Poly(A) polymerase (PAP) is the enzyme catalyzing the synthesis of the adenine tail to the 3’-end of mRNA. This A-tail is present on the majority of the primary RNA transcripts of protein-coding genes, and is important for mRNA stability, export to the cytoplasm and translation. Therefore, PAP is a key regulator of eukaryotic gene expression. This thesis describes the heterogeneity of PAP and the functional significance of multiple isoforms of PAP. PAP exists in many different isoforms generated by three different mechanisms, gene duplication, alternative mRNA processing and post-translational modification. In HeLa cell extracts three different forms of PAP being 90, 100 and 106 kDa in size have been detected, where the 106 kDa isoform is a phosphorylated version of the 100 kDa species. It is shown that the N-terminal region of PAP contains a region required for catalysis, while the C-terminal end is important for the interaction with the cleavage and polyadenylation specificity factor (CPSF). Interestingly, it was found that also the extreme N-terminal end is important for the interaction with CPSF. This region is post-translationally modified by phosphorylation. Five alternatively spliced forms of PAP mRNAs are encoded by the PAPOLA gene while one unique species is encoded by the PAPOLG gene. The analysis showed that the exact structure of the alternatively spliced C-terminal end of PAP played an important role for catalytic efficiency. Thus, the C-terminal end contains a region important for modulating the catalytic efficiency of PAP. Aminoglycoside antibiotics inhibit PAP activity, most likely by displacement of catalytically important divalent metal ions. Data shows that different aminoglycosides inhibit PAP activity by different mechanisms suggesting that the binding sites for the different aminoglycosides do not completely overlap. It is concluded that aminoglycosides interfere with enzymes important for housekeeping functions in mammalian cell, which may explain some of the toxic side effects caused by aminoglycoside antibiotics in clinical practice.

Background: In Canada, the prevalence of cervical cancer in Aboriginal women continues to increase with a significantly higher mortality rate than women of non-Aboriginal ancestry. Despite that invasive cervical cancer is highly preventable with regular cervical cancer screening, participation in screening remains lower among Aboriginal women. Since limited information exists on the way cervical cancer screening is perceived and experienced, the purpose of this study was to gain an understanding of Saskatchewan Aboriginal womens perceptions related to and experiences with cervical cancer screening.<p> Methodology: This qualitative exploratory study used an interpretive descriptive approach. Perceptions related to and experiences with cervical cancer screening were elicited through individual interviews with eleven Dakota First Nation women. Women were recruited through purposive sampling techniques. Initially direct quotes from individual transcripts were coded, and then organized with other participant quotes that reflected thematic similarities.<p> Findings: Shared insights reflected a main theme that described perceptions of, experiences with, and factors influencing cervical cancer screening participation among Saskatchewan Aboriginal women. This theme, transitioning from experiencing the negatives of cervical cancer screening participation to living healthier, consisted of the womens mind-set (attitudes and cultural beliefs), knowledge, encounters with the health care system, and sharing information across the generations, which included patterns of communication and a community oriented approach.<p> Discussion: Findings of this study suggest that improving knowledge about cervical cancer screening and cervical cancer may increase screening utilization. Emphasis on health promotion and prevention should be considered when designing education programs. Interventions designed to improve screening rates are more effective when community members are involved in each phase, ensuring cultural relevance. Improving knowledge about, experiences with, and stories shared regarding cervical cancer screening among Aboriginal women may increase screening rates.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

AtPAP15 is one of the purple acid phosphatases expressed by Arabidopsis thaliana that has been extensively studied. Purified AtPAP15 has been shown to exhibit both phytase and phosphomonoesterase activities in acidic pH with maximal activity at pH 4.5. AtPAP15 is a phosphorus starvation inducible (PSI) gene that is expressed highly during phosphorus deficient conditions. In the current study, AtPAP15 was overexpressed in Nicotiana tabaccum under cauliflower mosaic virus (CaMV35S) constitutive promoter. After PCR confirmation of the gene, plants were transferred to the greenhouse and allowed to grow in pots. The pots contained Sta-Green potting mix (Lowe’s Inc., Mooresville, North Carolina,U.S.). Individual flowers were covered to ensure selfpollination. Seeds that were generated (T1 generation) were used for functional analyses. The T1 seeds were germinated in different phosphorus conditions- phosphorus deficient (0mM), optimum inorganic phosphorus (1.25mM), high inorganic phosphorus (20mM) and high organic phosphorus (20mM AMP and IHP)- and analyzed for total biomass, primary root length, soluble phosphorus (Pi) content and acid phosphomonoesterase (Apase) activity. Results demonstrated that under high organic phosphorus conditions, transgenic lines presented increased biomass, longer primary root length, high total soluble phosphorus and high Apase activity than the wild-type. These findings will have important implications for soils with high phosphorus content since most phosphorus in soil is available in organic form. As part of this thesis, Agrobacterium-mediated in planta transformation protocol for transformation of Medicago sativa (alfalfa) was standardized. The protocol would be used to develop transgenic alfalfa expressing AtPAP15. In this protocol, shoot apical position of 2d old seedling is excised followed by infection by Agrobacterium tumefaciens strain EHA 105 for TDNA transfer. The construct used was pCAMBIA1302 harboring mGFP reporter gene. The construct also contained kanamycin, rifampicin and chloramphenicol selection markers. Confirmation of mGFP expression in the transformed alfalfa seedling was performed using Zeiss microscope fitted with GFP filters. Results showed that this protocol is replicable and efficient. In planta transformation method, therefore, may be used for the development transgenic alfalfa.

