In recent years, microarray technology has revealed the widespread presence of submicroscopic deletions and duplications throughout the human genome termed copy number variants (CNVs). CNVs have a profound effect on gene expression and are an important source of normal genetic variation. In addition, a small proportion of CNVs contribute to genetically simple and complex disease. This thesis focuses on the identification of pathogenic CNVs contributing to the etiology of diseases with "missing heritability" using a well-planned study design individually tailored to each disease cohort to optimize CNV detection and interpretation. We performed a genome-wide analysis for CNVs in five disease cohorts with genetic etiology: autism, age-related macular degeneration (AMD), glaucoma, clubfoot, and Bardet-Biedl syndrome (BBS). Our results indicate that CNVs likely account for a proportion of cases for each disease cohort reported in this thesis. Approximately 20% of our cohort of individuals with autism from trio pedigrees harbors a CNV known to confer risk to develop autism and we identified other novel and rare variants that may play a role in autism pathogenesis. We also characterized a duplication of 2p25.3 identified in two male half-siblings with autism and determined that their mother was somatic mosaic for the duplication. Our work provides evidence that this novel CNV disrupting the genes PXDN and MYT1L are the autism-causing mutation in this pedigree. A comparative cases experimental design was used in the study of AMD and glaucoma. While no common "risk CNVs" were identified for either eye disorder, we did identify several rare overlapping CNVs disrupting genes known to play a role in the eye that may confer risk to disease in a small proportion of individuals. In a fourth genetically complex disease, clubfoot, we identified a duplication of 17q23.2 disrupting the genes TBX4, NACA2, and BRIP1 that segregates with the autosomal dominant clubfoot phenotype in a large pedigree with 16 affected individuals. In addition, the duplication is within the linkage interval identified for this family. We also applied microarray technology to analyze the genomes of individuals with BBS, an autosomal recessive disorder, for the presence of CNVs in known BBS genes as well as CNVs that elucidate novel candidate genes for BBS. From 34 BBS patients with an unidentified mutation, we observed one CNV, a heterozygous deletion of BBS10, unmasking a BBS10 frameshift mutation. A promising BBS candidate gene also emerged from our studies, implicated by an intragenic deletion of the gene MARK3 predicted to result in a frameshift and premature truncation of the protein. Functional studies utilizing antisense morpholino gene knockdown in the zebrafish provide additional evidence that MARK3 is a BBS gene as knockdown of zebrafish mark3 results in a Kupffer's Vesicle defect and a melanosome transport delay, two cardinal BBS phenotypes in the zebrafish. In addition to identifying CNVs involved in disease, the work outlined in this thesis provides valuable insight into the study design and interpretation of a genome-wide analysis of CNV. This includes the appropriate use of controls and publicly available control databases, methods for enriching for CNVs in a patient cohort to maximize efficiency and discovery, and the importance of analyzing all patient cohorts with heritable disease for the presence of CNVs disrupting known disease genes and CNVs that implicate novel genetic candidates. As the reliability and resolution of CNV detection continue to improve, allowing detection of > 1,000 CNVs in each individual genome, it becomes more important than ever to have a well-defined study design for both the detection and interpretation of CNVs.

