Common anticancer strategies often fail resulting in tumour recurrences and metastasis that cause the majority of cancer-related deaths. Only about 6% of colorectal cancer (CRC) metastatic patients live more than 5 years after diagnosis. Thus, elucidating the crucial mechanisms underlying drug resistance and metastasis is an urgent need in cancer biology and therapy. The tumour suppressor gene FBXW7 (also called hCDC4, FBW7, Sel10, Ago) is one of the most frequently mutated genes in CRC. FBXW7, as a receptor subunit of the SCF-E3 ligase complex, drives the ubiquitination and degradation of many oncogenic proteins influencing cell proliferation, differentiation, senescence and other crucial pathways. Our group and others recently demonstrated that murine intestinal Fbxw7 loss of function induced tumorigenic activity, however its exact position in the multistep model of colorectal carcinogenesis remains to be clarified. More recent data from Dr Nateri’s laboratory (Li et al., under review) suggested that ZEB2, transcriptional inducer of epithelial-mesenchymal transition (EMT), represents a specific target of FBXW7 and FBXW7 loss induces an EMT-like phenotype. We have therefore proposed that FBXW7 suppression may confer resistance to chemotherapy, stem-like cell properties and invasive and metastatic activity via EMT, following accumulation of ZEB2. To test our hypothesis, we investigated FBXW7 role in the occurrence of drug resistance in human CRC cells and three-dimensional murine organoids, after treatment with 5-fluorouracil (5-FU) and oxaliplatin, DNA damaging chemotherapeutic agents. We explored EMT activation due to loss of FBXW7 by examining epithelial and mesenchymal markers in response to drug treatment as well as stem-like cell properties by colonosphere formation assay. EMT-associated properties were also analysed following stable down-regulation of ZEB2 in CRC cells and organoids. Moreover, to explore a putative involvement of FBXW7 through accumulation of ZEB2 in invasive and metastatic potential, we performed in vivo xenograft analyses by intra-splenic (for liver and lymph node metastasis) and intravenous (migration/invasion in lungs) injection of human CRC cells deleted for FBXW7 and stably down-regulating ZEB2. Herein, we show that CRC cell lines and murine organoids carrying deletion of FBXW7 are significantly less sensitive to 5-FU and oxaliplatin treatments. FBXW7 loss of function by accumulation of ZEB2 confers EMT phenotype to CRC cells including reduction of E-cadherin, up-regulation of Vimentin, stem-like properties and induction of invasive and metastatic activity. This suggest that FBXW7-deleted CRC cells may represent cancer stem cells. Moreover, this study also aimed to employ and establish an innovative in vitro model to evaluate the cell type-specific responses of gut epithelium to chemotherapeutic agents and how these responses are influenced by FBXW7. Chemotherapeutic sensitivity of the different cell types in normal gut and tumour tissues is poorly understood, impeding the development of improved targeted therapies. We have therefore established the organoid culture which mimics the structure of both gut and colorectal tumours comprising all the cell types likewise the human in vivo situation. To further investigate the mechanism(s) of action of Fbxw7, we grew organoids from isolated small intestinal crypts of mice in which fbxw7 gene is conditionally knocked-out in the intestine (fbxw7∆G), in parallel with wildtype floxed-fbxw7 (fbxw7fl/fl) organoids. The fbxw7∆G organoids exhibited rapid budding events in the crypt region. To test organoids for drug response, we exposed organoids from fbxw7fl/fl and fbxw7∆G mice to various concentrations of 5-FU for 72 hours. 5-FU triggers phenotypic differences in organoids including changing shape, survival, resistance, and death. 5-FU, however, rescues the drug resistance phenotype of fbxw7∆G through the induction of terminal differentiation. Our results support the hypothesis that a differentiating therapy successfully may target FBXW7-mutated CRC cells.

