming. In hierdie studie is dus na mutasies in eksons 5 - 8 van TP53 gesoek in tumor biopsie weefsel (n=44) en bloed isolate (n=42) van Kleurling LK pasiënte d.m.v. polimerase ketting reaksie - enkelstring konformasie polimorfisme (PKR-ESKP) analisering en direkte volgorde bepaling. Bloed monsters van "n vergelykbare groep (n=40) is ook in die studie ingesluit as "n kontrole. Betekenisvolle positiewe korrelasies is gevind tussen die voorkoms van LK en ouderdom sowel as rook. Daarmee saam is daaglikse vleisinname as potensiële beskermende faktor gevind. In tumor biopsies is mutasies in 3 van die ondersoekte eksons gevind, wat 25% van die biopsie monsters verteenwoordig. Hierdie mutasies is uniek aan die tumor biopsie weefsels en dui op "n somatiese oorsprong van mutasies. Hierdie bevindinge bevestig dat die gedeelte tussen kodons 173 - 273 van TP53 "n hipermuteerbare gebied geassosieer met kankers is. Hierdie studie bevestig 6 nuwe mutasies. Daar is 'n hoë insidensie van slukderm kanker (SK) in Suid Afrika relatief tot die res van die wêreld. Hierdie soort kanker word veral gevind by die Swart populasie van die Transkei. Die doel van hierdie studie was om verskille tussen die TP53 mutasie patroon van die Kleurling gemeenskap van die Wes Kaap en die Swart gemeenskap van die Transkei te vergelyk. Hiervoor is die molekulêre struktuur van TP53, veral eksons 5 - 8, in 'n groep Kleurling SK pasiënte (n=42) wat behandel is by Tygerberg Hospitaal, Kaapstad, Suid Afrika, geanaliseer. Analisering is gedoen deur DNS van tumor, bloed en ook oënskynlike gesonde aangrensende weefsel van dieselfde pasiënte te onderwerp aan PKR-ESKP analise en direkte volgorde bepaling. Slegs 4 nukleotied veranderings is gevind in 124 volgorde bepalings, waarvan 2 nuwe veranderings is in SK. Hierdie 4 nukleotied veranderinge verteenwoordig 9% van al die tumors wat ondersoek is in die studie. Hierdie studie bewys dat die mutasiespektrum van TP53 in die Kleurling gemeenskap van die Wes Kaap grootliks verskil van die Swart gemeenskap van die Transkei. Dit impliseer dat verskillende etiologiese faktore moontlik 'n rol mag speel op die tumorvormingsproses in die 2 afsonderlike geografiese gemeenskappe. Hierdie is die onderwerp van 'n epidemiologiese studie wat deur die MNR onderneem word. Die laaste deel van hierdie tesis handel oor die kwantifisering en vergelyking van TP53 transkripsievlakke in SK tumor weefsel teenoor TP53 vlakke in gesonde slukderm weefsel van pasiënte in 'n unieke geografiese en etniese agtergrond. Die studie populasie in hierdie projek het bestaan uit Kleurling pasiënte (n=2) wat by Tygerberg hospitaal behandel is. Die "LightCycler" sisteem is gebruik vir die akkurate kwantifisering van TP53 boodskapper RNS vlakke. Ongelukkig is die verlangde resultate nie gekry nie as gevolg van onvoorsiene probleme wat ondervind is tydens die studie. Hierdie probleme sluit in die onvoldoende preserv RNS studies. Hierdie inleiers maak dit nou moontlik om die teenwoordigheid van TP53 RNS spesies sowel as die afwesigheid van DNS kontaminasie in een PKR amplifikasie stap te kan identifiseer. 'n Ander belangrike bydrae is die ontwikkeling van 'n goed geoptimaliseerde RNS ekstraksie metode vir moeilike starre weelfsel tipes (soos menslike slukderm weefsel in hierdie studie) en maak die ekstraksie van groot hoeveelhede RNS uit klein hoeveelhede van moeilik hanteerbare weefsel tipes moontlik. Om saam te vat, hierdie studie het betekenisvolle bydraes gemaak tot die veld van kankernavorsing deur die ontrafeling van die TP53 mutasiespektrum in beide laringeale sowel as slukderm kanker, in 'n populasie uniek aan die Wes Kaap. Die eerste deel van hierdie tesis is gepubliseer in Cancer Geneties and Cytogenetics (Barnard, D., K. Lehmann, E. G. Hoal, P. D. van Heiden, and T. C. Victor. 2003. The spectrum of mutations in TP53 in laryngeal cancer patients from a high-incidence population shows similarites to many of the known mutational hotspots. Cancer Genetics and Cytogenetics 145: 126-132) en 'n afskrif van die artikel is ingesluit in Appendix I. Hierdie werk is ook voorgedra (deur D. Barnard) by 'n internasionale kongres getiteld "Cancer of the Esophagus and Gastric Cardia: From Gene to Cure", wat in Amsterdam, Nederland gehou is gedurende 13 - 15 Desember 2002