There have been several techniques developed in recent years to develop computer models of a variety of disease behaviors. Agent-based modeling is a discrete-based modeling approach used agents to represent individual cells that mechanically interact and secrete, consume or react to soluble products. It has become a powerful modeling approach, widely used by computational researchers. In this research, we utilized agent-based modeling to study and explore disease development, particularly in two applications, breast cancer and bioengineering experiments. We further proposed an error-minimization search approach and used it to estimate cellular parameters from multicellular in vitro data. In this dissertation, in the first study, we developed a 2D agent-based model that attempted to emulate the in vivo structure of breast cancer. The model was applied to describe the progression from DCIS into DCI. This model confirms that the interaction between tumor cells and the surrounding stroma in the duct plays a critical role in tumor growth and metastasis. This interaction depends on many mechanical and chemical factors that work with each other to produce tumor invasion of the surrounding tissue. In the second study, an in silico model was developed and applied to understanding the underlying mechanism of vascular-endothelial growth factor (VEGF) auto-regulation in REP and emulate the in vitro experiments as part of bioengineering research. This model may provide a system with robust predictive modeling and visualization that could enable discovery of the molecular mechanisms involved in age-related macular degeneration (AMD) progression and provide routers to the development of effective treatments. In the third and final study, a searching approach was applied to estimate cellular parameters from spatiotemporal data produced from bioengineered multicellular in vitro experiments. We applied a search method to an integrated cellular and multicellular model of retinal pigment epithelial cells to estimate the auto-regulation parameters of VEGF.

Age-related macular degeneration (AMD) is a chronic, irreversible disease that robs individuals of vision, quality of life, and independence. It is the leading cause of blindness in industrialized countries. Sleep-disordered breathing (SDB) is a condition characterized by repeated episodes of apnea and/or hypopnea, insomnia, short sleep duration, and/or sleep disturbances (snoring, gasping, etc.). Because SDB has been shown to cause chronic hypoxia resulting in oxidative stress on the retina, it has been proposed that SDB may be associated with AMD. Based on the life course theory of chronic disease, this quantitative, cross-sectional study used data from the 2005-2008 National Health and Nutrition Examination Survey to study whether there was an association between SDB and AMD, including neovascular AMD and geographic atrophy in adults 40 years and older. Descriptive statistics and logistic regression analyses were used. The results suggest that AMD is associated with diagnosed sleep disorders, including sleep apnea and insomnia, as well as sleep apnea symptoms of gasping snoring, snorting, and stopping breathing. The findings of this study highlight the importance of diagnostic screening and therapeutic intervention to treat SDB. Early diagnosis and therapy for SDB could address not only the comorbidities associated with SDB, but could also prevent or slow the progression of AMD. In turn, this would yield lower rates of vision loss, reduced comorbidities associated with vision loss, and reduced impact of AMD on the health care system and social and financial costs to society.

<p>Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The <i>VMD2</i> gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD.</p><p>In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a <i>C. elegans</i> protein family, expressed in retina, was identified as the <i>VMD2</i> gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the <i>VMD2</i> gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. <i>VMD2</i> expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation.</p><p>Analysis of mouse <i>Vmd2</i> and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. <i>Vmd2</i> expression levels were highest around birth.</p>

