A naphthoquinone, 7-methyljuglone and some of its 5-hydroxy, 5-acetoxy-, 5-alkoxy- and 1,2,4,5-tetra-O-acetate derivatives were tested for their activity in four human cancer cell lines: breast adenocarcinoma, cervical epithelial carcinoma, oesophageal carcinoma and prostate epithelial carcinoma. Compound 2,5-dihydroxy-7-methyl-1,4-naphthoquinone was found to be the most effective one (exhibited a fifty percent inhibitory concentration (IC50) in the range of 5.3 to 14.7 μM), while the parent compound 7-methyljuglone was less active than several of these derivatives. The IC50 values of 5-hydroxy-6-methyl-1,4-naphthoquinone were found to be between 19.1 and 15.4 μM on the four cell lines. However this compound showed toxicity on peripheral blood mononuclear cells. Six derivatives were selected for mechanistic studies. Considering the findings from cell cycle analysis, caspase 3/7 activation and annexinV-FITC dual labelling, 5-hydroxy-6-methyl-1,4-naphthoquinone was found to have antitumour effect by inducing apoptosis. Two derivatives namely, ‘8-fluoro-5-hydroxy-7- methyl-1,4-naphthoquinone’ and ‘2,5-dihydroxy-7-methyl-1,4-naphthoquinone’ were found to be not toxic on peripheral blood mononuclear cells suggesting their action is specific for tumour cells. Compound 2,5-dihydroxy-7-methyl-1,4-naphthoquinone was found to induce apoptosis through caspase 3/7 activation. In view of the enhanced potencies associated with these derivatives, these analogues may hold considerable therapeutic potential for the treatment of leukaemia cancers. The ethanol extracts of seven plant species (ethnobotanically selected) were also tested for their cytotoxicity, assayed by the XTT assay, against four human cancer cell lines at concentrations ranging from 0.78 to 100 μg/ml. Of all the ethanol extracts, Foeniculum vulgare was found to have the best activity on HeLa cells, which exhibited an IC50 value of 19.97± 0.048 μg/ml. Therefore, it was selected for isolation of the bioactive principles. The extract of Foeniculum vulgare was fractionated using column chromatography with hexane and ethyl acetate at different ratios as eluent. Two known compounds, ‘4-methoxycinnamyl alcohol’ and ‘syringin’ were isolated. The IC50 values of ‘4-methoxycinnamyl alcohol’ and ‘syringin’ were found to be 7.82 ± 0.28 μg/ml and 10.26 ± 0.18 μg/ml respectively on HeLa cells. Both compounds were tested for their cytotoxicity against U937 cells and also on peripheral blood mononuclear cells. At the concentrations of 10 and 100 μg/ml ‘4- methoxycinnamyl alcohol’ showed similar cell proliferation as that of the positive control ‘cisplatin’. ‘Syringin’ however, had much lower cytotoxicity on the U937 cells than ‘4- methoxycinnamyl alcohol’. IC50 was found to be 91.14 ± 0.63 μg/ml. Both ‘syringin’ and ‘4- methoxycinnamyl alcohol’ were not cytotoxic at concentrations of 1 and 10 μg/ml on the PBMCs as compared to cisplatin. ‘4-Methoxycinnamyl alcohol’ was selected based on its activity on the cancer cells, for further investigation with regard to its mechanism of action. On gel electrophoresis it did not show a typical ladder pattern, instead a characteristic smear resulted which indicated necrosis. Two best derivatives of 7-methyljuglone (‘8-fluoro-5-hydroxy-7-methyl-1,4-naphthoquinone’ and ‘2,5-dihydroxy-7-methyl-1,4-naphthoquinone’) and the ethanol extract of F. vulgare warrant further investigation to be considered for their potential as anticancer agents. / Dissertation (MSc)--University of Pretoria, 2011. / Plant Science / unrestricted