Thesis (MsCMedSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The gastrointestinal morphology and the distribution of the different types of mucin secreting goblet cells were investigated in three mammalian insectivorous species, namely A. spinosissimus, C. cyanea and A. hottentotus. The aim of the study was to provide a comprehensive morphological comparison between the different species. Another aim was to illustrate and compare the distribution of mucins (neutral, sulfo- and sialomucins) in the gastrointestinal tracts (GITs) of these species, in order to better understand the quality of the biofilm in the GIT. Mucins secreted onto the surface of the GIT have an effect on the colonisation of microflora in the mucosal layer, constructing a biofilm which protects the GIT surface from opportunistic pathogens. The shape, proportional length, and proportional surface areas of the different gastrointestinal regions were recorded and compared in the three species. Histochemical staining methods were used to detect and to distinguish between neutral, sulfo- and sialomucins. The number of goblet cells in the GIT containing each of the above mucins in the epithelium lining the surface or crypts was quantified, and the data expressed as the number of neutral, sulfo- or sialomucin containing goblet cells per mm2 of the surface or crypt epithelium. In all three species the stomach was uncompartmentalised. The internal aspect of the stomach in A. spinosissimus was hemi-glandular, containing stratified squamous epithelium in the fundus, with glandular epithelium in the body and pyloric region. However, C. cyanea and A. hottentotus had wholly glandular stomachs. A. spinosissimus was the only species studied that had a caecum which demonstrated transverse mucosal folds and V-shaped mucosal folds in the proximal colon. Both C. cyanea and A. hottentotus had villi up to the distal part of the GIT. Longitudinal mucosal folds were present in the distal colon. The GITs of both C. cyanea and A. hottentotus showed little morphological differentiation namely a simple, glandular stomach and the lack of a caecum. Mixed (neutral and acid) mucins and mixed acid (sulfo- and sialomucins) mucin secreting goblet cells were prominent mucin cell types in all three mammalian insectivorous species. Despite these general similarities, marked differences were observed in the qualitative expression and distribution of the three types of mucins throughout the GIT. The overall similarity between the three insectivores and other distantly related mammalian species suggests that mixed mucin secreting goblet cell types are prominent contributors to the maintenance of the intestinal biofilm in the majority of mammals, irrespective of their diet or taxonomy. / AFRIKAANSE OPSOMMING: Die bestudering van die morfologie van die spysverteringskanaal (SVK) en die verspreiding van die verskillende musien produserende bekerselle was in drie insek-etende soogdier spesies uitgevoer, naamlik in A. spinosissimus, C. cyanea en A. hottentotus. Die doel van die studie was om „n omvattende morfologiese vergelyking te maak tussen die drie spesies, sowel as om die verspreiding van die verskillende musiene te beskryf in die SVK. Kennis van die verspreiding van die verskillende tipes musiene (neutral, sulfaat en nie-sulfaat bevattende musiene) kan moontlik inligting verskaf aangaande die kwaliteit van die biofilm in the SVK. Die laasgenoemde musiene wat gesekreteer word op die oppervlak van die SVK, bepaal die kolonisasie van die mikroflora in die mukosale laag wat „n biofilm vorm en die SVK