published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

p21 C/P 1/"El and p 2 7K/P 1 are cyclin-dependant kinase inhibitors that fonn an integral part of the cell cycle process. These proteins function as cell-cycle inhibitors, and are able to induce cell cycle arrest by binding to cyclin complexes at key stages. p21 and p27 have been found to be down-regulated in various cancers. This study investigated aberrations at microsatellite markers linked to the p21 and p27 cell cycle genes, in a large cohort of oesophageal squamous cell carcinomas in South Africa. Fluorescent-based PCR were performed on markers linked to both the p21 and p27. The products were run with a 50-500hp marker on 6% denaturing polyacrylamide gels, on the ALFexpresstm' DNA sequencer. The detection and analysis of PCR products was achieved using the AL F e xp res sT M and Fragment M an a aerTm software programmes. Our findings indicate that markers linked to p27 display infrequent aberrations, with loss of heterozygosity ranging from 19% to 37%, and microsatellite instability at 3% to 7%. However, significant relationships between decreased survival time, and aberrations in markers DI2S391 and Dl2S364, were found to exist. Marker D6S1575 linked to p21 displayed frequent allelic loss at 47%, and was comparable to similar studies on the 6p region Further, LOH-Al in this marker was found to be significantly associated with poorly differentiated tumours. The findings from our study indicate that microsatellite aberrations occur infrequently at the p21 and p27 loci in oesophageal cancer. with the exception of marker D6S1575. In addition,this study clearly demonstrates the accuracy and sensitivity of the technology employed. This is the first microsatellite-based investigation of the p21/p27 gene loci in oesophageal cancer in South Africa, using a fluorescent-based PCR assay. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Durban, 2008.

Multidrug resistance (MDR) in small cell lung cancer is one of the major causes of failures of chemotherapy. MDR is a means of protection of tumor cells against chemotherapeutic drugs. Although the molecular basis of MDR is not fully understood, genes involved in apoptosis may be mutated. Recent finding of a link between over-expression of an apoptotic gene, cyclooxygenase 1 (cox 1), and MDR suggests that cox 1 is involved in the development of MDR phenotype. This research was an attempt to observe whether up-regulation of cox 1 contributes to the MDR phenotype in small cell lung cancer cells. This research ultimately may provide a mechanism to reverse the abberant up-regulation of apoptosis genes associated with multidrug resistance to either eliminate or control reproduction of cancer cells. Real time RT PCR was used to confirm the up-regulation of cox 1 in cultured MDR resistant small cell lung cancer cells (GLC4). The up-regulated cox 1 expression was down-regulated using RNA interference technology (RNAi) by transfection with an anti-cox 1 siRNA. More than 90% transfection of cells was confirmed using confocal microscopy. Down-regulation of cox 1 was validated as the protein expression significantly decreased (P=0.004) from multidrug resistant small cell lung cancer transfected cells compared to multidrug resistant nontransfected cells. There was decrease level of expression of cox 1 in multidrug resistant cells after the knockdown with siRNA specific to cox 1. The decreased level of cox 1 expression and, therefore, Cox 1 production increased the rate of apoptosis in small cell lung cancer cells as indicated by its sensitivity to the doxorubicin. / Department of Biology

