Introduction: Nosocomial infections remain a major cause of mortality and morbidity. The problem is most apparent in intensive care units (ICUs). Most ICU patients are compromised and vulnerable as a result of disease or severe trauma. One in ten people admitted to hospital is given an antibiotic for infection. The risk of acquiring a nosocomial infection in a European ICU is approximately 20%. It is vitally important that ways are found to prevent transmission between patients and personnel, and that local hygiene routines and antibiotic policies are developed. This thesis is a holistic work focused particularly on antimicrobial antibiotic resistance, antibiotic consumption and to some extent on hygiene in Swedish ICUs. Aims: The general aim of this thesis was to investigate bacterial resistance and antibiotic consumption in Swedish ICUs and to try to correlate ICU demographic data with antibiotic consumption and antibiotic resistance. Additional aims were to investigate on which clinical indications antibacterial drugs are prescribed in the ICU, and to investigate the emergence of resistance and transmission of Pseudomonas aeruginosa in the ICU using cluster analysis based on antibiograms and genotype data obtained by AFLP. Material and methods: In paper 1-3, antibiotic consumption data together with bacterial antibiotic resistance data and specific ICU-demographic data were collected from an increasing number of ICUs over the years 1997-2001. Data from ICUs covering up to six million out of Sweden’s nine million inhabitants were included. In paper 4, the indications for antibiotic prescribing were studied during two weeks in 2000. Paper 5 investigated Pseudomonas aeruginosa isolates in order to detect cross-transmission with genotype obtained by AFLP, and antibiogram-based cluster analysis was also performed in order to see if this could be a quicker and easier substitute for AFLP. Results: This thesis has produced three important findings. Firstly, antibiotic consumption in participating ICUs was relatively high during the study period, and every patient received on average more than one antimicrobial drug per day (I-IV). Secondly, levels of antimicrobial drug resistance seen in S. aureus, E. coli and Klebsiella spp remained low when data were pooled from all ICUs throughout the study period, despite relatively high antibiotic consumption (I-V). Thirdly, the prevalence of antibiotic resistance in CoNS and E. faecium, cefotaxime resistance in Enterobacter, and ciprofloxacin and imipenem resistance in P. aeruginosa was high enough to cause concern. Conclusion: For the period studied, multidrug resistance in Swedish ICUs was not a major problem. Signs of cross-transmission with non-multiresistant bacteria were observed, indicating a hygiene problem and identifying simple improvements that could be made in patient care guidelines and barrier precautions. A need for better follow up of prescribed antibiotics was evident. With further surveillance studies and monitoring of antibiotics and bacterial resistance patterns in the local setting as well as on a national and international level, some of the strategic goals in the prevention and control of the emergence of antimicrobial-resistant microbes may be achievable.

Background: In childhood acute lymphoblastic leukemia (ALL), the cure rate has now reached 80% in the western world. Even so, 15¬–20% will die from the disease or treatment-related causes, among them children who did not present any known unfavorable features at diagnosis. Treatment of childhood ALL is risk-adapted, meaning that certain factors that are related to the child or the leukemic blasts stratifies to more or less intensive treatment. In this thesis, characteristics of the bone marrow (BM) stroma, reflecting the interaction between the leukemic cells and their microenvironment, were evaluated. The aims were to investigate these factors in relation to other known data in order to further understand the biology of leukemia, and to suggest additional risk factors that would further improve decision making for the treatment of individual children diagnosed with ALL. Methods: We retrospectively investigated microvessel density (MVD), blast-congested vessel fraction (BCVF), and degree of fibrosis – reticulin fiber density (RFD) – in sections from diagnostic BM biopsies from children diagnosed in Umeå, Uppsala, and Stockholm. RFD was also studied in BM sections from treatment day 29. Results: RFD had prognostic impact in patients with high-hyperdiploid (HeH) leukemia. Moreover, rapid reduction of RFD during induction treatment was associated with a favorable prognosis compared to slow reduction, in B-cell precursor (BCP) ALL patients. There was also a correlation between RFD at diagnosis and minimal residual disease (MRD) measured by flow cytometry on treatment day 29 in BCP patients. BCP patients with high RFD and high MVD had an unfavorable outcome compared to all other BCP patients. In addition, MVD and RFD were both associated with immunophenotype, and MVD with cytogenetic aberrations. There was a correlation between MVD and WBC count in BCP high-risk patients. There was also a strong correlation between BCVF and WBC count in all BCP patients, but not between BCVF and MVD or RFD. There was a negative correlation between MVD and in vitro cellular resistance to several drugs in BCP patients. A drug-resistance score combining the drugs most strongly correlated to MVD – cytarabine, doxorubicin, and dexametasone (ADD score) – identified the prognostic potential of ADD score in HeH patients with no unfavorable features. Conclusions: Taken together, these studies indicate that stroma factors in leukemia are related to both phenotypic and genotypic features of acute leukemia. Stroma factors also seem to influence the response to induction treatment, in vitro drug resistance, and outcome in certain subgroups of childhood ALL patients. The results emphasize the importance of BM stroma in leukemia and the need for greater use of BM biopsy at diagnosis.

