<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

<p>&nbsp / </p> <p align="left">One of the most common HIV-associated opportunistic infections is candidiasis, caused by <i><font face="TimesNewRoman,Italic">Candida albicans </font></i><font lang="KO" face="TimesNewRoman">or other </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. In immune suppressed subjects, this commensal organism can cause an increase in patient morbidity and mortality due to oropharyngeal or systemic dissemination. Limited information exists on the prevalence and antifungal susceptibility of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species in the African continent, the most HIV-affected region globally and home to new and emerging drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species. The mechanisms of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">drug resistance in the African continent have also not been described. In this study, 255 </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species isolated from the oral mucosa of HIV-positive South African and Cameroonian patients were identified using differential and chromogenic media and their drug susceptibility profiles tested using the disk diffusion method and the TREK Sensititre system, an automated broth microdilution method. </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">cell wall fractions were run on SDSPAGE and HPLC-MS with the aim of identifying peptides specifically expressed by antifungal drug resistant isolates. Comparisons between the two groups of isolates revealed differences in </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">species prevalence and drug susceptibility with interesting associations observed between specific drug resistance and duration of ARV therapy. This study showed that fluconazole, the drug of choice for the treatment of candidiasis in the African continent, is not an effective therapy for most cases of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">infection, and suggests that regional surveillance be implemented in the continent. A multiple-drug resistant </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">strain was identified in this study, a finding that has not previously been documented. The use of proteomics tools allowed for the identification of peptides involved in drug resistance and the elucidation of </font><i><font face="TimesNewRoman,Italic">Candida </font><font lang="KO" face="TimesNewRoman">colonization mechanisms in HIV-infected African patients.</font></i></i></i></i></i></i></i></i></i></i></p>

Breast cancers with overexpression or amplification of the HER2 tyrosine kinase receptor are more aggressive, resistant to chemotherapy, and associated with a worse prognosis. Currently, these breast cancers are treated with the monoclonal antibody trastuzumab (Herceptin®). Unfortunately, not all patients respond to trastuzumab drug therapy; some patients show de novo resistance, while others acquire resistance during treatment. This thesis describes our RNAi studies to identify novel regulators of the HER2 signaling pathway in breast cancer. Three kinome-wide siRNA screens were performed on five HER2 amplified and seven HER2 non-amplified breast cancer cell lines, two normal breast cell lines, as well as two HER2-positive breast cancer cell lines with acquired trastuzumab resistance and their isogenic trastuzumab-sensitive controls. To understand the main kinase drivers of HER2 signaling, we performed a comprehensive screen that selected against growth inhibitors of the non-HER2 amplified breast cancer cell lines. This screen identified the loss of the HER2/HER3 heterodimer as the most prominent selective inhibitor of HER2-amplified breast cancers. In a trastuzumab sensitization screen on five trastuzumab-treated breast cancer cell lines, we identified several siRNA against the PI3K pathway as well as various other signaling pathways that inhibited proliferation. Finally, in a screen for acquired trastuzumab resistance, PKCη and its downstream targets were identified. Loss of PKCη resulted in a decrease in G1/S transition and upregulation of the cyclin dependent kinase inhibitor p27. Initial data suggest that PKCη promotes p27 ubiquitination and degradation. Taken together, these studies provide novel insight into the complex signaling of HER2-positive breast cancers and the mechanisms of resistance to trastuzumab therapy. This work describes how various kinases can modulate cell proliferation, and points to possible novel drug targets for the treatment of HER2-positive breast cancers.

