Seasonal variations of mood, behavior and physiology have been of increasing interest. At least two different seasonal rhythms seem to exist: Descriptions of Seasonal Affective Disorder (SAD) with increased weight, increased sleep and fatigue during winter have attracted attention in academic psychiatry and in the general public the last two decades. In addition to such a difference in mood, weight and sleep between summer and winter, many studies describe a spring and fall increase in frequency of suicides and of admissions to hospital for mood disorders. In searching for a possible etiology for these seasonal changes, the main focus has been on variations in length of day. The objective of this thesis was to study the existence and pattern of seasonal variation in some forms of behavior and of psychiatric illness among children and adults in Norway. Possible statistical connections between seasonal variations of behavior and changes in length of day and the influences of latitude, sex and age were also studied. The monthly numbers of incidents in different groups were studied: All suicides in Norway 1969-96 (N=14.503), admissions to hospital for depression and mania in some hospitals 1992-96 (N=4.341), all violent episodes recorded by the police in Norway 1991-97 (N=82.537), all patient-staff incidents in a psychiatric department 1990-97 (N=502), all telephone calls to the Red Cross help-line for children and adolescents in Norway 1996-98 (N=691.787calls, 220.602 conversations) and in Trondheim, Norway 1991-97 (N=80.983 calls, 22.698 conversations) were included in the thesis. The monthly frequencies of these incidents were compared to an expected equal daily frequency of incidents through the year. Changes with increasing age and increasing latitude were examined. Correlations between the monthly frequencies of incidents and the length of day, with maximum impact at midsummer, and correlations between the monthly frequencies of incidents and the speed of change in length of day, with maximum impact at the equinoxes, were also studied. In this thesis, an increased activity in April-June and in October-November is described for all the groups that were studied. In summer and winter there is less activity than in the rest of the year. Among children calling the help-line, a steady diminishing seasonal variation in number of calls with increasing age from 7 to 17 years of age and an increasing seasonal variation in number of calls with increasing latitude were found. Also the seasonal variation of violence increases with increasing latitude in Norway. Among men there is a correlation between the monthly number of suicides and the monthly number of admissions for mania and a correlation between the monthly number of suicides and the monthly number of admissions for depression. Among women there is a diminishing seasonal variation of admissions for depressions with increasing age. The monthly frequency of violence in Norway and the monthly frequency of calls to the Red Cross help-Line for children and adolescents correlated with a delay of 1-2 months with the monthly change in length of day with maximum impact at the equinoxes. The results in the thesis correspond with earlier studies describing an increase in the frequency of suicides and an increase in admissions for depressions in spring and fall. A corresponding rhythm for other forms of human behavior is described in the present thesis, indicating that the seasonal rhythm of psychiatric illness reflects a seasonal rhythm of behavior in greater parts of the population. The seasonal variation in behavior seems to increase with increasing latitude, to be more dramatic in the northern than in the southern parts of Norway. In this thesis results supporting a hypothesis of human behavior being influenced by changes in length of day are given. Changes in length of day may induce changes in sleep and other disturbances in the daily rhythm that could change mood and other emotional qualities in vulnerable individuals. The demands on our capability to adapt to changes in length of day are largest at the equinoxes. / <b>Årstidsvariasjon av sinnstemning og adferd.</b> Det er økende interesse for årstidsvariasjon av adferd og av forekomsten av psykiske lidelser. Det synes å foreligge minst to ulike årstidsrytmer i befolkningen; Størst oppmerksomhet har oppdagelsen av vinterdepresjon karakterisert ved tristhet, tretthet, økt vekt og forlenget søvn vakt. I tillegg til en slik forskjell i humør, vekt og søvn mellom sommer og vinter, er det en rekke beskrivelser av overhyppighet av selvmord og av innleggelser i sykehus for depresjoner vår og høst. Årsakene til disse to ulike årstidsrytmene er ikke kjent, men man har antatt at variasjon i dagslengde gjennom året spiller en rolle. Hensikten med denne avhandlingen har vært å undersøke om det er årstidsvariasjon i forekomsten av ulike former for adferd og av psykiske lidelser hos barn og voksne i Norge. Videre er eventuelle statistiske sammenhenger mellom adferd og dagslengde gjennom året undersøkt. Til sist er forskjeller i årstidsrytme knyttet til breddegrad, alder og kjønn undersøkt. Antallet hendelser pr måned i ulike grupper ble studert; Alle selvmord i Norge 1969-96 (N=14.503), innleggelser for depresjon og mani i en del sykehus 1992-96 (N=4.341), alle registrerte voldsepisoder i Norge 1991-97 (N= 82.537), personalskader i et psykiatrisk sykehus 1991-97 (N=502), alle telefoner til Røde Kors Kontakttelefon for barn og unge i Norge 1996–98 (N=691.787 oppringninger, 220.602 samtaler) og i Trondheim 1991-97 (N=80.983 oppringninger, 22.698 samtaler) ble inkludert i arbeidet. Hyppigheten av alle disse hendelsene i hver måned ble sammenlignet med en forventet lik fordeling av hendelsene året igjennom. Endringer med økende alder og med økende breddegrad ble undersøkt. Videre ble det gjort sammenligninger med dagslengde som er lengst ved sommersolverv og kortest ved vintersolverv, og sammenligninger med endringer av dagslengde som er hurtig ved vår og høstjamndøgn og sakte ved solvervene. I alle disse materialene er det en økt aktivitet april – juni og oktober – november, videre er det stille perioder om vinteren og om sommeren. Blant barn som ringer kontakttelefonen er det gradvis avtagende årstidsvariasjon av henvendelser med økende alder fra 7 til 17 år og økende årstidsvariasjon i antallet henvendelser jo lenger nord man kommer i Norge. Også årstidsvariasjonen av vold i Norge endrer seg jo lengre nord man kommer i landet. Blant menn er der en korrelasjon mellom det månedlige antallet av selvmord og av innleggelser for mani og mellom antallet selvmord og innleggelser for depresjon. Blant kvinner er det en avtagende årstidsvariasjon av innleggelser for depresjon med økende alder. Den månedlige endring av dagslengde som er raskest ved jamndøgnene korrelerer med en viss forsinkelse med forekomsten av vold i Norge og med antallet oppringninger til Barn og Unges kontakttelefon. Funnene i avhandlingen er i samsvar med tidligere beskrivelser av en markert økning av suicid og av innleggelser for depresjoner om våren og til dels om høsten. I avhandlingen er en tilsvarende rytme funnet for annen adferd. Dette tyder på at årstidsrytmen av psykiatrisk sykelighet avspeiler en årstidsrytme av adferd i store deler av befolkningen. Videre ser det ut til at forskjellene i adferd gjennom året blir mer markerte jo lengre nord man kommer i landet. I avhandlingen er det funn som støtter en hypotese om at endringer i dagslengde påvirker mennesket, det er mulig at dette skjer gjennom endret søvn og andre forstyrrelser i døgnrytmen. Vår døgnrytme er utsatt for størst krav til å tilpasse seg hurtige endringer i lysforhold rundt jamndøgnene.