Despite major advances in our understanding of the biology of childhood acute myeloid leukemia (AML) and the development of new cytotoxic drugs, the prognosis of long-term survival is still only 60-65 %. In the present research, we studied the pharmacokinetics of drugs used in the induction therapy of childhood AML and performed in vitro drug sensitivity testing of leukemic cells from children with AML. The aims of the studies were to correlate the results of the analysis to biological and clinical parameters and to identify subgroups of AML with specific drug sensitivity profiles in order to better understand why treatment fails in some patients and how therapy may be improved. Blood samples were analysed to study the pharmacokinetics of doxorubicin (n=41), etoposide (n=45) and 6-thioguanine (n=50). Doxorubicin plasma concentration and total body clearance were correlated to the effect of induction therapy, and doxorubicin plasma concentration was an independent factor for complete remission, both in univariate and multivariate analysis including sex, age, and white blood cell count at diagnosis. For etoposide and 6-thioguanine no correlation was found between pharmacokinetics and clinical effect. Children with Down syndrome (DS) tended to reach higher blood concentrations of etoposide and thioguanine nucleotides, indicating that dose reduction may be reasonable to reach the same drug exposure as in children without DS. Leukemic cells from 201 children with newly diagnosed AML, 15 of whom had DS, were successfully analysed for in vitro drug sensitivity by the fluorometric microculture cytotoxicity assay (FMCA). We found that samples from children with DS were highly sensitive to most drugs used in AML treatment. In non-DS children, the t(9;11) samples were significantly more sensitive to cytarabine (p=0.03) and doxorubicin (p=0.035) than other samples. The findings might explain the very favorable outcome reported in children with DS and t(9;11)-positive AML. A specific drug resistance profile was found for several other genetic subgroups as well. A detailed study of MLL-rearranged leukemia showed that cellular drug sensitivity is correlated both to partner genes and cell lineage, findings that support the strategy of contemporary protocols to include high-dose cytarabine in the treatment of patients with MLL-rearrangement, both in AML and acute lymphoblastic leukemia (ALL). Our results indicate that drug resistance and pharmacokinetic studies may yield important information regarding drug response in different sub-groups of childhood AML, helping us to optimize future chemotherapy in childhood AML.

