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. 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. 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. 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. 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. 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.

A proteomic approach was applied to examine the protein composition of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in humans. LDL and HDL were isolated by density gradient ultracentrifugation, and proteins were separated with twodimensional gel electrophoresis (2-DE) and identified with peptide mass fingerprinting, using matrix-assisted laser desorption/ionization-time of flight mass spectrometry, and with amino acid sequencing using electrospray ionization tandem mass spectrometry. To improve the identification of low abundant proteins in silver stained 2-DE gels, 2,5-dihydroxybenzoic acid was used instead of α-cyano-4-hydroxycinnamic acid as matrix in the peptide mass fingerprinting procedure; this was demonstrated to give more matching peptide peaks, higher sequence coverage, and higher signal to noise ratio. Altogether 18 different proteins were demonstrated in LDL and/or HDL: three of these (calgranulin A, lysozyme C and transthyretin) have not been identified in LDL before. Apo C-II, apo C-III, apo E, apo A-I, apo A-IV, apo J, apo M, serum amyloid A-IV and α1-antitrypsin were found in both LDL and HDL, while apo B-100 (clone), calgranulin A, lysozyme C and transthyretin were found only in LDL, and apo A-II, apo C-I, and serum amyloid A only in HDL. Salivary α-amylase wass identified only in HDL2, and apo L and glycosylated apo A-II only in HDL3. Many of the proteins occurred in a number of isoforms: in all, 47 different isoform identities were demonstrated. A 2-DE mobility shift assay and deglycosylation experiments were used to demonstrate, for the first time, that apo M in LDL and HDL occurs in five isoforms; three that are both N-glycosylated and sialylated, one that is N-glycosylated but not sialylated and one that is neither N-glycosylated nor sialylated. LDL from obese subjects was found to contain more apo J, apo C-II, apo M, α1-antitrypsin and serum amyloid A-IV than LDL from controls,, and also more of an acidic isoform (pI/Mr; 5.2 / 23 100) of apo A-I. In addition, the new LDLassociated protein transthyretin, was found to be significantly more abundant in LDL from obese subjects. On the other hand, the amounts of apo A-IV and the major isoform of apo A-I (pI/Mr; 5.3 / 23 100) were significantly less. Altogether, these findings (i) illustrate the power of 2-DE and mass spectrometry for detailed mapping of the proteins and their isoforms in human lipoproteins; (ii) demonstrate the presence of a number of new proteins in LDL (calgranulin A, lysozyme C and transthyretin); (iii) give precise biochemical clues to the polymorphism of apo M in LDL and HDL, and; (iv) indicate that obesity is associated with significant changes in the protein profile of LDL. It is concluded that new information on lipoproteins can easily be obtained through a proteomic approach, thus facilitating the development of a new proteomic field: lipoproteomics. Much further investigation in this field is warranted, particularly because newly discovered LDL and HDL proteins may play hitherto unknown role(s) in inflammatory reactions of the arterial wall and evolve as useful biomarkers in cardiovascular disease.

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. 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. 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. 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. In conclusion, antibodies against HER-2, especially pertuzumab radiolabelled with 177Lu, show promise as TRT agents.

Parkinson's disease is the second commonest neurodegenerative disease currently treated symptomatically. It is a multifactorial disease involving mechanisms ranging from protein aggregation to mitochondrial dysfunction, oxidative stress and dopamine dysregulation. The levels of α-synuclein have been causatively linked to the development and progression of Parkinson's disease. Therefore α-synuclein lowering strategies are valid approaches in Parkinson's disease. Neuropathologically, Lewy Bodies in the vulnerable substantia nigra of Parkinson's disease patients are less ubiquitinated and specifically less K-63 ubiquitinated than Lewy bodies in the cortex, suggesting differential activation or regulation of ubiquitin interactors. A targeted screen for such interactors revealed that the Deubiquitinating enzyme Usp8 is upregulated in the substantia nigra of Parkinson's disease brains and is inversely correlated with the degree of total and K-63 ubiquitination. Using genetic knockdown and overexpression techniques, Usp8 was found to colocalize and directly interact with α-synuclein. It was found to de-ubiquitinate α-synuclein and increase its half-life. Its knockdown increased the total and K-63 α-synuclein ubiquitination and decreased its levels by 35% at least partly by increasing its degradation via the lysosome. In vivo in the Drosophila melanogaster, Usp8 knockdown demonstrated protection against α-synuclein toxicity. It rescued in a specific manner the rough eye phenotype, the age-dependent locomotive defect and the loss of dopaminergic neurons caused by the expression of α-synuclein. Specific and effective pharmacological Usp8 inhibition also has the potential to lower α-synuclein levels. Collectively, the evidence produced in my thesis suggests that Usp8 could be a potential target for the future disease-modifying therapies in Parkinson's disease.

