published_or_final_version / Pathology / Master / Master of Medical Sciences

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

published_or_final_version / Biological Sciences / 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.

Squamous cell carcinoma of the oesophagus (OSCC) is a common malignancy that occurs with high frequency in certain parts of the world, including South Africa. The aetiology of OSCC has remained unclear although many studies suggest that it is caused by a combination of variable risk factors. Recent reports implicate a variety of genetic factors in the carcinogenesis of OSCC but their involvement is yet to be defined. / Thesis (M.Med)-University of KwaZulu-Natal, Durban, 2010.

[Truncated abstract] The Phase II clinical candidate, [(trans-Pt(NH3)2Cl)2{μ-trans-Pt(NH3)2(H2N(CH2)6NH2)2}]4+ (BBR3464 or 1,0,1/t,t,t) shows a unique binding profile when compared to the anticancer agent cis-[Pt(NH3)2Cl2] (cisplatin) and dinuclear platinum complexes of the general formula [(trans-Pt(NH3)2Cl)2(H2N(CH2)nNH2)]2+. There is evidence that the increased efficacy of 1,0,1/t,t,t results from the presence of the charged central linker, which can alter the mode of binding to DNA. This alternate binding mode may be due to an electrostatic and hydrogen bonding association of the central platinum moiety in the minor groove that occurs prior to covalent binding (termed “pre-association”) . . . This research shows that 0,0,0/t,t,t is an adequate model to study the pre-association process of 1,0,1/t,t,t and that it binds in the minor groove of DNA. Therefore it is likely that 1,0,1/t,t,t pre-associates in the minor groove of DNA prior to covalent binding. This work supports the conclusions reached in NMR studies of the binding of 1,0,1/t,t,t with the 1,4-GG sequence (Qu et al. JBIC. 8, 19-28 (2003)), which showed simultaneous binding in the major and minor groove. The findings of the current work may also explain the observed binding mode of 1,0,1/t,t,t, which can bind to DNA in both the 3',3' and 5',5' directions (Kasparkova et al. JBC. 277, 48076-48086 (2002)). These unique binding characteristics are thought to be responsible for the increased efficacy of 1,0,1/t,t,t, and in light of the current results the observed binding mode most likely stems from the electrostatic pre-association of the central platinum moiety.

Prostate cancer is the most common form of non-skin cancer and the second leading cause of cancer deaths within the United States. The five year survival rate has increased from 69% to 99% over the last 25 years for the local and regional disease, but has remained fairly low (approximately 34%) for the advanced disease. Therefore, current research is aimed at finding complementary or alternative treatments that will specifically target components of the signal transduction, cell-cycle and apoptosis pathways to induce cell death, with little or no toxic side effects to the patient. In this study we investigated the effect of genistein on expression levels of genes involved in these pathways. Genistein is a (4 , 5 , 7-trihydroxyisoflavone) is a major isoflavone constituent of soy that has been shown to inhibit growth proliferation and induce apoptosis in cancer cells. The mechanism of genistein-induced cell death and potential molecular targets for genistein in LNCaP prostate cancer c ells was investigated using several techniques. The chemosensitivity of genistein towards the prostate cancer cells was investigated using the ATP and MTS assays and apoptosis induction was determined using apoptosis and caspase assays. Several molecular targets were also identified using cDNA microarray and RT-PCR analysis. Our results revealed that genistein induces cell death in a time and dose-dependent manner and regulates expression levels of several genes involved in carcinogenesis and immunogenicity. Several cell cycle genes were down-regulated, including the mitotic kinesins, cyclins and cyclin dependent kinases, indicating that genistein is able to halt cell cycle progression through the regulation of genes involved in this process. / Several members of the Bcl-2 family which are involved in apoptosis were also affected and a number of genes involved in immunogenicity were up-regulated including the DefB1 and HLA membrane receptors. The results of this study provide evidence of genistein's ability to inhibit growth proliferation and induce apoptosis and indicates its potential as an adjuvant in chemotherapy and immunotherapy. / by Kendra Merchant. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Metastasis is the primary cause of mortality in women with breast cancer. Recently, elevated serum levels of a glycoprotein known as chitinase-3 likeprotein- 1 (CHI3L1) has been correlated with poor prognosis and shorter survival of patients with cancer and inflammatory diseases. The biological and physiological functions of CHI3L1 in tumor progression have not yet been elucidated. In this document, we describe the role of CHI3L1 in tumor growth and metastasis and its relationship with inflammation. Using well-established models of breast cancer, we show that CHI3L1 is increased in the serum of tumor bearing mice. We found that CHI3L1 levels are increased at both the “pre-metastatic” and “metastatic stage” and that tumor cells, splenic, alveolar and interstitial macrophages; and myeloid derived population produce CHI3L1. Furthermore, we demonstrated that CHI3L1 has an inhibitory role on the expression of interferon-gamma (IFN γ) by T cells, while enhancing the production of pro-inflammatory mediators by macrophages such as Cchemokine ligand 2 (CCL2/MCP-1), Chemokine CX motif ligand 2 (CXCL2/IL-8) and matrix metalloproteinase-9 (MMP-9), all of which promote tumor growth and metastasis. We demonstrated that in vivo treatment of tumor-bearing mice with chitin microparticles, a TH1 adjuvant and a substrate for CHI3L1, promoted immune effector functions with increased production of IFN-γ but decreased CCL2/MCP-1, CXCL2/IL-8 and MMP-9 expression by splenic and pulmonary macrophages. Significantly, in vivo administration of chitin microparticles decreased tumor growth and pulmonary metastasis in mammary tumor bearing mice. These results suggest that CHI3L1 may play a role in tumor progression. Inflammation plays a pivotal role during tumor progression and metastasis by promoting the production of pro-inflammatory molecules such as CHI3L1. However, little is known about how CHI3L1 expression can affect secondary sites to enhance metastasis. In these studies, we demonstrated that CHI3L1 alters the cellular composition and inflammatory mediators that aid in the establishment of a metastatic niche for the support of infiltrating tumor cells leading to accelerated tumor progression. Since previous studies showed that CHI3L1 modulates inflammation, we determined the role of CHI3L1 in the context of pre-existing inflammation and metastasis. We found that CHI3L1 deficient mice with preexisting inflammation had decreased pro-inflammatory mediators, and significant reduction in tumor volume and metastasis compared to wild type controls. Preexisting inflammation and CHI3L1 may be driving the establishment of a premetastatic milieu in the lungs and aiding in the establishment of metastasis. Understanding the role of CHI3L1 in inflammation during tumor progression could result in the design of targeted therapies for breast cancer patients. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015. / FAU Electronic Theses and Dissertations Collection