HIV-1 protease is the most effective target for drugs to treat AIDS, however, the long-term therapeutic efficiency is restricted by the rapid development of drug resistant variants. To better understand the molecular basis of drug resistance, crystallographic and kinetic studies were applied to wild-type HIV-1 protease (PR) and drug-resistant mutants, PRV82A, and PRI84V, in complex with substrate analogues, the current drug saquinavir and the new inhibitor UIC-94017 (TMC-114). UIC-94017 was also studied with mutants PRD30N and PRI50V. The drug-resistant mutations V82A, I84V, D30N and I50V participate in substrate binding. Eighteen crystal structures were refined at resolutions of 0.97-1.60A. The high accuracy of the atomic resolution crystal structures helps understand the reaction mechanism of HIV-1 PR. Different binding modes are observed for different types of inhibitors. The substrate analogs have more extended interactions with PR subsites up to S5-S5', while the clinical inhibitors maximize the contacts within S2-S2'. Hydrophobic interactions are the major force for saquinavir binding since it was designed with enhanced hydrophobic groups based on substrate side-chains. In contrast, the new clinical inhibitor UIC-94017 was designed to mimic the hydrogen bonds between substrates and PR. UIC-94017 forms polar interactions with the PR main-chain atoms of Asp29/30, which have been proposed to be critical for its potency against resistant HIV. The mutants showed different structural and kinetic effects, depending on the inhibitor and location of the mutations. The observed structural changes were consistent with the relative inhibition data. Both PRI84V and PRI50V lost favorable hydrophobic interactions with inhibitor compared with PR. Similarly, in PRD30N the UIC-94017 had a water-mediated interaction with the side-chain of Asn30 rather than the direct interaction observed in PR. However, PRV82A compensated for the mutation by shifts of the backbone of Ala82. Furthermore, the complexes of PRV82A showed smaller shifts relative to PR, but more movement of the peptide analog, compared to complexes with clinical inhibitors. The structures suggest that substrate analogs have more flexibility than the drugs to accommodate the structural changes caused by mutation, which may explain how HIV can develop drug resistance while retaining the ability of PR to hydrolyze natural substrates.

Resistance to antimicrobials is a well-known phenomenon leading to difficulties in the treatment of infectious diseases. The genetic determinants of such resistance are in general well understood: plasmids, transposons, insertion sequences and integrons are the most frequently related genetic elements. The word epigenetics refers to changes in the phenotype or in the gene expression caused by mechanisms other than underlying DNA sequence. In some cases these changes can remain for generations. Serratia marcescens is an enterobacterium characterized by its natural (intrinsic) resistance to most antibiotics. It is also a relevant opportunistic pathogen which has been involved in several pathologies such as urinary tract infections, prostheses infections, cellulitis, bacteremia and others. P. aeruginosa is a Gram-negative bacterium considered one of the major nosocomial pathogens worldwide. It causes several infections such as wound and burn infections as well as respiratory tract infections mostly affecting cystic fibrosis patients. An increasing prevalence of infections caused by multidrug-resistant (MDR) isolates has been reported in many countries and is actually a cause of concern. Both, P. aeruginosa and S. marcescens are relevant nosocomial pathogens. Some of the classic antimicrobials used to treat these pathogens are out-of-date and several of the new drugs available have already become targets for bacterial mechanism of resistance. Environmental conditions exert high pressure not only in the selection of genes encoding resistance to antibiotics or integron fixation in bacterial genomes or plasmids and other mobile elements transmission, but also in the expression of these potentialities that leads to resistance. Thus the role of epigenetics remains to be investigated. In addition it is well