Nicotine from smoking or from other products containing nicotine has adverse effects on the fetus during pregnancy, such as respiratory problems. Our laboratory has previously shown that exposure to nicotine during development (DNE) alters hypoglossal motor neuron (XII MN) function, including decreased excitatory synaptic input, desensitized nicotinic acetylcholine receptors, increased input resistance, and differences in the precision and reliability of spike timing in XIIMNs. Evidence of DNE effects on XIIMN function prompted us to test the hypothesis that DNE will affect the development of primary dendrites and the soma. Brainstem slices were collected from neonates and motoneurons were filled with neurobiotin via whole-cell patch clamp. Filled cells were visualized with heavy metal intensified-3,3'-Diaminobenzidine (DAB) reaction. DAB-stained cells were analyzed using Neurolucida hardware and software. On average, the maximum soma diameter of more rostral XIIMNs was larger than that in more caudal cells. Also, caudal XIIMNs had more primary nodes than rostral XIIMNs, and there was a significant treatment effect on minimum soma diameter (Control, 13.76 ± 0.71 µm; DNE, 18.09 ± 1.22 µm). The results from this study uncovered potential effects of nicotine on XIIMNs found in rostral and caudal regions of the hypoglossal nucleus.

<p>A novel technology for protein detection, proximity ligation, has been developed along with improved methods for <i>in situ</i> synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis.</p><p>Microarrays synthesized <i>in situ</i> using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.</p>

<p>The mature vascular system is composed of a network of blood vessels organized into arteries, capillaries, and veins. The vessels are composed of endothelial cells surrounded by smooth muscle cells and embedded in a specialized basement membrane. The demand for oxygen during embryonal development regulates vessel formation through a process denoted vasculogenesis. These primitive vessels are further remodeled through proliferation, sprouting and migration of endothelial cells in a process denoted angiogenesis. Vasculogenesis and angiogenes are regulated by growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF).</p><p>To study vasculogenesis and angiogenesis, we employed differentiating embryonal stem cells (embryoid bodies). Vascularization of embryoid bodies follows a vascular pattern highly reminiscent of the in vivo pattern, leading to expression of a set of endothelial cell markers. Treatment of the embryoid bodies with different angiogenic growth factors led to distinct vascular morphologies. Expression of VEGF receptor-2 was an absolute demand for proper vascular development. PDGF-BB was shown to be potent in regulating vascular plexus formation in embryoid bodies. PDGF-BB induced capillary formation by promoting endothelial cell migration and differentiation. Hypoxia is a powerful inducer of angiogenic growth factors, such as VEGF-A, leading to angiogenesis. Hypoxia treatment induced an extensive vascular network that covered the entire embryoid body. Hypoxia-induced vascularization still occurred when VEGF receptor function was blocked, indicating that other pathway than VEGF/VEGF receptors may be critical for hypoxia-driven vessel formation. </p><p>Heparan sulfated proteoglycans (HSPGs) are present in the vascular basement membrane and are known to modulate angiogenic growth factor effects on endothelial cells in normal and pathological conditions such as tumor growth and formation of metastases. We employed heparin as an HSPG equivalent to show that PDGF-BB stimulation of PDGF a-receptor phosphorylation was augmented by heparin, resulting in increased mitogen activated protein kinase (MAPK) and protein kinase B PKB/Akt activation, and enhanced cellular migration towards PDGF-BB.</p>

A novel technology for protein detection, proximity ligation, has been developed along with improved methods for in situ synthesis of DNA microarrays. Proximity ligation enables a specific and quantitative transformation of proteins present in a sample into nucleic acid sequences. As pairs of so-called proximity probes bind the individual target protein molecules at distinct sites, these reagents are brought in close proximity. The probes consist of a protein specific binding part coupled to an oligonucleotide with either a free 3’- or 5’-end capable of hybridizing to a common connector oligonucleotide. When the probes are in proximity, promoted by target binding, then the DNA strands can be joined by enzymatic ligation. The nucleic acid sequence that is formed can then be amplified and quantitatively detected in a real-time monitored polymerase chain reaction. This convenient assay is simple to perform and allows highly sensitive protein detection. Parallel analysis of multiple proteins by DNA microarray technology is anticipated for proximity ligation and enabled by the information carrying ability of nucleic acids to define the individual proteins. Assays detecting cytokines using SELEX aptamers or antibodies, monoclonal and polyclonal, are presented in the thesis. Microarrays synthesized in situ using photolithographic methods generate impure products due to damaged molecules and interrupted synthesis. Through a molecular inversion mechanism presented here, these impurities may be removed. At the end of synthesis, full-length oligonucleotides receive a functional group that can then be made to react with the solid support forming an arched structure. The 3’-ends of the oligonucleotides are then cleaved, removing the impurities from the support and allowing the liberated 3’-hydroxyl to prime polymerase extension reactions from the inverted oligonucleotides. The effect of having pure oligonucleotides probes compared to ones contaminated with shorter variants was investigated in allele specific hybridization reactions. Pure probes were shown to have greater ability to discriminate between matched and singly mismatched targets at optimal hybridization temperatures.