Background: Lymphangioleiomyomatosis (LAM) is a rare progressive neoplastic cystic lung disease that primarily affects women of child bearing age leading to lung destruction, respiratory failure and death. Thought to be a consequence of dysregulated protease expression, cells of unknown origin accumulate in the lung, often forming clusters or nodules of cells with both melanocytic and smooth muscle properties. Some of these cells, known as LAM cells, have bi-allelic mutations in TSC2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures and genotyping analyses suggest that cells without LOH for TSC2 including wild-type fibroblasts are also common within LAM nodules. Hypotheses and aims: We hypothesise that LAM cells recruit wild-type fibroblasts and modify their properties to generate a permissive microenvironment, akin to a tumour stroma including the production and activation of matrix-degrading proteases which contribute to the destruction of the lung parenchyma. This study has therefore deigned in vitro co-culture models with an aim to study the expression patterns and activation of proteases in a LAM lung leading to matrix destruction. Another aim was also to characterise transcriptional differences normal human lung fibroblasts (NHLFs) and LAM-associated fibroblasts (LAFs) and to investigate changes in their gene expression when cultured together with a model LAM cell line, 621-101 angiomyolipoma cells which were derived from a LAM patient and have bi-allelic loss of TSC2. Methods: In vitro 2-dimensional (2D) and 3-dimensionalD (3D) co-culture models were designed and validated using fibroblasts characterised and isolated from 4 LAM lung donors, now termed LAFs, and 621-101 cells. The 3D extracellular matrix (ECM) incorporated the two cell types in a 10:1 ratio embedded in a basement membrane extract (BME) mimicking the lung matrix. An organotypic spheroid model was also developed incorporating both cell types thereby mimicking a LAM nodule. 6 LAM lung and 3 normal lung tissue donors were screened for candidate proteases in LAM pathology using qRT-PCR and identified upregulated proteases which may contribute to a role in LAM pathology. These findings were verified in the 2D and 3D in vitro models as well as ex vivo tissue using a variety of immunostaining techniques, activity assays and ELISA. Lastly, commercially bought NHLF (n=3) and LAF (n=3) were cultured in the presence or absence of 621-101 cells in the 2D Boyden chamber co-culture model. LAF and NHLF RNA was analysed using Affymetrix Human Genome U133 Plus 2.0 Arrays and Genomics Suite and Pathway (Partek). Findings were validated by multiplex assay and immunohistochemistry in 2D and 3D in vitro models and tissue respectively. Inflammatory cell migration and function was examined in co-culture model and LAM tissue. Results: The 3D BME model showed that TSC2-/- 621-101 cells and fibroblasts spontaneously form aggregates and clump together akin to a LAM nodule. The two cell types exhibited strong heterotypic cell-cell adhesive forces and resulted in strictly spherical spheroids. The 3D models designed all showed expression of markers of LAM nodules thereby representing LAM nodules in a dish. Of 30 proteases screened, cathepsin K gene expression was increased almost 15-fold in LAM lung compared to normal tissue and was also found to be elevated in 3D BME model. Cathepsin K in LAM tissue was expressed in the LAM nodules associated with cysts and was expressed exclusively by fibroblasts in the 3D spheroid model. As cathepsin K requires low pH for activity it was determined if LAFs and TSC2-/- cells can acidify the extracellular space. TSC2-/- cells but not LAFs decreased extracellular pH, over 24 hours and pH values < 7 were associated with increased cathepsin K activity in co-cultures. TSC2-/- cells expressed membrane transporters associated with extra-cellular acidification and inhibitors of the sodium bicarbonate co-transporters, carbonic anhydrases and mTOR reduced the pH gradient and decreased CTSK activity in co-cultures. Transcriptomic analysis using the 2D co-culture model showed 148 genes were significantly altered in both NHLF and LAF by 621-101 cells. Soluble factors from 621-101 cells induce pro-inflammatory transcriptional changes in both NHLFs and LAFs and pathway analysis showed enhanced chemokine signalling which highlighted stimulation of mainly the C-X-C motif chemokines and chemokine receptor signalling. The analysis identified 6 C-X-C motif chemokines all possessing a cognate receptor. The gene and protein expression of these chemokines was validated in the in vitro models and in ex vivo LAM lung tissue. Conclusions: The in vitro models are versatile and mimic the LAM lung nodule and environment. A potent matrix degrading protease possibly playing a role in LAM has been identified and using the in vitro models a possible mechanism of activation of CTSK resulting from a synergistic relationship between TSC2-/- cells and LAFs has been demonstrated. Also, soluble factors from the TSC2-/- LAM cell line elicit changes in gene expression in co-cultured fibroblasts. Chemokine signalling is associated with cell migration; elevated chemokine expression may be associated with the recruitment of inflammatory cells to the LAM nodule. The identification of these mechanisms and pathways opens up new avenues for therapeutic interventions in LAM.