The CD40 ligand (CD40L) molecule is central to innate and adaptive immunity. CD40L expression is very tightly regulated whereas its CD40 receptor is constitutively expressed by many different cell types. CD40L is expressed transiently on helper T cells (Th) only after activation by specific immune recognition molecules carried by professional antigen presenting cells, in particular, dendritic cells (DC). CD40L subsequently binds to CD40 on DC to enable full Th activation. CD40 ligated DC produce interleukin-12 (IL-12) and contribute both to the development of IFNγ-secreting natural killer cells, a vital component of innate immunity, and of IFNγ-secreting type 1 Th (Th1) cells. CD40 ligated DC also contribute to the development of IL-4- and IL-10-secreting Th2 cells. CD40L on Th cells also binds CD40 on macrophages to enhance their cytotoxic functions. CD40L-expressing Th cells provide the ‘help’ pivotally required to activate other components of adaptive immunity responsible both for clearing invading pathogens and generating the memory cells required to prevent re-infection. Th-supplied CD40L binds (i) B cell CD40 to switch production of antibodies to more potent effector molecules that have higher avidity for antigen, and (ii) DC CD40 to prime then expand antigen-specific cytotoxic T lymphocytes (CTL). Activated NK cells and CTL are required both to eradicate malignant cells and cells infected with viruses or other intracellular pathogens. Genetic CD40L deficiency causes the very rare HyperIgM Syndrome Type 1 (HIGM1), which is realistically modelled by genetically engineered CD40L-deficient mice. Neither CD40L-deficient patients nor mice make effective antibodies or mount cellular immune responses that would defend them against intracellular pathogens such as parasites. Consequently, the only potentially curative therapy is allogeneic stem cell transplantation or CD40L gene replacement. Here, we used a retroviral vector, which constitutively expressed CD40L, to genetically modify CD40L-deficient bone marrow cells, which were used to reconstitute partially the immunity of CD40L-deficient mice. The crucial importance of tight regulation of CD40L expression was revealed when these mice later developed lethal thymic T cell malignancy. Growing tumours escape immune vigilance by genetic alterations that reduce their sensitivity to IFNγ. Using murine tumour models, we incorporated transgenic CD40L expression in therapeutic tumour vaccines to show that CD40L gene transfer augmented the immunogenicity of the host’s tumour thus reducing its tumorigenicity. We translated this finding clinically to safety and immunogenicity testing of a transgenic CD40L- and IL-2- expressing leukaemia vaccine. Finally, the common viral respiratory pathogen, respiratory syncytial virus (RSV) mainly infects young infants and the elderly to cause potentially lethal pneumonia. Both groups have reduced cellular and humoral immunity, which predisposes them to re-infection with RSV. Using a murine model, we showed first that simultaneous adenoviral expression of CD40L augmented primary RSV-specific Th1 responses that were associated with accelerated pulmonary viral clearance. Second, we showed that expression of CD40L in RSV-F and RSV-G subunit DNA vaccines elevated antibody and cellular immune responses to RSV challenge four and eight months after the initial immunisation. These results demonstrate the potent ability of CD40L gene transfer to solve the absolute immune deficiency caused by genetic lesions of CD40L. However, physiological regulation of the transgene is required to prevent serious adverse consequences. In contrast, no adverse effects were observed after transgenic CD40L expression was used to overcome relative immune deficiencies imposed by malignancy and RSV infection. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298200 / Thesis (Ph.D.) - University of Adelaide, School of Medicine, 2007

Introduction: Gastric cancer (GC) is ranked as the second most common fatal malignancy worldwide. Although Helicobacter pylori is recognized as a major predisposing factor for non-cardia GC, infection alone is not sufficient to cause cancer. This thesis aimed to determine the variation in host genetic polymorphisms in subjects from Malaysia and Singapore and to examine the role of H. pylori infection, host genetic factors and dietary factors in the etiology of non-cardia GC in Chinese subjects resident in Malaysia. Methods: Functional dyspepsia (FD) controls from three ethnic groups in Malaysia, Chinese (123), Indian (110) and Malay (84) and Singaporean Chinese (127) plus Malaysian Chinese gastric cancer cases (55)were examined. Polymorphisms in IL-1B-511, IL-1RN, IL-10 cluster, TNFA-308 and TLR5+1174 were determined by PCR-RFLP or PCR; H. pylori status by serology, dietary intake by questionnaire and gastric IL-1b levels by real time PCR. Results: 1) Significant differences existed in the frequency of all polymorphisms, except IL-1B-1473 and TNFA-308, in the three Malaysian ethnic groups and in the IL-1B-511 polymorphism in Malaysian and Singaporean Chinese FD 2) Globally, two distinct patterns of IL-1B-511, IL-1RN, IL-10-1082, IL-10-592 and TNFA-308 exist, Western and East-Asian 3) In Malaysian Malays, the IL-10 ATA haplotype was associated with H. pylori susceptibility 4) In Malaysian Chinese an increased risk of GC was associated with carriage of the IL-1B-1473 G allele {OR=4.4(1.3-15.3)} and the IL-1B-511 C allele {OR=1.8(0.8-4.1)} 5) Increased levels of IL-1b were observed in Singaporean and Malaysian Chinese FD subjects carrying the IL-1-511C and IL-1-1473G alleles 6) Malaysian Chinese not consuming fresh fruit and vegetables had the highest risk of GC {OR=10.2 (3.4-30.6)} 7) The highest risk of GC {OR=37.3(3.3-424.8)} was observed in H. pylori positive Malaysian Chinese who carried both the IL-1B-511C and IL-1B-1473G alleles and did not consume fresh fruit and vegetables. Conclusions: In Malaysian Chinese, H. pylori infection, host genetic and dietary factors all contribute to the risk of GC. However the significant difference observed in the frequency of host genetic polymorphisms within and between ethnic groups suggests that a single group of risk factors cannot be used to determine GC risk across all populations.

