Mastergradsoppgaver i biologi (Helsefak)

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Mastergradsoppgaver i biologi (Helsefak)

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• Partial characterization of predicted ABCC5 inhibitors by the aid of human erythrocyte inside-out vesicles

  Ørvoll, Elin (Master thesis; Mastergradsoppgave, 24-Jun-2011)

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  Abstract:

  ABCC5 is a member of the superfamily of ABC-transporters, and it has been identified as an efflux transporter of cGMP. This transporter is also involved in export of antibiotic and cytostatic drugs from target cells, and as such represents a challenge in treatment of cancer and infectious diseases. In order to find inhibitors to ABCC5 mediated drug efflux, compounds predicted as potent inhibitors by virtual ligand screening (VLS) were chosen for in-vitro studies by the use of human erythrocyte inside-out vesicles (IOV). The procedure for IOV preparation was improved, and transport assays were performed where the inhibiting effects of the various compounds on transport of cGMP into inside-out vesicles were measured. Several of these compounds showed a potent inhibiting effect on cGMP transport, and the few that were chosen for further characterization showed more potent inhibition of ABCC5 than the known ABCC5 and PDE5 inhibitor sildenafil.

  URI:

  http://hdl.handle.net/10037/5137

  File(s) in this item: 1

  thesis.pdf   (2.443Mb)
• Molecular characterization of Norwegian clinical isolates of Escherichia coli hyperproducing the chromosomal AmpC beta-lactamase : a regional spread of an IS911-mediated blaAmpC-hyperexpressing ST131 clone

  Ramberg, Cathrine Caspersen (Master thesis; Mastergradsoppgave, 15-May-2012)

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  Abstract:

  The worldwide dissemination of antimicrobial resistance is a growing problem causing increased morbidity, mortality, and financial costs. β-lactams are an important family of antimicrobial agents and accounts for ~46% of the total antibiotic use for systemic infections in Norway. Resistance to β-lactams can be caused by several factors where the production of enzymes, β-lactamases, is the major mechanism. Escherichia coli naturally produce small amounts of the chromosomally encoded AmpC β-lactamase. The expression blaAmpC is noninducible and regulated by a weak promoter and an attenuator. Insertion sequence (IS) elements inserted into the promoter region have been described as one reason for the hyperexpression of blaAmpC conferring resistance to β-lactams such as penicillins and cephalosporins, but not 4th generation cephalosporins and carbapenems. In this study 111 E. coli isolates with a hyperexpressed chromosomal AmpC profile were submitted to the Reference Center for Detection of Antimicrobial Resistance (K-res) from Haukeland University Hospital during 2006-2010 and a control group representing the same years from other Norwegian clinical microbiological laboratories (n=100) were included. The isolates were initially screened for an insertion of an element in the blaAmpC. A subset of isolates with an insertion was further molecularly characterized by sequencing of the region and linkage to IS911. Molecular typing was performed using multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Multi-resistance profiles were identified by antimicrobial susceptibility testing and further investigated by PCR and sequencing methods. The results from the study shows a regional clonal spread of ST131 E. coli blaAmpC-IS911 isolates in the Bergen region of Norway. The spread of these isolates were identified both in isolates from Hospital 1 and 2 but also from other medical institutions such as nursing homes and general practitioners. In contrast, no isolates from the control group from other Norwegian hospitals harbored the blaAmpC-IS911 linkage. In the control group only three isolates from two Norwegian counties, Vestfold and Rogaland were identified with an insertion in the blaAmpC region. However, in these isolates another IS-element, IS10 was identified. In the ST131 isolates multi-resistance was observed towards important antibiotics such as ciprofloxacin, gentamicin, tobramycin, and trimethoprim-sulphamethoxazole. Resistance to ciprofloxacin was caused by mutations in the quinolone-resistance determining region of the parC and gyrA genes. The resistance mechanism to the aminoglycosides gentamicin and tobramycin were not identified, but the isolates were negative for the aminoglycoside modifying enzyme AAC(6’)-Ib.

  URI:

  http://hdl.handle.net/10037/4228

  File(s) in this item: 1

  thesis.pdf   (1.964Mb)
• Effects of exogenous hydrogen sulfide administration on cardiac function and reactive oxygen species production : a study in hearts from normal rats and rats with heart hypertrophy or ischemia

  Paunas, Teodora Ioana (Master thesis; Mastergradsoppgave, Sep-2011)

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  Abstract:

  Coronary heart disease is the leading cause of death worldwide. Infarct size can be limited by interventions used after the ischemic event like the use of thrombolytic therapy or primary percutaneous coronary intervention. Paradoxically, however, the return of blood flow can also result in additional cardiac damage and complications, referred to as reperfusion injury. It has been shown that reperfusion injuries can be decreased by postconditioning- rapid intermittent interruptions of blood flow in the early phase of reperfusion, or post-treatment using various drug therapies which applied during reperfusion can reduce infarct size. H2S, a gas that is synthesized in mammalian tissue, has been reported to be cardioprotective during ischemia-reperfusion injury. The means by which H2S is cardioprotective during I/R are believed to be: the opening of the sarcolemmal KATP channel, the generation of antiapoptotic effects inside the cells as well as a direct antioxidant effect. Low levels of reactive oxygen species (ROS) are constantly produce within cells and play important roles in cell signaling, cellular homeostasis, differentiation and apoptosis. However an excessive increase in the level of ROS can be harmful and has been proposed to play crucial roles or contribute in the development of various diseases. The aim of our study was to investigate the effects of H2S in an acute ischemia-reperfusion model and to determine whether exogenous administration of H2S in both healthy rats and rats exposed to experimental models of cardiac disease influenced the production of ROS. In order to do this we established a method trough which we were able to measure the presence of ROS in heart tissue samples harvested from normal rats and rats with heart hypertrophy and ischemic heart disease.