Harnessing the actions of the resolvin pathways has the potential for the treatment of a wide-range of conditions associated with overt inflammatory signalling. Aspirin-triggered resolvin D1 (AT-RvD1) is a potent anti-inflammatory lipid derived from docosahexaenoic acid (DHA). In rodent, the biological effects of AT-RvD1 are mainly mediated by its specific G-protein coupled receptor, formyl peptide receptor 2 (ALX/FPR2). AT-RvD1 has been demonstrated potent anti-inflammatory and tissue resolution promoting (pro-resolving) activities in preclinical model of inflammatory diseases. Continuous peripheral activity of nociceptive fibres induces neuroinflammatory responses and alters the excitability neurones in both peripheral and central pain pathways. This thesis focused on spinal neuroinflammation which plays important roles in the sensitisation of nociceptive processing of the spinal dorsal horn neurones and contributes to the nociceptive hypersensitivity of the central pain pathway in acute and chronic pain. Since AT-RvD1 possess a potent anti-inflammatory activity, therefore, enhancing AT-RvD1 signalling in the spinal cord may attenuate the spinal nociceptive sensitisation and provide analgesia. AT-RvD1 also has robust antinociceptive effects in behavioural models of pain, however the potential underlying spinal neurophysiological mechanisms contributing to these inhibitory effects in vivo are yet to be determined. The purposes of this thesis were to investigate the differential effects of spinal AT-RvD1 on evoked responses of spinal neurones in vivo in well characterised pain models include carrageenan-induced acute inflammatory pain, monosodium iodoacetate (MIA)-induced OA pain and paclitaxel (PCX)-induced peripheral neuropathic pain and to investigate the alterations in the expression of spinal genes relevant to the resolvin system and neuroinflammation that may underlie the differential effects of AT-RvD1 among these pain models. Following model induction, pain behaviour was assessed and then rats were prepared for single unit extracellular recordings of dorsal horn wide dynamic range (WDR) neurones on the following day post carrageenan or day 28-32 post MIA or PCX induction. At the time-matched the spinal recording, ipsilateral dorsal quadrants of spinal cord form separate groups of rats were collected for gene expression quantification using TaqMan Low Density Arrays (TLDA). AT-RvD1 clearly demonstrated differential inhibition on evoked responses of WDR neurones in the pain models of interest. Low dose AT-RvD1 (15 ng/50ul) selectively produced a robust inhibition of electrical-evoked responses (Adelta-, C-fibre, post-discharge, input, wind-up) of WDR neurones in carrageenan rats but not in control rats. These effects were abolished by spinal pre-administration of a FPR2/ALX antagonist, butoxy carbonyl-Phe-Leu-Phe-Leu-Phe (BOC-2) (50 ug/50ul). In MIA rats, AT-RvD1 given at a high dose (150 ng/50ul) produced only mild inhibition of electrical evoked Adelta responses but not at a low dose (15 ng/50ul). AT-RvD1 (15 and 150 ng/50ul) had no significant effects on electrical evoked responses of PCX rat WDR neurones. Interestingly, AT-RvD1 produced a dose dependent and selective inhibition of low intensity mechanical evoked responses PCX rats whereas it had no effects in control rats. At high dose (150 ng/50ul), the magnitude of inhibition of 8g mechanical evoked responses was comparable to morphine (1 ug/50ul) applied at the end of the experiments. AT-RvD1 produced a dose dependent inhibition of acetone-evoked responses in PCX rats, however WDR neurones in the control rats were also inhibited to a similar degree. TLDA revealed the distinct alteration of resolvin genes in spinal cord underpinning the differential inhibition of AT-TvD1. Upregulated 5-lipoxygenase activating protein (FLAP) gene, encoding protein determining endogenous resolvin synthesis, in carrageenan and PCX rats may underlie the robust inhibition of AT-RvD1 on WDR neurones in these two models. The minimal effects AT-RvD1 of MIA rats were associated with upregulated 15-hydroxyprostaglandin dehydrogenase (15-HPGD) gene, encoding a major enzyme responsible for D-series resolvin inactivation. Data presented in this thesis provide for the first time the differential inhibition of AT-RvD1 on spinal nociceptive processing in different types of pain and the evidence of heterogeneous spinal alterations of the resolvin signalling potentially underlie such inhibition. This thesis strongly supports further investigation of AT-RvD1 as a novel analgesic. Another part of this thesis demonstrated changes in the responses of spinal WDR neurones in PCX rats. Spinal WDR neurones from PCX rats displayed characteristics representing pain sensitisation including: reduced the thresholds for Abeta and C-fibre responses; increased proportion of neurones exhibiting spontaneous activity, acetone responsiveness and post-discharge following low intensity mechanical stimuli and a more convergence in stimulus processing. A remarkable upregulation of multiple genes in proinflammatory signallings, toll-liked receptor 4 (Tlr4), interleukin-1 (Nlrp1a) and tumour necrosis factor-alpha receptors (Tnfrsf1a and Tnfrsf1b) and chemokines (Cxcl6, Cxcr1, Cxcr5, Ccr1, Cx3cr1), was found in the spinal cord of PCX rats. This suggests that a high inflammatory component in the spinal cord could contribute to the changes in the responses of spinal WDR neurones in the PCX rats. This thesis supports further investigation of targeting neuroinflammation as a promising approach for the treatment of PCX-induced neuropathic pain.

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2010 / Fructosamine is a generic name given to a compound known as plasma protein ketoamines. It is formed by a non-irreversible enzymatic reaction between glucose and serum proteins, mainly albumin. Fructosamine together with glycated haemoglobin (HbA1c) are used to monitor the state of hyperglycaemia in diabetics. It provides the clinician with an index of glycaemia over a shorter period of time than HbA1c, about 4 weeks due to the high turnover of human serum albumin in blood and the degree of glycation in serum proteins. The evolvement of automation in Clinical Chemistry necessitates that each pathology laboratory provides relevant sets of reliable reference values that are population and analyzer or method specific. Currently, the reference range for fructosamine at PathCare ranges between 200 to 285 μmol/L. Four hundred and forty six (120 white females, 117 white males, 114 mixed ancestry females, 95 mixed ancestry males) apparently healthy participants visiting the PathCare, Somerset West practice, were recruited for this study. Fructosamine, random blood glucose, HbA1c, total protein, albumin, and lipid profile was preformed on all individuals. Nonparametric methods, whereby the sample 2.5 and 97.5 percentiles are used to form the 95% reference interval, were used to determine the reference values for fructosamine. Though no significant differences (p = 0.086) were observed between males and females in the total population group the mixed ancestry males had significantly higher fructosamine levels (p = 0.01) compared to their female counterparts. The reference range of the entire sample was 223 – 295 μmol/L, however differed in the different population groups (white females = 228 - 291 μmol/L, white males= 223 – 296 μmol/L, mixed ancestry females = 217 - 293 μmol/L and mixed ancestry males = 222 – 304 μmol/L). The new fructosamine reference range obtained in this study showed a significant difference than the one used at Pathcare, which is 200 – 285 μmol/L. Our results further strengthen the recommendations by pathology bodies that laboratories must establish reference values that are representative of local populations. The single reference range (226 - 293 μmol/L) for the Caucasian males and females is recommended, however due to the significant differences observed between the mixed ancestry males and females it is recommended that gender specific references ranges be used for this population group, which are: 1) 222 - 304 μmol/L for the mixed ancestry males and 2) 217 - 293 μmol/L for the mixed ancestry females respectively.

Introduction: Significant developments have been made in the treatment and prevention of cancer. Despite these developments, a third of the population of the UK will get cancer in their lifetime and a quarter of the population are likely to die from it, making it a leading cause of death in the UK. There is an increase in the numbers of cancers being largely driven by the ageing and expanding population. Conventional anti-cancer and anti-metastatic approaches such as chemotherapy and radiotherapy are effective because these approaches mostly inhibit cell division in proliferating cancer cells or make the tissue environment hostile to cancer cell growth and migration. However, the non-selectiveness of these approaches often causes serious side effects leading to the damage of healthy tissue. Significant gains have been made through the development of targeted therapies and early detection with the benefit of personalising medicine for a specific target thereby minimising harm. However, there is a problem of drug resistance in about 50% of patients with these forms of treatment. Progress in developing these treatments for disease remain slow, with traditional approaches inadequate and rational new therapies needed to tackle cancer. As a result, an alternative strategy for drug development such as using previously approved drugs for new medical indications is beginning to be explored. This strategy will potentially remove substantial risks, costs and time from the pathway of drug development. Recent evidence has shown the potential anti-neoplastic effects of some cheaper and safer medications. Among the best examples of this is the near 50% reduction in cancer specific mortality from colorectal cancer recently shown in those starting Aspirin after diagnosis, which has already led to a randomised controlled trial. In vitro and in vivo studies suggest a role for beta blockers and alpha blockers in inhibiting the proliferation and migration of cancer cells. A series of case-control and cohort studies was therefore conducted using the large population based Clinical Practice Research Datalink (CPRD) and associated datasets as the data source to explore the impact of beta and alpha blockers on cancer incidence and overall cancer mortality. Epidemiological studies on the effect of adrenergic blockers on cancer incidence have proved inconclusive with particularly limited evidence from large population based studies on cancer incidence. A case-control study was therefore conducted to assess the effect of adrenergic blockers upon incidence of cancers of the prostate, lung, bowel and breast. Additionally, epidemiological studies have examined the potential beneficial effects of adrenergic blocker on cancer survival, but these are still inconclusive with limited evidence from large population-based studies. Cohort studies were therefore carried out to examine the effect of beta and alpha blocker exposure post diagnosis on cancer specific and overall mortality. Furthermore, laboratory studies have demonstrated the effect of alpha blockers in reducing induced angiogenesis and suppress metastasis in mouse models. A study was therefore conducted to examine in detail the effect of alpha blockers on mortality outcomes in a cohort of prostate cancer patients and additionally considering their indication of use. Finally observational studies have investigated the anti-proliferative effects of beta blocker use on survival outcomes but not specifically in those without metastases who might be most likely to benefit. This study therefore investigated the effect of adrenergic blockers on mortality outcomes in a large population based UK cohort of non-metastatic colorectal cancer patients. The objectives of this thesis were: To test the hypothesis that: β-blocker use is associated with a reduced incidence of breast, lung, prostate and colorectal cancer. To test the hypothesis that: α-blocker use is associated with a reduced incidence of breast, lung and colorectal cancer. To test the hypothesis that: α-blocker use is associated with a reduction in cancer specific and overall mortality in prostate cancer patients. To test the hypothesis that: β-blocker & α-blocker use is associated with a reduction in cancer specific and overall mortality in patients with non-metastatic colorectal cancer. To test the hypothesis that: β-blocker use is associated with a reduction in cancer specific and overall mortality in those diagnosed with prostate, breast, lung and colorectal cancer. To test the hypothesis that: α-blocker use is associated with a reduction in cancer specific and overall mortality in those diagnosed with breast, lung and colorectal cancer. To test the hypothesis that: β-blocker & α-blocker use is associated with a reduction in cancer specific and overall mortality in those diagnosed with non-metastatic breast, lung and prostate cancer. Methods: A frequency matched case-control study was carried out using the Clinical Practice Research Datalink to assess the effect of adrenergic blockers upon incidence of prostate, lung, bowel and breast cancer. Amongst patients aged 18 years or older contributing at least 2 years of usable data between 01/01/1987 – 31/12/2012. Incident cases of relevant cancers and controls were selected and frequency matched 10:1 by age. Those with 2 or more prescriptions for alpha or beta blockers in the 2 years prior to cancer diagnosis were considered exposed and also assessed effect of the dose and duration of use. Logistic regression was used to adjust effect estimates for age, sex, smoking, alcohol use, and a number of potentially confounding co-morbidities and co-prescriptions. A cohort study of colorectal, lung, breast and prostate cancer patients was conducted and selected from linked UK Clinical Practice Research Datalink, Hospital Episode Statistics and National Cancer Intelligence data between 1998 and 2010. Beta blocker and alpha blocker exposure were assessed in the 6 months post cancer diagnosis and its effect on all cause and cancer specific mortality was assessed. Data were analysed using cox proportional hazards modelling. Confounding by age, sex, cancer stage, grade and important comorbidities and co-prescriptions was adjusted for. The indication of use, dose and pre-diagnosis exposure was also examined. Additionally, from the linked data sources above, a cohort of 3164 non-metastatic colorectal cancer patients was selected and conducted a cohort study using similar methods but additionally considering the cardio-selectivity of beta blocker drugs. Results: For the case-control study 18968 colorectal, 19082 lung, 21608 prostate and 29109 breast cancers were identified. There was no evidence of a protective effect of α or β blockade in lung and prostate cancer and found a slightly increased risk of colorectal and breast cancer in users. This was largely explained by the effects of confounding in a multivariate analyses with final OR estimates of lung, colorectal, breast and prostate cancer of 0.99, 95% CI [0.96-1.04]1.14, 95% CI [1.09 – 1.18]1.10, 95% CI [1.06 – 1.14]1.01, 95% CI [0.98-1.05] respectively for beta blocker exposure and 1.03, 95% CI [0.97 – 1.09]1.13, 95% CI [1.07 – 1.20]1.08, 95% CI [1.00 – 1.17] for alpha blocker exposure. Stratification by dose and duration did not reveal any statistically significant findings. For the cohort study of common solid cancers 15636 colorectal, 13646 lung, 23877 breast and 18654 prostate cancer patients were selected with a median follow up of 3.7, 0.6, 5.5 and 4.4 years respectively. There were no significant effects observed on all-cause mortality in any cancers and similarly no significant effects observed on cancer specific mortality in patients on betablockers compared to those who were not. For alpha blocker exposure, there were no significant effects observed on all-cause mortality in any cancers and similarly no significant effect on cancer specific mortality except in prostate (HR0.874, 95% CI [0.781 – 0.978]) and colorectal (HR1.878, 95% CI [1.108 – 3.182]) cancer patients. There were no clear significant effects observed by dose or prediagnosis exposure. Conclusion: In these large population-based case-control and cohort studies investigating the impact of beta and alphablocker use on cancer incidence and mortality, limited evidence was found to suggest that adrenergic blocker use prevents the incidence of common cancers. Indeed, a slight increased risk of colorectal and breast cancer was found which may reflect residual confounding and health seeking behaviours. Furthermore, beta or alpha blocker use post diagnosis was not associated with a decreased risk of cancer-specific or all-cause mortality in colorectal, lung or breast cancer patients or in those with non-metastatic colorectal cancer. However, our results do provide evidence that alphablockers are associated with a decreased risk of prostate cancer.

The highly polymorphic DlS80 locus has no known genetic function. This variable number of tandem repeat (VNTR) has been valuable in forensic identification. We have obtained allelic and genotypic frequencies for five African populations (Benin, Cameroon, Egypt, Kenya and Rwanda), which could be employed as databases to identify individuals. The polymerase chain reaction, followed by vertical polyacrylamide gel electrophoresis and silver staining was our method of analysis. Allele frequencies were used to infer genetic associations using Phylip 3.5, Principal Component and G-test statistical programs. Tests for Hardy-Weinberg equilibrium were employed. Fst estimates and power of discrimination values were also determined for each of our populations. Our analyses of 28 additional populations demonstrated that the D1 S80 locus alone provided for the discrimination of major racial groups. Genetic homogeneity between the African groups was observed. We have generated a database useful for human differentiation and phylogenetic studies.

Colonisation by many bacteria and viruses is now thought to depend upon their ability to adhere to host cells via proteinacious surface appendages called adhesins. Information relevant to the prevention and cure of many diseases therefore is supplied by knowledge of this adhesive process, especially the chemistry of the binding and the structure of the binding molecules. At this time, the structure of very few adhesin receptors is known. Similarly, the quaternary and primary structure of only a small number of adhesins is currently available. Those associated with Escherichia coli are known in some cases to be arranged as helical coils with repeating proteinacious subunits with molecular weights of 10-30 kDa, however there is conflicting information on the distribution along these coils of the polypeptide involved in adhesion. Thermodynamic binding studies have not yet been used to clarify this problem because of the size of the receptors for the adhesins. This thesis presents a thermodynamic study of the binding between the adhesin from an F41+ E.coli and its receptor, glycophorin, from the human red blood cell membrane using an aqueous polymer two-phase system. The study shows that 2.4±0.8 glycophorin molecules bind to the predominant subunit of this adhesin, suggesting that this subunit has one binding site, since glycophorin dissolves as a dimer. It is proposed that the assay could be used, in addition, to obtain information on the chemical specificity and the thermodynamics of this particular reaction, in order to obtain a broader understanding of the colonisation and infection by this particular pathogen. / Science, Faculty of / Chemistry, Department of / Graduate