known that bacteria causing infections are naturally forming part of biofilms instead the planktonic way of life normally assumed to be in laboratory conditions. The aim of this thesis was the study of unconventional mechanisms of antimicrobial resistance contributing to MDR phenotypes in both S.marcescens and P. aeruginosa. Also the exploration of changes in antimicrobial susceptibilities of Serratia marcescens in the last 50 years by comparing isolates collected between 1945 and 1950, and current isolates. ¬The main conclusions obtained from this study are: 1. The resistome of Serratia marcescens did not change significantly during the antibiotic era. 2. Antibiotic withdrawing tends to restore original susceptible phenotypes, irrespective to the molecular mechanism involved in resistance. 3. None of Serratia strains studied presented integrons, any extended spectrum ß-lactamases. 4. Phenotypically determination of susceptibilities of old strains inactive during the last 60 years have confirmed results obtained by metagenomics i.e. the genes of resistance already existed before antibiotics discovery and use. 5. Multiresistant Pseudomonas aeruginosa harbored class 1 integrons containing a cassette encoding aminoglycoside adenylyltransferase (aadB). 6. Multiresistant Pseudomonas aeruginosa overexpressed MexAB-OprM and MexXY efflux machinery. 7. CCCP use should be avoided in experiments performed with P. aeruginosa and probably in other aerobic bacteria. 8. Meropenem induces the formation of aberrant long rods which can survive, accumulate less antibiotic than normal bacteria, and can revert to normal forma when antibiotic pressure disappears. 9. Colistin, the last therapeutic option to fight against Pseudomonas infections in cystic fibrosis patients, is normally active although cases of resistance have arisen recently. 10. Resistance to colistin seems to be mediated by lipopolysaccharide singular properties. 11. Colistin induces injuries in lipid bilayers, which can be studied by means of planar black lipid bilayer techniques. Preliminary results showed the ability of colistin to induce transient channels in the bilayers, with some dependence to voltage. 12. Recovery of susceptibility to imipenem is slower than acquision of resistance, since the selective advantage conferred by imipenem resistance in the presence of the antimicrobial is strong whereas OprD expression is likely evolutionarily advantageous only under certain and unknown environmental conditions. / Es van explorar els mecanismes no convencionals de resistència als agents antimicrobians que contribueixen a l’aparició de fenotips multiresistents en els bacteris Gram-negatius Serratia marcescens i Pseudomonas aeruginosa. Es van examinar també els canvis en la susceptibilitat als agents antimicrobians en S. marcescens durant els darrers 50 anys, comparant soques aïllades entre els anys 1945-1950 y soques actuals. ¬Les principals conclusions obtingudes d’aquest estudi són les següents: 1. El resistoma de Serratia marcescens no ha canviat significativament des de l’era pre-antibiòtica fins l’actualitat. 2. La retirada dels antibiòtics tendeix a recuperar els fenotips de susceptibilitat originals, independentment del mecanisme molecular implicat en la resistència. 3. Cap de las soques de Serratia estudiades va presentar integrons ni tampoc ß-lactamases d’espectre estès. 4. La determinació fenotípica de les susceptibilitats de las soques “antigues” de Serratia inactives durant 60 anys ha confirmat els resultats obtinguts mitjançant metagenòmica, és a dir, els gens de resistència als antibiòtics ja existien amb anterioritat al descobriment i ús dels antibiòtics. 5. En les soques clíniques de P. aeruginosa multiresistents es va detectar un integró de classe 1 que contenia el cassette gènic aadB, que codifica l’enzim aminoglicòsid 2’-O- adeniltransferasa, que confereix resistència a gentamicina, tobramicina i kanamicina. 6. Les soques multiresistents de P. aeruginosa van sobreexpressar els sistemes de reflux MexAB-OprM i MexXY-OprM. 7. L’ús de l’inhibidor de bombes de reflux CCCP s’hauria d’evitar als experiments realitzats amb P. aeruginosa i probablement amb altres bacteris de metabolisme aeròbic. 