  URI:

  http://hdl.handle.net/10037/3726

  File(s) in this item: 1

  thesis.pdf   (1.535Mb)
• Study of interaction between BK virus large T-antigen and agnoprotein

  Adou, Koman Mireille Sophie Chinan (Master thesis; Mastergradsoppgave, Aug-2011)

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  Abstract:

  Human polyomavirus BK (BKV) is a non enveloped virus with a double-stranded, circular DNA genome. BKV infects >70% of the human population world-wide. Infection occurs predominantly during childhood and the virus remains in a latent state throughout life in the immune competent individuals. In the context of immunosuppression, however, reactivation occurs and can lead to renal stenosis and interstitial nephritis in kidney transplant patients, and hemorrhagic cystitis in bone narrow transplant patients. Moreover, BKV has been associated with several human cancers, but its causal role remains disputed. One of BKV’s protein known as agnoprotein may play a role in these pathogenic processes. To develop antiviral therapy it is required to elucidate the exact biological function of this protein. One way to examine the function of agnoprotein is by identifying possible cellular interaction partners. Another way is to understand agnoprotein’s role in the viral life cycle. Thereto, we examined the interaction of agnoprotein with another viral protein, large T-antigen (LT-ag) and the functional implication of this interaction. First, we investigated the effect of agnoprotein on the transcriptional activity of LT-ag on the BKV early promoter by transient transfection studies in HEK293. Our results revealed that LT-ag affects BKV early promoter in a concentration-dependent manner with low concentrations of LT-ag inhibiting, while high concentrations stimulated BKV early promoter activity. Co-expression of agnoprotein repressed LT-ag-induced activation of the BKV early promoter, suggesting that agnoprotein may exert a negative regulatory effect on transactivation by LT-ag. To test whether agnoprotein mediates its effect through direct interaction with LT-ag, we studied a possible association between these proteins. GST pulldown, co-immunoprecipitation (in vivo and in vitro), and mammalian two hybrid studies confirmed an interaction between LT-ag and agnoprotein.

  URI:

  http://hdl.handle.net/10037/3568

  File(s) in this item: 1

  thesis.pdf   (2.113Mb)
• Directed evolution of Escherichia coli LacZ gene to create diversity in glycosidic bonds hydrolysis

  Bohra, Pallavi (Master thesis; Mastergradsoppgave, 16-May-2011)

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  Abstract:

  Starting with LacZ of Escherichia coli, coding for β-galactosidase, the aim of the thesis project is to apply in vitro directed evolution techniques to help create other glycosidic bond hydrolysis activities. This was done using the main β-galactosidase backbone with limited amino acid sequence change. Any altered glycosyl hydrolase activity would lead to changed substrate specificity. Moreover, genetic changes leading to improved beta-galactosidase activity was also investigated. Error-prone PCR was applied to the LacZ gene (β-galactosidase) to achieve the desired aims. The technique used to introduce random mutagenesis was based on modifications of method developed by Xu et al., 1999.Optimization was performed with DNA polymerase selection, PCR conditions and various Mn and dITP concentrations to obtain best amplified PCR product for random mutagenesis library construction. Plasmid pTZ1 containing the entire coding sequence of LacZ was used a whole plasmid random Mutagenesis library construction strategy. The complete pTZ1 plasmid sequence had to be done in order to help establish a framework for primer design and establish a complete restriction map of the plasmid including the lacZ gene. The sequence analysis of the plasmid revealed that it has 5,502bp. Screening of random mutagenesis libraries was based on the colour development resulting from the glycosidic hydrolysis of chromogenic substrate to identify any glycosidic activity towards particular glycosyl hydrolase on LB plates or M9 plates. We have screened random mutagenesis libraries for any possible activity for β-glucosidase, β-xylosidase or for an improved β-galactosidase activity. Colonies that showed colour development on substrate even after retransformation of plasmid DNA for β-xylosidase activity were selected and its mutated plasmid DNA was sequenced. Two of the variants in which one has mutation at K552E position and another at N959Y were isolated, from two different clear blue colonies on β-xylosidase substrate. However, to the issue of change in substrate specificity (colour development on plates) was not clear. The direct evolution method applied here is seems simpler and promising in creating random mutagenesis libraries in order to select variants with useful novel properties.

  URI:

  http://hdl.handle.net/10037/3567

  File(s) in this item: 1

  thesis.pdf   (2.624Mb)

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