Mutation of the p53 gene appears to be an early event in skin cancer development. The present study is based on morphology and represents a cellular and genetic investigation of p53 alterations in normal human skin and basal cell cancer. Using double immunofluorescent labelling, we have demonstrated an increase in thymine dimers and p53 protein expression in the same keratinocytes following ultraviolet radiation. Large inter-individual differences in the kinetics of thymine dimer repair and subsequent epidermal p53 response were evident in both sunscreen-protected and non-protected skin. The formation of thymine dimers and the epidermal p53 response were partially blocked by topical sunscreen. We have optimized a method to analyze the p53 gene in single cells from frozen tissue sections. In chronically sun-exposed skin there exist clusters of p53 immunoreactive keratinocytes (p53 clones) in addition to scattered p53 immunoreactive cells. Laser assisted microdissection was used to retrieve single keratinocytes from immunostained tissue sections, single cells were amplified and the p53 gene was sequenced. We have shown that p53 mutations are prevalent in normal skin. Furthermore, we detected an epidermal p53 clone which had prevailed despite two months of total protection from ultraviolet light. Loss of heterozygosity in the PTCH and p53 loci as well as in the sequenced p53 gene was determined in basal cell cancer from sporadic cases and in patients with Gorlin syndrome. Allelic loss in the PTCH region was prominent in both sporadic and hereditary tumors, while loss of heterozygosity in the p53 locus was rare in both groups. p53 mutations found in the hereditary tumors differed from the typical mutations found in sporadic cases. In addition, we found genetically linked subclones with partially different p53 and/or PTCH genotypes in individual tumors. Our data show that both genes are important in the development of basal cell cancer.

The main purpose of the theses is to explore the significance of anxiety and depression in patterns of pain, fatigue, quality of life. Lifestyle, functional disability, co-morbidity and gender among individuals given the diagnosis of fibromyalgia by their doctor.

All-trans retinoic acid (ATRA), a biologically active metabolite of vitamin A, is a powerful inducer of terminal differentiation and growth arrest of several myeloid cell lines in vitro. Although the efficacy of ATRA as an anti-cancer drug has been demonstrated by the successful treatment of acute promyelocytic leukemia (APL), knowledge concerning the molecular mechanisms directing ATRA-induced differentiation and cell cycle arrest of myeloid cells is lacking. Our results show, for the first time, that the complex regulation of cell cycle proteins and myeloid-specific transcription factors induced by ATRA relies on functional Stat1. We found that Stat1 is activated by both tyrosine-701 and serine-727 phosphorylation upon ATRA-induced differentiation of the human monoblastic cell line U-937. Expression of phosphorylation deficient mutants of Stat1 (Stat1Y701F or Stat1S727A) inhibited both ATRA-induced differentiation and cell cycle arrest of U-937 cells, pointing to a requirement of active Stat1 in these processes. Detailed analysis of the molecular mechanism of ATRA-induced cell cycle arrest and differentiation showed that the onset of cell cycle arrest was associated with a decrease in c-Myc and cyclin E levels and upregulation of p27Kip1 and p21WAF1/CIP1. This was followed by a rapid fall in cyclin A and B and a coordinate dephosphorylation of the retinoblastoma protein (pRb). The inhibition of ATRA-induced cell-cycle arrest by constitutive expression of Stat1Y701F or Stat1S727A was associated with impaired regulation of these cyclins and p27Kip1, positioning Stat1 activation upstream of these events. To further understand the process of ATRA-induced differentiation, the regulation of myeloid-specific transcription factors was investigated during ATRA-treatment. Notably, ATRA-induced upregulation of Stat2, ICSBP and C/EBP-ε was selectively impaired in sublines expressing Stat1Y701F or Stat1S727A, suggesting an important function of these factors downstream Stat1. Taken together, the work in this thesis clearly demonstrates that Stat1 plays a key role in ATRA-induced terminal differentiation of myeloid cells, through regulation of cell cycle proteins and myeloid-specific transcription factors.

A series of novel methods for detection of known sequence variants in DNA, in particular single nucleotide polymorphism, using padlock probes and rolling circle replication are presented. DNA probes that can be circularized – padlock probes – are ideal for rolling circle replication. Circularized, but not unreacted probes, can generate powerful signal amplification by allowing the reacted probes to template a rolling circle replication (RCR) reaction. However, when hybridized and ligated to a target DNA molecule with no nearby ends, the probes are bound to the target sequence, inhibiting the RCR reaction is. This problem can be solved by generating a branched DNA probe with two 3’ arms such that the probes may be circularized while leaving the second 3’ arm as a primer for the RCR reaction. We describe how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5’ end but two distinct 3’ ends that extend from the 2’ and 3’ carbons of an internal nucleotide. An even stronger approach to circumvent the topological problem that can inhibit RCR is to restriction digest the template downstream of the padlock recognition site. By using Phi 29 DNA polymerase with efficient 3’ exonuclease and strand displacement activity, the template strand can then be used to prime the RCR reaction. The amplified molecule is contiguous with the target DNA, generating an anchored localized signal. The kinetics of the reaction was investigated by following the reaction in real-time using molecular beacon probes. Localized RCR signal were obtained on DNA arrays, allowing detection of as little as 104-105 spotted molecules, of either single- or double-stranded M13 DNA, in a model experiment. We have also established a serial rolling circle amplification procedure. By converting rolling circle products to a second and even third generation of padlock probes the signal was amplified thousand-fold per generation. This procedure provides sufficient sensitivity for detection of single-copy gene sequences in 50 ng of human genomic DNA, and large numbers of probes were amplified in parallel with excellent quantitative resolution.