Spirocerca lupi is a nematode that infects the dog’s oesophagus and promotes the formation of an inflammatory fibroblastic nodule that progresses to sarcoma in approximately 25% of cases. Differentiating neoplastic from non-neoplastic cases ante-mortally is challenging and has major therapeutic and prognostic implications. More importantly, spirocercosis-associated oesophageal sarcoma is an excellent and under-utilized spontaneous model of parasite-associated malignancy and the pathogenesis of the neoplastic transformation is poorly understood. The current study objective was to investigate potential clinical, clinicopathological, radiological and tissue biomarkers for the malignant transformation and an attempt to use these biomarkers to gain a deeper understanding of the pathogenesis of the neoplastic transformation. Our central hypothesis was that the parasite produces excretory product(s) which diverts the immune response from a T helper 1 (Th1) to Th2 cell response, typical of many nematode infections, and further to an immunoregulatory (immunosuppressive), FoxP3+ regulatory T cell-predominated response which then facilitates neoplastic transformation. The following parameters were studied and compared between cases with non-neoplastic and neoplastic spirocercosis: clinical presentation, haematology, serum albumin and globulin, thoracic radiology, haematoxylin-eosin (H&E) histology, Immunohistochemistry for expression of vascular endothelial growth factor (VEGF)-A, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), MAC387 (myeloid cells), CD3 (T cells), Pax5 (B cells) and FoxP3 (T regulatory cells) and plasma cytokine concentrations including IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, GM-CSF and MCP-1. Hypertrophic osteopathy showed 100% specificity for neoplastic transformation but relatively poor sensitivity (40%). Female gender, anaemia, leukocytosis, thrombocytosis, spondylitis and bronchial displacement were significantly more common in neoplastic cases, but appeared in non-neoplastic cases as well. The H&E study revealed 2 stages in the non-neoplastic nodules: early inflammation, characterized by fibrocytes and abundant collagen, and a pre-neoplastic stage, characterized by activated fibroblasts and reduced collagen. The neoplastic cases were all sarcomas, primarily osteosarcoma with very aggressive features comparable to other appendicular osteosarcoma in the dog. The inflammation in spirocercosis is characterized by pockets of pus (MAC387+ cells) surrounded by organized lymphoid foci (CD3+ and to a lesser degree Pax5+ cells). There was no evidence of a local accumulation of FoxP3+ cells, unlike many previous studies which have reported an increase in Foxp3+ T cells in both malignancies and parasite infections. Interleukin-8 plasma concentration was higher in the neoplastic group compared to the non-neoplastic and the control groups. Interleukin-18 concentration was higher in the non-neoplastic group followed by the control group and finally the neoplastic group. As with most similar studies, no ideal biomarker with high sensitivity and specificity was identified. However, if examined together, a panel of the biomarkers that were identified more commonly in the neoplastic cases should substantially increase the index of suspicion for neoplastic transformation in a diagnosed spirocercosis case. The inflammatory response showed features of increased myeloid (innate) response and lymphocytic response with pro-inflammatory cytokines. This was not our initial hypothesis and the question remains whether the response is secondary to the worm infection, or to a symbiotic bacterium that is carried by the worm. The role of such a reaction in neoplastic transformation remains to be elucidated. / Thesis (PhD)--University of Pretoria, 2012. / Companion Animal Clinical Studies / 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.