8. El meropenem indueix la formació de llargs bacils aberrants capaços de sobreviure en presència de l’antibiòtic. Aquests bacils acumulen menys antibiòtic que els bacteris normals, i poden revertir a la forma normal quan s’elimina la pressió selectiva. 9. La colistina, la última alternativa terapèutica per lluitar contra P. aeruginosa en pacients amb fibrosis quística, és normalment efectiva, encara que recentment han sorgit casos de resistència a aquest agent antimicrobià. 10. La resistència a colistina sembla estar causada per propietats singulars del lipopolisacàrid. 11. La colistina produeix danys en les membranes lipídiques que poden ser estudiats mitjançant tècniques de Black lipid bilayer. Estudis preliminars van mostrar la capacitat de la colistina para induir canals transitoris en les bicapes lipídiques, amb certa dependència de voltatge. 12. La recuperació de la susceptibilitat a l’imipenem en P. aeruginosa és més lenta que l’adquisició de resistència, donat que l’avantatge selectiva conferida per la resistència a l’imipenem en presència de l’agent antimicrobià és forta, mentre que l’expressió d’ OprD és probablement avantatjosa solament sota certes i desconegudes condicions ambientals. / Se exploraron los mecanismos no convencionales de resistencia a agentes antimicrobianos que contribuyen a la aparición de fenotipos multiresistentes en las bacterias Gram-negativas Serratia marcescens y Pseudomonas aeruginosa. Se examinaron también los cambios en la susceptibilidad a los agentes antimicrobianos en S. marcescens en los últimos 50 años, comparando cepas aisladas entre los años 1945-1950 y cepas actuales. ¬Las principales conclusiones obtenidas de este estudio son las siguientes: 1. El resistoma de Serratia marcescens no ha cambiado significativamente desde la era pre-antibiótica hasta la actualidad. 2. La retirada de los antibióticos tiende a recuperar los fenotipos de susceptibilidad originales, independientemente del mecanismo molecular implicado en la resistencia. 3. Ninguna de las cepas de Serratia estudiadas presentó integrones ni tampoco ß-lactamasas de espectro extendido. 4. La determinación fenotípica de las susceptibilidades de las cepas “antiguas” de Serratia inactivas durante 60 años ha confirmado los resultados obtenidos mediante metagenómica, es decir, los genes de resistencia a los antibióticos ya existían con anterioridad al descubrimiento y uso de los antibióticos. 5. En las cepas clínicas de P. aeruginosa multiresistentes se detectó un integrón de clase 1 que contenía el cassette génico aadB, que codifica la enzima aminoglicósido 2’-O- adeniltransferasa, que confiere resistencia a gentamicina, tobramicina y kanamicina. 6. Las cepas multiresistentes de P. aeruginosa sobreexpresaron los sistemas de reflujo MexAB-OprM y MexXY-OprM. 7. El uso del inhibidor de bombas de reflujo CCCP se debería evitar en los experimentos realizados con P. aeruginosa y probablemente con otras bacterias de metabolismo aeróbico. 8. El meropenem induce la formación de largos bacilos aberrantes capaces de sobrevivir en presencia del antibiótico. Estos bacilos acumulan menos antibiótico que las bacterias normales, y pueden revertir a la forma normal cuando se elimina la presión selectiva. 9. La colistina, la última alternativa terapéutica para luchar contra P. aeruginosa en pacientes con fibrosis quística, es normalmente efectiva, aunque recientemente han surgido casos de resistencia a este agente antimicrobiano. 10. La resistencia a colistina parece estar mediada por propiedades singulares del lipopolisacárido. 11. La colistina produce daños en las membranas lipídicas que pueden ser estudiados mediante técnicas de Black lipid bilayer. Estudios preliminares mostraron la capacidad de la colistina para inducir canales transitorios en las bicapas lipídicas, con cierta dependencia de voltaje. 12. La recuperación de la susceptibilidad al imipenem en P. aeruginosa es más lenta que la adquisición de resistencia, dado que la ventaja selectiva conferida por la resistencia al imipenem en presencia del agente antimicrobiano es fuerte, mientras que la expresión de OprD es probablemente ventajosa sólo bajo ciertas y desconocidas condiciones ambientales.

Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.

