Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification. In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype. In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21WAF1/CIP1 (CDKN1A) and p27KIP1 (CDKN1B). TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.

An accumulation of sudden unexpected cardiac deaths (SUCD) occurred in young Swedish orienteers, most of whom were elite athletes. From 1979 to 1992 the incidence in 18 to 34 year old male elite orienteers ranked on the national level the same year as death was calculated to 30 (per 100,000), which represents a 20 to 40 fold increase from the expected rate. From 1989 to 1992, the incidence was 50. There were, however, no indications on any similar clusters of SUCD in other sports. A special program to alter behaviour in orienteers was implemented in 1992-1993, after which there have been no more cases of SUCD in orienteers below 35 years of age. A histopathological re-evaluation of 16 cases of SUCD revealed myocarditis in 75% of these cases. In parallel, four of those cases also had changes mimicing arrhythmogenic right ventricular cardiomyopathy (ARVC). The combination of an increased incidence and myocarditis suggested that infection may be a pathogenetic factor. A broad search for different microorganisms in archival sera from five cases and tissues from the autopsies in two of those cases revealed the only common finding that all had antibodies to Chlamydia pneumoniae. DNA from C. pneumoniae was detected in the lung and heart in one of two cases. The intimate contact with nature of orienteers suggested possible zoonotic/vectorborne pathogens. Bartonella is such a pathogen and known to cross-react with C. pneumoniae. The use of PCR to test for DNA from the gltA gene of Bartonella in the two formerly mentioned cases of SUCD, and in three additional cases, gave positive bands from the hearts in four cases and the lung in a fifth case. The PCR products were sequenced and found to be identical to B. henselae in three cases and almost identical to B. quintana in the remaining two cases. Four of the five cases had antibodies to Bartonella when using micro immunofluorescence test with the antigens B. henselae, B. quintana, and B. elizabethae. The total prevalence of antibodies to Bartonella was 31% in 1,136 elite orienteers vs. 6.8% in 322 healthy blood donors (p<0.001), suggesting widespread exposure in the elite. It is hypothesized that subacute or reactivated Bartonella infection has a pathogenetic role in SUCD in orienteers, and may be involved in the development of ARVC-like disease.

The aim was to contribute to the optimal use of the resting ECG by exploring, in middle-aged and elderly men, the development and regression of ECG abnormalities; the prognostic value of the ECG for cardiovascular disease compared to conventional risk factors; and the impact of age at baseline and follow-up time for prediction of cardiovascular disease. It was based on the Uppsala Study of Adult Men cohort that was started in 1970. Participants were examined at ages 50, 70, 77, and 82, with annual updates on mortality and in-hospital morbidity using national registries. The studies indicated that the prevalence of silent MI and frequency of regression of major Q/QS patterns may be higher than previously believed. Considering that persistent T wave abnormalities and ST segment depression carried twice as high a risk for future cardiovascular disease (CVD) mortality as new or reverted abnormalities, the results suggested that serial electrocardiograms (ECG) would contribute to proper risk assessment. Also, the inclusion of ischemic ECG findings significantly increased the predictive power of the Framingham score at age 70 for CVD. While hypertension and dyslipidemia were consistent long-term risk factors for myocardial infarction at ages 50 and 70, the length of follow-up period and age at baseline affected the predictive power of ECG abnormalities, fasting insulin, BMI, and smoking. For stroke, midlife values for blood pressure and ECG abnormalities retained prognostic value over long follow-up periods, even though they improved when re-measured in elderly participants. ApoB/apoA1 ratio, driven by apoA1, was associated with stroke in elderly but not middle-aged men. Hyperinsulinemia and diabetes mellitus were more specifically associated with ischemic stroke than with any-cause stroke. In summary, the resting ECG carried prognostic information beyond conventional risk factors. Even though the low prevalence of ECG abnormalities at the age of 50 calls into question the role of the ECG as a screening tool, the additional risk information it carries with it justifies its regular and repeated registration above the age of 50.

