Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disease characterized by cerebellar ataxia and visual problems due to a progressive and selective loss of neurons within the cerebellum, brainstem and retina. The disease is caused by the expansion of a CAG repeat in the first coding exon of the SCA7 gene, resulting in an expanded polyglutamine domain in the N-terminal part of ataxin-7, a protein of unknown function. To expand our knowledge of the ataxin-7 protein and the mechanism by which mutant ataxin-7 causes disease, we have studied the expression and function of both the normal and the mutated ataxin-7 protein. Ataxin-7 expression was examination in brain and non-CNS tissues from SCA7 patients and age-matched controls. Expression was predominantly nuclear in neurons throughout the brain of both healthy and SCA7 individuals. We also observed aggregation of mutant ataxin-7 in the nuclei of neurons. No obvious difference in the expression level of ataxin-7 or the formation of aggregates could be observed between affected and non-affected brain regions in SCA7 patients. Based on these findings, we could conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression levels or to the formation of ataxin-7 aggregates. To widen our studies on ataxin-7 expression, we isolated and characterized the mouse SCA7 gene homolog. Cloning of the mouse SCA7 gene revealed two SCA7 mRNA isoforms that were highly homologous to their human counterparts. Immunohistochemical analysis also revealed a conserved expression pattern of ataxin-7 in adult mouse brain. In addition, ataxin-7 expression was observed during embryonic development in brain as well as in several non-neuronal tissues such as heart, liver and lung. Besides SCA7, eight neurodegenerative disorders are known to be caused by expanded polyglutamine repeats, including SCA 1-3, 6 and 17, DRPLA, SBMA and Huntington’s disease. The polyglutamine disorders have many features in common and a common pathological disease mechanism involving transcriptional dysregulation has been proposed. To investigate the possible involvement of transcriptional dysregulation in SCA7 pathology, we analyzed the effects of both wild-type and expanded ataxin-7 on transcription driven by the co-activator CBP, the Purkinje cell-expressed nuclear receptor RORα1 or a basic TATA promoter. As previously shown for other polyglutamine disease proteins, expansion of the polyglutamine domain in ataxin-7 leads to reduced transcription. Surprisingly, strong repression of CBP-mediated, RORα1-mediated and basal transcription was also observed with wild-type ataxin-7, suggesting that the normal ataxin-7 protein may have a role in transcriptional regulation.

Human Papillomavirus (HPV) is a key factor in the development of cervical cancer. Out of the more than 100 known HPV types 13 are considered oncogenic. In addition to presence of the virus several other factors have been proposed to influence risk of cervical cancer. This thesis focuses on viral load and HLA class II alleles as risk factors for cervical cancer. To enable quantification of the most common oncogenic HPV types, a real-time PCR-based assay was developed and evaluated in terms of technical sensitivity and specificity. This assay was then employed on archival smears from 457 cases and 552 controls to assess associations between viral load and cervical carcinoma in situ (CIS). Whereas the data indicate a pronounced dose dependent effect of HPV 16 load on the risk of CIS, other HPV types only seem to increase CIS risk at higher viral loads. These effects were observed even when cytology indicated that cells were normal. We then investigated viral load as a risk factor for invasive cervical carcinoma (ICC) in a retrospective study comprising 139 cases and 550 controls. Viral load contributed similarly to the risk of ICC as to the risk of CIS. Finally, associations between HLA class II alleles, viral load and CIS were investigated. Carriers of the DRB1*1301 allele were less prone to infections and high viral loads of HPV 31 and -18/45. Moreover, DRB1*1301 had a protective effect against CIS among women infected by HPV 31 or -18/45. In contrast, carriers of DRB1*1501 and DQB1*0602 were more susceptible to infections and high viral loads of HPV 16. These results indicate that HPV load may have HPV-type specific effects on cervical cancer risk. Furthermore, HLA class II alleles may confer either susceptibility or protection against cervical cancer by acting on the HPV infections preceding tumor development.

The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment. We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002. In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes. The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.

Globally of today, acquired immunodeficiency syndrome (AIDS) and malaria are two of the most threatening diseases known to mankind. The World Health Organization estimated that AIDS and malaria together claimed nearly 4 million lives in 2003 and many more were infected by the causative agent human immunodeficiency virus (HIV) and the Plasmodium falciparum (P. falicparum) parasite. Current treatment regims for HIV and P. falicparum infections are undermined by rapid emergence of drug-resistant strains and severe drug side-effects. A resistance mechanism of the commonly selected K103N RT mutant towards three second generation non-nucleoside RT inhibitors (NNRTIs) is presented based on X-ray structures. Subtle changes in contacts between inhibitor and residue in position 103 aided the design of improved inhibitors. For the PR target, attempts have been made to structurally assist the development of diol-based protease inhibitors (PIs) with the aim of improving the anti-viral potency without reducing the inhibitory efficacy. It was shown that ortho- and meta-fluoro-substituted P1/P1’-benzyloxy side chains improved the anti-viral potency without affecting the accommodation to the S1/S1’ subsites. The apparent increase in malaria resistance makes drug interventions of current targets increasingly complicated. A prominent new drug target is found in the parasite’s hemoglobin degradation pathway – the aspartic protease plasmepsin II (Plm II). The usefulness of Plm II as an anti-malarial target is presented supported by Plm II complexed with a novel inhibitor. Structurally it is shown that bulky P1- and P3-side-chains adopt a novel binding mode to the Plm II binding cleft with implications for further inhibitor development.

Ribosomes stalled on problematic mRNAs can be rescued by a mechanism called trans-translation. This mechanism employs a dual transfer-messenger RNA molecule (tmRNA) together with a helper protein (SmpB). In this work we have used an in vitro translation system with pure components to further clarify the roles of tmRNA and SmpB in trans-translation. We found that SmpB binds ribosomes in vivo and in vitro independently of tmRNA presence and is essential for tmRNA binding and trans-peptidation. We show that two SmpB molecules can bind per ribosome, that SmpB does not leave the ribosome after trans-peptidation and that SmpB pre-bound to the ribosome can trigger trans-translation. We demonstrated that the rate of trans-transfer of a peptide from the P-site tRNA to Ala-tmRNA and the efficiency by which Ala-tmRNA competes with peptide release factors decrease with increasing the mRNA length downstream from the P site of the ribosome. We showed that trans-translation is strongly stimulated by RelE cleavage of A-site mRNA. We concluded that tmRNA action in vivo must always be preceded by mRNA truncation. We showed that rapid release of truncated mRNAs from the ribosome requires translocation of the peptidyl-tmRNA into the ribosomal P site, which is strictly EF-G dependent. mRNA release is slowed down by strong Shine and Dalgarno like sequences upstream the A site and by long 3’-extensions downstream from the P-site codon. Footprinting was used to monitor SmpB binding to tmRNA, ribosomes and subunits and to study tmRNA interactions with the ribosome at distinct trans-translation stages. We confirmed that two SmpB molecules bind per ribosome and interact with nucleotides below the L7/L12-stalk on the 50S subunit and near the subunit interface on the 30S. We showed that tmRNA is mostly in complex with SmpB in vivo and during trans-translation. Specific cleavage patterns of tmRNA were observed at different stages of trans-translation, but the overall tmRNA conformation seems to be maintained during the whole process.

Resistance to antimicrobials is an increasing problem in the world of today, and develops faster than man can counter. It is therefore of importance to study metabolic pathways in order to develop new antibiotics, but also to understand how resistance spreads and stabilizes in microbial populations. The commensal flora could be an important factor in the spread of antimicrobial resistance, as drugs aimed at other targets also hit the harmless commensal bacteria. If stable resistance develops in such a population, it could seriously impair a later treatment with the same drug. After a treatment with the macrolide clarithromycin, resistance to this antibiotic increased markedly in the untargeted throat flora, and resistance levels did not recede until at least one year later. Another example of stable resistance can also be seen in sulfonamide resistant Streptococcus pyogenes. Sequence determinations of the dihydropteroate synthase (dhps) gene conferring this resistance revealed a mosaic organisation implying that the it had been brought there by horizontal transfer. Molecular characterization of this gene showed that the sulfonamide resistance was due to mutations of structurally important amino acids in position 65 and 213. The folate synthesis pathway has potential for being exploited further as a drug target. One possible new drug target is hydroxymethyl-dihydropterin pyrophosphokinase (hppk). In the malaria parasite Plasmodium falciparum this enzyme is part of a polyfunctional entity, also encoding dhps. The HPPK part can be separated from DHPS, but that the opposite is not possible. The PfHPPK has two insertions: one also present in other plasmodia, and one apparently unique to P. falciparum. Both are crucial for enzyme activity. To further characterize HPPK, we developed a spectrophotometric activity assay and a method to measure substrate channelling of hydroxymethyl-dihydropterin diphosphate.

Haptoglobin (Hp) is a serum protein known for its ability to form a tight complex with hemoglobin (Hb) and thereby inhibiting the oxidative activity of Hb. Mammalian Hp is synthesized as a precursor (proHp) that undergoes proteolytic cleavage by a previously unidentified enzyme in the endoplasmic reticulum (ER). In this study, a proHp-cleaving enzyme was isolated from human serum and identified as complement C1r-like protein (C1rLP). Co-expression of C1rLP with proHp in mammalian cells resulted in cleavage of the latter protein in the ER. Mutation of either the active site serine residue in C1rLP or the arginine residue in the cleavage site of Hp abolished the cleavage of proHp by C1rLP. RNAi studies in mammalian cells identified the proHp-cleaving enzyme as C1rLP. Hp has been found in all mammals studied to date but its presence in non-mammalian species has not been unambiguously shown. By searching currently available genomic DNA and cDNA sequence databases, a gene orthologous to mammalian Hp was found in bony fish. Hp-like protein expressed from this gene was demonstrated to be a major Hb protein in fish serum. Surprisingly, no Hp-like gene was found in the genomes of either frog or chicken. In chicken, a protein previously described as Hp was identified as PIT54, a member of a scavenger receptor cysteine-rich family of proteins. Interestingly, ostrich serum seemed to contain two Hb-binding proteins; one similar to PIT54 and one to mammalian Hp. We are not aware of any other case where the function of one gene has been taken over by another, completely unrelated gene Fish Hp (fHp) is composed of a serine proteinase-related domain preceded by an extension consisting of several aminoa acids and a signal peptide. The extension contains a consensus motif for cleavage by subtilisin-like proprotein convertases (SPCs). fHp was found to be cleaved by SPCs in the Golgi complex. Collectively, this thesis presents evidence that Hp has undergone significant changes during evolution with respect to its molecular organization and to the mechanism of its proteolytic cleavage.

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is expressed in a parent of origin specific manner. Most of the imprinted genes are located in clusters. Genetic evidences suggest that genes in imprinted clusters are regulated by Imprinting Control Regions (ICRs). To elucidate the mechanisms by which the imprinting is maintained in clusters, we have chosen a well characterized cluster at the distal end of mouse chromosome 7. This cluster contains 15 imprinted genes and they have been shown to be regulated by H19 and Kcnq1 ICRs. The mouse H19 ICR, which is shown to have a chromatin insulator function, is implicated in the regulation of H19 and Igf2 genes by interacting with the CTCF protein. It has been documented that CTCF is also involved in the maintenance of differential methylation at the ICR. In this investigation we demonstrated that CTCF maintained differential methylation is lost when we subjected the ICR containing episomal plasmids to de novo methylation machinery of the human choriocarcinoma cell line, JEG3, suggesting that the H19 ICR looses its methylation privilege property under neoplastic conditions. The Kcnq1 ICR has been implicated in the regulation of 11 imprinted genes. The Kcnq1 ICR is methylated on the active maternal allele but unmethylated on the inactive paternal allele and overlaps an oppositely oriented and paternally expressed gene known as Kcnq1ot1. In this investigation, we documented that the Kcnq1 ICR controls the imprinting of neighboring genes by behaving as a bidirectional silencer and that this function is regulated by antisense RNA Kcnq1ot1. Furthermore, we have documented that duration of antisense transcription plays a critical role in the antisense RNA- mediated silencing. In conclusion, this thesis provides more insights into the complex mechanistic aspects by which ICRs, control imprinting of genes in clusters during development and neoplasia.

The members of the ubiquitously expressed E-protein subfamily of basic Helix-Loop-Helix (bHLH) transcription factors, E12/E47, SEF2-1 and HEB, have important roles as regulators of gene expression in various differentiation processes, including lymphocyte development and myogenesis. In myogenesis, E-proteins are proposed to function as obligate heterodimer partners for members of the MyoD family of muscle-specific bHLH transcription factors. The calcium ion (Ca2+) is a universal cellular messenger involved in regulation of a variety of cellular functions, including transcription. The Ca2+-bound form of the Ca2+-binding protein calmodulin (Ca2+/CaM) has been shown to inhibit DNA binding of E-proteins, but not tissue specific bHLH transcription factors, through direct physical interaction with the DNA binding basic sequence. The main focus of this thesis is on the role of Ca2+-binding proteins in regulation of bHLH transcription factors. Solution structure analysis of CaM in complex with the CaM-binding basic sequence of an E-protein revealed a novel type of protein-protein interaction with alternative binding modes in a complex of a CaM dimer surrounding the dimer of the E-protein sequence. This model for the interaction was further supported by mutational analysis, since every amino-acid substitution in the CaM binding basic sequence of E12 only partially affected the interaction with CaM. The mechanism of Ca2+/CaM regulation of transcriptional activation by E-proteins was studied using a cell culture system. CaM overexpression inhibited transcriptional activation by E12, E47 and SEF2-1 but not by MyoD. Ca2+/CaM inhibition of DNA binding in vitro directly correlated with the inhibitory effects of Ca2+ stimulation and CaM overexpression on transcription in vivo in a series of E12 basic sequence mutants. Furthermore, in vivo DNA binding of E12, but not a CaM resistant mutant of E12, was inhibited by overexpression of CaM. The data indicate that Ca2+/CaM can inhibit transcriptional activation by E-proteins through formation of a CaM-E-protein complex that can not bind DNA. An in vitro myogenesis system was used to investigate the potential role of the CaM-E-protein interaction in regulation of differentiation. CaM resistant mutants of E12 were inhibitory in MyoD initiated myogenic conversion of transfected fibroblasts, and inducers of intracellular Ca2+ activated, and Ca2+-channel blockers inhibited, transcriptional activation by E12, but not by a CaM resistant mutant of E12, with MyoD. The data support a model that Ca2+/CaM plays a role in initiation of myogenic differentiation through inhibition of E-protein dimers that can function as competitors to the CaM resistant MyoD/E-protein heterodimers required for myogenesis. The potential involvement of the Ca2+-binding calretinin proteins in regulation of bHLH transcription factors was also studied. Calretinin and the alternative splice variant calretinin-22k have been proposed to function as Ca2+-buffer proteins. Calretinin expression is restricted primarily to neuronal tissues. Calretinin and calretinin-22k are also found expressed in colon cancers, but not in normal colon tissue, and a role for calretinins in tumorigenesis has been proposed. We show that calretinins can inhibit DNA binding and transcriptional activation by E12 through basic sequence interaction. Endogenous E12/E47 and calretinin co-localize in a subset of cells in a proliferating colon cancer cell line and can be co-immunoprecipitated from the cell extract. A model is proposed in which calretinin overexpression can contribute to tumorigenesis through inhibition of the anti-proliferative function of E-proteins. The role of the E-protein E2-2 in lymphocyte development was studied using genetically altered mice with mosaic deletion of the E2-2 gene. The proportion of cells with a functional E2-2 allele was increased in the B- and T-lymphocyte populations, indicating a role for E2-2 not only in B-cell development, as reported before, but also in T-cell development.

Fungi are present everywhere in indoor and outdoor environments. Many fungi are toxigenic or pathogenic that may cause various public health concerns. Rapid detection, quantification and characterization of fungi in living and working environments are essential for exposure risk assessment to safe guard public health. Rapid and accurate detection and identification of fungi using molecular method require specific markers. In this thesis, partial mt SSU and LSU rDNA were amplified and sequenced from 31 fungal species of 16 genera. Sequence alignments showed that fungal mt SSU and LSU rDNA contained sufficient amount of variation for the development of markers that can discriminate even among closely related species. Forty-eight probes were designed and were verified as highly specific to 25 fungal species commonly detected in living and working environments. These specific probes would have potential applications in clinical diagnosis and public health-related environmental monitoring. Nested PCR is a highly sensitive and specific method. Based on the nuclear 18S rDNA sequence variation pattern, three nested PCR systems were developed to detect the conifer tree pathogen Gremmeniella abietina, an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The three nested PCR systems showed high specificity and sensitivity. These methods could have broad applications in forest protection and disease management programs. Quantitative real-time PCR offers the ability of simultaneous detection and quantification of DNA of a specific microbe in one reaction. Based on the 18S rDNA sequence, two real-time PCR assays were developed to detect and quantify Wallemia sebi, a deuteromycete fungus commonly found in agricultural environments and is suspected to be a causative agent of farmer’s lung disease. Both PCR systems proved to be highly specific and sensitive for W. sebi detection even in a high background of other fungal DNAs. Application of the real-time PCR methods in the quantification of W. sebi in the aerosols of a farm revealed a high concentration of W. sebi spores (107/m3). The study indicates that W. sebi is a dominant fungus in agriculture environments. Cladosporium spores are important aeroallergens, and prolonged exposure to elevated spore concentrations can provoke chronic allergy and asthma. A TaqMan probe and a SYBR Green I based real-time PCR assay were developed to detect and quantify Cladosporium in aerosols. The two real-time PCR systems proved to be highly specific and sensitive for Cladosporium. These methods were employed to quantify Cladosporium in aerosols of five different indoor environments. High spore concentration of Cladosporium (107/m3) was observed in a cow barn. Cladosporium spore concentration in paper and pulp factory and countryside house also exceeded threshold value for clinical significance. Prolonged exposure in these environments could impose certain health risk. Thus, monitoring Cladosporium spore concentration in indoor environments is important for indoor air quality control.