Pancreatic cancer is a devastating disease that leaves patients with a very poor prognosis and few therapeutic options. Many of the treatment options available are the same that have been used for almost 2 decades. There is a dire need for both novel treatments for this disease as well as novel strategies of treatment. This body of work will introduce and provide evidence in support of a novel combination therapy for pancreatic cancer treatment, a novel strategy of modifying currently used chemotherapeutics for pancreatic cancer therapy, and a novel transgenic preclinical mouse model of pancreatic cancer. Sabutoclax, an antagonist of the anti-apoptotic Bcl-2 proteins, and Minocycline, a commonly used antibiotic, show potent synergy when used in combination in both pancreatic cancer cells and in multiple immune-deficient and immune-competent mouse models of pancreatic cancer. Sabutoclax alone is capable of inducing cell cycle arrest and apoptosis in cells and its cytotoxicity is enhanced significantly when combined with Minocycline. This combination results in the loss of Stat3 activation both in vitro and in vivo, which is essential for its toxicity. It also inhibits tumor growth and prolongs survival in the KPC transgenic mouse model of pancreatic cancer. Also presented here are studies that demonstrate efficacy in vivo of modified versions of Gemcitabine and Paclitaxel. These drugs are linked to a peptide that shows specificity for the EphA2 receptor, which is overexpressed on the surface of pancreatic cancer cells and only minimally on normal cells. This peptide results in increased cellular uptake of drug, as it is bypassing its normal mechanism of entry. These normal mechanisms are often dysregulated in cancer, leading to decreased uptake and drug resistance. The use of these modified drugs show significantly increased tumor growth inhibition as compared to the parent drug alone. Finally, we provide data on the characterization of a novel transgenic mouse model of pancreatic cancer. This model, the Pan Met View (PMV) mouse, combines the commonly used KPC transgenic mouse model of pancreatic cancer and a mouse that expresses a Luciferase reporter gene under the control of the cancer-specific promoter, CCN1. Our data shows that double transgenic PMV mice can now be used to follow primary tumor and metastasis development in real time by Bioluminescent imaging (BLI) through disease progression and potentially therapy. This strategy will enhance the use of genetically engineered mouse models (GEMMS) to study cancer initiation and progression with potential to non-invasively monitor therapy. These chapters present novel and exciting data that have the potential to open multiple avenues of translational study and result in significant advances in pancreatic cancer therapy.

Thesis (DPhil (Biomedical Science))--Cape Peninsula University of Technology, 2019. / Knowledge of population vulnerabilities to infectious diseases is key in managing many public health problems and for mapping appropriate strategies for prevention or intervention. A number of genes associated with resistance to HIV infection, such as the double deletion of 32 base pairs in the CCR5 gene , have been described and potentially account for lower HIV infections in some populations. The magnitude of the HIV pandemic in Sub-Saharan Africa warrants an investigation of the peculiar genetic factors that may have exacerbated its spread. An understanding of the genetic factors that are involved may aid in the development of specific strategies for prevention such as vaccine development, genetic counselling as well as gene therapy. The aim of this project was therefore to study the relationship between blood groups and HIV-infection in Botswana. HIV infection in Africa has not been linked to particular blood groups. The project was undertaken in two phases from December 2012 to December 2017. In the first phase, 346 subjects of known HIV status (negative or positive) were phenotyped for 23 erythrocyte antigens via standard scientific procedures. A Chi-square analysis was used to determine those antigens associated with increased or reduced risk of HIV infection. In the second phase, 120 samples were phenotyped for the protective blood group (RhC) and the risk-associated groups (Lub and P1). The samples were also characterized according to their laboratory results for viral load, lymphocyte sub-populations, complete blood count and blood chemistry, including total cholesterol. Some of the samples were also assessed for erythrocyte-associated viral RNA. Generally, the prevalence of the blood groups in the general population in Botswana did not differ with the known prevalence for Africans broadly. Three novel findings were established. First, the blood group Rh(C) was associated with a 40% risk reduction for HIV infection. Immunologically, carriage of the C antigen was associated with a more robust cell-mediated immunity as evidenced by enhanced cytotoxic T cell counts. Moreover, this antigen occurred with a frequency lower than 30% in all countries where HIV prevalence was high. There was therefore an inverse relationship between Rh(C) frequency and HIV prevalence. An examination of reports from previous studies revealed that the pattern was consistent in Africa, Europe, Asia, South America and Caribbean countries. It appears that the population frequency of this antigen explains, at least in part, a genetic factor that puts some African populations at higher risk for HIV infection. These results are novel in that Rh antigens have not been previously associated with immunity in any reports. Novel findings regarding the P1 blood group was its association with a double risk for HIV infection. While the plasma viral load did not differ between P1-positive and P1-negative subjects, P1-positive erythrocyte lysate yielded more viral RNA than P1-negative cells, implying more intracellular HIV RNA. Intra-erythrocytic viral RNA was detected even in patients with an undetectable plasma viral load. Glycosphingolipids, of which P1 is an example, have been documented to promote viral fusion to cells independent of CD4 receptors or other ligands. In at least one report, the presence of sphingolipids in lipid rafts was considered to be sufficient for viral fusion. The presence of viral RNA even in erythrocyte lysates corroborates this phenomenon and potentially explains the double risk of HIV infection observed. The occurrence of HIV RNA in erythrocyte lysate is a novel finding that suggests a new viral reservoir. Apparently, P1 has a high frequency among Africans and low in other races.

<p>Innate immunity has received impressive attention in the past decade owing to the discovery of the Toll like receptors (TLRs) and the NOD-like receptors (NLRs). While the TLRs specialize in fighting microbes at the cell surface, the NLRs complement by detecting and responding to intracellular microbes. Recently, the non-microbe sensing NLR called inflammasomes, have been identified, which senses metabolic stress as well as certain pathogenic microbes and elicits host’s inflammatory response. <strong></strong></p><p>The NLR, NALP3 (formerly known as cryopyrin) forms a large cytoplasmic complex called the ‘inflammasome’ when NALP3, activated by a stimuli, associates with the adaptor proteins ASC and CARD-8. This interaction leads to the activation of pro-inflammatory caspase-1 which subsequently results in the formation of Interleukin (IL)-1β and IL-18. Mutations in the gene encoding NALP3, termed <em>NLRP3</em> can lead to its constitutive activation resulting in an uncontrolled production of IL-1β. These mutations have been implicated in hereditary inflammatory diseases, often grouped under cryopyrin associated periodic syndromes (CAPS).</p><p>This thesis describes a patient with a long history of arthritis and antibiotic resistant fever, but without the typical symptoms of CAPS. The patient was found to be a heterozygous carrier of two common polymorphisms Q705K in <em>NLRP3 </em>and C10X in the <em>CARD-8</em>. Experimental studies showed elevated levels of caspase-1 and IL-1β in the patient, and a total clinical remission was achieved by IL-1β blockade. These two polymorphisms combined, were found to occur in approximately 4% of the control population, suggesting the possibility of a genetic predisposition for inflammation in these individuals. Therefore, a cohort of rheumatoid arthritis (RA) patients, where elevated IL-1β could be one of the reasons behind chronic inflammation, was investigated. We found that carrying the combined polymorphisms resulted in increased RA susceptibility and a more severe disease course. Hypothetically, this subgroup of patients might benefit from IL-1β blockade. Additional studies are warranted to elucidate the functional effects of the two polymorphisms and to determine whether they identify a subgroup of patients that could benefit from IL-1 targeted therapy. Given the structural similarity of NALP3 to other NALPs, the possibility of involvement of the alternative, homologous genes cannot be eliminated.</p>

Thesis (M. Phil.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 86-111).

The rapidly developing sequencing technology has brought up an opportunity to scientists to look into the detailed genotype information in human genome. Computational programs have played important roles in identifying disease related genomic variants from huge amount of sequencing data. In the past years, a number of computational algorithms have been developed, solving many crucial problems in sequencing data analysis, such as mapping sequencing reads to genome and identifying SNPs. However, many difficult and important issues are still expecting satisfactory solutions. A key challenge is identifying disease related mutations in the background of non-pathogenic polymorphisms. Another crucial problem is detecting INDELs especially the long deletions under the technical limitations of second generation sequencing technology. To predict disease related mutations, we developed a machine learning-based (Random forests) prediction tool, EFIN (Evaluation of Functional Impact of Nonsynonymous mutations). We build A Multiple Sequence Alignment (MSA) for a querying protein with its homologous sequences. MSA is later divided into different blocks according to taxonomic information of the sequences. After that, we quantified the conservation in each block using a number of selected features, for example, entropy, a concept borrowed from information theory. EFIN was trained by Swiss-Prot and HumDiv datasets. By a series of fair comparisons, EFIN showed better results than the widely-used algorithms in terms of AUC (Area under ROC curve), accuracy, specificity and sensitivity. The web-based database is provided to worldwide user at paed.hku.hk/efin. To solve the second problem, we developed Linux-based software, SPLindel that detects deletions (especially long deletions) and insertions using second generation sequencing data. For each sample, SPLindel uses split-read method to detect the candidate INDELs by building alternative references to go along with the reference sequences. And then we remap all the relevant reads using both original references and alternative allele references. A Bayesian model integrating paired-end information was used to assign the reads to the most likely locations on either the original reference allele or the alternative allele. Finally we count the number of reads that support the alternative allele (with insertion or deletions comparing to the original reference allele) and the original allele, and fit a beta-binomial mixture model. Based on this model, the likelihood for each INDEL is calculated and the genotype is predicted. SPLindel runs about the same speed as GATK and DINDEL, but much faster than DINDEL. SPLindel obtained very similar results as GATK and DINDEL for the INDELs of size 1-15 bps, but is much more effective in detecting INDELs of larger size. Using machine learning method and statistical modeling technology, we proposed the tools to solve these two important problems in sequencing data analysis. This study will help identify novel damaging nsSNPs more accurately and efficiently, and equip researcher with more powerful tool in identifying INDELs, especially long deletions. As more and more sequencing data are generated, methods and tools introduced in this thesis may help us extract useful information to facilitate identification of causal mutations to human diseases. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

No description available.

No description available.

Copies of author's previously published articles inserted. / Amendments pasted onto back-end paper. / Bibliography: leaves 255-286. / ix, 286, [15] leaves, [5] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to isolate the gene(s) responsible for Fancomi anaemia and breast cancer using a positional cloning strategy / Thesis (Ph.D.)--University of Adelaide, Dept. of Cytogenetics and Molecular Genetics, 1999

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics, 1985. / Offprints of several author's journal articles inserted. Includes bibliographical references.