Mitochondrial helicase Twinkle in mtDNA copy number regulation and a late-onset disease model

Mitochondrial diseases are caused by disturbances of the energy metabolism. The disorders range from severe childhood neurological diseases to muscle diseases of adults. Recently, mitochondrial dysfunction has also been found in Parkinson s disease, diabetes, certain types of cancer and premature aging. Mitochondria are the power plants of the cell but they also participate in the regulation of cell growth, signaling and cell death. Mitochondria have their own genetic material, mtDNA, which contains the genetic instructions for cellular respiration. Single cell may host thousands of mitochondria and several mtDNA molecules may reside inside single mitochondrion. All proteins needed for mtDNA maintenance are, however, encoded by the nuclear genome, and therefore, mutations of the corresponding genes can also cause mitochondrial disease. We have here studied the function of mitochondrial helicase Twinkle. Our research group has previously identified nuclear Twinkle gene mutations underlying an inherited adult-onset disorder, progressive external ophthalmoplegia (PEO). Characteristic for the PEO disease is the accumulation of multiple mtDNA deletions in tissues such as the muscle and brain. In this study, we have shown that Twinkle helicase is essential for mtDNA maintenance and that it is capable of regulating mtDNA copy number. Our results support the role of Twinkle as the mtDNA replication helicase. No cure is available for mitochondrial disease. Good disease models are needed for studies of the cause of disease and its progression and for treatment trials. Such disease model, which replicates the key features of the PEO disease, has been generated in this study. The model allows for careful inspection of how Twinkle mutations lead to mtDNA deletions and further causes the PEO disease. This model will be utilized in a range of studies addressing the delay of the disease onset and progression and in subsequent treatment trials. In conclusion, in this thesis fundamental knowledge of the function of the mitochondrial helicase Twinkle was gained. In addition, the first model for adult-onset mitochondrial disease was generated.

Primary polyestradiol phosphate versus orchiectomy for advanced prostate cancer : Studies on the efficacy, cardiovascular complications and prognostic factors

Paikallisesti levinnyttä (T3-4 M0) ja luustoon levinnyttä (T1-4 M1) eturauhassyöpää sairastaneet potilaat satunnaistettiin kirurgiseen kastraatioon (orkiektomia) tai lääkkeelliseen kastraatioon lihaksensisäisellä polyestradiolifosfaatilla (PEP) annoksella 240 mg/kk. Verrattiin hoitojen kliinistä tehoa sekä sydän- ja verisuonikomplikaatioita (SV-komplikaatioita). Verrattiin myös hoitoa edeltäviä plasman testosteroni (T) ja estradioli (E2) pitoisuuksia T3-4 M0 ja T1-4 M1 potilaiden välillä sekä selvitettiin potilaiden yleistilan vaikutusta näihin hormonitasoihin. Lopuksi luotiin T1-4 M1 potilaille eturauhassyövän aiheuttaman kuoleman ennusteellinen riskiluokittelu kolmeen riskiryhmään käyttämällä hoitoa edeltäviä ennustetekijöitä. Kliinisessä tehossa ei orkiektomian ja PEP-hoidon välillä todettu tilastollisesti merkitsevää eroa. Odotetusti T1-4 M1 potilaiden ennuste oli huonompi kuin T3-4 M0 potilaiden. T1-4 M1 potilailla ei ollut SV-kuolemissa hoitoryhmien välillä tilastollista eroa, mutta ei-tappavia SV-komplikaatioita oli PEP ryhmässä (5.9%) enemmän kuin orkiektomia ryhmässä (2.0%). T3-4 M0 potilailla PEP-hoitoon liittyi tilastollisesti merkitsevä SV-kuolleisuus riski orkiektomiaan verrattuna (p = 0.001). PEP ryhmässä 67% kuolemista oli akuutteja sydäninfarkteja. Tämä PEP hoitoon liittyvä sydäninfarktiriski (mukaan lukien myös ei-tappavat sydäninfarktit) oli merkitsevästi pienempi potilailla, joiden hoitoa edeltävä E2 taso oli vähintään 93 pmol/l (p = 0.022). E2 taso oli merkitsevästi matalampi T1-4 M1 potilailla (74.7 pmol/l) kuin T3-4 M0 potilailla (87.9 pmol/l), mutta vastaavaa eroa ei ollut T tasoissa. Sekä T3-4 M0 että T1-4 M1 potilailla yleistilan lasku osittain selitti yksilöllisen T ja E2 tasojen laskun. Eturauhassyövän aiheuttaman kuoleman riskiryhmäluokittelu (Rg) kolmeen ryhmään luotiin käyttämällä alkalista fosfataasia (AFOS), prostata spesifistä antigeenia (PSA), laskoa (La) ja potilaan ikää. Yksi riskipiste annettiin, jos AFOS > 180 U/l (tällä hetkellä käytössä olevalla menetelmällä AFOS > 83 U/l), PSA > 35 µg/l, La > 80 mm/h ja ikä < 60 vuotta. Lopuksi pisteet laskettiin yhteen. Muodostettiin seuraavat ryhmät: Rg-a (0 -1 riskipistettä), Rg-b (2 riskipistettä) ja Rg-c (3 – 4 riskipistettä). Eturauhassyövän aiheuttama kuoleman riski lisääntyi merkitsevästi siirryttäessä riskiryhmästä seuraavaan (p < 0.001). Rg-luokittelu oli kliinisesti käytännöllinen ja hyvä havaitsemaan huonon ennusteen potilaat.