Purpose: This study evaluated the effect of lung volume at the catch position to force and power outputs during single maximal effort strokes in rowing. Responses were compared when the participants were ‘fresh’ and following specific inspiratory muscle fatigue (IMF). In addition, a single subject pilot study was performed to characterise the changes in intra-thoracic (ITP), intra-abdominal (IAP) and trans-diaphragmatic (Pdi) pressures during a 30 second maximal effort piece on a rowing ergometer. Methods: Nine male rowers of international standard participated in the research. Static force, as well as the power produced during a single stroke were assessed at residual volume (RV), 25%TLC, 50%TLC, 75%TLC, total lung capacity (TLC), and a self-selected lung volume (S-S). Lung volumes were derived from maximal flow-volume loops (MFVLs) and achieved using online real-time feedback. Inspiratory muscle fatigue (IMF) was induced by breathing against an inspiratory load equivalent to 80% baseline maximal inspiratory pressure (MIP), at a breathing frequency (fB) of 15 breaths per minute, and a duty cycle of 0.6. Expiration was unimpeded. The single subject pilot study was undertaken using balloon catheters to measure ITP, IAP, and Pdi during a 30 second maximal effort free-rating piece on the ergometer. Results: There was no significant effect of lung volume upon either force or power production. The RMF protocol induced a significant reduction in MIP (159.9 ± 70.8 vs. 106.8 ± 58.7 cmH2O; p = 0.000), but not maximal expiratory pressure (MEP; 159.9 ± 79.2 vs. 166.6 ± 53.0 cmH2O; p = 0.376). RMF induced a significant reduction in force output with increasing lung volume, across all lung volumes (mean force 1313.4 ± 31.9 vs. 1209.6 ± 45.0N; p < 0.008), but not power (mean power 598.6 ± 31.9 vs. 592.7 ± 45.0W; p > 0.05). Self-selected lung volumes were consistent across all tests for force and power (mean 38.1 ± 6.9% [Force] vs. 28.2 ± 0.6% [Power]; p > 0.017). The pilot study indicated that internal pressures fluctuate markedly during maximal effort rowing (pressure, [max, min, average] cmH2O; IAP [144.69, 7.46, 73.59], ITP [75, -22.65, 15.34], Pdi [111.84, 7.09, 58.83]), suggesting that the trunk muscles play an active role in power production during rowing. Conclusion: The present study suggests that there is no significant effect of lung volume on force or power when athletes are in a fresh condition. However, a decrement in force production is present with inspiratory muscle fatigue. Combined with evidence of high internal pressures during maximal effort rowing, these data may indicate a role for the inspiratory muscles in force production during rowing.

Background: Altered autonomic nervous system (ANS) function has been proposed as a mechanism in the development of central sensitisation (CS) and visceral pain hypersensitivity (VPH). The contribution of the parasympathetic nervous system (PNS) and the factors that mediate differences in sensitisation to acid are unclear and their study will clarify risk factors for oesophageal pain hypersensitivity (OPH) in gastrooesophageal reflux disease. Aims: To investigate psychophysiological and pharmacological manipulation of PNS tone in the development of OPH, and to determine factors which predict the development of OPH to acid infusion in healthy volunteers in a validated model of acid induced OPH. Methods: Pain thresholds to electrical stimulation in the proximal oesophagus were determined before and after a 30-minute distal oesophageal infusion of 0.15 mol/L hydrochloric acid in subjects. Sympathetic (SNS) and PNS parameters were measured at baseline and continuously thereafter. Subjects underwent psychological profiling for anxiety, depression, attachment vulnerability and personality type. Using this model, five studies were undertaken: Study 1 a pilot study to trail modulation suitability for further study used. In Study 2, subjects who demonstrated secondary hyperalgesia in the proximal non-acid-exposed oesophagus performed deep or sham breathing. Study 3 subjects, who did not sensitise to acid, underwent a validated stress test to induce OPH. With Study 4, deep breathing with IV saline (placebo) or atropine (PNS antagonist) was used to evaluate deep breathing’s induced PNS tone in OPH reduction. Study 5, a genetic pilot study, exploring the role of the GCH-1 haplotype in VPH. Results: ANS control’s key role in CS was clarified. Deep breathing increased PNS tone and prevented acid-induced OPH in comparison to sham breathing and confirmed increased PNS tone’s reversal of OPH. Psychological factors of anxiety, alexithymia and attachment status influence ANS modulation of CS. Individuals’ predisposition to VPH due to psychogenetic profiles were clarified and their biopsychosocial role illustrated. Conclusions and Inferences: A mechanistic explanation for the analgesic effect of deep breathing is provided with potential therapeutic implications in the treatment of VPH syndromes. Further clinical study is warranted to develop cost-effective treatments for chronic VPH syndromes.