[Truncated abstract] Erythropoietin (Epo) is a glycoprotein hormone involved in the formation of erythrocytes, by controlling survival, differentiation and proliferation. The technique of cDNA Representational Difference Analysis was utilized to investigate J2E-NR cells that demonstrate a viability response to Epo, but not differentiation or proliferation. The aim of this project was to identify genes that may be upregulated in response to Epo-induced survival; however, no change in gene expression was detected. This was most probably because any changes were below the limit of detectability for the cDNA RDA technique, or the viability effect was mediated post-transcriptionally. Next, it was decided to investigate HLS5, a putative tumour suppressor that was identified in a myeloid variant of the J2E cell line and had been shown to cause apoptosis. A number of HeLa cell lines inducible for Hls5 expression using the tet-off system were produced; despite extremely low expression, Hls5 was shown to produce marked suppression of growth and proliferation, particularly in colony assays colony size and numbers were halved for one induced clone. … A number of haemopoiesis-associated genes were downregulated (viz. globin genes and the Epo receptor gene), which suggested Hls5s role in the myeloid variant of J2E cells, may be to suppress genes expressed in the erythroid lineage. In addition, several interferon-responsive genes were decreased in cells with elevated HLS5, suggesting it may play a role in negatively regulating interferon signaling. Online databases were also searched for information on HLS5, and showed that it is significantly downregulated in liver, lung and uterine cancers, supporting the proposition that HLS5 is a tumour suppressor gene. In summary, a number of approaches were taken to identify the effects of the Hls5 protein. It appears that it strongly suppresses proliferation and that this is likely mediated through an effect on mitosis. This may also result in apoptosis of overexpressing cells. It is possible that this is the mechanism through which HLS5 exerts its potential tumour suppressor function, as a number of tumour suppressors appear to be associated with mitosis/apoptosis control. Hls5 is also likely to have other functions in haemopoietic cells, which includes downregulation of erythroid-specific genes and suppression of interferon responses.

Made available in DSpace on 2014-08-13T14:50:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-19Bitstream added on 2014-08-13T18:01:35Z : No. of bitstreams: 1 000742882.pdf: 1109001 bytes, checksum: 8541a66a007dafecd4081452f2930cc6 (MD5) / O câncer gástrico é mundialmente a segunda causa de morte por câncer. No Brasil está entre os cinco neoplasias mais incidentes, mostrando-se como um grave problema de saúde pública. O conhecimento do padrão de expressão gênica de genes supressores tumorais e protooncogenes é fundamental para o esclarecimento dos mecanismos desta carcinogênese. O gene FOXE1 (forkhead box E1) pertence a uma grande família de fatores de transcrição envolvidos em processos de crescimento e diferenciação celular. Pesquisas recentes têm demonstrado o papel determinante de FOXE1 na gênese de certos tipos de neoplasias. Assim, a análise da expressão gênica de FOXE1 em amostras de tumores gástricos faz-se necessária para esclarecer seu possível envolvimento neste carcinogênese. O DNA foi extraído de 89 amostras de tumores gástricos, com seus respectivos tecidos normais adjacentes. Após o tratamento com bissulfito de sódio, o padrão de metilação foi determinado por PCR específica para metilação (MSP-PCR) com a visualização dos resultados em poliacrilamida 6% corado com nitrato de prata. 86,5% das amostras apresentaram metilação em FOXE1, sendo o mesmo padrão encontrado em tecidos normais adjacentes 74,2%. Não houve significância estatística quando os resultados foram agrupados de acordo com o sexo, tipo histológico e estadiamento. A expressão gênica foi avaliada através de qPCR para 13 dos 89 pacientes incluídos no estudo. Obteve-se para cada paciente a razão (T/N) comparando a expressão gênica da amostra de tecido tumoral (T) em relação à expressão do tecido normal adjacente ao tumor (N). 8 amostras (61,5%) apresentaram um aumento da expressão do FOXE1, com uma média de 1,6. Dentre as 5 (38,5%) amostras com QR abaixo de 1, isto é, com expressão do FOXE1 abaixo do tecido normal, a média foi de 0,7. Quanto à concordância entre o estado de metilação em FOXE1 e a sua expressão ...