Thrombin regulation in newborn infants

The coagulation system of newborn infants differs markedly from that of older children and adults. The activities of most coagulation factors and anticoagulants are low, leading to altered regulation in the formation of the key enzyme, thrombin. Timely and adequate generation of thrombin is essential, as thrombin activates platelets and many coagulation factors, cleaves fibrinogen into fibrin and activates the antithrombotic and anti-inflammatory protein C pathway. On the other hand, excess thrombin may promote thrombotic complications and exacerbate harmful inflammatory reactions. Despite the characteristic features, the newborn coagulation system can be considered physiological, since healthy newborns rarely show haemorrhagic or thrombotic complications. Sick newborns, however, often encounter clinical situations that challenge their coagulation system. The aim of this study was to clarify the behaviour of the neonatal coagulation system in selected clinical situations, with a special emphasis on the generation of thrombin. Thrombin was measured by in vivo thrombin generation markers and by thrombin generation potential in vitro. The patient groups included sick newborns undergoing intensive care and receiving fresh-frozen plasma (FFP), requiring exchange transfusions (ET) or presenting with a congenital heart defect requiring open heart surgery. Additionally, healthy newborns with inherited heterozygous factor V Leiden (FVL) mutation were studied. Thrombin generation potential was also analysed in cord plasma of healthy infants and in adults. Healthy as well as sick newborn infants showed lower total thrombin generation potential in vitro but faster initiation of thrombin generation than adults. These findings were qualitatively similar when plasma was supplemented with platelets. Platelets, however, significantly altered the effect of the major anticoagulant, activated protein C (APC), on thrombin generation potential. In accordance with previous studies, thrombin generation in healthy newborn platelet-poor plasma was resistant to the anticoagulant effects of APC, but when the plasma was supplemented with platelets APC attenuated thrombin generation significantly more in newborns than in adults. In vivo generation of thrombin was elevated in nearly all of the sick newborn infants. The low-volume FFP transfusion as opposed to the change from neonatal to adult blood in ET exerted markedly different effects on neonatal thrombin generation. FFP reduced the in vivo generation of thrombin in those newborns with the highest pretransfusional thrombin generation, thus acting as an anticoagulant agent. In those infants with lower pretransfusional thrombin generation, the effect of FFP on thrombin generation was fairly neutral. On the other hand, the combination of red blood cells and FFP, used to perform ET, significantly increased the in vivo thrombin formation and shifted the balance in the newborn coagulation system to the procoagulant direction. Cardiopulmonary bypass (CPB) also significantly increased the in vivo thrombin generation, but the thrombin generation profile during CPB differed from that previously observed in adults. Escalation of thrombin at early reperfusion was not observed in newborns; in adults, its occurrence is associated with postoperative myocardial damage. Finally, in healthy newborns with FVL heterozygosity, faster initiation of thrombin generation was observed compared with controls. Interestingly, FV level was lower in FVL-heterozygous infants, possibly to counteract the procoagulant effects induced by FVL. In conclusion, unique features regarding thrombin regulation in newborn infants were observed. These features included a novel platelet effect on the regulation of the protein C pathway. The clinical challenges mainly seemed to shift the balance in the coagulation system of newborns to the procoagulant direction. Blood component transfusions markedly affected coagulation in a manner specific to the product but that could also be altered by the clinical situation. Overall, the results highlight the need for understanding developmental haemostasis for both diagnostic and therapeutic purposes.

