The incidence of gastrointestinal (GI) cancers has been steadily increasing in the UK since the mid 1970’s. These include cancers of the colon and oesophagus. Colon cancers have a high incidence rate, being the fourth most common cancer in the UK for both men and women. Oesophageal cancers in comparison are much rarer, however they have a poor survival rate primarily due to a late diagnosis. The key to improving survival for these cancers and many others is to detect and remove the disease at the early stages, to prevent the cancer from advancing. At present the ‘gold standard’ for diagnosis is a biopsy followed by histopathology. This technique is invasive, time consuming and highly subjective. It is therefore important to look towards non-invasive methods for early and rapid diagnosis. Optical techniques have begun to show such promise. By probing the interactions of tissues with light, diagnostic information is able to be obtained non-invasively. Techniques such as Raman spectroscopy utilise inherent molecular vibrations to extract biochemical information from tissues. Raman spectroscopy, however, is currently fundamentally limited by long acquisition times, due to the inherently weak signals produced. Using coherent Raman techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS), the molecular vibrations are coherently driven to provide an enhancement in signal. This thesis explored spectral signatures from snap frozen oesophageal sections in the fingerprint (450 cm-1 to 1850 cm-1) and high wavenumber (2800 cm-1 to 3050 cm-1) regions using spontaneous Raman and compared with spectra from hyperspectral SRS. The diagnostic potential for each technique was assessed for four major pathology groups, normal, Barrett’s oesophagus, dysplasia and adenocarcinoma. Samples were classified using a principal component fed linear discriminant analysis (PCA-LDA) approach with a leave-one-out cross validation. Comparisons were made to haematoxylin and eosin (H&E) stained sections. Raman in the fingerprint region was found to be the most promising for diagnosis. There were minimal changes in the high wavenumber region between pathology groups which was also reflected in the SRS spectra and proved to be insufficient for classification. Further comparisons were made between spontaneous and coherent Raman techniques using frozen colon sections. The morphological and structural information available was explored using a k-means cluster analysis. Both spontaneous and coherent Raman were able to distinguish important structural features in the colon, such as the epithelial cells that form the colonic glands and surrounding connective tissue. Both are important visual markers for cancer diagnosis in the current approach. SRS demonstrated higher spatial resolution and faster acquisition times in comparison to spontaneous Raman. This work has discussed the many advantages of using coherent Raman techniques for tissue applications, but has also highlighted some of the limitations for spectral measurements, arising from the complexity of the system.

A Distrofia Muscular de Duchenne (DMD) é um tipo de miopatia grave, degenerativa e progressiva, geneticamente determinada e ligada ao cromossomo X. Além dos graves distúrbios cardiorrespiratórios e da motricidade, o paciente apresenta disfunções do sistema digestório, caracterizadas pelas desordens da motilidade. Entretanto, sabe-se que tais disfunções não ocorrem no camundongo MDX. O objetivo deste estudo foi estimar a densidade numérica por área da população total de neurônios (QA[T]) e dos neurônios nitrérgicos (QA[N]); assim como a área de secção transversal média do corpo celular destes neurônios (A[T] e A[N]) do plexo mioentérico esofágico e a largura média das fibras estriadas (L) das camadas musculares. Foram utilizados 40 camundongos machos da linhagem C57BL/10 nas idades de 4 e 10 semanas, distribuídos entre grupos experimentais (MDX4 e MDX10), e controles (C4 e C10). As estimativas foram analisadas em preparados de membrana dos esôfagos, e técnicas histoquímicas de NADH-diaforase (NADH-d) e NADPH-diaforase (NADPH-d) foram utilizadas para evidenciar toda a população de neurônios e os neurônios nitrérgicos, respectivamente. Os resultados mostraram que a QA[T] foi significativamente maior no grupo MDX10 em relação ao C10 (p<0.05); enquanto a QA[N] foi menor no grupo MDX4 em relação ao C4 (p<0.05). A A[T] foi menor no grupo MDX10 em relação ao MDX4 e ao C10 (p<0.05); enquanto que para a A[N] não houve diferença significativa entre os grupos controle e experimental, e tampouco entre os grupos de 4 e de 10 semanas. A variável L foi maior nos grupos MDX4 e MDX10 em relação aos seus respectivos controles de mesma idade (p<0.05). Concluímos que no plexo mioentérico esofágico de camundongos MDX existe uma redução dos neurônios mediadores do relaxamento, especialmente em animais jovens (MDX4), provavelmente porque o organismo tenta manter íntegra a função peristáltica do órgão. Esse fato pode explicar a adaptação e ausência de disfunções esofágicas durante quase toda a vida desses animais. / Duchenne Muscular Dystrophy (DMD) is the most common and the most severe muscular dystrophy of childhood. DMD is degenerative, progressive and a genetic X-linked disease. Besides the cardiomyopathy and the movement disorders, the patients have serious disfunctions in the alimentary system, characterized by motility disorders. However, it is known that such changes do not exist in MDX mice. The aim of this study was to estimate the neuronal numerical density/area of total (QA[T]) and nitregic neurons (QA[N]), the average cross-sectional area of total (A[T]) and nitregic (A[N]) neurons in myenteric plexus of the oesophagus and the average width of striped muscle of muscular layer (L). Forty C57BL/10 male mice were studied from four to ten weeks of age, divided into four groups: MDX mice formed the experimental groups (MDX4 and MDX10) and C57BL/10 male mice without the mutation formed the control groups (C4 and MDX10). Whole mounts preparations were obtained from the samples and histochemistry for NADH-diaphorase (NADH-d) and NADPH-diaphorase (NADPH-d) were used for morphometric evaluation. The results showed a significant increase of the QA[T] in the MDX10 than C10 (p<0.05), and a decrease of the QA[N] in MDX4 comparing to C4 (p<0.05). The A[T] decreased significantly in MDX10 comparing to both MDX4 and C10 (p<0.05), while no significant differences were observed among all the groups regarding the A[N]. The groups MDX4 and MDX10 showed a significant increase in L when compared to its controls at the same age (p<0.05). We conclude that in the myenteric plexus of the oesophagus in MDX mice there is a reduction of the inhibitory neurons, manly in the young animals (MDX4), probably to keep normal the peristaltic functions. Thus, it may explain the adaptation and the absence of oesophageal disfunction during almost the whole life in this animal model.

Please read the abstract in the section 02back of this document / Thesis (DVSc)--University of Pretoria, 2010. / Surgery / unrestricted

Objective: Gastro-oesophageal reflux disease (GERD) has heterogeneous aetiology primarily attributable to its symptom-based definitions. GERD genome-wide association studies (GWASs) have shown strong genetic overlaps with established risk factors such as obesity and depression. We hypothesised that the shared genetic architecture between GERD and these risk factors can be leveraged to (1) identify new GERD and Barrett's oesophagus (BE) risk loci and (2) explore potentially heterogeneous pathways leading to GERD and oesophageal complications. Design: We applied multitrait GWAS models combining GERD (78 707 cases; 288 734 controls) and genetically correlated traits including education attainment, depression and body mass index. We also used multitrait analysis to identify BE risk loci. Top hits were replicated in 23andMe (462 753 GERD cases, 24 099 BE cases, 1 484 025 controls). We additionally dissected the GERD loci into obesity-driven and depression-driven subgroups. These subgroups were investigated to determine how they relate to tissue-specific gene expression and to risk of serious oesophageal disease (BE and/or oesophageal adenocarcinoma, EA). Results: We identified 88 loci associated with GERD, with 59 replicating in 23andMe after multiple testing corrections. Our BE analysis identified seven novel loci. Additionally we showed that only the obesity-driven GERD loci (but not the depression-driven loci) were associated with genes enriched in oesophageal tissues and successfully predicted BE/EA. Conclusion: Our multitrait model identified many novel risk loci for GERD and BE. We present strong evidence for a genetic underpinning of disease heterogeneity in GERD and show that GERD loci associated with depressive symptoms are not strong predictors of BE/EA relative to obesity-driven GERD loci.