La bléomycine est un antibiotique cytotoxique, son potentiel génotoxique est plus important quand elle est utilisée en combinaison avec des agents antinéoplasiques sur le cancer testiculaire, que sur les autres types qui développent souvent une résistance envers la drogue. Notre but consiste alors de mettre en évidence ce mécanisme de résistance en utilisant l’organisme modèle Saccharomyces cerevisiae. Nous avons démontré au sein de notre laboratoire, que les levures délétées au niveau de leur coactivateur transcriptionnel Imp2, présentent une hypersensibilité à la bléomycine, en raison de son accumulation toxique dans la cellule. Ceci suggère que Imp2 pourrait réguler l’expression d’une ou de plusieurs pompes à efflux, capables d’expulser la bléomycine à l’extérieur de la cellule. Pour tester notre hypothèse, nous avons recherché des suppresseurs multicopies capables de restaurer la résistance à la bléomycine chez le mutant imp2, et c’est ainsi que nous avons identifié l'activateur transcriptionnel Yap1. Ce dernier se lie à une région spécifique localisée au niveau du promoteur et permet d’activer l'expression d'un sous-ensemble de gènes, codant pour des pompes à efflux, impliquées dans la résistance aux drogues. Selon la littérature, au moins 27 pompes à efflux ont été identifiées chez la levure Saccharomyces cerevisiae, certaines d’entre elles disposent du site de liaison pour Yap1, tels que Qdr3, Tpo2 et Tpo1. Afin de déterminer si une de ces pompes expulse la bléomycine, nous avons créé des mutations simples et doubles en combinaison avec IMP2, aussi nous avons verifié si les mutants étaient sensibles à la drogue et enfin, nous avons testé si la surexpression de Yap1 pouvait restaurer le phénotype sauvage chez ces mutants, via l’activation de pompes à efflux. / Bleomycin is a cytotoxic antibiotic that, when used in combination with antineoplastic agents, has more genotoxic potential on testicular cancer than other types of cancer, which often develop resistance to the drugs. Our goal is to identify the resistance mechanism, using the organism Saccharomyces cerevisiae as a model. In our laboratory, we have demonstrated that deleted yeast strains on their transcriptional coactivator Imp2 have presented hypersensitivity to bleomycin due to the toxic accumulation inside the cell. This led us to believe that Imp2 might regulate the expression of one or more efflux pumps capable of expelling bleomycin outside the cell. To test our hypothesis, we sought multi-copy of suppressors capable of restoring bleomycin resistance in the mutant imp2. As a result we identified the transcriptional activator Yap1, which binds to a specific region within the promoter and activates the expression of subset of genes, encoding efflux pumps that are involved in drug resistance. Based on the literature, at least 27 efflux pumps have been identified in Saccharomyces cerevisiae. Some of these efflux pumps have binging sites for Yap1; such as Qdr3, Tpo2 and Tpo1. To determine whether or not one of these pumps expelled bleomycin, we proceded by single and double mutations in combination with IMP2. We also verified if these single and double mutants were sensitive to the drug, and then we have examined whether the overexpression of Yap1 could restore the wild phenotype in these mutants through the activation of efflux pumps.

The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Amplification of the receptor tyrosine kinase ErbB-2 has been linked to the proliferation of breast cancer cells.1,2 Trastuzumab targets the extracellular domain of ErbB-2, leading to growth inhibition of approximately 15% of the breast cancers with genomic amplification of the ERBB2 gene.3 Clinical studies have demonstrated its efficacy in both early4 and metastatic breast cancers. 5,6 However, many tumors with ERBB2 amplification are not responsive to treatment.7 Moreover, the ones that initially respond, eventually progress and acquire drug resistance.8 An in vitro model for this acquired resistance was established by Chan & al.9 The breast cancer cell line, BT474, containing amplified ERBB2, was grown in the presence of trastuzumab for several months until subclones outgrew. Gene expression profiling was performed on these clones to determine differentially expressed genes between the parental and resistant cells. DARPP-32 (Dopamine and cAMP regulated phosphoprotein of 32kDa) was, by far, the most overexpressed transcript. DARPP-32 is coamplified with ERBB2 on the same amplicon of chromosome 17.10 This protein has been mostly described in neurobiology, but DARPP-32 overexpression was recently reported in gastrointestinal, esophageal, prostate and breast cancer.11 Therefore, we suggest that overexpression of DARPP-32 can cause acquired resistance of breast cancer cells to trastuzumab. The in vitro knockout of DARPP-32, using stable shRNA transfection, abolishes the resistance to trastuzumab in the clones, while overexpression of DARPP-32 in the parental cells results in de novo resistance. Overall, our results suggest that DARPP-32 may be a potential therapeutic target in breast cancer patients who develop acquired trastuzumab resistance.