Soy supplementation and role of equol production capability in postmenopausal women using tibolone: effects on cardiovascular risk markers

Tibolone, a synthetic steroid, is effective in the treatment of postmenopausal symptoms. Its cardiovascular safety profile has been questioned, because tibolone reduces the levels of high-density lipoprotein (HDL) cholesterol. Soy-derived isoflavones may offer health benefits, particularly as regards lipids and also other cardiovascular disease (CVD) risk factors. The soy-isoflavone metabolite equol is thought to be the key as regards soy-related beneficial effects. We studied the effects of soy supplementation on various CVD risk factors in postmenopausal monkeys and postmenopausal women using tibolone. In addition, the impact of equol production capability was studied. A total of 18 monkeys received casein/lactalbumin (C/L) (placebo), tibolone, soy (a woman s equivalent dose of 138 mg of isoflavones), or soy with tibolone in a randomized order for 14 weeks periods, and there was a 4-week washout (C/L) in between treatments. Postmenopausal women using tibolone (N=110) were screened by means of a one-week soy challenge to find 20 women with equol production capability (4-fold elevation from baseline equol level) and 20 control women, and treated in a randomized cross-over trial with a soy powder (52 g of soy protein containing 112 mg of isoflavones) or placebo for 8 weeks. Before and after the treatments lipids and lipoproteins were assessed in both monkeys and women. In addition, blood pressure, arterial stiffness, endothelial function, sex steroids, sex hormone-binding globulin (SHBG), and vascular inflammation markers were assessed. A 14% increase in plasma low-density lipoprotein (LDL) + very low-density lipoprotein (VLDL) cholesterol was observed in tibolone-treated monkeys vs. placebo. Soy treatment resulted in a 18% decrease in LDL+VLDL cholesterol, and concomitant supplementation with tibolone did not negate the LDL+VLDL cholesterol-lowering effect of soy. A 30% increase in HDL cholesterol was observed in monkeys fed with soy, whereas HDL cholesterol levels were reduced (48%) after tibolone. Interestingly, Soy+Tibolone diet conserved HDL cholesterol levels. Tibolone alone increased the total cholesterol (TC):HDL cholesterol ratio, whereas it was reduced by Soy or Soy+Tibolone. In postmenopausal women using tibolone, reductions in the levels of total cholesterol and LDL cholesterol were seen after soy supplementation compared with placebo, but there was no effect on HDL cholesterol, blood pressure, arterial stiffness or endothelial function. Soy supplementation decreased the levels of estrone in equol producers, and those of testosterone in the entire study population. No changes were seen in the levels of androstenedione, dehydroepiandrosterone sulfate, or SHBG. The levels of vascular cell adhesion molecule-1 increased, and platelet-selectin decreased after soy treatment, whereas C-reactive protein and intercellular adhesion molecule-1 remained unchanged. At baseline and unrelated to soy treatment, equol producers had lower systolic, diastolic and mean arterial pressures, less arterial stiffness and better endothelial function than non-producers. To conclude, soy supplementation reversed the tibolone-induced fall in HDL cholesterol in postmenopausal monkeys, but this effect was not seen in women taking tibolone. Equol production capability was associated with beneficial cardiovascular changes and thus, this characteristic may offer cardiovascular benefits, at least in women using tibolone.

Parathyroid hormone as an outcome indicator in old age

Serum parathyroid hormone (PTH) and vitamin D are the major regulators of extracellular calcium homeostasis. The inverse association between PTH and vitamin D and the common age-related elevation of the PTH concentration are well known phenomena. However, the confounding or modifying factors of this relationship and their impact on the response of PTH levels to vitamin D supplementation need further investigation. Clinical conditions such as primary hyperparathyroidism (PHPT), renal failure and vitamin D deficiency, characterized by an elevation of the PTH concentration, have been associated with impaired long-term health outcomes. Curative treatments for these conditions have also been shown to decreases PTH concentration and attenuate some of the adverse health effects. In PHPT it has also been commonly held that hypercalcaemia, the other hallmark of the disease, is the key mediator of the adverse health outcomes. In chronic kidney disease the systemic vascular disease has been proposed to have the most important impact on general health. Some evidence also indicates that vitamin D may have significant extraskeletal actions. However, the frank elevation of PTH concentration seen in advanced PHPT and in end-stage renal failure have also been suggested to be at least partly causally related to an increased risk of death as well as cognitive dysfunction. However, the exact mechanisms have remained unclear. Furthermore, the predictive value of elevated PTH in unselected older populations has been less well studied. The studies presented in this thesis investigated the impact of age and mobility on the responses of PTH levels to vitamin D deficiency and supplementation. Furthermore, the predictive value of PTH for long-term survival and cognitive decline was addressed in an unselected population of older people. The hypothesis was that age and chronic immobility are related to a persistently blunted elevation of PTH concentration, even in the presence of chronic vitamin D deficiency, and to attenuated responses of PTH to vitamin D supplementation. It was also further hypothesized that a slightly elevated or even high-normal PTH concentration is an independent indicator of an increased risk of death and cognitive decline in the general aged population. The data of this thesis are based on three samples: a meta-analysis of published vitamin D supplementation trials, a randomized placebo controlled six-month vitamin D supplementation trial, and a longitudinal prospective cohort study on a general aged population. Based on a PubMed search, a meta-analysis of 52 clinical trials with 6 290 adult participants was performed to evaluate the impact of age and immobility on the responses of PTH to 25-OHD levels and vitamin D supplementation. A total of 218 chronically immobile, very old inpatients were also enrolled into a vitamin D supplementation trial. Mortality data for these patients was also collected after a two-year follow-up. Finally, data from the Helsinki Aging Study, which followed three random age cohorts (75, 80 and 85 years) until death in almost all subjects, was used to evaluate the predictive value of PTH for long-term survival and cognitive decline. This series of studies demonstrated that in older people without overt renal failure or severe hypercalcaemia, serum 25-OHD and PTH were closely associated, but this relationship was also affected by age and immobility. Furthermore, a substantial proportion of old chronically bedridden patients did not respond to vitamin D deficiency by elevating PTH, and the effect of a high-dose (1200 IU/d) six-month cholecalciferol supplementation on the PTH concentration was minor. This study demonstrated longitudinally for the first time that the blunted PTH also persisted over time. Even a subtle elevation of PTH to high-normal levels predicted impaired long-term health outcomes. Slightly elevated PTH concentrations indicated an increased risk of clinically significant cognitive decline and death during the last years of life in a general aged population. This association was also independent of serum ionized calcium (Ca2+) and the estimated glomerular filtration rate (GFR). A slightly elevated PTH also indicated impaired two-year survival during the terminal years of frail elderly subjects independently of Ca2+, GFR, and of 25-OHD levels. The interplay between PTH and vitamin D in the regulation of calcium homeostasis is more complex than has been generally considered. In addition to muskuloskeletal health parathyroid hormone is also related to the maintenance of other important domains of health in old age. Higher PTH concentrations, even within conventional laboratory reference ranges, seem to be an independent indicator of an increased risk of all-cause and of cardiovascular mortality, independently of established cardiovascular risk factors, disturbances in mineral metabolism, and renal failure. Limited and inconsistent evidence supports the role of vitamin D deficiency-related lack of neuroprotective effects over the causal association between PTH and impaired cognitive functions. However, the causality of these associations remains unclear. The clinical implications of the observed relationships remain to be elucidated by future studies interfering with PTH concentrations, especially by long-term interventions to reduce PTH.