Increasing symptom awareness, encouraging help-seeking, and facilitating timely referral are key for improving cancer outcomes, particularly for cancers such as oesophageal and gastric (stomach), where five-year survival is less than 20%. In this research, I used multiple methods to explore factors that influence timely diagnosis of these cancers from a patient's perspective, with a particular focus on health literacy (accessing, understanding and using health information, and navigating healthcare systems). I started by exploring current knowledge in this field before conducting a systematic review investigating health literacy in the timely diagnosis of symptomatic cancer. Literature was searched from January 1990-May 2017 using six bibliographic databases. I screened 2304 titles/abstracts, assessed 26 full-text papers and included three, although they were methodologically weak, therefore limiting the conclusions. To examine pathways to diagnosis for oesophageal and gastric cancer, I conducted a questionnaire study of newly diagnosed patients across two hospitals in the East and North East of England. 127 participants were recruited (39.6% recruitment rate), aged 44-96 (median 71); 102 male (80%). Most had oesophageal cancer (n=102, 80%); 64 (50%) of the total cohort were late-stage at diagnosis. Common pre-diagnostic symptoms varied between cancers (oesophageal: difficulty swallowing (n=66, 65%), painful swallowing (n=55, 54%); gastric: fatigue/tiredness (n=20, 80%), weight loss (n=13, 52%)). The questionnaire included two domains (engagement, understanding) of the Health Literacy Questionnaire with participants demonstrating high health literacy (mean 4.18 and 4.28, score 1-5). The median time from noticing the trigger symptom (prompting help-seeking) to diagnosis was 81 days (IQR 45-137.5, n=107). Twenty-six participants were purposively sampled, from questionnaire respondents, for face-to-face interviews (aged 55-88, 18 male, 15 with oesophageal cancer). I undertook thematic analysis to explore participant accounts of their pathways to diagnosis, identifying that the symptom nature was important for appraisal, while health literacy ability influenced the health system interval. Descriptions of 'heartburn', 'reflux' and 'indigestion' differed between participants, suggesting these terms may introduce uncertainty in relation to symptom experience. This is the first study to explore the role of health literacy in the timely diagnosis of symptomatic cancer, and pathways to diagnosis for oesophageal and gastric cancers, from a patient's perspective. Findings provide important insights for the development of targeted awareness campaigns and strategies enhancing GP symptom exploration.

<p>The extension of laparoscopic procedures into the chest may induce specific pathophysiologic effects.</p><p>In pigs, we have demonstrated how devastating a combined thoraco-laparoscopic approach can be for gas exchange. Furthermore, the transmission of elevated pressure intra-cranially is a potential danger. The application of positive end-expiratory pressure (PEEP) was found to improve gas exchange and, more importantly, hypoxemia could be avoided. The application of PEEP did not increase intra-cranial pressure further; nor did it adversely affect cerebral circulation.</p><p>Even before the introduction of the laparoscopic technique, there was a substantial increase in the annual number of antireflux procedures. Therefore, the threefold increase of the incidence of antireflux surgery recorded during the past decade cannot solely be explained by the introduction of minimal access surgery. However, a clear shift in the preferred methodology took place. This change was not scientifically supported at the time of the transition and, surprisingly, it is still not supported today. In comparison with open surgery, patients do not seem to derive significant long-term benefits from having the antireflux procedure done laparoscopically. As was demonstrated, laparoscopy might even be an inferior approach in some patients. Nevertheless, it is reasonable to assume that laparoscopy can yield equally good results as open surgery despite our failure to confirm that in our studies. Determination of the effectiveness of minimal access surgery in the treatment of GORD is critical, before minimal access techniques become the standard for antireflux surgery in the community.</p>

