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Studies of the pathophysiology of Barrett's oesophagusMoyes, Lisa Helen January 2013 (has links)
Barrett’s oesophagus is a common condition in which the normal stratified squamous oesophageal epithelium is replaced by metaplastic reflux-induced glandular (“columnar”) mucosa (Jankowski, Barr, Wang et al. 2010;Playford 2005). Over the last three decades, the incidences of oesophageal adenocarcinoma (OA) and Barrett’s oesophagus have risen to the point that OA is now common in the United Kingdom, with Scotland having one of the highest rates in the world (Jankowski, Provenzale, & Moayyedi 2002). Unfortunately most cancers present at an advanced stage with five year survival less than 30% (Holmes and Vaughan 2007). Barrett’s oesophagus is associated with malignant progression via a recognised metaplasia-dysplasia-carcinoma sequence (Jankowski, Wright, Meltzer et al. 1999). The premalignant nature of Barrett’s oesophagus has powered intense clinical interest in the hope of eventually having an impact on the earlier diagnosis and treatment of dysplasia, and ultimately the prognosis of oesophageal adenocarcinoma. Despite years of research interest, Barrett’s oesophagus remains an enigmatic condition. The exact incidence is unknown, and it is recognised that not all patients with Barrett’s oesophagus will progress to adenocarcinoma. Current strategies aim to ascertain the presence of dysplasia, the current gold standard marker of malignant progression. However although Barrett’s mucosa is visible at endoscopy, the presence of dysplasia is difficult to diagnose as these areas tend to be focal and inconspicuous to the naked eye. Current systematic biopsy regimes are recommended, but can be fraught with sampling errors. Furthermore, the molecular mechanisms underlying Barrett’s metaplasia and progression to dysplasia remain unclear. Molecular risk biomarkers have been sought with modest success, and at present dysplasia remains the most reliable clinical marker. However dysplasia itself is not without limitations: focal dysplasia can be difficult to ascertain, with many biopsies sometimes necessary to detect it reliably (Abela, Going, Mackenzie et al. 2008). Inter-observer variability may cause over or under diagnosis, especially regarding LGD (Flejou 2005). Moreover, although patients with HGD are at elevated risk of progression to OA, few studies provide reliable data on rates of progression from HGD to OA, with estimates varying between 16-59% at five years (Reid, Blount, Feng et al. 2000;Schnell, Sontag, Chejfec et al. 2001;Shaheen and Richter 2009;Spechler SJ 2011). There is a real need, therefore, to be able to identify and treat those patients at greatest risk of malignant transformation, and reassure those at low risk. Without an improved molecular understanding of Barrett's metaplasia and progression to neoplasia, clinically useful prognostic biomarkers (allowing appropriate targeting of surveillance and therapy) will be delayed. The current challenges associated with Barrett’s oesophagus are 1) to accurately determine the rate of malignant progression of Barrett’s oesophagus and identify clinical risk factors, 2) to improve the endoscopic detection of dysplasia and early neoplasia allowing earlier diagnosis and treatment and, 3) to understand the molecular mechanisms involved in the initiation of Barrett’s metaplasia, and the pathways involved in disease progression. In an attempt to improve the care of patients with Barrett’s oesophagus within the West of Scotland, my thesis will address each of the main challenges associated with this puzzling condition at clinical, endoscopic and molecular levels. The hypotheses of my thesis are threefold - a) Patients with Barrett's oesophagus in the West of Scotland have high rates of progression to high grade dysplasia and oesophageal adenocarcinoma. b) The WavSTAT optical biopsy system will be able to correctly identify non-dysplastic and dysplastic Barrett's oesophagus. c) The Wnt signalling pathway is upregulated in Barrett's oesophagus and dysplasia. The aims of my thesis are as follows: 1)To present a general overview of the Barrett’s literature highlighting current clinical challenges 2)To examine the incidence of dysplasia and oesophageal adenocarcinoma in the West of Scotland by analysing a cohort of patients undergoing surveillance endoscopy 3)To review the current endoscopic imaging adjuncts for the diagnosis of Barrett’s oesophagus and dysplasia, and assess the role of optical biopsy forceps in determining the presence of dysplasia 4)To evaluate the role of Wnt signalling in Barrett’s oesophagus, from metaplasia to carcinoma in a mouse model, with complementary human studies Chapter 1 introduces the reader to Barrett’s oesophagus and highlights current areas of clinical challenge and debate. A universal definition of Barrett’s oesophagus does not exist and Chapter 2 explores the need for the presence of intestinal metaplasia in the diagnosis of Barrett’s oesophagus. Chapters 3 and 4 present original data from a West of Scotland Barrett’s oesophagus database, specifically analysing rates of dysplasia and adenocarcinoma and cause of death. This study suggests patients with Barrett’s oesophagus in the West of Scotland are at high risk of disease progression with almost 10% of patients dying from oesophageal adenocarcinoma. The results highlight the importance of a comprehensive surveillance in our “high risk” population - an ideal niche for future chemopreventative and molecular studies. In an attempt to improve the diagnosis of dysplasia in our West of Scotland population, Chapter 5 reviews current endoscopic imaging adjuncts used in research and clinical practice while Chapter 6 presents original data from a pilot study assessing the use of innovative optical biopsy forceps in the endoscopic diagnosis of dysplasia. While this technology is in its infancy and further changes in the algorithm are required, the optical forceps could be a promising tool for ongoing surveillance in high risk Barrett’s patients. Chapter 7 summarises the role of biomarkers in Barrett’s oesophagus, reviewing the literature and highlighting the lack of clinically useful markers of disease progression to date. The Wnt signalling pathway plays an important role in normal oesophageal (and intestinal) development, yet when aberrantly activated leads to carcinogenesis. To date, very little is known about the role of Wnt signalling in Barrett’s oesophagus. Chapter 8 presents the results of a mouse model of upregulated Wnt signalling and the interesting finding of dysplasia within the oesophageal mucosa. Chapter 9 therefore translates these results to the human population by assessing the role of Wnt signalling in Barrett’s metaplasia and dysplasia by immunohistochemical analysis of a panel of markers. The results suggest Wnt signalling is upregulated in Barrett’s dysplasia, particularly in high grade, and this may have a future role as a biomarker. Chapter 10 summarises the main findings of the thesis, and presents future directions.
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The role of Src kinase in renal cell carcinomaQayyum, Tahir January 2014 (has links)
Renal cancer is a malignancy which is not only increasing in incidence but there has also been an increase in mortality rates. There are various prognostic factors in renal cell cancer. We have demonstrated that some of these such as nuclear grading, tumour necrosis and systemic inflammatory response can be further refined to aid in prognosis but cannot be utilised at present to assess which would benefit from therapeutic agents when recurrence occurs. We investigated if SFK members are expressed in renal cancer. Eight SFK members were found to be expressed in renal cancer and were present to varying degrees. Furthermore, expression differed in organ confined disease and metastatic disease. Immunohistochemistry was employed to assess protein expression and activation of c-Src and SFK activity as well as the downstream marker FAK Y861. Analysis demonstrated that c-Src expression was associated with improved survival and expression of the downstream marker FAK Y861 was associated with poor survival and demonstrated a positive relationship with known prognostic factors. This would suggest that another SFK member was associated with poor survival. Dasatinib, a SFK inhibitor was utilised on renal cell lines, demonstrating a dose dependant reduction on cellular metabolic activity as well an increase in apoptotic rates. This would support that Dasatinib may be a useful therapeutic drug for RCC. Treatment with Dasatinib also demonstrated that expression of c-Src, SFK activity and FAK Y861 reduced in a dose dependant manner. It was necessary to further assess that another SFK member was responsible for poor prognosis and this was undertaken by silencing c-Src. Cellular metabolic activity rates increased following silencing c-Src and assessment of SFK activity (Src Y416) and FAK Y861 on cell pellets demonstrated no change suggesting that another SFK member is responsible for the phosphorylation of FAK Y861 and therefore responsible for poor survival. This would suggest that another SFK inhibitor and not c-Src inhibitors may play a role in the treatment of renal cell cancer and further work is required to ascertain which SFK member is responsible so that this can be targeted for treatment.
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Mechanisms of central nervous system disease in childhood acute lymphoblastic leukaemiaYousafzai, Yasar Mehmood January 2015 (has links)
Acute lymphoblastic leukaemia (ALL) is the commonest childhood malignancy. Once a universally fatal disease, modern therapy has achieved excellent outcome and the majority of children achieve long term cure. Yet relapse remains a challenge. One of the major hurdles in achieving complete cure is the relapse of ALL at extramedullary sites such as the central nervous system (CNS). Despite significant advances in understanding leukaemia biology, most predictors of leukaemic behaviour are accurate for the bone marrow disease only. The precise timing, frequency and properties of CNS infiltrating leukaemic cells are not elucidated. Therefore, the broad aim of this thesis is to develop a better understanding of the mechanisms of leukaemic entry and infiltration patterns of leukaemic cells in the CNS. In order to address the frequency and pattern of CNS infiltration, a xenograft model using primary leukaemic cells from children with B-cell precursor (BCP) ALL in NOD/Scid IL2Rγ null (NSG) mice was established. The majority of samples from children with and without overt CNS disease were able to infiltrate the CNS in NSG mice. CNS infiltration was seen in mice engrafted with small numbers of cells and distinct immunophenotypic subpopulations. The leukaemic samples followed a distinct and reproducible pattern of CNS infiltration with leukaemic infiltrates in the meninges while sparing the CNS parenchyma. To investigate whether a distinct set of leukocyte trafficking molecules provided tissue specificity for entering the CNS, leukaemic cells retrieved from the bone marrow and the CNS were assessed for expression levels of selected chemokine receptors and P-selectin glycoprotein ligand-1 (PSGL1). Additionally, chemotaxis assays were utilized to investigate the function of the chemokine receptor CXCR4. Despite surface expression of chemokine receptors on leukaemic cells and presence of chemokines in the CNS, no evidence for positive selection of a high-expressing subpopulation was seen. Overall, it appears that, unlike the bone marrow, chemokine receptors do not direct leukaemic cell trafficking to the CNS. To investigate the published observation that interleukin-15 (IL-15) expression in leukaemic samples correlates with the risk of CNS disease, the effects of IL-15 stimulation on BCP-ALL cells were assessed. IL-15 and IL-15 receptor subunits were noted to be expressed at mRNA level in samples from BCP-ALL patient and cell lines. Exogenous IL-15 stimulated leukaemic cell proliferation and upregulated genes associated with migration and invasion in SD1 cells. A higher proliferative advantage was observed at low-serum conditions which mimics conditions found in the CNS. Therefore a plausible mechanistic link was established for the association of CNS disease with high IL-15 expression levels. In cerebrospinal fluid (CSF) samples from patients, quantitative PCR (qPCR) was utilized to detect submicroscopic levels of CNS disease. In approximately 40% patients, qPCR using patient specific primers tested positive for the presence of leukaemic DNA. Therefore this test is much more sensitive than conventional diagnostic techniques which only detect CNS disease in 2-5% of patients. CSF supernatants were also tested to assess whether the levels of chemokines could be used to diagnose patients with qPCR positive disease. Although differences in the levels of chemokines between qPCR positive and negative patients were noted, the values are not sufficiently discriminatory to be clinically useful. In conclusion, CNS entry appears to be a much more frequent property of leukaemic cell than previously appreciated. Leukocyte trafficking molecules do not appear to play an instructive role in the CNS entry and therefore, it is unlikely that expression levels of leukocyte trafficking molecules or the levels of chemokines in the CSF will be useful biomarkers of CNS disease. In addition, CNS disease appears to be present at diagnosis in at least 40% of patients. Therefore, attempts at blocking leukaemic entry into the CNS are unlikely to be therapeutically useful. Instead, analysing and targeting factors that allow long-term survival of leukaemic cells in the CNS may be a better strategy to eradicate CNS disease and prevent leukaemic relapse.
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An investigation into the relationships between host and tumour related factors and their influence on survival in patients with colorectal cancerRoxburgh, Campbell S. D. January 2011 (has links)
Colorectal cancer is the second commonest cause of cancer death in the western world. In addition to tumour factors such as depth of invasion, lymph node involvement and venous invasion it is increasingly recognised that host factors, are important determinants of survival. In particular the host local and systemic inflammatory responses are stage independent predictors of survival in operable disease. The present thesis further examines the prognostic importance of host and tumour factors in colorectal cancer, specifically: 1. An examination of the prognostic importance of venous invasion (detected using elastica stains) in colorectal cancer. 2. Detailed analysis of the determinants (including age, comorbidity and deprivation) of the systemic inflammatory response and their relationship with survival. 3. The application and validation of a prognostic score providing a measure of the local inflammatory response in colorectal cancer. 4. Detailed analysis of the determinants (including all white cells, lymphocytes and macrophages) of the local inflammatory response and their relationship with survival. 5. The inter-relationships between the local and systemic inflammatory responses in colorectal cancer specifically: early stage disease (node negative) and in patients receiving adjuvant chemotherapy. 6. Mediators (including immunological parameters and vitamin antioxidants) of the local and systemic inflammatory responses and their relationship with survival.
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Effects of heat shock, hypoxia, post-mortem interval and glioma disease state on heat shock gene HSPA expressionBeaman, Glenda Marie January 2012 (has links)
Heat shock protein 70 (HSPA/HSP70) gene expression is induced by a wide range of cellular stress conditions. This study investigated HSPA/HSP70 expression in human cell lines exposed to hypoxic conditions, in cancerous and non-cancerous brain tissue specimens from 18 patients (gliomas and normal conditions), and in post mortem rat brain samples exposed to heat shock. Three human glioma cell lines were chosen for this study, each representing various types of glioma: (astrocytoma, oligodendroglioma and glioblastoma), with a normal human astrocyte cell line used as a control. In addition, 18 clinical brain tissue samples were also examined. HSPA RNA transcripts and proteins were examined in these samples using qRT-PCR, immunofluorescence and flow cytometry techniques. The average HSPA mRNA copy numbers detected in glioblastoma tissue were 1.8 and 8.8 fold higher respectively than in lower grade glioma and control tissues, which is suggestive of a grade related transcription profile. Similar patterns of grade related expression were also observed in corresponding cell lines. The percentage of cells showing positive for HSPA protein in normal cell lines increased from 0 to 33% immediately after exposure to hypoxia, and gradually declined to 11% 24 h after treatment. However, the effects of hypoxia were marginal in glioma cells, due to the already elevated levels of HSPA. Although hypoxia induced HSPA expression in normal cells, it did not achieve the same level of induction in cancer cells, suggesting that there are other factors which contribute to the induction of HSPA. These results suggest that HSPA is induced in cancer cells, not only by hypoxia, but also by other factors. In addition, this study indicated for the first time that HSPA expression in glioma cells may possibly be grade related, and thus may have value as a prognostic marker. However a greater sample size is needed to validate such findings. This study showed that HSPA is expressed at low levels in normal brain tissue, but was more highly expressed in brain tissue subjected to mild heat shock. The levels of HSPA transcripts in heat shocked post mortem brain tissue showed a marked increase in HSPA expression. GAPDH was used as a control gene for these studies, and exhibited a consistent level of expression in normal and tumourous cell lines and tissue samples under normal and hypoxic conditions, and also in post mortem tissues exposed to heat shock. For Homo sapiens GAPDH, the average transcript numbers for normal and tumourous cell lines and brain tissue samples were approximately 145,000 copies per sample. For Rattus norvegicus GAPDH, levels were higher than for human samples, at an average of 268,300 copies per sample. The consistency of these results confirms that GAPDH was a suitable candidate gene for the purpose of this study. Early in the post-mortem period, HSPA is expressed more highly in tissues subjected to single and multiple heat shocks compared to controls. However, later post-mortem intervals of between 3 - 24 h demonstrated inconsistent and irregular results, with no predictive or reproducible patterns. Therefore, although there is demonstrable de novo expression of HSPA in post mortem brain tissue in response to heat shock, it is difficult to predict the full parameters of this induction, probably as a result of other forms of cellular stress affecting these tissues under our experimental methodology. These initial studies indicate that the use of HSPA with the methodologies employed here are not suitable as an accurate indicator of post-mortem interval.
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The role of Src kinase and Src kinase family members in breast cancerElsberger, Beatrix January 2010 (has links)
This project highlighted that Src and Src kinase family (SFK) members play a definitive role in breast cancer. Due to the paucity of translational studies, we investigated if SFK members are expressed in human breast tissue. Eight SFK members were present with distinct mRNA expression patterns in normal, non-malignant and malignant breast tissue. Immunohistochemistry was employed to investigate protein expression and activation of Src and SFK members. Survival analysis revealed that c-Src and activated Y419Src were associated with worse patient outcome, confirming current in vitro literature, whereas a different phosphorylation site of Src (Y215) and expression of Lck was associated with improved clinical outcome. Dasatinib was employed in different breast cancer cell lines to establish its effect on those phosphorylation sites. Decreased expression of c-Src and Y419Src was observed, whilst Y215Src expression stayed unchanged, providing a rationale for using this Src kinase inhibitor in clinical trials.
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New approaches to fluorescence-based diagnostics for human African trypanosomiasisGiordani, Federica January 2011 (has links)
In the absence of any vaccine, prophylactic drug and effective vector control, the fight against human African trypanosomiais (HAT) is based on the the combination of active case-finding and consequent drug treatment of identified positive cases. Unfortunately, low sensitivity and specificity of current diagnostic techniques often result in misdiagnosis, leaving infected patients without cure or exposing them to inappropriate chemotherapy protocols, which use dangerous and expensive drugs. The development of more efficient, simple, cheap and field-robust diagnostic tests is, therefore, urgently needed. In the field, direct observation by light microscopy of trypanosomes in human fluids (blood, lymph node aspirate, cerebrospinal fluid) is considered the ideal way of confirming HAT infection. However, in practice this approach is problematic, especially for the Gambian form of the disease, where patients may present with very low parasitaemia. Detection limits of parasitological techniques can be improved by adding a preliminary step of sample concentration, although this further increases the laboriousness of HAT diagnostic algorithm. Recent advances in fluorescence microscopy could be exploited to facilitate trypanosome detection. The introduction and implementation of fluorescence microscopy in HAT endemic countries would offer the advantages of an increased overall sensitivity of microscopical examination and a more rapid screening of the specimen. In contrast to traditional, expensive and fragile fluorescence microscopes, new LED-illuminated instruments are relatively cheap, very efficient and portable, lending themselves to utilisation in poorly equipped rural settings. In order to design a new diagnostic tool that exploits LED technology, however, selective and reliable fluorescent markers to label trypanosomes in human fluids are needed. The development of new tools to assist in the diagnosis of African trypanosomiasis by use of LED fluorescence microscopy was the overall objective of this project. The work was mainly focused on testing various fluorescent compounds for their ability to selectively stain trypanosomes. Fluorophores were otained from commercial and academic sources, or else directly synthesised during the project. An important requirement evaluated was the compounds’ compatibility with the currently available SMR LED Cytoscience fluorescence microscope, developed and kindly provided by our collaborator Prof. D. Jones (Philipps University, Marburg). The utility of a UV LED-driven microscope in performing the arsenical drug resistance test was also assessed. This assay, developed in our laboratory to detect trypanosome strains resistant to arsenical and diamidine compounds, could represent a useful tool for chemotherapeutic decision making in the field, where resistance to arsenical drugs is a rising problem.
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Investigating the roles of RKIP and p53 in colorectal carcinomaDoyle, Brendan January 2010 (has links)
Raf Kinase Inhibitor Protein (RKIP) was originally described as an inhibitor of the Ras-Raf-MEK-ERK pathway, exerting its action by the physical inhibition of the interaction of Raf with MEK. It has subsequently been shown to play important roles in a number of other signalling pathways, including the NFκB pathway and in the stability of the mitotic spindle. Not surprisingly given that it impacts on many important signalling pathways RKIP levels have been shown to be important in the progression of a number of different cancers. RKIP expression is lost or decreased in a number of common human cancers and decreased still further in tumour metastases. One of the tumours in which RKIP is downregulated is colorectal cancer (CRC). Importantly it has been shown that not only is RKIP depleted in tumour tissue when compared with normal tissue but that the level of RKIP within a tumour is inversely correlated with the likelihood of metastatic relapse and with patient prognosis. Although we already have a number of very good prognostic indicators in CRC, one group of patients for whom new prognostic indicators would be useful are patients with Dukes B CRC. These are patients with locally advanced but non-metastatic disease and at present there is no firm consensus on their correct post-operative management. Therefore we set out to examine whether RKIP is a useful prognosticator in this particular group using a tissue microarray (TMA) with samples from over 200 patients with Dukes B CRC. The analysis revealed a strong inverse correlation between RKIP levels and disease specific survival. Moreover, in a multivariate analysis RKIP emerged as an independent prognostic indicator along with lympho-vascular invasion and peritoneal invasion, two well-known and powerful prognosticators. This allowed for the generation of a simple prognostic index, using information from the different independent indicators, allowing for improved patient risk stratification. This led us to examine whether RKIP could also function as a predictive marker in CRC. To do this we again used a TMA, this time consisting of a much larger cohort of patients across the whole range of tumour stages. The results confirmed the prognostic utility of RKIP and indicated that patients whose tumours have low levels of RKIP may derive a greater benefit from chemotherapy than those patients whose tumours have high levels, although this result did not reach statistical significance. In the second part of the thesis I have examined the effect of RKIP in previously characterised mouse models of CRC. To do this I have used a germline RKIP knockout mouse and in the first instance crossed it to the APC580S mouse. In this mouse APC is lost conditionally within the intestine and liver. RKIP knockout did not have any effect on the rate of tumourigenesis or on the invasiveness of tumours in this model. However, in the setting of acute homozygous deletion of APC, RKIP knockout resulted in a decrease in apoptoses in the small intestine and an increase in aberrant mitotic activity in the liver. To follow this up I have examined the effect of RKIP knockout in a mouse model of superficially invasive CRC, specifically to see if RKIP knockout can promote invasive and metastatic behaviour. In this model the APC580S mouse is crossed to mice which conditionally express oncogenic KRas. Although RKIP knockout did not result in an increase in invasive tumours in this model there was a shift in tumour location from the small intestine to the colon. This shift appeared to be due, at least in part to an increase in chromosomal instability in the tumours. The final aim of the thesis was to develop a mouse model of CRC which more closely recapitulates the late stages of the human disease, specifically invasion and metastasis. To do this we have crossed the APC580S mouse with either a conditional p53 knockout or with a mouse that conditionally expresses a point mutation of p53 (p53R172H). In human tumours the majority of abnormalities of p53 are point mutations that result in the production of mutant protein that accumulates in tumour cells. There is evidence that this mutant protein may have oncogenic properties beyond the simple loss of normal p53 protein function. Therefore we have also used this model to study the differing effects of p53 loss and point mutation in CRC. We found that mice homozygous for p53 deletion (p53fl/fl) and those expressing a single copy of the mutant allele with loss of the second copy (p53R172H/fl) developed invasive tumours with nearly 100% penetrance and indeed metastasis was observed. Remarkably, although mice that were heterozygous for p53 deletion (p53fl/+) only rarely developed invasive tumours almost 100% of mice expressing a single copy of the mutant allele (p53R172H/+) developed invasive tumours. We went on to show that the increase in invasion seen in this model is related to an increase in Wnt signalling, which is associated with increased expression of pro-invasive Wnt targets such as fascin. We also showed a novel pro-invasive role for ARF in this process. This is also an excellent model of Dukes B CRC and therefore the ideal model to test the effect of RKIP deletion on invasion and metastasis. These studies led us to examine the differences in effect between knockout and mutant p53 in another tumour model. In this we used a novel model of the aggressive tumour pleomorphic rhabdomyosarcoma to demonstrate that mutant p53 can both promote both tumourigenesis and metastasis more potently than p53 knockout. These studies have demonstrated the value of RKIP in the clinically important Dukes B CRC population and shown its possible utility as a predictive marker in this group. Although we have not seen an effect of RKIP knockout in traditional mouse models of CRC we have developed a novel model which closely recapitulates Dukes B CRC and may be useful in elucidating the effect of RKIP knockout. We have also used this model to gain novel insights into the invasive process, in particular into the role played by mutant p53.