The aims of this dissertation were to investigate alcohol-related seizures in clinical neurological practice. We wanted to assess the extent of this problem, to classify the seizures, and to investigate methods to improve the clinical diagnosis of such seizures. We propose an arbitrary but simple and reproducible way of diagnosing alcohol-related seizures and alcohol withdrawal seizures. Papers I and II relate to seizure classification and the extent of the problem in relation to the level and weekly pattern of alcohol use. Paper III investigates the performance of various biological markers as aids in the diagnosis of alcohol-related seizures. Paper IV explores pitfalls in the result interpretation for two methods for detection of CDT in patients with neurological disorders. Paper V investigates the utility of standard EEG for the identification of alcohol-related seizures. Even though the general alcohol consumption in our region is low, every third patient with an epileptic seizure leading to hospitalisation had hazardous alcohol consumption. Evidence of focal lesions or focal seizure start was found in a high proportion of alcohol-related seizures. All such seizures were secondarily generalized and thus, we challenge the establishment impression that the vast majority of alcohol-related seizures are primarily generalized. Binge drinking (more than six drinks for men or four drinks for women, in a single drinking occasion) was common, but had little influence on seizure susceptibility or timing of seizures. In contrast to prior knowledge, we found that in some patients there was no time lag from cessation of drinking to the occurrence of a seizure, but falling intake levels prior to withdrawal seizures were demonstrated. This indicates that a state of relative withdrawal while still drinking may be sufficient to induce a seizure. Carbohydrate-deficient transferring (CDT) is the most accurate biomarker for alcohol use and good adjunct to the diagnosis of alcohol-related seizures, but its accuracy does not compete with a good clinical investigation. Generally poor accuracy should be expected for fertile women. Women on enzyme-inducing antiepileptic drugs who drink no or little alcohol seem to be at risk of having false positive CDT. Other variables associated with increased CDT were low body mass index, or having total transferring levels outside normal range. A definitely abnormal EEG suggests epilepsy or symptomatic seizures unrelated to alcohol use. The predictive value of a normal EEG is limited, but the typical post-ictal finding in alcohol-related seizures is nevertheless a normal low-amplitude EEG record. The best method for identification of alcohol-related seizures is a clinical work-up based on a thorough medical history. The Alcohol Use Disorders Identification Test (AUDIT) provides a reliable measure of drinking habits. CDT is a good supplement to the clinical diagnosis when there is doubt, if factors associated with false-positive values are appreciated. The diagnostic value of EEG is limited.