Heme oxygenase-1 in cardiovascular diseases

Myocardial infarction (MI) and heart failure are major causes of morbidity and mortality worldwide. Treatment of MI involves early restoration of blood flow to limit infarct size and preserve cardiac function. MI leads to left ventricular remodeling, which may eventually progress to heart failure, despite the established pharmacological treatment of the disease. To improve outcome of MI, new strategies for protecting the myocardium against ischemic injury and enhancing the recovery and repair of the infarcted heart are needed. Heme oxygenase-1 (HO-1) is a stress-responsive and cytoprotective enzyme catalyzing the degradation of heme into the biologically active reaction products biliverdin/bilirubin, carbon monoxide (CO) and free iron. HO-1 plays a key role in maintaining cellular homeostasis by its antiapoptotic, anti-inflammatory, antioxidative and proangiogenic properties. The present study aimed, first, at evaluating the role of HO-1 as a cardioprotective and prohealing enzyme in experimental rat models and at investigating the potential mechanisms mediating the beneficial effects of HO-1 in the heart. The second aim was to evaluate the role of HO-1 in 231 critically ill intensive care unit (ICU) patients by investigating the association of HO-1 polymorphisms and HO-1 plasma concentrations with illness severity, organ dysfunction and mortality throughout the study population and in the subgroup of cardiac patients. We observed in an experimental rat MI model, that HO-1 expression was induced in the infarcted rat hearts, especially in the infarct and infarct border areas. In addition, pre-emptive HO-1 induction and CO donor pretreatment promoted recovery and repair of the infarcted hearts by differential mechanisms. CO promoted vasculogenesis and formation of new cardiomyocytes by activating c-kit+ stem/progenitor cells via hypoxia-inducible factor 1 alpha, stromal cell-derived factor 1 alpha (SDF-1a) and vascular endothelial growth factor B, whereas HO-1 promoted angiogenesis possibly via SDF-1a. Furthermore, HO-1 protected the heart in the early phase of infarct healing by increasing survival and proliferation of cardiomyocytes. The antiapoptotic effect of HO-1 persisted in the late phases of infarct healing. HO-1 also modulated the production of extracellular matrix components and reduced perivascular fibrosis. Some of these beneficial effects of HO-1 were mediated by CO, e.g. the antiapoptotic effect. However, CO may also have adverse effects on the heart, since it increased the expression of extracellular matrix components. In isolated perfused rat hearts, HO-1 induction improved the recovery of postischemic cardiac function and abrogated reperfusion-induced ventricular fibrillation, possibly in part via connexin 43. We found that HO-1 plasma levels were increased in all critically ill patients, including cardiac patients, and were associated with the degree of organ dysfunction and disease severity. HO-1 plasma concentrations were also higher in ICU and hospital nonsurvivors than in survivors, and the maximum HO-1 concentration was an independent predictor of hospital mortality. Patients with the HO-1 -413T/GT(L)/+99C haplotype had lower HO-1 plasma concentrations and lower incidence of multiple organ dysfunction. However, HO-1 polymorphisms were not associated with ICU or hospital mortality. The present study shows that HO-1 is induced in response to stress in both experimental animal models and severely ill patients. HO-1 played an important role in the recovery and repair of infarcted rat hearts. HO-1 induction and CO donor pretreatment enhanced cardiac regeneration after MI, and HO-1 may protect against pathological left ventricular remodeling. Furthermore, HO-1 induction potentially may protect against I/R injury and cardiac dysfunction in isolated rat hearts. In critically ill ICU patients, HO-1 plasma levels correlate with the degree of organ dysfunction, disease severity, and mortality, suggesting that HO-1 may be useful as a marker of disease severity and in the assessment of outcome of critically ill patients.

Functional role of the Mso1p-Sec1p complex in membrane fusion regulation

Sec1/Munc18 (SM) protein family members are evolutionary conserved proteins. They perform an essential, albeit poorly understood function in SNARE complex formation in membrane fusion. In addition to the SNARE complex components, only a few SM protein binding proteins are known. Typically, their binding modes to SM proteins and their contribution to the membrane fusion regulation is poorly characterised. We identified Mso1p as a novel Sec1p interacting partner. It was shown that Mso1p and Sec1p interact at sites of polarised secretion and that this localisation is dependent on the Rab GTPase Sec4p and its GEF Sec2p. Using targeted mutagenesis and N- and C-terminal deletants, it was discovered that the interaction between an N-terminal peptide of Mso1p and the putative Syntaxin N-peptide binding area in Sec1p domain 1 is important for membrane fusion regulation. The yeast Syntaxin homologues Sso1p and Sso2p lack the N-terminal peptide. Our results show that in addition to binding to the putative N-peptide binding area in Sec1p, Mso1p can interact with Sso1p and Sso2p. This result suggests that Mso1p can mimic the N-peptide binding to facilitate membrane fusion. In addition to Mso1p, a novel role in membrane fusion regulation was revealed for the Sec1p C-terminal tail, which is missing in its mammalian homologues. Deletion of the Sec1p-tail results in temperature sensitive growth and reduced sporulation. Using in vivo and in vitro experiments, it was shown that the Sec1p-tail mediates SNARE complex binding and assembly. These results propose a regulatory role for the Sec1p-tail in SNARE complex formation. Furthermore, two novel interaction partners for Mso1p, the Rab GTPase Sec4p and plasma membrane phospholipids, were identified. The Sec4p link was identified using Bimolecular Fluorescence Complementation assays with Mso1p and the non-SNARE binding Sec1p(1-657). The assay revealed that Mso1p can target Sec1p(1-657) to sites of secretion. This effect is mediated via the Mso1p C-terminus, which previously has been genetically linked to Sec4p. These results and in vitro binding experiments suggest that Mso1p acts in cooperation with the GTP-bound form of Sec4p on vesicle-like structures prior to membrane fusion. Mso1p shares homology with the PIP2 binding domain of the mammalian Munc18 binding Mint proteins. It was shown both in vivo and in vitro that Mso1p is a phospholipid inserting protein and that this insertion is mediated by the conserved Mso1p amino terminus. In vivo, the Mso1p phospholipid binding is needed for sporulation and Mso1p-Sec1p localisation at the sites of secretion at the plasma membrane. The results reveal a novel layer of membrane fusion regulation in exocytosis and propose a coordinating role for Mso1p in connection with membrane lipids, Sec1p, Sec4p and SNARE complexes in this process.

Sterol-binding proteins in late endosomes : Regulation of endosome motility and lipid metabolism

Despite its bad reputation in the mass media, cholesterol is an indispensable constituent of cellular membranes and vertebrate life. It is, however, also potentially lethal as it may accumulate in the arterial intima causing atherosclerosis or elsewhere in the body due to inherited conditions. Studying cholesterol in cells, and research on how the cell biology of cholesterol affects on system level is essential for a better understanding of the disease states associated with cholesterol and for the development of new therapies for these conditions. On its way to the cell, exogenous cholesterol traverses through endosomes, transport vesicles involved in internalizing material to cells, and needs to be transported out of this compartment. This endosomal pool of cholesterol is important for understanding both the common disorders of metabolism and the more rare hereditary disorders of cholesterol metabolism. The study of cholesterol in cells has been hampered by the lack of bright fluorescent sterol analogs that would resemble cholesterol enough to be used in cellular studies. In the first study of my thesis, we present a new sterol analog, Boron-Dipyrromethene (BODIPY)-cholesterol for visualizing sterols in living cells and organism. This fluorescent cholesterol derivative is shown to behave similarly to cholesterol both by atomic scale computer simulations and biochemical experiments. We characterize its localization inside different types of living cells and show that it can be used to study sterol trafficking in living organisms. Two sterol binding proteins associated with the endosomal membrane; the Niemann-Pick type C disease protein 1 (NPC1) and the Oxysterol Binding Protein Related Protein 1 (ORP1) are the subjects of the rest of this study. Sensing cholesterol on endosomes, transporting lipids away from this compartment and the effects these lipids play on cellular metabolism are considered. In the second study we characterize how the NPC1 protein affects lipid metabolism. We show that this cholesterol binding protein affects synthesis of triglycerides and that genetic polymorphisms or a genetic defect in the NPC1 gene affect triglyceride on the whole body level. These effects take place via regulation of carbon fluxes to different lipid classes in cells. In the third part we characterize the effects of another endosomal sterol binding protein, ORP1L on the function and motility of endosomes. Specifically we elucidate how a mutation in the ability of ORP1L to bind sterols affects its behavior in cells, and how a change in ORP1L levels in cells affects the localization, degradative capacity and motility of endosomes. In addition we show that ORP1L manipulations affect cholesterol balance also in macrophages, a cell type important for the development of atherosclerosis.