beskerm teen patogene. Die vorm, proportionele lengte en proportionele oppervlaks areas van die verskillende SVK gebiede is opgeteken, waarna dit vergelyk is tussen die drie insektivore spesies. Histochemiese kleurings tegnieke is gebruik om die musiene waar te neem en om te onderskei tussen die neutraal, sulfaat en nie-sulfaat bevattende musiene. Die aantal beker selle wat elk van die bogenoemde musiene bevat het, is getel in die oppervlaks epiteel- en kript areas van die SVK. Hierdie data is weergegee as die aantal neutraal, sulfaat en nie-sulfaat bevattende beker selle per oppervlaks epiteel- of kript area (mm2). Die vorm van die maag in al drie spesies was eenvoudig en nie gekompartementaliseer nie. Die interne aspek van die maag in A. spinosissimus het meerlagige plaveisel epiteel in die fundus gehad en klieragtige epiteel in die liggaam en pilorus gedeeltes. Daarbenewens het C. cyanea en A. hottentotus slegs klieragtige epiteel in die maag gehad. A. spinosissimus was die enigste spesie in hierdie studie wat „n sekum gehad het met dwars voue, asook V-vormige mukosale voue in die proksimale kolon. Beide C. cyanea en A. hottentotus het villi tot in die distale gedeelte van die SVK gehad. Longitudinale mukosale voue was teenwoordig in die distale gedeelte van die kolon. Die SVK van beide C. cyanea en A. hottentotus het min morfologiese differensiasie getoon deurdat die spesies „n eenvoudige, klieragtige maag gehad het en geen sekum nie. Gemenge (neutral en suur) musiene asook gemengde suur (sulfaat en nie-sulfaat bevattende) musiene was die dominante musien tipes in the SVK van al drie insek-etende soogdier spesies. Ten spyte van die algemene ooreenkomste, was daar merkwaardige verskille in die getalle en verspreiding van die verskillende musiene in die SVK. Die algemene ooreenkomste tussen die drie insektivore soogdier spesies met soogdiere van ander families, stel voor dat die gemende musien sekreterende beker selle „n prominente bydrae maak tot die onderhoud van die biofilm in the SVK in die meerderheid van soogdiere, ongeag van hul dieet of spesie klassifikasie.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Background: Irritable bowel syndrome (IBS) is a common gastrointestinal (GI) disorder. GI symptoms and impaired quality of life affect between 10-20% of all adults, corresponding to about 25-50% of all patients who visit a gastroenterologist’s clinic. In recent years, several novel mechanisms of IBS that likely relate to previously established theories have been identified. Inflammation, postinfectious low-grade inflammation, immunological and genetic predisposition along with altered microbiota are critical in IBS development, while several dietary factors may also play a role in this syndrome. However, none of these factors accounts for the full repertoire of IBS symptoms, and the pathophysiology of this condition is not fully understood. The overarching aim of this study was to investigate the nutrient intakes, GI microbiota and the effect of Lactobacillus plantarum (L.plantarum) 299v in IBS patients. Sub-aims: 1) Update healthcare professionals on current probiotic information and provide an overview of probiotic treatment approaches, with special emphasis on IBS, 2) conduct a well designed randomised, double blind, placebo-controlled trial (RCT) with L. plantarum 299v as part of an intervention and establish whether a course of probiotics may alleviate undesirable symptoms of IBS and improve quality of life, 3) assess nutrient intake in patients with irritable bowel syndrome (IBS) compared to dietary recommendations, 4) validate and assess the reproducibility of food records and 5) identify possible nutrient risk components for establishing GI microbiota involved in IBS and as part of an intervention, determine whether a course of probiotics may alter stool microbiota. Results: 1) A review article published by the author provides an overview of current probiotic