Die Anastomoseninsuffizienz, als postoperative Komplikation der Kontinuitätswiederherstellung nach Oesophagusresektionen bei malignen Erkrankungen der Speiseröhre, stellt einen wesentlichen Faktor der erhöhten Patientenmortalität- und Morbidität dar. Ziel der Arbeit war es, den Einfluss von VEGF-A als Wachstumsfaktor, in Kombination mit einer, bereits als Medizinprodukt zugelassene Versiegelungsmatrix (TachoSil®) aus Kollagen, mit Fibrinogenbeschichtung, als Augmentation einer End-zu-End Oesophagogastrostomie im Tiermodell zu untersuchen. Es konnten mittels ELISA gezeigt werden, dass eine Liberation von VEGF, aus der damit inkubierten Versiegelungsmatrix, über die als kritisch für die postoperative Anastomosenheilung geltende Dauer von fünf Tagen, möglich ist. Es konnte nach Auswertung von drei Verdünnungsreihen eine wirksame Konzentration für die nachfolgenden Versuche ermittelt werden. Danach wurde in vitro, im Zellversuch an HUVECs, die Auswirkung auf die Zellproliferation unter Anwesenheit der Versiegelungsmatrix und VEGF-A untersucht. Hier zeigte sich wie erwartet die proliferationsfördernde Wirkung des VEGF-A auf Endothelzellen, jedoch auch ein unklarer antiproliferativer Effekt der Versiegelungsmatrix. Im nachfolgenden Tierversuch wurden 34 Brown Norway Rats einer Oberbauch- Medianlaparotomie mit nachfolgender Gastrolyse, Resektion des proximalen, kleinkurvaturseitigen Magenanteils, des Vormagens und des oesophagogastralen Übergangs unterzogen und im gleichen Eingriff eine Schlauchmagenbildung mit einer End-zu-End oesophagogastralen Anastomose durchgeführt. Bei 19 Tieren wurde die Anastomosenregion mittels TachoSil® 360° zirkulär augmentiert, bei 14 Tieren erfolgte die Augmentation der Anastomosenregion mit VEGF inkubiertem TachoSil® in gleicher Weise. Sechs Tiere verstarben postoperativ, die Todesursache blieb, trotz Obduktion unklar. Bei a.e. entzündlichen Veränderungen der Lunge schien eine pulmonale Komplikation wahrscheinlich. Am fünften postoperativen Tag wurden die bis dahin überlebenden Tiere relaparatomiert, der distale Oesophagus, der Schlauchmagen und das Duodenum wurden en-bloc reseziert und die Tiere intraoperativ euthanasiert. Anhand der Resektate erfolgte die manometrische Dichtigkeitsprüfung der Anastomosenregion, hier zeigte sich lediglich bei einem Tier, der nur mit TachoSil® augmentierten Gruppe, eine Anastomoseninsuffizienz. Alle anderen Resektate zeigten sich manometrisch suffizient, signifikante Unterschiede zwischen den verschiedenen Gruppen bestanden nicht. Abschließend wurde die notwendige Bruchkraft der verschiedenen Anastomosen ermittelt. Dies gelangt durch Einspannen der Resektate in ein Tensiometer. Hier wurden durchschnittlich 2,08 N bis zur Zerreißung der Anastomose gemessen. Signifikante Unterschiede zwischen den verschiedenen Gruppen bestanden nicht. Im Vergleich zum TachoSil® freien Vorversuch, der von Drescher et al. 2012 publiziert wurde, konnte abschließend gezeigt werden, dass die Anastomosenaugmentation mit TachoSil® die Insuffizienzrate signifikant senkte (0-8% vs. 33%). Die Anastomosenbruchkraft war in den TachoSil® Gruppen signifikant höher (2,08 N vs 1,53 N). Hinsichtlich der Verwendung von VEGF gab es keine statistisch signifikanten Unterschiede.:Inhalt Abkürzungsverzeichnis 3 1. Einleitung 5 1.1. Hypothesen 7 1.2. Anatomie des Oesophagus 8 1.3. Anatomie des Oesophagus bei Rattus norvegicus und vergleichende Anatomie der Vertebraten 11 1.4. Kurze Historie der Oesophaguschirurgie 11 1.5. Das Oesophaguskarzinom 15 2. Anastomosenheilung 19 2.1. Die Anastomoseninsuffizienz 21 2.2. Klassifikation der Anastomoseninsuffizienz 24 2.3. Prävention von Anastomoseninsuffizienzen 24 2.4. Erkennen von Anastomoseninsuffizienzen 25 2.5. Diagnostik von Anastomoseninsuffizienzen 26 2.6. Therapie von Anastomoseninsuffizienzen 26 2.7. Spezielle Situation oesophagogastraler Anastomosen 27 3. VEGF 34 4. TachoSil 40 5. Versuchsaufbau 42 6. Materialien und Methoden 43 6.1. Geräte und Software 43 6.2. Verbrauchsmaterialien 44 6.3. Reagenzien 44 6.4. Operative Versuche 46 6.5. Statistik 47 6.6. Liberationsversuch 47 6.7. HUVEC Anzuchtversuch 49 6.8. Konfokale Lasermikroskopie 52 6.9. Operative Versuchsdurchführung 52 6.9.1. Manometrie der Anastomosenregion 57 6.10. Tensiometrie der Anastomosenregion 58 7. Ergebnisse 59 7.1. VEGF Liberationsversuch 59 7.2. Auswirkung von TachoSil und VEGF-A auf die HUVEC Proliferation 60 7.3. Konfokale Lasermikroskopie 62 7.4. Operativer Versuch 64 7.5. Interpretation und Diskussion 65 7.6. Limitationen 71 7.7. Ausblick 71 8. Zusammenfassung der Arbeit 73 9. Literaturverzeichnis 75 10. Anlagen: 101 11. Abbildungsverzeichnis: 103 Erklärung über die eigenständige Abfassung der Arbeit 104 Danksagung 106