Significant progress has been made in the development of new agents against cancer and new ways of delivering existing and new agents. Yet, the major challenge to target and selectively kill cancer cells while affecting as few healthy cells as possible remains. When linked with tumor targeting moieties such as tumor-specific ligands or monoclonal antibodies, nanoparticles can be used to target cancer-specific receptors, tumor biomarkers as well as tumor vasculatures with high affinity and precision. Recently, the use of nanoparticles for drug delivery and targeting has emerged as one of the most exciting and clinically important areas in cancer nanotechnology. In this work, we tested the hypothesis that our novel ternary biomolecular nanostructures of folic acid (FA), biodegradable polymer, and paclitaxel will improve the delivery and tumor-specific distribution of the anticancer drug. The design was based on three principles: 1) Passive targeting via enhanced permeation and retention (EPR) effect; 2) active targeting via a tumor-specific ligand; and 3) prodrug that would release the drug upon delivery. First, self-assembled polymer-paclitaxel-FA nanostructures were synthesized. Their physicochemical properties were examined and biological efficacy was tested. The conjugates had significantly improved solubility in water, enabling cremophor-free formulation. Second, in vitro cellular toxicity and targeting ability of the nanostructures were investigated. In cancer cell lines with high folate receptor (FR) expression, the ternary conjugates were efficiently taken up whereas no detectable association was found in cells with minimal or no FR expression. Third, in vivo investigation in human xenograft mice models was carried out. Ternary nanostructures drastically inhibited tumor growth without inducing systemic toxicity or side effects. The ternary nanostructures displayed remarkable anti-angiogenic effect on tumor vasculature. Heparin-paclitaxel-FA was also very effective in drug resistant tumors, potentially overcoming multidrug resistance. Studies in other cancer models are in progress to determine the spectrum of applicability of these ternary nanostructures. The design principles applied in these nanoparticles can be extended to delivery and targeting of diagnostic and imaging agents. The ability to engineer multifunctional nanostructures will have a significant impact on cancer diagnostics, molecular profiling, and the integration of cancer therapy and imaging.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

The development of resistance to chemotherapeutic drugs is the main obstacle to the successful treatment of many cancers, including childhood neuroblastoma, the most common solid tumour of infants. One factor that may play a role in determining response of neuroblastoma tumours to therapeutic agents is the p53 tumour suppressor gene. A number of previous studies have suggested that this tumour suppressor protein is inactive in neuroblastoma due to its cytoplasmic sequestration. This thesis therefore has examined the functionality of p53 and its role in determining drug response of neuroblastoma cells. An initial study was undertaken that characterised an unusually broad multidrug resistance (MDR) phenotype of a neuroblastoma cell line (IMR/KAT100). The results demonstrated that the MDR phenotype of the IMR/KAT100 cells was associated with the acquisition of mutant p53. To explore the role of p53 in drug resistance further, p53-deficient variants in cell lines with wild-type p53 were generated by transduction of p53-suppressive constructs encoding either shRNA or a dominant-negative p53 mutant. Analysis of these cells indicated that: (i) in contrast to previous reports, wild-type p53 was fully functional in all neuroblastoma lines tested, as evidenced by its activation and nuclear translocation in response to DNA damage, transactivation of target genes and control of cell cycle checkpoints; (ii) inactivation of p53 in neuroblastoma cells resulted in establishment of an MDR phenotype; (iii) knockdown of mutant p53 did not revert the drug resistance phenotype, suggesting it is determined by loss of wild-type function rather than gain of mutant function; (iv) p53-dependent cell senescence, the primary response of S-type neuroblastoma cells to DNA damage, is replaced, after p53 inactivation, by mitotic catastrophe and subsequent apoptosis. In contrast to neuroblastoma, p53 suppression had no effect or increased drug susceptibility in several other tumour cell types, indicating the importance of tissue context for p53- mediated modulation of tumour cell sensitivity to treatment. Taken together, these data provide strong evidence for p53 having a role in mediating drug resistance in neuroblastoma and suggest that p53 status may be an important prognostic marker of treatment response in this disease.