Angiogenesis can be defined as the formation of new blood vessels from pre-existing ones. Angiogenesis is required for development and maintenance of our vascular system and thus of fundamental importance to our existence. The endothelial cells that line the inside of the vessels de-differentiate, migrate, proliferate and re-differentiate during angiogenesis. Angiogenesis is tightly regulated, controlled by several angiogenic factors of various classes that promote angiogenesis but also by anti-angiogenic factors that counteract the effect of the pro-angiogenic factors. We have examined three factors involved in angiogenesis regulation, Vascular endotelial growth factor (VEGFR) -3, the matrix protein laminin-1 and the collagen XVIII derived fragment endostatin. Five tyrosine phosphorylation sites in the cytoplasmic tail of VEGFR-3 were identified by phosphopeptide mapping (PPM). The data was confirmed by PPM using point-mutated receptors generated by site-directed mutagenesis. Laminin-1 was found to promote angiogenesis in the chicken chorioallantoic membrane assay and in a synergistic fashion together with suboptimal levels of fibroblast growth factor 2 (FGF-2) in embryoid bodies. Laminin-1 also promoted endothelial tubular morphogenesis in vitro, and upregulated the expression of the endothelial differentiation marker Jagged-1. Endostatin was shown to affect endothelial FGF-2-induced cell survival and morphogenesis. This was a result of direct binding to endothelial cells and induction of tyrosine phosphorylation of many proteins including the adaptor protein Shb. The apoptotic and morphogenic responses induced by endostatin was shown to be dependent on Shb. Further, endostatin inhibited endothelial migration and affected molecules implicated in migration. In particular, FGF-2 induced actin reorganization, and β-catenin regulation was modulated by endostatin.

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.

<b>Pressemelding:</b> Behandling av type 2 diabetes har trolig best effekt i en tidlig fase av sykdommen. Dette skriver assistentlege Elisabeth Qvigstad (36) fra Grimstad i doktoravhandlingen sin ved Norges teknisk-naturvitenskapelige universitet NTNU. Arbeidet kan bidra til at det utvikles nye medisiner mot diabetes. Avhandlingen tar utgangspunkt i type 2 diabetes, som rammer 105-120 000 nordmenn. Tidligere forskning i form av celle- og dyreforsøk har vist at vedvarende høye nivåer av fettsyrer i blodet og langvarig stimulering av insulinfrigjøring kan svekke funksjonen til de insulinproduserende beta-cellene i bukspyttkjertelen. Avhandlingen ville teste om lignende forhold er til stede hos mennesker og om korrigerende tiltak ville bedre insulinfrigjøringen ved type 2 diabetes. Nivået av frie fettsyrer hos personer med type 2 diabetes er oftest forhøyet. Langvarig faste hos friske gir også forhøyet fettsyrenivå og kan ses på som en modellsituasjon for type 2 diabetes. Qvigstad fant redusert insulinfrigjøring hos friske forsøkspersoner etter 58 timer faste. Fettsyrenivået i blod under testing ble senket ved hjelp av et nikotinsyrederivat hos friske personer og personer med type 2 diabetes. Hos friske påvirket ikke medikamentet insulinfrigjøring eller -følsomhet. Imidlertid virket behandlingen positivt på insulinfrigjøring hos de diabetikerne som hadde best blodsukker-kontroll. Derimot, når type 2 diabetikere reduserte fett i kosten, ga dette ingen utslag på insulinfrigjøringen, men noe nedsatt insulinfølsomhet. Nivået av fettvevshormoner (leptin, adiponectin) ble redusert. Den egne insulinfrigjøringen ble hemmet med medikamentet diazoxid, og insulininjeksjoner ble brukt som erstatning. Insulinfrigjøringen økte uten å endre insulinbehov eller blodsukkerkontroll sammenliknet med placebo. Disse resultatene tyder på at "betacelle-hvile" er gunstig ved type-2 diabetes. Qvigstads doktorgradsarbeid bidrar til økt forståelse av betydningen av fettsyrer for insulinfrigjøring og insulinfølsomhet hos friske og ved type 2 diabetes. I tillegg støtter funnene betydningen av "betacelle-hvile», som kan bidra til utvikling av nye medisiner mot diabetes. http://www.ntnu.no/doktorgrader/dr.med/02.03/qvigstad.htm