Human papillomavirus type 16 (HPV-16) is the major causative agent of cervical cancer. The life cycle of this oncogenic DNA tumour virus is strictly associated with the differentiation program of the infected epithelial cells. Expression of the viral capsid genes L1 and L2 can only be detected in the terminally differentiated epithelial cells. The studies here focus on the regulation of HPV-16 late gene expression, which is under tight regulation. Our experimental system consisted of almost the full length HPV-16 genome driven by a strong CMV promoter. This plasmid and mutants thereof could be transfected into HeLa cells and RNA levels monitored. Using this system, we identified an hnRNP A1-dependent splicing silencer between positions 178 and 226 of the L1 gene. This silencer inhibited the use of the 3' splice site, located immediately upstream of the L1 AUG. We speculate that this splicing silencer plays an essential role in preventing late gene expression at an early stage of the viral life cycle. We subsequently identified a splicing enhancer located in the first 17 nucleotides of L1 that may be needed to counteract the multiple hnRNP A1 dependent splicing silencers in the L1 coding region. A 55kDa protein specifically bound to this splicing enhancer. We also demonstrated that binding of the cellular factors to the splicing silencer in the L1 coding region had an inhibitory effect on expression from L1 cDNA expression plasmids. The HPV-16 genome is divided into the early region and the late region, separated by the early poly(A) signal (pAE). pAE is used preferentially early in infection, thereby efficiently blocking late gene expression. We demonstrated that a 57 nucleotide U-rich region of the early 3’untranslated region (3’eUTR) acted as an enhancing upstream element on the usage of pAE. We demonstrated that this U-rich region specifically interacts with hFip1, CstF-64, hnRNP C1/C2 and PTB, suggesting that these factors were either enhancing or regulating polyadenylation at the HPV-16 pAE. In conclusion, two regulatory RNA elements that both act to prevent late gene expression at an early stage in the viral life cycle and in proliferating cells were identified: a splicing silencer in the late region and an upstream u-rich element at the pAE.

The sick building syndrome (SBS) comprises eye, nose, skin, and throat symptoms, headache and fatigue. Gender, personality aspects, and psychosocial factors at work have lately been at focus in health research. The main aim of this thesis was to investigate the relationships between SBS and personal factors, personality traits, and psychosocial work stress. Two different populations were studied: a cohort of 195 subjects working in suspected “sick buildings” followed 1988-1999, and a random sample of 695 subjects from the Swedish population, 20-65 y. Two personality scales were used; the Karolinska Scales of Personality (KSP) and the Sense of Coherence (SOC). Two stress questionnaires were used: the 3-dimensional demands-control-support model (DCS) and the Assessment of Perceived Psychosocial Work Environment (PPWE). SBS and personal factors were assessed by a postal symptoms questionnaire and a symptom score (SC) was calculated. Personality was assessed by means of two verbal personality scales, the KSP and the SOC. Females in problem buildings had more SBS than men, and females had a lower SOC and more anxiety and aggressivity in the KSP. Females and males in the general working population reported differences in psychosocial work environment. Moreover, the associations between personality traits and SBS, and between DCS and SBS, differed between males and females. Over all, the investigated occupants of suspected sick-buildings were within the normal range for both KSP and SOC norms. However, those with symptoms showed prominent personality vulnerability, especially in anxiety, aggressivity, and lower SOC, compared to those that were not reporting symptoms. Female gender, low age, asthma, and a history of atopy were related to SBS in the general working population. A combination of low social support and either passive, strained, or active work situation, as well as a combination of high social support and active work situation, were associated with SBS. In conclusion, measurements of personality and psychosocial work environment could be of value in future studies on environmental syndromes. Moreover, the demans-control-support model can predict SBS, but in a more complex way than indicated by earlier research.

Microarray technology is a promising approach for the simultaneous analysis of multiple single nucleotide polymorphisms (SNPs), which are the most abundant form of genetic variation. In this thesis enzyme-assisted microarray-based methods were developed to improve the accuracy and genotype discrimination power of the current methods for SNP genotyping. The improved technology was applied for analysing recessively inherited disease mutations, for Y-chromosomal SNPs in a population study, for an evolutionary analysis of SNPs in flycatchers and for multiplexed quantitative determination of SNP-allele frequencies in pooled DNA samples. A robust attachment chemistry for immobilising oligonucleotides on glass surface was established, based on an evaluation of eight covalent coupling methods. A four-colour fluorescence detection strategy, which enabled a multiplexed quantitative analysis for as little as 2% of a minority allele frequency in pooled samples was generated. Twenty-five Y-chromosomal SNPs were screened in a collection of 300 samples from five Finno-Ugric-speaking populations using minisequencing on microarrays. In these populations six distinct haplotypes were defined by the six SNPs that were polymorphic. Data from five microsatellite markers was combined with the SNP data, revealing shared Y-chromosomal haplotypes between the Finns and the Saami, indicating, in accordance with earlier data, at least two founding Y-chromosomal lineages in these populations. Database screening and subsequent validation of 125 potential SNPs in the highly repetitive type 1 interferon genes and genes coding for proteins in the interferon-related regulatory pathways revealed 25 informative SNPs in the Finnish and Swedish populations. These SNPs were included in a panel for microarray based genotyping that should find a variety of applications in genetic studies due to the important immunoregulatory functions of the IFN family. The significance of sex-chromosome evolution on speciation was investigated in two naturally hybridising flycatcher species (N=459) by analysing a panel of 20 SNPs using minisequencing on microarrays. A strong selection against gene flow across the species boundary of sex-linked genes was observed, as well as a sex-chromosomal influence on male plumage characteristics that have previously been shown to reinforce isolation in these birds. The results suggest a major role for sex-chromosome-mediated isolation of the two flycatcher species.

<p>This thesis describes the use of a new technology, the Cytosensor^® microphysiometer, in the in vitro evaluation of cytotoxic drugs, using the lymphoma cell line U-937 GTB and primary cultures of tumour cells from patients as model systems. The method was specifically applied to study the metabolic effects of the novel cyanoguanidine N-(6-(4-chlorophenoxy)hexyl)-N’-cyano-N’’-4-pyridylguanidine, CHS 828, currently in phase I/II clinical trials. </p><p>The Cytosensor^® measures metabolic effects as changes in the rate of extracellular acidification of cells exposed to a drug by perfusion. A number of standard cytotoxic drugs were found to produce typical and reproducible acidification response patterns during observation times up to 20 h. There seemed to be a relationship between a decrease in acidification and cytotoxicity, measured in the fluorometric microculture cytotoxicity assay (FMCA), after 20-24 h of continuous drug exposure.</p><p>In U-937 cells, CHS 828 induced a cytotoxic effect characterised by a steep concentration-response relationship followed by a plateau. After 24 h of incubation the DNA and protein synthesis were turned off. CHS 828 was found to produce a rapid and prolonged increase in extracellular acidification and lactate production similar to that of the structurally related mitochondrial inhibitor m-iodobenzylguanidine (MIBG). The CHS 828 induced acidification was observed in cell lines as well as in cells from various tumour types from patients and probably originates from increased glycolytic flux. The effects may be secondary to block of oxidative phosphorylation in the mitochondria, but the relevance of the early acidification is not clear. CHS 828 seemed to induce a late, at approximately 15 h, inhibition of the glycolysis followed by loss of ATP and subsequent cell death. After exposure to MIBG the loss of ATP and cell death occurred earlier and in parallel. The effects of CHS 828 were not found to resemble those of the structurally related polyamine biosynthesis inhibitor methylglyoxal-bis(guanyl-hydrazone) (MGBG). Thus, CHS 828 may represent a new and, thus, interesting mode of cytotoxic action worthwhile for further development.</p><p>In combinatory studies, a synergistic interaction was demonstrated between CHS 828 and the non-toxic drug amiloride. Additive-to-synergistic effects were also seen between CHS 828 and the bioreductive cytotoxic drug mitomycin C. In U-937 cells as well as in tumour cells from patients, CHS 828 demonstrated synergistic interactions in combination with melphalan and etoposide. </p><p>It is concluded that measurement in the Cytosensor^® microphysiometer of early cellular metabolic changes is a feasible and potentially valuable complement to more conventional methods used in the evaluation of anticancer agents. </p>

<p>The overall aim of this thesis was to describe (1) needs for occupational therapy among persons with self-perceived activity limitations and/or participation restrictions due to long-term/recurrent pain, and (2) treatment interventions in occupational therapy to meet demonstrated needs. The Liss’ model for assessing health care needs was used as a structural scheme. A randomly selected sample (n=10,000) from the Swedish general population aged 18-58 years was the foundation for the study population with and without pain. In addition, occupational therapists were included (n=109). Data collection was made by postal questionnaires. The results showed a prevalence of 26 % and an incidence rate of 0.07. Demographic characteristics of the sample were female gender, ages 40-58 years and fewer years of education than those without pain. Pain in shoulders/lower back of searing/aching/gnawing character was the most frequently reported. A majority of the respondents reported affective/emo-tional effects of pain mainly of depressive character and they had previously been on sick leave due to pain. Women reported higher frequencies of self-perceived activity limitations/ participation restrictions due to pain, more difficulties with intermediate ADL, perceived higher job demands and had longer sick leave than men. Men perceived poorer social support than did women. Needs for occupational therapy were reported mainly as a consequence of activity and temporal imbalance. High health care consumers reported higher frequencies of needs/problems than did low health care consumers. The main goals and interventions suggested by occupational therapists to meet the needs in pain management focused on increased knowledge of handling daily occupations with the purpose to reduce pain, maintain competence/improve performance of home maintenance, reduce consequences of pain and increase knowledge how to handle effects of pain.</p>

<p>Cardiovascular risk in primary hyperparathyroidism (HPT) is controversial, and studies mainly from Europe associate HPT with increased cardiovascular morbidity and mortality. Cardiovascular morphology and function were evaluated prospectively in 31 consecutive HPT patients (mean serum calcium 2.97±0.04) and randomly enrolled controls matched for age and sex. Patients were re-examined at normocalcemia about one year after parathyroidectomy. </p><p>HPT patients showed an operatively reversible disturbance in endothelial vasodilatory function that seemed unrelated to an early sign of atherosclerosis, i.e. thickness of carotid artery intima-media complex. Acute hypercalcemia in healthy subjects induced a similar impairment in endothelial function, which suggests a dependence on biochemical rather than structural vascular changes in HPT. Echocardiography showed left ventricular diastolic dysfunction and supernormal systolic performance being reversed after operation. Left ventricular mass tended to be irreversibly increased. During exercise HPT patients exhibited greater rise in systolic blood pressure compared to controls and an increased number of premature ventricular beats. This indicated increased work load and a propensity for fatal cardiac events. Following surgery, an improvement with less pronounced ST-segment depression was seen. 24-hour ambulatory blood pressure monitoring showed irreversibly increased levels despite maintained diurnal rhythm, while 24-hour heart rate variability analysis displayed blunted nocturnal increase of low and very low frequency bands that was corrected postoperatively. </p><p>Parathyroidectomy seems to alleviate most of the cardiovascular disturbances in HPT, except for hypertension. This is consistent with the normalised longevity in HPT treated with parathyroidectomy and supports active treatment of HPT. </p>

<p>Atrial fibrillation is a common disease. With pharmacological therapy most patients with atrial fibrillation have moderate or little symptoms, but a number of patients have severely symptomatic disease. This study evaluates two new non-pharmacological therapies for atrial fibrillation, the Maze procedure and atrial overdrive pacing.</p><p>In the patients planned for Maze surgery the quality of life, assessed with the SF-36 questionnaire, was very low before the operation. The quality of life was markedly improved 6 and 12 months after the Maze operation, and was comparable to values of the general Swedish population.</p><p>In the patients with sinus rhythm before surgery, the atrial size and transport function was assessed with echocardiography, and the autonomic balance was assessed with heart rate variability (HRV). The sizes of both atria were reduced and the transmitral early filling / atrial filling (E/A) ratio was increased at 6 months after the operation compared to before. A progressive increase of the E/A ratio was seen during the 24 months follow-up period, indicating a progressive decline of the left atrial transport function.</p><p>All components of HRV, including the parameters expressing sympathetic and parasympathetic modulation, were markedly decreased early after the Maze procedure compared to before. Late after the operation all components of HRV were still markedly depressed. This is interpreted as a partial autonomic denervation of the heart</p><p>Single-site right atrial overdrive pacing with two different levels of overdriving was compared with no pacing in patients with paroxysmal atrial fibrillation in a cross-over study. Overdrive pacing reduced the median number of episodes of atrial fibrillation with 50% compared to no pacing. There was no difference between medium rate overdrive pacing and high rate overdrive pacing.</p>