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.

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.

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.

Doctor of Philosophy(PhD) / ABSTRACT Despite the extensive quantitative and qualitative knowledge about therapeutic regimens and the molecular biology of HIV/AIDS, the eradication of HIV infection cannot be achieved with available antiretroviral regimens. HIV drug resistance remains the most challenging factor in the application of approved antiretroviral agents. Previous investigations and existing HIV/AIDS models and algorithms have not enabled the development of long-lasting and preventive drug agents. Therefore, the analysis of the dynamics of drug resistance and the development of sophisticated HIV/AIDS analytical algorithms and models are critical for the development of new, potent antiviral agents, and for the greater understanding of the evolutionary behaviours of HIV. This study presents novel computational methods for the analysis of drug-resistance dynamics, including: viral sequences, phenotypic resistance, immunological and virological responses and key clinical data, from HIV-infected patients at Royal Prince Alfred Hospital in Sydney. The lability of immunological and virological responses is analysed in the context of the evolution of antiretroviral drug-resistance mutations. A novel Bayesian algorithm is developed for the detection and classification of neutral and adaptive mutational patterns associated with HIV drug resistance. To simplify and provide insights into the multifactorial interactions between viral populations, immune-system cells, drug resistance and treatment parameters, a Bayesian graphical model of drug-resistance dynamics is developed; the model supports the exploration of the interdependent associations among these dynamics.

Platinum based chemotherapy is widely used to treat cancer. Cisplatin (diamminedichloroplatinum) combination treatments provide cure for metastatic testicular cancer and prolong survival for patients suffering from ovarian, head and neck, bladder and non small cell lung cancer. Tumors that initially respond to treatment may eventually acquire resistance, resulting in treatment failure. Cisplatin resistant cells are crossresistant to arsenite and antimonite and these metalloids are exported from bacteria by the ars-operon. In this thesis, we describe the human ArsA homolog, ASNA1, as a protein involved in a novel resistance mechanism to cisplatin, arsenite and antimonite. ASNA1 was downregulated by antisense and siRNA techniques in human melanoma and ovarian carcinoma cell lines. These cells displayed increased sensitivity to arsenite and the platinum based drugs cisplatin, carboplatin and oxaliplatin. In both melanoma and ovarian carcinoma, cisplatin resistant cells overexpressed ASNA1. Blockage of ASNA1 resulted in increased apoptosis and retarded growth, complicating further characterization of ASNA1 in human cell lines. ASNA1 also promotes insulin signaling and mediates membrane insertion of tail-anchored proteins. To explore different aspects of ASNA1 function with respect to cisplatin resistance, we used the model organism C. elegans. In the nematode C. elegans, asna-1 (rnai) treated larvae were hypersensitive to cisplatin, arsenite and antimonite. Adult asna-1 mutant worms were cisplatin sensitive and this hypersensitivity was seen even when apoptosis was blocked. Expression of human ASNA1 rescued the cisplatin hypersensitivity in asna-1 mutants, showing conservation of function. Transgene expression of mutated forms of asna-1 separated the cisplatin hypersensitivity phenotype from the insulin signaling phenotype of asna-1 mutants. Three ASNA-1 residues, His164, Cys285 and Cys288 were identified as essential for ASNA-1 promoted cisplatin resistance but not for insulin signaling. Finally, studies of the C. elegans germline revealed increased numbers of apoptotic cells in asna-1 mutants. In conclusion, C. elegans is a suitable model organism to identify and characterize cisplatin response mechanisms. A targeted therapy against ASNA1 could sensitize cisplatin resistant cells and improve outcome for cancer patients.

Treating fungal infections is challenging due to the emergence of drug resistance and the limited number of clinically useful antifungal drugs. To improve clinical outcome it will be necessary to develop new antifungal drugs with different mechanisms of action and to discover drugs that improve the fungicidal activity of current antifungals. This study reveals a new role for fungal protein kinase C (PKC) signaling in resistance to drugs targeting the ergosterol biosynthesis pathway in the pathogenic fungus, Candida albicans, and the model yeast, Saccharomyces cerevisiae. PKC signaling enabled survival of antifungal-induced cell membrane stress in part through the mitogen-activated protein kinase (MAPK) cascade and through cross-talk with calcineurin signaling in both species. The molecular chaperone Hsp90, which stabilizes client proteins including calcineurin, also stabilized the terminal C. albicans MAPK, Mkc1. This establishes new circuitry connecting PKC with Hsp90 and calcineurin, and suggests that inhibiting fungal Pkc1 can be a promising strategy for treating life-threatening fungal infections.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Treating fungal infections is challenging due to the emergence of drug resistance and the limited number of clinically useful antifungal drugs. To improve clinical outcome it will be necessary to develop new antifungal drugs with different mechanisms of action and to discover drugs that improve the fungicidal activity of current antifungals. This study reveals a new role for fungal protein kinase C (PKC) signaling in resistance to drugs targeting the ergosterol biosynthesis pathway in the pathogenic fungus, Candida albicans, and the model yeast, Saccharomyces cerevisiae. PKC signaling enabled survival of antifungal-induced cell membrane stress in part through the mitogen-activated protein kinase (MAPK) cascade and through cross-talk with calcineurin signaling in both species. The molecular chaperone Hsp90, which stabilizes client proteins including calcineurin, also stabilized the terminal C. albicans MAPK, Mkc1. This establishes new circuitry connecting PKC with Hsp90 and calcineurin, and suggests that inhibiting fungal Pkc1 can be a promising strategy for treating life-threatening fungal infections.

Several families of highly effective anticancer drugs are selectively toxic to cancer cells because they interfere with nucleic acids synthesis. Many such drugs are pumped out of cells faster than they can reach their targets, which limits efficacy and renders many tumors drug-resistant. By delivering a drug to the mitochondria of mammalian cells – an organelle where nucleic acids synthesis also occurs – efflux could be prevented through sequestration. Doxorubicin, a topoisomerase II inhibitor, was used as proof-of-principle for this concept due to its susceptibility to resistance. When doxorubicin is attached to a peptide that specifically targets mitochondria, its efficacy is not attenuated by various resistance mechanisms to which doxorubicin is normally susceptible. These results indicate that targeting drugs to the mitochondria provides a means to evade the most common mechanism of drug resistance.

Human Immunodeficiency Virus (HIV) has become a global pandemic with at least 25 million deaths and no cure. One of the most important targets to inhibit this virus is HIV-1 protease (PR), which is required to cleave the viral proteins needed for maturation of the virus after it invades and replicates in the host cell. There are nine protease inhibitors that are used in AIDS treatment. The virus loses susceptibility to these inhibitors by drug resistance due to mutations. The goal of the project is to examine the highly drug resistant HIV PR P51 in its complex with inhibitors. In this experiment we expressed and purified HIV PR P51 protein. We performed protein crystallization with inhibitors Tipranavir, Amprenavir, Darunavir, and Saquinavir to obtain the structure of the protease and the inhibitors in their complexes. Future analysis of the crystal structures will help with the development of successful therapeutic inhibitors.