Cervical cancer is a disease that annually claims the lives of over a quarter of a million women. A substantial number of these deaths could be prevented if population wide cancer screening, based on the Papanicolaou test, were globally available. The Papanicolaou test involves a visual review of cellular material obtained from the uterine cervix. While being relatively inexpensive from a material standpoint, the test requires highly trained cytology specialists to conduct the analysis. There is a great shortage of such specialists in developing countries, causing these to be grossly overrepresented in the mortality statistics. For the last 60 years, numerous attempts at constructing an automated system, able to perform the screening, have been made. Unfortunately, a cost-effective, automated system has yet to be produced. In this thesis, a set of methods, aimed to be used in the development of an automated screening system, are presented. These have been produced as part of an international cooperative effort to create a low-cost cervical cancer screening system. The contributions are linked to a number of key problems associated with the screening: Deciding which areas of a specimen that warrant analysis, delineating cervical cell nuclei, rejecting artefacts to make sure that only cells of diagnostic value are included when drawing conclusions regarding the final diagnosis of the specimen. Also, to facilitate efficient method development, two methods for creating synthetic images that mimic images acquired from specimen are described.

Bacterial Vaginosis (BV) is a disorder of unknown etiology, characterized by a foul smelling vaginal discharge, loss or reduction of the normal vaginal Lactobacilli, and overgrowth of other anaerobic bacteria. Thus, it presents a formidable problem for clinicians as well as microbiologists researching its etiology, clinical course, treatment, and epidemiology. The present work focuses on the unresolved issues of the epidemiology and treatment of BV in order to provide valid methods for treatment studies of this condition and to describe the prevalence of BV in defined populations. The first study validates the use of PAP-stained smears in the diagnosis of BV. The study assesses the methods of Amsel’s clinical criteria and Nugent criteria on Gram-stain smears, against Pap-stained smears and also validates different observers. The result shows that the PAP-staining of vaginal smears is a good method in BV diagnosis; the kappa value is 0.86 (interobserver weighted kappa index) compared to 0.81 for Gram-stained smears, and 0.70 for rehydrated air-dried smears using the mean Nugent score as the criterion standard. This enables population based studies on archived PAP-stained smears from the screening of cervical cancer. In the second study, we use the knowledge gained from study one to investigate the prevalence of BV in a cohort from the population of Åland. The prevalences of BV on the Åland Islands were: 15.6 %, 11.9 %, 8.7 %, and 8.6% in 1993, 1998, 2003, and 2008, respectively. This means that the prevalence of BV decreased between1993-2008 from 15.6% to 8.6%. The confidence intervals are not overlapping, thus indicating a significant decrease in prevalence from 1993 to 2008. The third study is a prospective, double-blind placebo controlled treatment study of BV. After conventional treatment with clindamycin, the patients were treated with adjuvant treatment of Lactobacilli-loaded tampons or placebo. The study showed no differences between the treatment and the placebo group, indicating that the tampon does not work at all. There are a variety of possible explanations for the result, which are analyzed in this thesis. The fourth study aimed to evaluate whether clindamycin is retained for a long time in the vaginal mucosa, thus disturbing the Lactobacilli in an attempt to reimplant Lactobacilli in the probiotic treatment studies. In conventional treatment, it is also useful to know whether clindamycin is retained, especially when considering the pressure from antibiotics on the antimicrobial sensitivity pattern. In the study, we found that the clindamycin disappears rapidly. Conclusion: BV research requires effort from many different scientific disciplines and the riddle of this condition and its treatment can only be resolved by concerted actions in research and treatment. The vision for the future includes, among other factors, better molecular biology based diagnostic tools, and knowledge of population based bacterial floras.

By 1970, Singapore’s urban landscape was dominated by high-rise blocks of planned public housing built by the People’s Action Party government, signifying the establishment of a high modernist nation-state. A decade earlier, the margins of the City had been dominated by kampongs, home to semi-autonomous communities of low-income Chinese families which freely built, and rebuilt, unauthorised wooden houses. This change was not merely one of housing but belied a more fundamental realignment of state-society relations in the 1960s. Relocated in Housing and Development Board flats, urban kampong families were progressively integrated into the social fabric of the emergent nation-state. This study examines the pivotal role of an event, the great Kampong Bukit Ho Swee fire of 1961, in bringing about this transformation. The redevelopment of the fire site in the aftermath of the calamity brought to completion the British colonial regime’s ‘emergency’ programmes of resettling urban kampong dwellers in planned accommodation, in particular, of building emergency public housing on the sites of major fires in the 1950s. The PAP’s far greater political resolve, and the timing of and state of emergency occasioned by the scale of the 1961 disaster, enabled the government to rehouse the Bukit Ho Swee fire victims in emergency housing in record time. This in turn provided the HDB with a strategic platform for clearing other kampongs and for transforming their residents into model citizens of the nation-state. The 1961 fire’s symbolic usefulness extended into the 1980s and beyond, in sanctioning the PAP’s new housing redevelopment schemes. The official account of the inferno has also become politically useful for the government of today for disciplining a new generation of Singaporeans against taking the nation’s progress for granted. Against these exalted claims of the fire’s role in the Singapore Story, this study also examines the degree of actual change and continuity in the social and economic lives of the people of Bukit Ho Swee after the inferno. In some crucial ways, the residents continued to occupy a marginal place in society while pondering, too, over the unresolved question of the cause of the fire. These continuities of everyday life reflect the ambivalence with which the citizenry regarded the high modernist state in contemporary Singapore.

Univ., Diss.--Köln, 2001.

Freiburg i. Br., Univ., Diss., 2008.