[Truncated abstract] Introduction: Breast cancer is a heterogeneous disease, resulting in very different outcomes for women with apparently similar tumour characteristics. In order for patients to have optimal treatment, a better understanding of the molecular nature of their disease is required. Aims: The aims of this thesis were: 1) To determine whether methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes are associated with phenotypic features of breast cancer and the prognostic significance of methylation of these genes. 2) To investigate for possible associations between the frequency of methylation at RARβ2, CDH1, ER, BRCA1, CCND2, p16 and TWIST genes and the presence of germ-line variants in the TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes, as well as for possible correlations between these polymorphisms and clincopathological features of breast cancer including patient outcome. 3) To determine whether PIK3CA mutations determined clinical phenotype and the prognostic significance of PIK3CA mutations in a large and well characterized cohort of breast cancer patients. Methods: A large and well characterized series of primary breast tumours were selected for methylation of RARβ2, ER, CDH1, BRCA1, CCND2, p16 and TWIST genes using MSP, and for polymorphisms in TS, MTHFR, MS, CBS, MTHFD1 and DNMT3B genes using PCR, PCR-RFLP and PCR-SSCP. Mutations to PIK3CA were detected using F-SSCP. Results and Conclusions: Methylation frequencies ranged from 11% for CCND2 to 84% for ER. More frequent hypermethylation was observed in tumours with poor histological differentiation compared to those with well/moderate differentiation, as well as trends for association with larger tumour size and mutant TP53. Tumours with ER and CDH1 methylation were associated with significantly lower hormone receptor levels, younger age at diagnosis and the presence of mutant p53. TWIST methylation is firstly reported to be associated with significantly older patient age at diagnosis and larger tumour size. Our data suggests that gene methylation may be linked to various pathological features of breast cancer. However, there appears to be little support for a distinctive CpG island methylator phenotype in breast cancer.

Submitted in the fulfillment of the requirement for the Degree of Master's in Chemistry, Durban University of Technology, 2017. / P53 is a tumour suppressor protein that is dysfunctional in most human cancer cells. Mutations in the p53 genes result in the expression of mutant proteins which accumulate to high levels in tumour cells. Several studies have shown that majority of the mutations are concentrated in the DNA-binding domain where they destabilize its conformation and eliminate the sequence- specific DNA-binding to abolish p53 transcription activities. Accordingly, this study involved an investigation of the effects of mutations associated with cancer, based on the framework of sequences and structures of p53 DNA-binding domains, analysed by SIFT, Pmut, I-mutant, MuStab, CUPSAT, EASY-MM and SDM servers. These analyses suggest that 156 mutations may be associated with cancer, and may result in protein malfunction, including the experimentally validated mutations. Thereafter, 54 mutations were further classified as disease- causing mutations and probably have a significant impact on the stability of the structure. The detailed stability analyses revealed that Val143Asp, Ala159Pro, Val197Pro, Tyr234Pro, Cys238Pro, Gly262Pro and Cys275Pro mutations caused the highest destabilization of the structure thus leading to malfunctioning of the protein. Additionally, the structural and functional consequences of the resulting highly destabilizing mutations were explored further using molecular docking and molecular dynamics simulations. Molecular docking results revealed that the p53 DNA-binding domain loses its stability and abrogates the specific DNA-binding as shown by a decrease in binding affinity characterized by the ZRANK scores. This result was confirmed by the residues Val143Asp, Ala159Pro, Val197Pro, Tyr234Pro and Cys238Pro p53-DNA mutant complexes inducing the loss of important hydrogen bonds, and introduced non-native hydrogen bonds between the two biomolecules. Furthermore, Molecular dynamics (MD) simulations of the experimental mutant forms showed that the structures of the p53 DNA-binding domains were more rigid comparing to the wild-type structure. The MD trajectories of Val134Ala, Arg213Gly and Gly245Ser DNA-binding domain mutants clearly revealed a loss of the flexibility and stability by the structures. This might affect the structural conformation and interfere with the interaction to DNA. Understanding the effects of mutations associated with cancer at a molecular level will be helpful in designing new therapeutics for cancer diseases. / M