Diabetic nephropathy (DN) is associated with morbidity and mortality due to cardiovascular disease and renal failure. This study focused on the impact of glycemic control on the development of DN and the metabolic consequences of DN. The euglycemic hyperinsulinemic clamp technique was used to assess insulin sensitivity and insulin clearance. Two different registries, the Diabetes Incidence Study in Sweden (DISS) and the Swedish Childhood Diabetes Registry, as well as questionnaires and data from medical records were used to study diabetic complications in population-based cohorts. Microalbuminuria is an early marker of DN and may also be associated with impaired insulin sensitiv-ity in diabetic and non-diabetic subjects. We studied the relationship between insulin sensitivity and the degree of albuminuria in patients with type 1 diabetes and micro- or macroalbuminuria but normal glomerular filtration rate (GFR). We did not find a direct quantitative association between the degree of albuminuria and insulin resistance, arguing against a cause-effect relationship. With progression of DN, a decline in GFR is seen. Patients with severe renal failure have both im-paired insulin sensitivity and insulin clearance. We studied insulin sensitivity and insulin clearance in type 1 diabetes patients with three different degrees of renal involvement (none, only albuminuria, and slightly reduced GFR, ~40-70 ml/min/1.73 m2, respectively). A clear reduction in insulin sensitivity in vivo, but not in insulin clearance, was seen in the group with reduced GFR, and concomitant changes in the levels of PTH, IGF-1, IL-6 and TNF-α were found. In parallel, cellular insulin sensitivity and insulin degradation were examined in vitro, in subcutaneous fat cells but no differences were found between the three groups of patients. To study the occurrence of renal involvement in patients with modern diabetes treatment we moni-tored a cohort of young adults from the DISS-registry with onset of diabetes in 1987-88 at age 15-34 years. We found that ~7% of the patients had signs of renal involvement, i.e. incipient nephropathy (5%) and overt nephropathy (2%), after a median follow-up of ~9 years and the strongest risk markers were poor glycemic control (HbA1c) and high blood pressure. Patients with type 2 diabetes were most prone to have renal involvement in this age group. Retrospectively, we studied 94 patients diagnosed with type 1 diabetes in 1981-1992 at age 0-14 years at the Umeå University Hospital. Incipient nephropathy and background retinopathy occurred in 18 and 45%, respectively, of the patients, during ~12 years of follow-up. Glycemic control, also during the first five years of diabetes, was a strong risk marker. Young age at onset of diabetes prolonged the time to development of microvascular complications. Conclusion: Despite modern diabetes treatment some patients with diabetes develop renal involvement within the first ten years. Inadequate glycemic control, also early in the disease, is a risk marker as well as type 2 diabetes and high blood pressure. In patients with type 1 diabetes and diabetic neph-ropathy a slightly reduced GFR, but not albuminuria, is associated with insulin resistance. Concomi-tant changes in insulin-antagonistic hormones and cytokines may be involved.

The phylogeography of a wide ranging temperate species, the adder, Vipera berus, was investigated using several genetic tools, with special emphasis on the post-glacial colonisation pattern of Fennoscandia. The area was colonised from two directions by adder populations representing different glacial refugia. The two populations meet in three places and the main contact zone is situated in Northern Finland. The two other contact zones are the result of dispersal across the Baltic Sea to the Umeå archepelago and South-Western Finland. Asymmetrically distributed nuclear genetic variation compared to mitochondrial DNA in the northern contact zone suggests a skewed gene flow from the east to the west across the zone. This pattern might reflect differences in dispersal among sexes and lineages, or may be accounted for by a selective advantage for nuclear variation of eastern origin among Fennoscandian adders. The phylogeographic pattern for adders across the entire species range was addressed by sequencing part of the mitochondrial genome and scoring microsatellite markers. The adder can be divided into three major genetic groups. One group is confined to the Balkan peninsula harbouring the distribution range of V. b. bosniensis. A second, well differentiated group is restricted to the Southern Alps. These two areas have probably served as refugia for adders during a number of ice ages for the adders. The third group is distributed across the remainder of the species’ range, from extreme Western Europe to Pacific Russia and can be further divided into one ancestral group inhabiting the Carpathians refugial area, and three more recent groups inhabiting areas west, north and east of the Alps. The adder provides an example of a species where the Mediterranean areas are housing endemic populations, rather than the sources for post-glacial continental colonisation. Continent-wide colonisation has instead occurred from up to three cryptic northern refugia.

The studies in the present thesis sought to define virus and host factors that can influence on the susceptibility to murine retrovirus infection. In addition, we wanted to study possible correlations between events of early infection and subsequent disease progression. For an extensive discussion of the major findings, the reader is referred to papers I-IV. The following section will give a general discussion concerning 1) some methodological aspects; 2) the course of FIS-2 infection; 3) determinants responsible for erythroleukaemia; 4) determinants responsible for immunosuppression; and, 5) does sex matter?