Depression and Cognition in Type 2 Diabetes from a Life Course Perspective

Background: Type 2 diabetes is linked to several complications which add to both physical and mental distress. Depression is a common co-morbidity of diabetes which can occur both as a cause and a consequence of type 2 diabetes. Depression has been shown to correlate with glucose regulation and treating depression might prove beneficial for glucose regulation as well as for mental well being. Another complication which might affect diabetes management is cognitive decline. Several risk factors and complications of diabetes might modify the risk for developing cognitive impairment, which is increased 1.5 times among subjects with type 2 diabetes. Type 2 diabetes, depression and impaired cognitive performance have all been linked to low birth weight. This thesis aimed to explore the effects and interactions of birth weight, depression and cognitive ability in relation to type 2 diabetes from a life course perspective. Subjects and methods: Studies I, II and V were part of the Helsinki Birth Cohort Study. 2003 subjects participated in an extensive clinical examination at an average age of 61 years. A standard glucose tolerance test (OGTT) was performed and depressive symptoms were assessed using the Beck Depression Inventory (BDI). In addition data was obtained from child welfare clinics and national registers. A subset of the cohort (n=1247) also performed a test on cognitive performance (CogState ®) at the average age of 64. Studies III and IV were randomised clinical trials where mildly depressed diabetic subjects were treated with paroxetine or placebo and the effect on metabolic parameters and quality of life was assessed. The first trial included 14 women and lasted 10 weeks, while the second trial included 43 subjects, both men and women, and lasted 6 months. Results: Type 2 diabetes was positively associated with the occurrence of depressive symptoms. Among diabetic subjects 23.6% had depressive symptoms, compared to 16.7% of subjects with normal glucose tolerance (OR = 1.77, p<0.001). Formal mediation analysis revealed that cardiovascular disease (CVD) is likely to act as a mediator in the association. Furthermore, low birth weight was found to modify the association between type 2 diabetes, CVD and depression. The association between BDI score and having type 2 diabetes or CVD was twice as strong in the subgroup with low birth weight (≤ 2500g) compared with the group with birth weight > 2500g (p for interaction 0.058). In the six months long randomised clinical trial (study IV) paroxetine had a transient beneficial effect on glycosylated haemoglobin A1c (GHbA1c) and quality of life when compared to placebo after three months of treatment. In study V we found that subjects with known diabetes had a consistently poorer level of cognitive performance than subjects with normal glucose tolerance in most of the tested cognitive domains. This effect was further amplified among those born with a small birth weight (p for interaction 0.002). Conclusions: Type 2 diabetes is associated with a higher occurrence of depressive symptoms compared to subjects with normal glucose tolerance. This association is especially strong among subjects with CVD and those born with a low birth weight. Treating depressed diabetic subjects with paroxetine has no long term effect on glucose regulation. Physicians should be aware of depression as an important co-morbidity of type 2 diabetes. Both depression and the cognitive decline often seen among diabetic subjects are increased if the subject is born with a low birth weight. Physicians should recognise low birth weight as an additional risk factor and modifier of diabetic complications.

Essays on Platforms: Business Strategies, Regulation and Policy in Telecommunications, Media and Technology Industries

The growth of the information economy has been stellar in the last decade. General-purpose technologies such as the computer and the Internet have promoted productivity growth in a large number of industries. The effect on telecommunications, media and technology industries has been particularly strong. These industries include mobile telecommunications, printing and publishing, broadcasting, software, hardware and Internet services. There have been large structural changes, which have led to new questions on business strategies, regulation and policy. This thesis focuses on four such questions and answers them by extending the theoretical literature on platforms. The questions (with short answers) are: (i) Do we need to regulate how Internet service providers discriminate between content providers? (Yes.) (ii) What are the welfare effects of allowing consumers to pay to remove advertisements from advertisement-supported products?(Ambiguous, but those watching ads are worse off.) (iii) Why are some markets characterized by open platforms, extendable by third parties, and some by closed platforms, which are not extendable? (It is a trade-off between intensified competition for consumers and benefits from third parties) (iv) Do private platform providers allow third parties to access their platform when it is socially desirable? (No.)