The mature vascular system is composed of a network of blood vessels organized into arteries, capillaries, and veins. The vessels are composed of endothelial cells surrounded by smooth muscle cells and embedded in a specialized basement membrane. The demand for oxygen during embryonal development regulates vessel formation through a process denoted vasculogenesis. These primitive vessels are further remodeled through proliferation, sprouting and migration of endothelial cells in a process denoted angiogenesis. Vasculogenesis and angiogenes are regulated by growth factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). To study vasculogenesis and angiogenesis, we employed differentiating embryonal stem cells (embryoid bodies). Vascularization of embryoid bodies follows a vascular pattern highly reminiscent of the in vivo pattern, leading to expression of a set of endothelial cell markers. Treatment of the embryoid bodies with different angiogenic growth factors led to distinct vascular morphologies. Expression of VEGF receptor-2 was an absolute demand for proper vascular development. PDGF-BB was shown to be potent in regulating vascular plexus formation in embryoid bodies. PDGF-BB induced capillary formation by promoting endothelial cell migration and differentiation. Hypoxia is a powerful inducer of angiogenic growth factors, such as VEGF-A, leading to angiogenesis. Hypoxia treatment induced an extensive vascular network that covered the entire embryoid body. Hypoxia-induced vascularization still occurred when VEGF receptor function was blocked, indicating that other pathway than VEGF/VEGF receptors may be critical for hypoxia-driven vessel formation. Heparan sulfated proteoglycans (HSPGs) are present in the vascular basement membrane and are known to modulate angiogenic growth factor effects on endothelial cells in normal and pathological conditions such as tumor growth and formation of metastases. We employed heparin as an HSPG equivalent to show that PDGF-BB stimulation of PDGF a-receptor phosphorylation was augmented by heparin, resulting in increased mitogen activated protein kinase (MAPK) and protein kinase B PKB/Akt activation, and enhanced cellular migration towards PDGF-BB.

<p>The malfunction of the transcriptional regulator RUNX1 is the major cause of several variants of acute human leukemias and its normal function is to regulate the development of the blood system in concert with other transcriptional co-regulators. RUNX1 belongs to a conserved family of heterodimeric transcription factors that share a conserved DNA binding domain, the Runt domain (RD), named after the first member of this group – Runt - found in Drosophila melanogaster. The binding partner CBFβ serves as a regulator of RUNX by enhancing its DNA binding affinity through an allosteric mechanism.</p><p>The main focus ofo my thesis work has been the crystallization and structural analysis of the RUNX1 RD and involved also more technical methodological aspects that can be applied to X-ray crystallography in general.</p><p>The high resolution crystal structure of the free RD shows that this immunoglobulin-like molecule undergoes significant structural changes upon binding to both CBFβ and DNA. This involves a large flip of the L11 loop from a closed conformation in the free protein to an open conformation when CBFβ and/or DNA are bound. We refer to this transition as the “S-switch”. Smaller but significant conformational changes in other parts of the RD accompany the “S-switch”. We suggest that CBFβ triggers and stabilizes the “S-switch” which leads to the conversion of the RD into a conformation enhanced for DNA binding.</p><p>During the structural analysis of the RD we identified two chloride ions that are coordinated by residues otherwise involved in DNA binding. In electrophoretic mobility-shift analyses (EMSA) we demonstrated a chloride ion concentration dependent stimulation of the DNA binding affinity of RUNX1. We further showed by NMR line width broadening experiments that the chloride binding occurred within the physiological range. A comparable DNA binding stimulation of RUNX1 was seen in the presence of negative amino acids. This suggests a regulation of the DNA binding activity of RUNX1 proteins through acidic amino acid residues possibly provided by activation domains of transcriptional co-regulators that interact with RUNX1.</p><p>The use of the anomalous signal from halide ions has become a powerful technique for obtaining phase information. By replacing the sodium chloride with potassium bromide in the crystallisation conditions of the RD, we could demonstrate in a single wavelength anomalous diffraction (SAD) experiment that the anomalous signal from 2 bromide ions were sufficient to phase a 16 kDa protein. Due to lack of completeness in the low-resolution shells caused by overloaded intensities, density modification schemes failed and the resulting electron density maps were not interpretable. By combining the highresolution</p><p>synchrotron data with low-resolution data from a native data set collected on a home X-ray source, the density modified bromide phases gave easily traceable maps.</p>