The extension of laparoscopic procedures into the chest may induce specific pathophysiologic effects. In pigs, we have demonstrated how devastating a combined thoraco-laparoscopic approach can be for gas exchange. Furthermore, the transmission of elevated pressure intra-cranially is a potential danger. The application of positive end-expiratory pressure (PEEP) was found to improve gas exchange and, more importantly, hypoxemia could be avoided. The application of PEEP did not increase intra-cranial pressure further; nor did it adversely affect cerebral circulation. Even before the introduction of the laparoscopic technique, there was a substantial increase in the annual number of antireflux procedures. Therefore, the threefold increase of the incidence of antireflux surgery recorded during the past decade cannot solely be explained by the introduction of minimal access surgery. However, a clear shift in the preferred methodology took place. This change was not scientifically supported at the time of the transition and, surprisingly, it is still not supported today. In comparison with open surgery, patients do not seem to derive significant long-term benefits from having the antireflux procedure done laparoscopically. As was demonstrated, laparoscopy might even be an inferior approach in some patients. Nevertheless, it is reasonable to assume that laparoscopy can yield equally good results as open surgery despite our failure to confirm that in our studies. Determination of the effectiveness of minimal access surgery in the treatment of GORD is critical, before minimal access techniques become the standard for antireflux surgery in the community.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Oesophageal cancer (OC) is the sixth leading cause of cancer related deaths in the world with approximately 300 000 new cases reported each year. OC may be characterized into two forms with 90% of cases presenting as squamous-cell carcinoma (SCC) and the remaining 10% as adenocarcinoma (ADC). Several factors have been attributed to the development of OC, including oesphageal injury and/or irritation, chronic inflammation and excess iron associated with enhanced tumour growth. The HAMP gene codes for a 25 amino-acid protein found to be primarily expressed in the liver and crucial to regulation of bodily iron status. Defects occurring in the HAMP gene could therefore lead to the dysregulation of the gene, resulting in an iron overload status. Iron overload is a previously described risk factor in the development of various cancers, including OC, and therefore the aim of this study was to investigate whether dysregulation of the HAMP gene may be involved in the cancer phenotype exhibition. The study cohort comprised of 48 unrelated patients presenting with SCC and a control group of 51 healthy, unrelated population-matched individuals. Mutation detection techniques included polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformation polymorphism (HEX-SSCP) analysis and bi-directional semi-automated DNA sequencing analysis. Screening of the 5’ regulatory region (5’UTR) of the HAMP gene revealed one known (-582A/G) and two novel (-188C/T and -429G/T) variants with the -429G/T variant showing statistically significant reduction in expression in patients relative to controls. Iron parameters were correlated between patient and control cohorts, as well as for variant presence and absence within individuals. Luciferase reporter constructs were used to investigate the functional implications of the presence of a variant on HAMP gene expression, and how these results correlated to the iron parameter statistics obtained. Luciferase reporter assay results indicated the -188C/T and -429G/T variants to result in under-, and the -582A/G variant to result in over-expression at the basal level, relative to the respective wild-type sequence constructs. Correlation of the luciferase data with the iron parameter statistics, indicate the -429G/T variant to be coupled to significantly higher levels of ferritin and C-reactive protein (CRP) and significantly lower levels of serum-iron and transferrin when compared to individuals without the variant. Considering only the patient group, the presence of the -188C/T and -429G/T variants were coupled to significantly lower levels of transferrin in patients with either variant, compared to patients without. The variants found within the HAMP promoter region are therefore able to alter gene regulation to an extent where iron parameters deviate between healthy and OC afflicted individuals, and also between patients with and without a variant. This dysregulation in iron homeostasis may play a role in the development and/ or progression of OC. Characterisation of the 5’ UTR of the HAMP gene may contribute to linking iron regulation to the establishment of an effective screening program, facilitating the early detection of OC. / AFRIKAANSE OPSOMMING: Slukdermkanker (SK) is die sesde grootste oorsaak van kanker-verwante sterftes in die wêreld, met sowat 300 000 nuwe gevalle wat aangemeld word elke jaar. SK kan geklassifiseer word in twee vorme, waar 90% van die gevalle plaveisel-selkarsinoom (SSC) vorm en die oorblywende 10%, adenokarsinoom (ADC). Verskeie faktore word toegeskryf aan die ontwikkeling van SK, insluitend slukderm beserings en/ of irritasie, chroniese inflammasie en oormatige ystervlakke wat geassosieer word met verhoogde gewasgroei. Die HAMP geen kodeer vir 'n 25 aminosuur proteïen wat hoofsaaklik in die lewer uitgedruk word en noodsaaklik is vir die regulering van ystervlakke in die liggaam. Defekte wat in die HAMP geen voorkom kan dus die onreëlmatige regulering van die geen tot gevolg hê, wat lei tot yster-oorlading. Yster-oorlading is voorheen beskryf as ‘n risiko faktor in die ontwikkeling van verskillende vorme van kanker, insluitend SK en gevolglik was die doel van hierdie studie om te bepaal of die wanregulering van die HAMP geen betrokke mag wees by die uitdrukking van die kanker fenotipe. Die studiepopulasie het bestaan uit 48 onverwante pasiënte met SSC en ‘n kontrole-groep van 51 gesonde, onverwante soortgelyke individue. Die mutasie opsporingstegnieke wat gebruik is, het polimerase kettingreaksie (PKR) amplifisering, heterodupleks enkelstring-konformasie polimorfisme (HEX-SSCP) analise en bidireksionele semi-outomatiese DNS volgordebepaling-analise van die geïdentifiseerde variante ingesluit. Sifting van die 5’ regulerende area (5'UTR) van die HAMP geen het een bekende (-582A/G) en twee nuwe (-188C/T en -429G/T) variante opgelewer, met die -429G/T variant wat statisties beduidend onderdruk is in pasiënt uitdrukkings vlakke relatief tot 'n gesonde kontole-groep. Yster-parameters van alle pasiënt en kontole individue is gekorreleerd tussen pasiënt en kontrole groepe, sowel as vir teenwoordigheid of afwesigheid van variante in elke individu. Luciferase verklikker konstrukte is gebruik om die funksionele implikasies van die teenwoordigheid van ‘n variant op HAMP geenuitdrukking te ondersoek, en hierdie resultate te korreleer met yster-parameter statistieke wat verkry is. Luciferase verklikkertoetse dui aan dat die -188C/T en -429G/T variante tot verminderde, en die -582A/G variant lei tot die verhoogte uitdrukking op die basale vlak lei, relatief tot die onderskeie wilde-tipe konstukte. Korrelasie van die luciferase data met die yster-parameter statistieke, dui aan dat die -429G/T-variant gekoppel is aan aansienlik hoër vlakke van feritien en C-reaktiewe proteïen (CRP) en beduidend laer vlakke van serum-yster en transferrien in vergelyking is met individue sonder die variant. Met oorweging van slegs die pasiënt-groep, is die teenwoordigheid van die -188C/T en -429G/T variante beduidend gekoppel aan laer vlakke van transferrien in pasiënte met die variant, in vergelyking met pasiënte daarsonder. Variante binne die HAMP promotor is dus in staat om geenregulasie te verander tot so 'n mate dat die yster-parameters afwyk tussen gesonde en SK geaffekteerde individue, sowel as tussen pasiënte met en sonder ’n variant. Hierdie wanregulering in yster homeostase kan 'n rol speel in die ontwikkeling en/ of die progressie van SK. Karakterisering van die 5’ regulerende area van die HAMP geen kan grootliks bydra om ysterregulasie te verbind met die implementering van ‘n effektiewe siftingsprogram, en sodoende die vroeë opsporing van SK fasiliteer.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22036号 / 医博第4521号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 松田 道行, 教授 山田 亮 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM