<p>In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA.</p><p>The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.</p>

In Molecular Medicine a wide range of methods are applied to analyze the genome to find genetic predictors of human disease. Apart from predisposing disease, genetic variations may also serve as genetic markers in the search for factors underlying complex diseases. Additionally, they provide a means to distinguish between species, analyze evolutionary relationships and subdivide species into strains. The development and improvement of laboratory techniques and computational methods was a spin-off effect of the Human Genome Project. The same techniques for analyzing genomic sequence variations may be used independent of organism or source of DNA or RNA. In this thesis, methods for high-throughput analysis of sequence variations were developed, evaluated and applied. The performance of several genotyping assays were investigated prior to genotyping 4000 samples in a co-operative genetic epidemiological study. Sequence variations in the estrogen receptor alpha gene were found to be associated with an increased risk of breast and endometrial cancer in Swedish women. Whole genome amplification (WGA) enables large scale genetic analysis of sparse amounts of biobanked DNA samples. The performance of two WGA methods was evaluated using four-color minisequencing on tag-arrays. Our in-house developed assay and “array of arrays” format allow up to 80 samples to be analyzed in parallel on a single microscope slide. Multiple displacement amplification by the Φ29 DNA polymerase gave essentially identical genotyping results as genomic DNA. To facilitate accurate method comparisons, a cluster quality assessment approach was established and applied to assess the performance of four commercially available DNA polymerases in the tag-array minisequencing assay. A microarray method for genotyping human group A rotavirus (HRV) was developed and applied to an epidemiological survey of infectious HRV strains in Nicaragua. The method combines specific capture of amplified viral sequences on microarrays with genotype-specific DNA-polymerase mediated extension of capture oligonucleotides with fluorescent dNTPs.

In this thesis, the four-color fluorescence tag-microarray minisequencing system pioneered by our group was further developed and applied for analysing genetic variation in human DNA and RNA. A SNP marker panel representing different chromosomal regions was established and used for identification of informative SNP markers for monitoring chimerism after stem cell transplantation (SCT). The success of SCT was monitored by measuring the allelic ratios of informative SNPs in follow-up samples from nine patients with leukaemia. The results agreed with data obtained using microsatellite markers. Further the same SNP marker panel was used for evaluation of two whole genome amplification methods, primer extension preamplification (PEP) and multiple displacement amplification (MDA) in comparison with genomic DNA with respect to SNP genotyping success and accuracy in tag-array minisequencing. Identical results were obtained from MDA products and genomic DNA. The tag-microarray minisequencing system was also established for multiplexed quantification of imbalanced expression of SNP alleles. Two endothelial cell lines and a panel of ten coding SNPs in five genes were used as model system. Six heterozygous SNPs were genotyped in RNA (cDNA) from the cell lines. Comparison of the relative amounts of the SNPs alleles in cDNA to heterozygote SNPs in genomic DNA displayed four SNPs with significant imbalanced expression between the SNP alleles. Finally, the tag-array minisequencing system was modified for detection of splice variants in mRNA from five leukaemia cell lines. A panel of 20 cancer-related genes with 74 alternatively splice variants was screened. Over half of the splice variants were detected in the cell lines, and similar alternative splicing patterns were observed in each cell line. The results were verified by size analysis of the PCR product subjected to the minisequencing primer extension reaction. The data from both methods agreed well, evidencing for a high sensitivity of our system.

High-risk human papillomaviruses (HPVs) cause 5% of all human cancers worldwide. The HPV capsid consists of 72 disulfide-linked pentamers of major capsid protein L1 and up to 72 molecules of minor capsid protein L2. The viral genome (vDNA) is 8KB circular dsDNA, condensed with histones and complexed with L2. HPV infection requires the virion particle to get access to basal layer keratinocytes, binding and entry of the cells, uncoating, and transport of the viral genomes to the host cell nucleus. During infection, L2 is important for transport of the viral genome from membrane bound vesicular compartments, through the cytosol and into the host cell nucleus. Previous work has identified a conserved disulfide bond between Cys22 and Cys28, which is necessary for HPV16 infection. We hypothesize that endosomal reduction of this disulfide might be important for L2 conformational changes that allow a hydrophobic transmembrane-like region in L2 to span across endosomal membranes, exposing sorting adaptor binding motifs within L2 to the cytosol. Prior research suggests that cytosolic glutathione (GSH) redox potential is important for reduction of disulfide-linked proteins within the lumen of endosomes. This is achieved by endosomal influx of cytosolic reduced cysteine, where it can reduce disulfide bonds in lumenal proteins. Cytosolic GSH regenerates the pool of reduced cysteine needed to maintain endosomal redox potential. Here we studied the relationship between cytosolic GSH and HPV16 infection. siRNA knockdown of critical enzymes of the GSH biosynthesis pathway or the endosomal cystine efflux pump cystinosin caused partial abrogation of HPV16 infection. Likewise, inhibition of the GSH biosynthesis pathway with L-buthionine sulfoximine (L-BSO) blocked HPV16 infection in multiple cell types, suggesting that cytosolic GSH redox may be important for HPV16 infection. Further studies have revealed that the decrease of HPV16 infection is not because of defects in binding, entry, L2 cleavage or capsid uncoating, but rather is due to inefficient cytosolic translocation of L2/viral genome from the trans-Golgi network (TGN). Contrary to our initial hypothesis, we show that L2 is able to span the endosomal membrane and direct TGN localization in the presence of BSO. Lack of cytosolic GSH causes L2/viral genome to become trapped in the TGN lumen. This suggests that there are redox-sensitive viral or cellular factors necessary for L2/viral genome translocation at the TGN. Future research will focus on the redox state of the Cys22-Cys28 disulfide bond during infection of normal and GSH-depleted cells.

Vascular endothelial growth factor-B (VEGF-B), when initially discovered, was thought to be an angiogenic factor, due to its intimate sequence homology and receptor binding similarity to the prototype angiogenic factor, VEGF-A. Studies demonstrated VEGF-B, unlike VEGF-A, did not play a significant role in angiogenesis or vascular permeability and has become an active area of interest because of its role as a survival factor in pathological processes in a multitude of systems, including the brain. By characterization of important downstream targets of VEGF-B that regulate different cellular processes in the nervous system and cardiovascular system, it may be possible to develop more effective clinical interventions in diseases such as Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS) and ischemic heart disease, which all share mitochondrial dysfunction as part of the disease. Here we summarize what is currently known about VEGF-B function in pathological processes, compare probable mechanisms of action and elude to its potential as a homeostatic protective factor to increase mitochondrial function in the setting of neurological disease and cardiovascular disease.

Human papillomaviruses (HPV) are among the most common sexually transmitted infections and are responsible for 5% of all human cancers. HPV type 16 is the most prevalent of the high-risk HPVs (a subgroup of HPVs with potential to cause cancer), accounting for ~55% of HPV-associated cancers. HPV16 is a nonenveloped virus, composed of the major capsid protein L1, the minor capsid protein L2, and a circular double-stranded DNA genome (vDNA) condensed with human histones. HPV initially infects undifferentiated basal keratinocytes and viral replication is dependent on epithelial differentiation. Like many other DNA viruses, HPV must deliver its vDNA to the host cell nucleus to successfully replicate. Initial binding of HPV16 to host cells is through L1 interactions with cell surface heparan sulfate receptors. Shortly after virus binding, L2 is believed to undergo furin cleavage-dependent conformational changes, resulting in spanning of the protein across the local membrane and exposure of the central and C-terminal regions of L2 (which was lumenal and and inaccessible before furin cleavage) to the host cell cytosol. L2 is critical for transport of the L2/vDNA from endosomes to the trans-Golgi network (TGN). We hypothesize that furin-dependent early L2 spanning, through the direct binding and recruitment of cytosolic sorting factors, may contribute to viral endocytosis and subcellular retrograde trafficking (trafficking from endosomes to Golgi) of vDNA. We have developed a Tac receptor (CD25 or IL2 receptor, a transmembrane cell surface protein) chimera system to study L2-dependent endocytosis and trafficking. In this system the Tac ecto- and transmembrane domains are fused to the ~400 amino acid portion of L2 that is likely cytosolic upon L2 spanning. Through transient expression of Tac-L2 chimera we use anti-Tac ectodomain antibodies to label and track cell surface populations by immunofluorescence and confocal microscopy. We have also adopted this system to study endocytosis through a cell surface biotinylation approach. Both approaches suggest that L2 may enhance endocytosis and preliminary evidence suggests that the Tac-L2 chimera may recruit the cytosolic retromer complex (the host cytosolic factors help protein retrograde trafficking) to preferentially traffic to the TGN. Retromer-dependent trafficking of cargo from early endosomes to the TGN is known to involve certain members of the sorting nexin family, specifically the SNX-BAR proteins. We performed a small siRNA screen and identify SNX6 and SNX32 (aka SNX6b) as SNX-BAR proteins that may be specifically involved in retrograde trafficking of HPV16 L2/vDNA during infection. Future work will focus on the mechanisms through which L2 and SNX6 influence HPV16 entry and trafficking.

<p>Targeted radionuclide therapy (TRT) holds great promise for the treatment of cancer. In TRT, radioactive nuclides are delivered specifically to tumours by molecules that recognise and bind to structures overexpressed by, or specific to, cancer cells. Human epidermal growth factor receptor like protein 2 (HER-2) is an oncogene product overexpressed in e.g. urological, breast, or ovarian cancers that have been correlated to poor prognosis and resistance to hormonal therapy. There is also evidence that tumour cells retain their HER-2 overexpression in metastases.</p><p>Trastuzumab and pertuzumab are two humanised monoclonal antibodies targeting different parts of HER-2. This thesis describes the radiolabelling of these antibodies for use in TRT and diagnostics. The thesis also investigates possible methods for modifying uptake and retention of radioactivity delivered with antibodies binding to HER-2. Modification of the cellular retention of ¹²⁵I by using polyhedral boron anion based linker molecules (DABI and NBI) is investigated, and it is shown that linking ¹²⁵I to trastuzumab using DABI increases cellular accumulation of radioactivity by 33%. It is also shown that trastuzumab can be efficiently coupled to the positron emitter ⁷⁶Br by using NBI. Furthermore, it is shown that cellular uptake of ¹²⁵I can be modified by stimulating EGFR (HER-1) with EGF.</p><p>When labelled with the alpha emitter ²¹¹At, trastuzumab could specifically kill cells in vitro. This cell killing effect could be prevented by saturating the receptors of the target cells with non-radiolabelled trastuzumab.</p><p>Pertuzumab was radiolabelled with the low energy beta emitter ¹⁷⁷Lu without losing affinity or immunocompetence. [¹⁷⁷Lu]pertuzumab was specific to HER-2 in vitro and in vivo. This targeting conjugate was shown to increase median time to tumour progression in mice bearing xenografts of the radioresistant SKOV-3 cell line. </p><p>In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with ¹⁷⁷Lu, show promise as TRT agents.</p>

<p>After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. </p><p>Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.</p>

<p>Osteoporosis is a disease that leads to decreased bone mineral density (BMD), an altered bone micro-architecture and fragile bones. The disease is highly heritable and numerous genes are thought to be involved, making it difficult to identify the causative genetic elements.</p><p>Animal models, mainly intercrosses between laboratory strains of mice, have been succesfully used to map genes affecting these traits, but may not mirror the multifactorial genetic etiology of highly complex traits such as osteoporosis.</p><p>Over the course of tens of thousand years humans have kept domestic animals whose phenotypic repertoires have been tailored to meet our needs. Wild-type red junglefowl (RJ) and domestic White Leghorn (WL) chicken differ for several bone traits. </p><p>In this thesis Quantitative Trait Loci (QTL) mapping was used to trace the inheritance of bone traits in two separate intercrosses between RJ and WL. In these studies we identified several QTL that contributed to differences in BMD, bone size and biomechanical strength of bone. In a comparison of QTL identified in the two intercrosses it was observed that nine QTL had overlapping genomic positions, implicating these loci as important to bone phenotypic variation in chicken.</p><p>In two separate studies, microarray technology was used to compare global gene expression in bone tissue from RJ and WL. In these studies, differential expression was observed for 779 and 560 genes, respectively. Many differentially expressed genes were co-localized with QTL, which implicates them as QTL-candidates. </p><p>Results presented in this thesis link several genomic regions and genes to variation in bone traits. Increased knowledge about these identified genes and regions will contribute to a better understanding of the mechanisms underlying inter-individual differences in bone metabolism, both in chicken and man.</p>

After the completion of the human genome project the human genome was annotated with the surprisingly small amount of 24 000 (www.ensemble.com) genes. This has focused research on the contribution of splice variants, posttranslational modifications and interactions of proteins at the proteome level and other regulatory elements in the cell to fully understand the complexity of functions in a higher organism. Proteomic oriented projects are currently aiming to investigate all the splice variants and posttranslational modifications of all the proteins present in an organism or cell type and annotate their function and interaction partners. Projects on this scale are at the moment difficult to achieve and new methodologies are needed. Proximity ligation assays (PLAs) are based on a novel protein detection strategy that converts the presence of a target molecule in a unique DNA tag through ligation reactions. PLA detection of proteins requires several independent recognition events by affinity reagents that have been converted into proximity probes. Different formats of the proximity ligation strategy have been developed in both heterogeneous and homogeneous format[1-4]. This thesis presents the development of an antibody based proximity ligation approach and the development of a novel proximity ligation based detection strategy named triple specific proximity ligation (3PLA). To extend the range of target molecules we adapted the proximity ligation assay for the use with antibodies by converting matched monoclonal antibody pairs and polyclonal antibody batches into proximity probes and used them for the detection of several cytokines in complex biological fluids. The novel 3PLA requires the simultaneous detection by three independent affinity binders to create one specific DNA based signal. This requirement for triple recognition extends the biological specificity of immunoassays and allows a proximity ligation design with reduced background signal and thus higher sensitivity. We have established proof of principle detection of the biomarkers troponin I and prostate specific antigen (PSA) alone and in complex with 1-alpha-antichymotrypsin (ACT) and detected as little as 100 molecules of vascular endothelial growth factor (VEGF). To further explore the extended biological specificity of 3PLA we adapted the assay for detection of protein complexes formed during NFκB signaling and used this system to profile the mode of action of three small molecular weight inhibitors of the IκB Kinase (IKK). The development of new protein detection methods hold promises for the investigation of complex interactions and mechanism on the proteome level which are not accessible with current technologies. We have developed tools and protocols useful for the development of new proximity ligation strategies and designs. These protocols allow the rapid and low cost custom set up of PLAs without the need for extensive conjugation protocols or purification procedures.