Neuroblastoma (NB) is the most common solid tumor of early infancy. Despite a significant improvement in the general survival rate for children with cancer, the prognosis of high-risk NB remains low, at about 30%, despite the use of intensive chemo-radiotherapy followed by differentiation therapy with retinoic acid (RA). Relapses in this category of NB are often due to the emergence of multi-drug and RA-resistant minimal residual cancer cells. The use of natural 13-cis RA, as a single chemo-preventive agent, has improved the survival rate to 50% for high-risk NB patients. However, the prevalence of RA-resistance is high in high-risk NB, and in solid cancers, in general. RA-resistance in cancer cells is mediated by a number of factors. Loss of RA-induced expression of the putative tumor suppressor gene, retinoic acid receptor-beta (RARβ), is one of the most common factors that have been reported in RAresistant phenotypes of a wide range of cancer cells. The transcriptional regulation of RAR(β) gene and other retinoid responsive-genes is believed to be regulated by the ligand-dependent transactivation of the homo- or heterodimer complexes of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) subtypes, namely alpha (α), beta (β) and gamma (γ). It is believed that the anti-cancer activities of natural all-trans RA and 13-cis RA are mediated through activation of RAR-complexes. The loss of RA-induced RAR β expression can be caused by aberrant recruitment of chromatin structure modifying enzymes, histone deacetylases (HDACs), which have major roles in the global regulation of gene transcription. However, the mechanism of RA-resistance in NB cells is unclear. This thesis set out to identify the molecular mechanism of RA-resistance and to develop a new therapeutic approach to overcome RA-resistance in NB cells. The data in this thesis demonstrated that deregulation or over-expression of proto-oncogene MYCN caused a total RA-resistance in NB cells in vitro and in vivo, despite the strong induction of RARI3 expression. The data also indicated that the activation of RAR-dependent pathways by aRA or 13RA alone is not sufficient to overcome MYCN-mediated RA-resistance in NB cells. In the light of this observation, this thesis went on to examine whether combined targeting activation of RAR and RXR subtypes with receptor specific ligands could enhance the therapeutic efficacy of the retinoid signaling pathway. NB cells were treated with a panel of receptor-specific retinoids, namely aRA, l3RA, 9RA (RAR-specific), CD 417, CD 2314 (RARβ-specific), CD 666 (RARγ-specific), CD 336 (RARα-specific), CD 3640, CD 2872 (RXR-specific), as a single agent or in combination at a low concentration of 0.1 ??M. The results showed that combined targeting activation of RARα and RXR was not only the most effective combination, but also overcame MYCN-mediated RA-resistance in NB cells in vitro.Collectively, these data demonstrated the combined targeting activation of RAR and RXRs as a new approach to enhance the efficacy of retinoid therapy and overcome RA-resistance in the treatment of high-risk NB, and other cancers. The emerging therapeutic potential of HDAC inhibitors (HDACi) as front line anti-cancer agents, or adjuvants to other agents such as RA, has suggested a new approach in the treatment of cancer. However, the molecular mechanism of the remarkably specific anticancer actions of HDACi is still largely speculation. The data presented in this study was the first to demonstrate a novel sequential order and the dosage-dependent roles of basal p21Wafl expression and G2/M arrest as protective mechanisms against HDACi-induced apoptosis. In addition, this thesis also examined and compared the therapeutic efficacy of HDACi as a single agent and in combination with other anti-cancer agents such as RA, IFNα and chemotherapeutic agents. Evaluation of the therapeutic effects of combinations of aRA, IFN and HDACi showed that combination of HDACi and IFNα exhibited the strongest synergy against NB cells in vitro. Treatment of MYCN transgenic mice, which consistently develop abdominal NB tumors that closely mirror the human disease in both physiological and biological aspects, with hydroxamic acid-based HDACi, trichostatin A (TSA), alone reduced tumor growth by nearly 50%, compared to the solvent control and IFNα alone, which had no effect on NB tumor growth. The most exciting finding was that the combination of HDACi and IFNα synergistically reduced tumor mass and angiogenesis by over 80% without any apparent systemic side-effects. The therapeutic effect of treatment with HDACi correlated with the induction of acetylation of histone 4 protein (H4) in both tumor and organ tissues, indicating a wide therapeutic index for HDACi in vivo. Collectively, the data in this study have demonstrated basal p21 Wafl expression as a potential marker of sensitivity to HDACi-based therapy, and the therapeutic efficacy of a novel combination of HDACi with IFNα in vivo. These preclinical data have provided an evidence-based rationale for a clinical trial of the combination of HDACi and IFNα in the treatment of high risk NB.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