Made available in DSpace on 2014-06-11T19:26:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-26Bitstream added on 2014-06-13T18:53:59Z : No. of bitstreams: 1 succi_m_me_sjrp_parcial.pdf: 268320 bytes, checksum: 47bca06cf986a823daf8462cd47bd92b (MD5) Bitstreams deleted on 2015-03-16T11:30:15Z: succi_m_me_sjrp_parcial.pdf,Bitstream added on 2015-03-16T11:31:00Z : No. of bitstreams: 1 000714071.pdf: 1014657 bytes, checksum: b46cd9f9aefc594fb472e9cf8b775085 (MD5) / Alterações nos níveis de expressão de moduladores da resposta inflamatória, como a Anexina-A1 (AnxA1/ANXA1) e a Galectina-1 (Gal-1/LGALS1) têm sido observadas em diversos tipos de câncer. O câncer colorretal, um dos modelos da associação inflamação-câncer progride, na sequência, do epitélio normal para adenoma (AD) e adenocarcinoma (ADC). Objetivos: Avaliar a expressão gênica e proteica da AnxA1 e Gal-1 e o índice de proliferação celular (IP) em amostras de AD, ADC esporádico e das mucosas normais adjacentes, assim como investigar a ocorrência de correlação entre os níveis de expressão de ambos os mRNA e de associação com fatores de risco (idade, gênero, tabagismo e etilismo) e sítio anatômico de origem da lesão. Materiais e Métodos: As análises foram realizadas pelas técnicas de PCR quantitativa em tempo real (qPCR), para quantificar os níveis de mRNA de ANXA1 e LGALS1 em 70 biópsias de lesão (27AD e 43ADC) e 58 de mucosa normal adjacente (19 e 39 respectivamente), e de imuno-histoquímica, para caracterizar a expressão proteica da AnxA1 e Gal-1 em 25 biópsias de lesão (10AD e 15ADC) e 16 de mucosa normal adjacente (6 e 10 respectivamente), como também investigar o IP pela detecção do antígeno Ki-67 em 44 biópsias de lesão (19AD e 25ADC) e 16 de mucosa normal adjacente (8 em ambas). Resultados: A expressão relativa de ANXA1 apresentou-se elevada em comparação à mucosa normal tanto no AD (RQ=1,11; P=0,040), como no ADC (RQ=2,33; P<0,001). Contudo, LGALS1 apresentou expressão relativa aumentada apenas no ADC em comparação à mucosa normal (RQ=1,85; P<0,001), enquanto no grupo AD foi observada expressão basal (RQ=0,90; P=0,319). A comparação entre as lesões mostrou que ambos os genes apresentam-se significantemente mais expressos no ADC em comparação... / Changes in expression levels of inflammatory response modulators, such as Annexin-A1 (AnxA1/ANXA1) and Galectin-1 (Gal-1/LGALS1) have been observed in several types of cancer. Colorectal cancer, one of the models of inflammation-cancer association, progresses, in sequence, from normal epithelium to adenoma (AD) and adenocarcinoma (ADC). Objectives: To evaluate the gene and protein expression of AnxA1 and Gal-1 and the cell proliferation index (IP) in samples of AD, sporadic ADC and adjacent normal mucosa, as well as to investigate the occurrence of correlation between the mRNA expression levels and association with risk factors (age, gender, smoking and drinking habits) and anatomic site of lesion origin. Materials and Methods: The analyzes were performed by quantitative real-time PCR (qPCR) technique to quantify the levels of ANXA1 and LGALS1 mRNA in 70 biopsies of lesions (27AD and 43ADC) and 58 adjacent normal mucosa (19 and 39 respectively), and immunohistochemistry technique to characterize the protein expression of AnxA1 and Gal-1 in 25 biopsies of lesions (10AD and 15ADC) and 16 adjacent normal mucosa (6 and 10 respectively), and also to investigate the IP by detection of Ki-67 antigen in 44 biopsies of lesions (19AD and 25ADC) and 16 adjacent normal mucosa (8 in both). Results: The relative expression of ANXA1 showed higher compared to adjacent normal mucosa in both AD (RQ=1.11; P=0.040) and ADC (RQ=2.33; P<0.001). However, LGALS1 mRNA showed overexpression only in ADC compared to normal mucosa (RQ=1.85; P<0.001), while in AD group it was observed basal expression (RQ=0.90; P=0.319). The comparison between lesions showed that both genes were significantly more expressed in ADC compared to AD (ANXA1: P=0.039; LGALS1: P=0.019). In both lesion groups it was observed positive correlation between the mRNA expression of these genes... (Complete abstract click electronic access below)