Masters of Science / There is a need for defined models of human nutritional disorders partly because serious misconceptions about models are common amongst researchers. Historically a large variety of species has been used including primates, pigs, rats, lagomorphs. Advantages various small carnivores and and disadvantages are not well known and availability is a major factor. In 1753 John Hunter used pigs to study bone growth in one of the first scientifically controlled nutrition experiments (Kobler 1960). Rats were most likely the first animals to be bred specifically for scientific purposes and there is evidence that they were used in nutrition experiments during the late eighteenth century (Kobler 1960). Experience with carcinogenesis in animals has shown the great diversity of results which may possibly be obtained from different species (Lave et al. 1988). This is pertinent to nutritional research as there is an established link between diet and cancer. The selection of a suitable substitute to attempt to model possible human response to a variety of procedures is dependent upon criteria among which the following are possibly the more important. Availability; this is of great importance in Southern Africa where the cost of importation of exotic species. must be taken into account. Du Plessis (1981) referred to the fact that our indigenous primates were a valuable resource. A second consideration must be the cost the selected animal in a scientifically acceptable environment. Keeping animals of maintaining and ethically for research purposes in an uncontrolled environment could well lead to erroneous conclusions being made. Thirdly the cost of a research program in which animals are used may be increased if there is insufficient knowledge of the model selected. A paucity of knowledge available about an animal may affect the viability of an experiment. The need for precise information regarding the effects of extended term dietary supplementation of experimental animals has been noted by Fincham et. al. (1987) . Additionally the selected animal should preferably have similar dietary requirements to man, and have a life span which will enable extended term investigations.

Oesophageal cancer is an aggressive malignancy with an overall five-year survival of 5-10% and two-thirds of patients have irresectable disease at diagnosis. Accurate staging of oesophageal cancer is important as survival closely correlates with the stage of the tumour, nodal involvement and presence of metastases (TNM staging). Endoscopic ultrasonography (EUS) is currently the most reliable modality for providing accurate T and N staging. Depending on findings of the staging, various treatment options including endoscopic, oncological, and surgical treatments may be performed. It was theorised that the development of three-dimensional radial endoscopic ultrasonography would reduce the operator dependence of EUS and provide accurate dimensional and volume measurements to aid planning and monitoring of treatment. This thesis investigates the development of a three dimensional endoscopic ultrasound technique that can be used with the radial echoendoscopes. Various agar-based tissue mimicking material (TMM) recipes were characterised using a scanning acoustic macroscope to obtain the acoustic properties of attenuation, backscatter and speed of sound. Using these results, a number of endoscopic ultrasound phantoms were developed for the in-vitro investigation and evaluation of 3D-EUS techniques. To increase my understanding of EUS equipment, the imaging and acoustic properties of the EUS endoscopes were characterised using a pipe phantom and a hydrophone. The dual ‘single element’ mechanical and ‘multi-element’ electronic echoendoscopes were investigated. Measured imaging properties included dead space, low contrast penetration, and pipe length. The measured acoustic properties included transmitted beam plots, active working frequency and peak pressures. Three-dimensional ultrasound techniques were developed for specific application to EUS. This included the study of positional monitoring systems, reconstruction algorithms and measurement techniques. A 3D-EUS system was developed using a Microscribe positional arm and frame grabber card, to acquire the 3D dataset. A Matlab 3D-EUS toolbox was written to reconstruct and analyse the volumes. The 3D-EUS systems were evaluated on the EUS phantom and in clinical cases. The usefulness of the 3D-EUS systems was evaluated in a cohort of patients, who were routinely investigated by conventional EUS for a variety of upper gastrointestinal pathology. 3D-EUS accurately staged early tumours and provided the necessary anatomical information to facilitate treatment. With regards to more advanced tumours, 3D-EUS was more accurate than EUS in T and N staging. 3D-EUS gave useful anatomical details in a variety of benign conditions such as varicies and GISTs.