treatment options to health care professionals and indicates certain probiotics are a promising therapeutic treatment option for management of IBS symtpoms, 2) the effects of the single strain probiotic, L. plantarum 299v, supplementation was evaluated in a RCT. Compared to placebo, the probiotic supplementation showed no significant reduction in GI symptom severity scores, particularly abdominal pain relief. Quality of life was also not improved in the treatment versus control group. Both the treatment and placebo groups improved significantly over the trial period, indicating a large placebo effect, 3) nutrient intakes of the IBS patients compared to current dietary reference recommendations indicates that this group of patients are at risk for nutrient inadequacies in key macro and micronutrients, 4) the validity and reliability of the dietary data showed good reliability but poor validity as measured by plasma fatty acids and 5) the GI microbiota composition in the phenotypically different diarrhoea-predominant IBS (D-IBS) vs. constipation-predominant IBS (C-IBS) showed that D-IBS patients had significantly lower counts of Lactobacillus plantarum compared to C-IBS patients. The probiotic had no significant effects on the GI microbiota as measured by quantitative polymerase chain reaction (qPCR). It was found that nutrient intakes had a significant impact on the microbiota. Lower fibre intakes were associated with higher Bacteroides spp., lower Bifidobacteria bifidum and Lactobacillus plantarum counts in both IBS groups. Conclusion: Taken together, L.plantarum 299v did not alleviate the GI symptoms of IBS, nor was it associated with significant changes in the GI microbiota. IBS patients may be at risk of key nutrient inadequacies. The influence of nutrient intakes on the GI microbiota provides an attractive explanation as a potential pathophysiological factor for IBS. / AFRIKAANSE OPSOMMING: Agtergrond: Prikkelbare derm-sindroom (PDS) is ‘n algemene gastro-intestinale (GI) stoornis. GI simptome affekteer die lewenskwaliteit van 10-20% van alle volwassenes. Dit stem ooreen met ongeveer 25-50% van alle pasiënte wat ‘n gastroënteroloog konsulteer. Verskeie oorspronklike meganismes vir die ontwikkeling van PDS is onlangs identifiseer. Inflammasie, post-infektiewe lae-graadse inflammasie, immunologiese en genetiese vatbaarheid tesame met veranderde mikrobiota is krities vir die ontwikkeling van PDS. Sekere dieetfaktore mag ook bydraend wees tot hierdie sindroom. Geen van hierdie faktore is egter verantwoordelik vir die volle spektrum van PDS simptome nie en die patofisiologie van die toestand word ook nog nie ten volle verstaan nie. Die oorkoepelende doel van hierdie studie is om nutriëntinname, GI mikrobiota en die uitwerking van L.plantarum 299v in PDS pasiënte bepaal. Sub-doelwitte: 1) Om gesondheidswerkers in te lig aangaande die nuutste inligting oor probiotika en om ‘n oorsig van probiotika behandelingsopsies te verskaf, met spesiale klem op PDS, 2) om ‘n goed beplande ewekansige, dubbel-blinde, plasebo-beheerde kliniese studie met L.plantarum 299v as deel van die intervensie uit te voer om sodoende te bepaal of ‘n kursus probiotika ongewensde simptome van PDS kan verbeter en lewenskwaliteit sodoende verhoog, 3) om nutriëntinname in pasiënte met PDS te bepaal vergeleke met dieet aanbevelings, 4) om die geldigheid en herhaalbaarheid van voedselrekords te bepaal en 5) om moontlike nutriënt risikokomponente vir die ontwikkeling van GI mikrobiota betrokke in PDS te identifiseer en om as deel van ‘n intervensie te bepaal of ‘n kursus probiotika stoelgang mikrobiota patrone verander. Resultate: 1) ‘n Oorsigartikel gepubliseer deur die kandidaat dui probiotika aan as ‘n belowende terapeutiese opsie in die behandeling van PDS simptome, 2) die effek van ‘n enkelstam probiotikum, L.plantarum 299v, is evalueer deur ‘n ewekansige, dubbel-blinde, plasebo-beheerde kliniese studie. Vergeleke