In the African Cape buffalo, circulating serum xanthine oxidoreductase (XOR) produces trypanotoxic levels of hydrogen peroxide (H2O 2). Previous, preliminary studies suggest that mammalian serum XOR H 2O2 production rates (“specific activities”) are species- and breed-associated. The research objectives pursued here were: (1) to verify that mammalian serum XOR specific activities were truly species- and breed-associated, and if found to be so (2) to determine if the noted activity differences were a product of translational and/or post-translational mechanism(s). The ultimate intent of this work was to reveal strategies by which trypanosome tolerance might be achieved in susceptible mammals. By a novel kinetic peroxidase assay, developed and used here, it was found that the serum XOR specific activities of Cape buffalo, Holstein and Hereford heifers, Sprague-Dawley rats, and humans were significantly different from one another (p < 0.025). A concurrent, comparative analysis of XOR nucleotide/amino acid sequences and tissue-specific transcription rates in Cape buffalo and representative bovine breeds yielded no points of variability that might explain observed activity differences. To determine if such differences were, instead, a product of distinct translational and/or post-translational regulatory mechanism(s), serum XOR was isolated, quantified, and characterized. Two immunoaffinity (IA) chromatography protocols were developed to isolate serum XOR. Mouse monoclonal antibodies directed against cow milk xanthine oxidase (α-CMXO) exhibited a recognition for serum XOR that was limited to bovid species. Polyclonal rabbit α-CMXO antibodies were found to recognize serum XOR from a greater range of mammalian species. However, IA eluent resolution by SDS-PAGE (under reducing conditions) elucidated polypeptide profiles that were species-specific, suggesting the presence of co-eluting contaminants. MALDI-TOF analysis identified the putative contaminants as components of serum IgG and IgM. These immunoglobulins (Igs) were found to exist in complex with the isolated serum XOR or were recognized directly by the polyclonal α-CMXO antibodies of the IA column, in an idiotypic manner. The consequences and implications of such Ig contamination are discussed, specifically with regard to: (1) the future use of α-XOR antibodies in extracting serum XOR, and (2) the possible roles of XOR-Ig complexes in systemic inflammatory pathologies.

Abstract not available

Neuroendocrine Neoplasms (NENs) of the Gastrointestinal Tract (GIT) are a heterogeneous group of tumours with varied biologic potential and clinical outcomes. They are classified as well differentiated neuroendocrine tumours (WD NET) or poorly differentiated neuroendocrine carcinomas (PD NECs) based on morphology. WD NETs are further subtyped (grade 1, 2 or 3) by evaluating the mitotic rate and Ki-67 proliferative index. The most recent grading system was published in 2019 by the World Health Organisation (WHO). Insulinoma-associated protein 1 (INSM1) is a transcription factor that is expressed in neuroendocrine cells, and recent studies have shown that it is a sensitive and specific marker for neuroendocrine differentiation. The aims of this study were to grade NENs of the GIT according to the 2019 WHO grading system, and to evaluate the expression of INSM1 in order to assess its sensitivity and specificity as a marker of neuroendocrine differentiation compared to chromogranin A (CgA) and synaptophysin (SYN). Sixty-nine GIT NENs diagnosed between 2003 and 2017 at Groote Schuur Hospital were included in this study. The mitotic rate and Ki-67 proliferation index were evaluated for each case. We identified 38 grade 1 NETs, 16 grade 2 NETs, 1 grade 3 NET, 13 small cell type NECs and 1 large cell type NEC. INSM1 immunohistochemical staining was performed on all cases. To assess specificity, we evaluated the expression of INSM1 in other GIT primary tumours (adenocarcinoma, gastrointestinal stromal tumour, lymphoma, leiomyoma and Kaposi sarcoma). Eighty percent of our NEN cases stained with INSM1. We found the sensitivity of INSM1 to be higher than CgA (68%), but lower than SYN (87%) and the combined use of CgA-SYN (94%) when considering all NENs. When evaluating only the PD NEC cases, INSM1 had a higher sensitivity than CgA (50%) and SYN (64%), and an equal sensitivity to the combined use of CgA-SYN (79%). We conclude that the high sensitivity and specificity of INSM1 make it a valuable standalone marker for assessing neuroendocrine differentiation. The nuclear reactivity and high sensitivity of INSM1 make it the preferred neuroendocrine marker for PD NEC.