La formation de néo-vaisseaux sanguins est un processus impliqué dans de nombreuses pathologies, telles que le développement de carcinomes cutanés et la néo-vascularisation choroïdienne caractéristique de la forme exsudative de la dégénérescence maculaire liée à lâge (DMLA). Dans ces deux cas, lactivation du réseau vasculaire est un facteur de mauvais pronostic. Lanalogie entre ces deux pathologies est renforcée par le développement récent dapproches thérapeutiques anti-angiogènes. Ces approches ciblent principalement le VEGF et les molécules associées. Malgré lefficacité de ces molécules sur la pathologie ciblée, leur administration systémique engendre des effets secondaires non négligeables. Notre travail a pour but détudier limplication dautres acteurs moléculaires dans ces pathologies, en vue de développer des stratégies thérapeutiques alternatives ou complémentaires. Parmi les principales molécules impliquées lors de langiogenèse, les protéases et leurs inhibiteurs sont des cibles potentielles pour le développement de nouveaux traitements. Il était initialement admis que les protéases (MMPs, protéases à sérine) sont des facteurs pro-angiogènes et leurs inhibiteurs (TIMPs, PAI-1) des facteurs anti-angiogènes. Dans ce contexte, lhypothèse thérapeutique évidente était dinhiber les protéases et de protéger ou dactiver leurs inhibiteurs. Cependant, lorsque nous avons entamé ce travail, ce concept a été remis en question. En effet, un niveau élevé de PAI-1 a été détecté dans de nombreux cancers et représente un facteur de mauvais pronostic. Par ailleurs, les inhibiteurs des MMPs nont présenté aucun effet lors dessais cliniques, certains stimulant même la croissance tumorale. Il est important de noter que ces premiers inhibiteurs étaient des inhibiteurs à large spectre bloquant laction non seulement des MMPs, mais aussi des membres de familles proches. Une détection fine des rôles joués par les MMPs et linhibiteur PAI-1 sest avérée indispensable. Nous avons, dès lors, focalisé notre travail sur deux thèmes : létude de PAI-1 et létude du rôle individuel de quelques MMPs. Il a précédemment été démontré que linhibiteur PAI-1 exerce un rôle pro-angiogène lors du développement de carcinomes cutanés et de néo-vascularisation choroïdienne. PAI-1 exerce donc un effet paradoxal et complexe sur langiogenèse. Bien que le rôle de PAI-1 au cours de langiogenèse bourgeonnante était bien documenté, son implication au cours de la vasculogenèse nétait pas connue. Nos résultats démontrent que le développement des carcinomes cutanés nécessite la migration des cellules stromales adjacentes aux cellules tumorales. Par contre, la néo-vascularisation choroïdienne, également dépendante de PAI-1, requiert le recrutement des cellules issues de la moelle osseuse. Publications 1 et 2. La seconde partie de notre travail est consacrée aux métalloprotéinases matricielles. Nos résultats montrent les rôles opposés ou synergiques des MMPs. Les MMP-2 et -9 sont des protéases pro-angiogènes agissant de concert au cours de linvasion des carcinomes. A lopposé, la MMP-19 exerce une fonction anti-angiogène et nos travaux suggèrent que cette MMP contribuerait à la stabilité des vaisseaux matures et au maintien physiologique des tissus, son expression étant diminuée lors de linvasion tumorale. Publications 3, 4 et 5.

Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The VMD2 gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD. In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a C. elegans protein family, expressed in retina, was identified as the VMD2 gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the VMD2 gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. VMD2 expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation. Analysis of mouse Vmd2 and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. Vmd2 expression levels were highest around birth.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

Current anti-VEGF biologics, such as bevacizumab and VEGF trap, have been successfully used as therapeutic agents for cancer and age-related macular degeneration (AMD). Since these strategies target VEGF systemically, their toxicity profile, including proteinuria and thromboembolic events, and need for frequent eye injections in AMD treatment, prevail. Therefore, the aim of this PhD thesis was to generate novel anti-VEGF biologics that inhibit VEGF activity specifically at the desired target site. Two classes of biologics were engineered that simultaneously bind VEGF and either: 1) the extracellular matrix (ECM) or 2) target-site specific antigens. The first subgroup, “sticky-traps”, is composed of VEGF trap linked to a sequence of hydrophobic amino acids, with affinity for heparin sulfate proteoglycans of the ECM. The second subgroup, “lassos”, is composed of a C-terminus positioned form of VEGF trap linked to single-chain variable domain antibodies specific for either HER2 (HER2/V lasso) or fibronectin extra domain B (EDB; EDB/V lasso), expressed on breast cancer cell surfaces or in the vascular bed of solid tumours, respectively. ii Using a novel transgenic method, piggyBac transposons, biologics were expressed in transgenic cancer cell lines in a doxycycline inducible manner. They were shown to inhibit VEGF activity and also retain the native function of their constituent domains. Specifically, the sticky-traps adhered to the ECM and the HER2/V lasso inhibited the proliferation of HER2 positive cancer cell lines. Sticky-traps as well as lassos were able to inhibit or delay tumour growth of A-673, Pc-3, SKOV-3 and HT-29 xenografts. In contrast to soluble VEGF trap, sticky-traps were retained at the tumour site and were undetectable in the circulation. Moreover, sticky-traps, in contrast to VEGF trap, did not delay wound healing and regression of trachea blood vessels. Furthermore, transgenic studies indicated that HER2/V lasso is more effective compared to anti-HER2 Ab and VEGF trap used alone or in combination. These novel classes of antiangiogenic molecules could be advantageous in a clinical setting. Using the principles established in my PhD thesis work, similar dual function biologics can be designed for inhibition of other molecules with disease relevance.