met die plasebo, het probiotiese aanvulling geen betekenisvolle vermindering in die GI simptome in PDS pasiënte tot gevolg gehad nie. Lewenskwaliteit het ook nie verbeter in die behandelde versus die kontrole groep nie. Beide die behandelde en plasebo groepe het aansienlik verbeter oor die studietydperk, wat ‘n groot plasebo effek aandui, 3) nutriëntinname van die PDS groep vergeleke met huidige dieetaanbevelings, dui daarop dat hierdie groep pasiënte ‘n risiko het vir die ontwikkeling van kern nutriënttekorte (makro- en mikronutriënte), 4) die geldigheid en betroubaarheid van die dieetdata dui op goeie betroubaarheid, maar swak geldigheid soos bepaal deur plasma vetsure en 5) die dermkanaal mikrobiotiese samestelling in die verskillende fenotipes, diarree-oorheersende PDS (D-PDS) vs. konstipasie-oorheersende PDS (K-PDS) dui daarop dat D-PDS pasiënte aansienlike minder Lactobacillus plantarum gehad het vergeleke met K-PDS pasiënte. Die probiotikum het geen beduidende uitwerking op die oorheersende mikrobiota gehad nie, soos gemeet deur kwantitatiewe polimerase kettingreaksie (kPKR). Daar is gevind dat dieet ‘n beduidende impak op die mikrobiota gehad het. Daar is ‘n verband tussen laer vesel inname en hoёr Bacteroides spp. en laer Bifidobacteria bididum en Lactobacillus plantarum tellings gevind in beide PDS groepe. Gevolgtrekking: Die L.plantarum 299v enkelstam probiotikum het nie die gastrointestinale simptome van PDS pasiënte verlig nie en daar is ook geen beduidende veranderinge in die mikrobiota gevind nie. PDS pasiënte mag ‘n verhoogde risiko toon vir kern nutriënttekorte. Die invloed van nutriëntinname op GI mikrobiota verskaf ‘n belowende verduideliking as ‘n potensiële patofisiologiese faktor in PDS.

Thesis (PhD)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: This study describes the use of a gastro-intestinal model to screen lactic acid bacteria isolated from the gastro-intestinal tract of post-weaned piglets (raised on six different diets) for probiotic properties. Intestinal bacteria were isolated from ,the stomach, duodenum, jejunum, caecum, ileum and colon. The highest cell numbers (6 x 107 cfulg) were isolated from the ileum. No significant differences in viable cell counts were recorded for piglets raised on the six diets. Isolates with the best overall probiotic properties were identified as members of Lactobacillus salivarius and Lactobacillus fermentum. The two strains selected for further studies were Lactobacillus plantarum 423 (originally isolated from sorghum beer) and Lactobacillus salivarius 241 (isolated from pig intestine). Enterococcus faecalis FAIR E 92 was originally isolated from pig intestine and was included in this study as a non-pathogenic challenge strain. L. plantarum 423 produces a bacteriocin plantaricin 423, active against E. faecalis FAIRE 92. L. plan/arum 423 and L. salivarius 241 were included in the gastro-intestinal model and their adhesion to the mucus of porcine ileum studied with fluorescent-in-si/u-hybridization (FISH). A decrease in viable cell numbers of L. plan/arum 423 was recorded in the duodenum, jejunum and ileum in the presence of bile and pancreatic juice. However, higher cell numbers were recorded in the caecum and anterior colon, which suggested that strain 423 recovered from these stress factors. Plantaricin 423 was detected for up to 28 hours in the duodenum, jejunum, ileum and middle colon. Lower cell numbers (one log unit) of L. salivarius 241 were recorded in the gastro-intestinal model over seven days, compared to strain 423. Piglets of one, 14 and 28-days-old were dosed with L. plan/arum 423 and L. salivarius 241, separately and in combination (1: 1). In a separate experiment, 14-day-old piglets were challenged twice with E. faecalis FAIRE 92, followed by dosage with strains 423 and 241. New-borne piglets dosed with L. plantarum 423 gained more weight (4 kg over 19 days) compared to piglets dosed with L. salivarius 241 (2.2 kg over 19 days), or a combination of the two strains (2 kg over 19 days). Piglets of 14 and 28-days-old, on the other hand, gained more weight when dosed with a combination of strains 423 and 241. The cell numbers of E. faecalis FAIR E 92 and other enterococci decreased drastically (two log units) when the piglets were dosed with the latter two strains. Overall, piglets of various ages reacted differently when administered L. plantarum 423 and L. salivarius 241, separately or in combination. Fluorescent-in-situ-hybridization (FISH) was used to study the in vivo adhesion of L. plantarum and L. salivarius to mucus in the stomach, duodenum, jejunum, ileum, caecum and colon. The highest number of L. plantarum cells was recorded in the ileum, whereas L. salivarius favoured adhesion to the duodenum. A decrease in cell numbers of E. faecalis in the ileum mucus was recorded when a combination of the probiotic strains 423 and 241 was administered. This study provided a reliable estimation of the presence and/or adhesion of L. plantarum and L. salivarius to various parts of the porcine gastro-intestinal tract, without the use of expensive cultivation techniques. Insight was gained into the co-evolution existing between probiotic bacteria and the porcine gastro-intestinal tract, emphasizing the use of gastro-intestinal models to study the dynamics of the gastro-intestinal tract. / AFRIKAANSE OPSOMMING: Hierdie studie beskryf die gebruik van 'n gastro-intestinale model, om melksuurbakterieë wat geïsoleer is uit die spysverteringskanaal (SVK) van reeds gespeende varkies (gevoed op ses verskillende diëte) vir probiotiese eienskappe te toets. Ingewandsbakterieë is uit die maag, duodenum, jejunum, caecum, ileum en kolon geïsoleer. Die hoogste aantal selle (6 x 107 kve/g) is geïsoleer uit die ileum. Geen betekenisvolle verskille in lewensvatbare seltellings, vir varkies gevoed op ses verskillende voere is aangeteken nie. Isolate met die beste algehele probiotiese eienskappe is as Lactobacillus salivarius en Lactobacillus fermentum geïdentifiseer. Vir verdere studie is twee isolate Lactobacillus plantarum (oorspronklik uit sorghum-bier geïsoleer) en Lactobacillus salivarius (uit die varkdermkanaal geïsoleer) geselekteer. Enterococcus faecalis FAIRE 92, oorspronklik uit die varkdermkanaal geïsoleer, is in hierdie studie as 'n nie-patogeniese indikator gebruik. L. plantarum 423 produseer 'n bakteriosien plantarisien 423 wat aktief is teen E. faecalis FAIR E92. L. plantarum 423 en L. sa/ivarius 241 is ingesluit in die gastro-intestinale model, en vashegting van die bakterieë aan die mukus van vark-ileum is met fluoresensie-in-si/uhibridisasie (FISH) bestudeer. 'n Afname in lewende selgetalle van L. plan/arum 423 in die duodenum, jejunum en ileum is aangetoon in reaksie tot die byvoeging van gal en pankreatiese sappe. Hoër selgetalle is nietemin aangeteken in die caecum en voorste gedeelte van die kolon, wat 'n aanduiding gee dat isolaat 423, ten spyte van hierdie stres-faktore, oorleef. Plantaricin 423 is vir 'n tydperk (28 uur) in die duodenum, jejunum, ileum en sentrale kolon gevind. Laer selgetalle (een logaritmiese eenheid) van L. salivarius 241 is in die gastro-intestinale modeloor 'n tydperk van sewe dae aangetoon, in vergelyking met isolaat 423. Een, 14 en 28 dag oud varkies is met L. plantarum 423 en L. salivarius 241 (afsonderlik en in kombinasie 1:1) twee keer gedaag met E. faecalis FAIR E 92, opgevolg met dosering van 423 en 241. Pasgebore varkies het die hoogste gewigstoename getoon (4 kg oor 19 dae) na dosering met L. plantarum 423 in vergelyking met varkies gedoseer met L. salivarius 241 (2.2 kg oor 19 dae) of 'n kombinasie van die twee isolate (2 kg oor 19 dae). Daarenteen het veertien- en 28 dag oud varkies beter gewigstoename getoon na dosering met 'n kombinasie van isolate 423 en 241. Die selgetalle van E. faecalis FAIRE 92 en ander enterococci het drasties afgeneem (twee logaritmiese eenhede) nadat die varkies met laasgenoemde twee isolate gedoseer is. Varkies van onderskeie ouderdom het verskillend gereageer na dosering met L. plantarum 423 en L. salivarius 241 afsonderlik of in kombinasie. Fluoresensie-in-situ-hibridisasie (FISH) is gebruik om die in vivo vashegting van L plantarum en L. salivarius tot die vark mukus in die maag, duodenum, jejunum, ileum, caecum en kolon te bestudeer. Die hoogste telling van L. plantarum selle is aangeteken in die ileum, terwyl L. salivarius aanhegting tot die duodenum verkies het. 