A solid-phase sequencing technique was applied to quantify the mitochondrial A3243G mutation in three families with maternally inherited diabetes and deafness. A correlation between the level of heteroplasmy and age at onset was found. The fluctuation of the heteroplasmy levels of the A3243G mutation was monitored from 4 to 18 years, in three female patients. Using the minisequencing method, the level of heteroplasmy was found to decrease over time in endothelial cell samples from all three patients. With a similar strategy, the heteroplasmy levels of two neutral polymorphisms in the non-coding region of the mtDNA in healthy individuals were monitored. It has recently been suggested that heteroplasmy occurs frequently at neutral nucleotide positions in the control region of mtDNA and that the heteroplasmy level changes with age. The level of heteroplasmy of the neutral polymorphisms was found to remain unchanged over a time period of up to 25 years in four individuals. Wilson disease (WD) is caused by mutations in the ATP7B gene that encodes a mitochondrial copper-transporting ATPase. The worldwide prevalence of WD has been estimated to 1 in 30 000. Based on the number of diagnosed patients the estimated prevalence in the Swedish population would be 1 in 300 000. The prevalence of WD in Sweden was estimated indirectly by quantitative minisequencing analysis of two WD-causing mutations in pooled DNA samples. In addition, the population frequencies of eight SNPs in the ATP7B gene were determined. Our results confirmed that WD is truly more rare in Sweden than in other populations. A previously nondetectable diversity of alleles at the KIT locus, determining the coat color of pigs, was found by using three quantitative methods, minisequencing, pyrosequencing and the "TaqMan" 5' exonuclease assay. A splice-site mutation and a duplication of the KIT gene, encoding the mast/stem cell growth factor receptor causes the allelic diversity. Despite of a strong selection for white color dating from the medevial era, the desired phenotype has not been fixed. This study provides tools for genotyping the complicated KIT locus in pigs, which may be used for the purpose of breeding true for white color.

Adenovirus infected cells provide a good model system for studying nuclear organization during RNA production and transport. This thesis is focused on the dynamic organization of splicing factors during the late phase of Adenovirus infection in HeLa cells, the nuclear localization of viral RNA, and the pathway used for viral RNA transport to the cytoplasm. Splicing factors are relocalized from interchromatin granule clusters to sites of transcription in Adenovirus infected cells at intermediate times of infection. Later, splicing factors and viral RNA accumulate posttranscriptionally in interchromatin granule clusters. The release of the splicing factors from transcription sites was energy dependent or preceded by energy requiring mechanisms. Our data indicated that phosphorylation events inhibited by staurosporine, and 3' cleavage of the transcript are two possible mechanisms involved prior to the release of the RNP complex from transcription sites. A viral protein derived from orf6 of early region 4, 34K, is important for the nuclear stability and transport of late viral mRNA derived from the major late transcription unit. A viral mutant lacking this region is defective for posttranscriptional accumulation of viral mRNA in interchromatin granule clusters, and for the accumulation of viral RNA in the cytoplasm. These results suggest that posttranscriptional accumulation of viral RNA in interchromatin granule clusters may contribute to the maturation of the RNP complex or sorting of RNAs and proteins, to prepare the final RNP complex for transport to the cytoplasm. A previous model suggested that adenoviral late mRNA is transported to the cytoplasm by utilizing the CRM-1 pathway. This pathway can be blocked by the drug leptomycin B. The data presented in paper IV suggests that this model might not be applicable, since leptomycin B did not inhibit adenoviral late gene expression.

Platelet-derived growth factor receptor α (PDGFRα) plays an important role during embryogenesis. After implantation, the patterns of expression of Pdgfrα and its ligand Pdgf-A undergo an "autocrine-paracrine transition", in that Pdgf-A becomes expressed in the ectoderm and epithelia, while Pdgfrα is expressed in the adjacent mesenchymal tissue. In human tumors, such as malignant glioma, both PDGF and PDGFRα are overexpressed within the same tissue, indicating that an autocrine PDGF loop is generated in the tumors. This thesis is focused on the in vivo functionality of the PDGFRα gene (PDGFRA) promoter, arid on the effect of autocrine PDGF-A stimulation in transgenic n-iice during embryogenesis. To test the in vivo promoter function of a human PDGFRA 2.2 kb 5' flanking fragment, we generated transgenic mouse lines and found that the 2.2 kb fragment was able to promote lacZ reporter gene expression in most of the endogenous Pdgfra expressing tissues. Absence of expression and "ectopic" expression of the transgenic lacZ were also observed. To investigate the autocrine PDGF effect, we produced autocrine PDGF-As (A short-chain) transient transgenic embryos. These transgenic embryos carried a 6 kb mouse Pdgfra 5' flanking sequence linked to a human PDGF-As cDNA. The pattern of expression of the PDGF-As transgene mRNA was similar to that of lacZ. Some of the transgenic embryos exhibited severe abnormal phenotypes, such as midline fusion defects in the cephalic and craniofacial region and small body size, and these embryos die at mid-gestation stage. These findings indicate that a paracrine pattern of expression and the dosage of PDGF are important for sustaining normal embryo development, especially with regard to the middline fusion in craniofacial regions. The possible signaling pathways that may be involved in regulating Pdgfra activity were also studied by comparison of patterns of mRNA expression of Gli, Ptc, and Paxl with that of Pdgfra. The results pointed to the possibility that the Shh signaling pathway may be involved in the regulation of Pdgfra expression for example during early bone and foregut development. The specific regulatory mechanisms may vary for different tissues.

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.