Intrinsic or acquired drug resistance of tumours is a major problem for successful therapy of breast cancer patients. The efficacy of doxorubicin, one of the most important and commonly used drugs in chemotherapy, can be severely compromised by a variety of unspecific mechanisms rendering tumours drug resistant. Little is known however, about the specific events taking place in response to doxorubicin treatment, which may repair doxorubicin-induced damage, leading to drug resistance. Doxorubicin is a topoisomerase II poison, which interferes with topoisomerase II enzymes during DNA replication, resulting in DNA double-strand breaks. Topoisomerase II enzymes mediate the passage of DNA strands by introducing transient DNA breaks, and are essential for changes in DNA topology during replication. The DNA lesions induced by the combination of topoisomerase II and doxorubicin can be repaired by either non homologous end-joining or homologous recombination repair, as both pathways are specifically responsible for the repair of DNA double-strand breaks. The DNA-dependent protein kinase catalytic subunit in non homologous end-joining and Rad51 in homologous recombination repair are essential for each of these pathways. If it was possible to specifically target these proteins or other antagonistic mechanisms of doxorubicin-induced cell death, which may be activated in response to doxorubicin treatment, chemosensitivity of tumours could be restored and chemotherapy made more effective. Hence it was the purpose of this study to investigate proteome-wide changes in protein expression in response to drug treatment, as well as specifically analysing alterations in the protein levels of the DNA-dependent protein kinase catalytic subunit and Rad51. Global changes in protein regulation of breast and breast cancer cells were investigated using mass spectrometric and electrophoretic analysis techniques. These experiments however, could not reproducibly identify any genuine drug-induced changes in protein levels, as only proteins of relatively high abundance could be analysed. Immunoblotting results however, showed that Rad51 was differentially regulated in a cell line- and drug dosage-dependent manner, while levels of the DNA-dependent protein kinase catalytic subunit remained largely unchanged. Furthermore, increased levels of topoisomerase II alpha protein were also detected. In addition, immunohistochemical analysis demonstrated that both Rad51 and the DNA-dependent protein kinase catalytic subunit could be independently overexpressed in breast tumours and therefore may represent potential targets for selectively enhancing chemosensitivity of breast cancers.

An epidemic of staphylococcal infections occurred in New Zealand hospitals and communities from 1955-1963. The 'H', or 'Hospital Bug', a strain of Staphylococcus aureus characteristic of the epidemic, was resistant to the most commonly used antibiotics. Post-operative patients, the frail elderly and mothers and babies were particularly vulnerable to staphylococcal colonization and infection. This thesis places the H-Bug epidemic in its historical context, discussing the ways in which the government and health professionals responded to the rising incidence of staphylococcal infection, and the major effects of the epidemic on medical and hospital practice. It also examines the impact of persistent staphylococcal infection on women and families in the community. Primary sources provided the basis for this thesis. The H-Bug epidemic has gone largely unrecorded except in contemporary documents. Health Department files and Auckland Hospital Board records as well as newspaper clippings were important sources. The New Zealand epidemic was clearly linked to the global pandemic of antibiotic resistant staphylococcal infection, 1946-1966, through medical literature and archival documents. International medical journals, including the New Zealand Medical Journal, published numerous articles on the epidemiology of antibiotic-resistant staphylococcal infection, providing an excellent record of research, case studies, current opinion, and recommended practice. The most valuable contribution to an understanding of the impact and experience of the H-Bug epidemic was, however, provided by the nineteen people who agreed to be interviewed for the study. Interviewees included a wide variety of health professionals and women and their children, all of whom had personal experience or association with the epidemic. In this thesis it is argued that the main focus of the medical response was the prevention and control of hospital cross-infection, both to protect patients and to preserve the public perception of the hospital as a safe venue for care. Although the emergence of resistant strains of staphylococci was widely attributed to the misuse of antibiotics, this thesis contends that the Health Department was reluctant to impose restrictions on medical prescribing and that Health Department official and senior clinicians chose instead to modify hospital environments and clinical practice. Rooming-in was widely introduced to counter the epidemic despite the fact that a trial in 1959, at National Women's Hospital, did not demonstrate a reduction in infection rates among neonates. The concept endured, however, as it held strong appeal for hospital administrators hard pressed to keep wards adequately staffed with trained personnel. It was also supported by women and health professionals who were convinced of the benefits of a close mother-baby relationship from birth. The H-Bug epidemic was eventually resolved by the introduction of the methicillin antibiotics in the early 1960s. As a consequence, confidence in a pharmaceutical solution to infectious disease remained intact until the emergence of multiple antibiotic resistant organisms in the 1980s. The lessons of the H-Bug epidemic had been largely forgotten in the intervening years, ignored until New Zealand clinicians were reminded once again that antimicrobial resistance would inevitably accompany the indiscriminate use of antibiotics and inadequate attention to infection prevention and control.