'n Afname in seltellings van E. faecalis in die ileum mukus was aangeteken na toediening met 'n kombinasie van probiotiese isolate 423 en 241. Hierdie studie het 'n betroubare bepaling van die voorkoms en/ofvashegting van L. plantarum en L. sa/ivarius isolate in verskeie gedeeltes van die varkspysverteringskanaal voorsien, sonder die hulp van duur kwekings tegnieke. Probiotiese bakterieë is in 'n gastro-intestinale model, wat die natuurlike omgewing verteenwoordig, bestudeer. Insig oor die ko-evolusie tussen probiotiese bakterieë en die SVK van die vark is verkry. Die gebruik van 'n gastro-intestinale model om die dinamika van die SVK te bestudeer is met hierdie studie beklemtoon.

published_or_final_version / abstract / Medicine / Master / Doctor of Medicine

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

published_or_final_version / Surgery / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy

The hormone glucagon-like peptide-2 (GLP-2) is important in the regulation of intestinal growth and blood flow in nonruminant animals. However, no research reports the existence of GLP-2 in ruminants. Therefore, this dissertation examined the existence of GLP-2 and its receptor, their response to physiological stimuli, and its ability to induce gastrointestinal growth and intestinal blood flow in ruminants. Experiments 1 and 2 established the gastrointestinal distribution of mRNA for proglucagon (the GLP-2 precursor) and the GLP-2 receptor. Furthermore, these experiments determined the effects of changing dietary energy intake on plasma GLP-2 concentrations and proglucagon and GLP-2 receptor mRNA expression. Experiment 3 examined the effect of exogenous bovine GLP-2 on splanchnic blood flow, splanchnic nutrient flux, and gastrointestinal growth. This research shows that ruminants possess a functional GLP-2 signaling system that responds to nutrient ingestion. Based on observed receptor distribution and growth changes with GLP-2 treatment, GLP-2 targets the small intestine and does not affect forestomach or large intestinal growth. Increases in ileal proglucagon mRNA expression and plasma GLP-2 with increasing energy intake demonstrate that GLP-2 responds to physiologic changes in nutrient intake and can be relevant to feeding practices. Furthermore, observed increases in small intestinal growth and blood flow with GLP-2 suggest that it could substantially affect the capacity of the gastrointestinal tract for nutrient absorption. Modification of GLP-2 through diet could allow for improvements in nutrient utilization and animal productivity. This research also has important implications for use of GLP-2 for human disease therapy as the observed downregulation in the blood flow response to 10-d GLP-2 administration has never been reported in any species prior to this dissertation. This research systematically characterized and evaluated the potential role of GLP-2 in the control of gastrointestinal growth and splanchnic blood flow in ruminants. While it extends the knowledge of hormonal control of the gastrointestinal tract in ruminants, it also adds crucial information to the larger body of work investigating the actions of GLP- 2. This dissertation research has contributed to the groundwork necessary to enable the use of GLP-2 in improving the health and productivity of a diverse group of mammalian species.