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The molecular basis of modern marker chemistryvon Ruhland, Christopher John January 2011 (has links)
This thesis focuses on empirical investigations and refinements of immunohistochemical marker chemistry to gain insights into the design of novel markers for light and electron microscopy. In Chapter 2, incorporation of d-block metals into polymerised biphenyl-3,3′,4,4′-tetramine (polyDAB) identified complexes of Ni(II), Pt(II), Pt(IV) and Au(III) to be powerful catalysts of silver reduction from physical developers. Na2S(aq) treatment increased the range and activity of catalytic complexes, allowing previously invisible immunohistochemical deposits of polyDAB to be clearly seen in diagnostically relevant samples. Chapter 3 refined this technique by manipulating reagent concentrations whilst suppressing tissue argyrophilia, increasing immunohistochemical sensitivity by an order of magnitude. Marker deposition and thus amplification, was dependent on conjugate quality and coupling method. In Chapter 4, scanning and transmission electron microscopy identified 8 d-block metals that increased the electron opacity of polyDAB, including W(VI), Os(VIII), Pt(II) and Au(III). The majority were detectable by energy dispersive X-ray analysis (EDX), but were present in insufficient quantities for use in analytical electron microscopical tomography (AEMT). In Chapter 5, immunohistochemical polymerisation of halogenated aromatic diamines and bis-diamines as AEMT markers was investigated. The 16 compounds studied produced deposits of varying properties and compositions, morphological criteria identifying those of 1,2-diamino-4-bromobenzene and 1,2-diamino-4,5-diiodobenzene as suitable candidates; EDX indicated that the latter might be applicable to AEMT. Chapter 6 investigated silver deposition from a physical developer by photoconversion. Photo-excitation of immunofluorescently-stained tissue sections in the presence of physical developer caused selective silver deposition at immunopositive sites, a novel method that might find application in AEMT. In Chapter 7, characterisation of polyDAB revealed a molecular weight range of 600 to over 100,000; IR spectra were consistent with an indamine- or phenazine-like polymer. Poor solubility restricted further characterisation. In Chapter 8, additional applications of halogenated compounds were investigated and results suggested potential applications in biological research and diagnosis.
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CD59a – A novel role in boneBloom, Anja Constanze January 2012 (has links)
The complement system has crucial functions in host defence. Novel data revealed a role for complement components in the pathology of osteoarthritis (OA). CD59a is a regulator of the terminal complement pathway in mice; the purpose of the study was to determine if CD59a-/- mice have an osteoarthritic bone phenotype. Osteoblast (OB) mineralisation, colony forming unit (CFU) and OCG assays were performed in vitro from bone marrow preparations of 8-20 week old mice. Decreased CFU differentiating towards osteoblasts and adipocytes (n=1 only), as well as an increased OCG, was revealed in male CD59a deficient (-/-) over wildtype (WT) mice. OCG in females were comparable. A human CD59 knockdown system utilising short hairpin (sh) ribonucleic acid (RNA) delivered by adenoviruses was established but did not differentiate into osteoclasts (OC). In vivo the bone phenotype of CD59a-/- mice was established for femora and vertebra L6 via X-ray, microcomputed tomography and histology. In male mice femoral length was increased in CD59a-/- versus WT mice at 8-10, 20 and 50 weeks. Cortical bone volume was increased whilst bone mineral density (BMD) was reduced in CD59a-/- versus WT mice at 8-10 and 20 weeks. Trabecular bone analysis of the distal femur (and spine) showed increased trabecular bone ratio, number, thickness, connectivity and total BMD in CD59a-/- over WT at 8-10 (and 20) weeks of age. In female mice there was no difference in femoral length and trabecular bone, but cortical BMD was raised at 50 weeks (CD59a-/- versus WT). Finally, histology revealed enhanced mineral apposition rate and OC surface as well as reduced osteoid surface in male CD59a-/- over WT mice at 8-10 weeks of age. Increased bone growth and turnover related to CD59a gene deletion were gender specific. These studies highlight CD59a as a potential target for OA treatment.
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