The Sjögren-Larsson syndrome (SLS) is a genetically determined syndrome with autosomal recessive inheritance originally and comprehensively described from Sweden. It is characterized by the three cardinal signs congenital ichthyosis, spastic di- or tetraplegia and mental retardation.The present investigation covers all 35 SLS patients alive in Sweden in 1978 and the genetic study all 58 SLS patients born in Sweden in 1886-1977. Forty-five of these were bom in a restricted area in the northeast of Sweden and five more had ancestors from this area. This concentration is probably the result of a founder effect — transmission of the SLS gene mutation from early immigrants to this area — followed by little migration to and from this area. Ancestors of SLS patients have been traced back to an early immigrant to this area in the 14th century.The mean yearly incidences of SLS per 100.000 newborn during the years 1901-1977 were 0.6 in the whole of Sweden, 10.2 in the county of Västerbotten and 2.7 in the county of Norrbotten. The corresponding prevalence figures for SLS on 31 December 1978 were 0.4, 8.3 and 2.6 per 100.000 in these areas, respectively.The ichthyosis in the SLS was found to be congenital in the true sense of the word, as it was always seen at birth. The characteristic skin findings indicate that the use of replicas in ichthyotic patients may be a good complement to the clinical and histopathological examinations.All SLS patients had mental retardation and spastic di- or tetraplegia. The first observation of these symptoms was made between the age of 4 and 30 months.Small white glistening dots located in the ocular fundus were noted in all 30 SLS patients examined in this respect and seem to be pathognomonic for SLS.The fatty acid patterns of plasma phospholipids, cholesteryl esters, triglycerides and free fatty acids in SLS patients were examined. The results suggest that SLS is possibly a disorder of fatty acid metabolism leading to an altered composition of polyunsaturated fatty acids due to a disturbance in the A6-desaturation of essential fatty acids. / <p>Some photos have been censored.</p><p>En del foton har blivit censurerade.</p> / digitalisering@umu

Eine lokale Aktivierung des Komplementsystems im RPE ist ein pathogener Faktor der AMD. Neben der Wirkung von angiogenen Faktoren wie VEGF könnte eine Aktivierung des Blutgerinnungssystems im RPE dazu beitragen, dass sich aus einer trockenen eine feuchte AMD entwickelt. Dies könnte auf mehreren Ebenen geschehen: Gerinnungsfaktoren könnten die Expression der Komplementfaktoren und der angiogenen Faktoren regulieren sowie Wirkungen auf die Proliferation und Migration der RPE-Zellen besitzen. Eine Stimulierung der Proliferation und Migration der RPE-Zellen trägt zur Ausbildung von CNV-Membranen bei. Es ist aber bis jetzt nichts darüber bekannt, ob RPE-Zellen Faktoren des Blutgerinnungssystems exprimieren und ob z.B. Thrombin (als zentrale Protease des Blutgerinnungssystems) die Genexpression von Komplementfaktoren und von VEGF im RPE beeinflusst. Die Ziele der vorliegenden Dissertation waren daher: ● Nachweis der mRNA-Expression von Blutgerinnungs- und Komplementfaktoren im RPE; ● Nachweis der Wirkung von Thrombin auf die Expression von VEGF und von Komplementfaktoren, sowie auf die Proliferation und Migration der RPE-Zellen; und ● Nachweis der Wirkung der Komplementfaktoren C5a und C9 auf die Sekretion von VEGF und die Proliferation und Migration der RPE-Zellen.