University Students' Approaches to Learning, Self-Regulation, and Cognitive and Attributional Strategies : Connections with Well-Being and Academic Success

This dissertation empirically explores the relations among three theoretical perspectives: university students approaches to learning, self-regulated learning, as well as cognitive and attributional strategies. The relations were quantitatively studied from both variable- and person-centered perspectives. In addition, the meaning that students gave to their disciplinary choices was examined. The general research questions of the study were: 1) What kinds of relationships exist among approaches to learning, regulation of learning, and cognitive and attributional strategies? What kinds of cognitive-motivational profiles can be identified among university students, and how are such profiles related to study success and well-being? 3) How do university students explain their disciplinary choices? Four empirical studies addressed these questions. Studies I, II, and III were quantitative, applying self-report questionnaires, and Study IV was qualitative in nature. Study I explored relations among cognitive strategies, approaches to learning, regulation of learning, and study success by using correlations and a K-means cluster analysis. The participants were 366 students from various faculties at different phases of their studies. The results showed that all the measured constructs were logically related to each other in both variable- and person-centered approaches. Study II further examined what kinds of cognitive-motivational profiles could be identified among first-year university students (n=436) in arts, law, and agriculture and forestry. Differences in terms of study success, exhaustion, and stress among students with differing profiles were also looked at. By using a latent class cluster analysis (LCCA), three groups of students were identified: non-academic (34%), self-directed (35%), and helpless students (31%). Helpless students reported the highest levels of stress and exhaustion. Self-directed students received the highest grades. In Study III, cognitive-motivational profiles were identified among novice teacher students (n=213) using LCCA. Well-being, epistemological beliefs, and study success were looked at in relation to the profiles. Three groups of students were found: non-regulating (50%), self-directed (35%), and non-reflective (22%). Self-directed students again received the best grades. Non-regulating students reported the highest levels of stress and exhaustion, the lowest level of interest, and showed the strongest preference for certain and practical knowledge. Study IV, which was qualitative in nature, explored how first-year students (n = 536 ) in three fields of studies, arts, law, and veterinary medicine explained their disciplinary choices. Content analyses showed that interest appeared to be a common concept in students description of their choices across the three faculties. However, the objects of interest of the freshmen appeared rather unspecified. Veterinary medicine and law students most often referred to future work or a profession, whereas only one-fifth of the arts students did so. The dissertation showed that combining different theoretical perspectives and methodologies enabled us to build a rich picture of university students cognitive and motivational predispositions towards studying and learning. Further, cognitive-emotional aspects played a significant role in studying, not only in relation to study success, but also in terms of well-being. Keywords: approaches to learning, self-regulation, cognitive and attributional strategies, university students

Methods and tools for mass spectrometric lipidome analysis

The analysis of lipid compositions from biological samples has become increasingly important. Lipids have a role in cardiovascular disease, metabolic syndrome and diabetes. They also participate in cellular processes such as signalling, inflammatory response, aging and apoptosis. Also, the mechanisms of regulation of cell membrane lipid compositions are poorly understood, partially because a lack of good analytical methods. Mass spectrometry has opened up new possibilities for lipid analysis due to its high resolving power, sensitivity and the possibility to do structural identification by fragment analysis. The introduction of Electrospray ionization (ESI) and the advances in instrumentation revolutionized the analysis of lipid compositions. ESI is a soft ionization method, i.e. it avoids unwanted fragmentation the lipids. Mass spectrometric analysis of lipid compositions is complicated by incomplete separation of the signals, the differences in the instrument response of different lipids and the large amount of data generated by the measurements. These factors necessitate the use of computer software for the analysis of the data. The topic of the thesis is the development of methods for mass spectrometric analysis of lipids. The work includes both computational and experimental aspects of lipid analysis. The first article explores the practical aspects of quantitative mass spectrometric analysis of complex lipid samples and describes how the properties of phospholipids and their concentration affect the response of the mass spectrometer. The second article describes a new algorithm for computing the theoretical mass spectrometric peak distribution, given the elemental isotope composition and the molecular formula of a compound. The third article introduces programs aimed specifically for the analysis of complex lipid samples and discusses different computational methods for separating the overlapping mass spectrometric peaks of closely related lipids. The fourth article applies the methods developed by simultaneously measuring the progress curve of enzymatic hydrolysis for a large number of phospholipids, which are used to determine the substrate specificity of various A-type phospholipases. The data provides evidence that the substrate efflux from bilayer is the key determining factor for the rate of hydrolysis.

Hantavirus glycoprotein GN in virion assembly and regulation of interferon response

Hantaviruses have a tri-segmented negative-stranded RNA genome. The S segment encodes the nucleocapsid protein (N), M segment two glycoproteins, Gn and Gc, and the L segment the RNA polymerase. Gn and Gc are co-translationally cleaved from a precursor and targeted to the cis-Golgi compartment. The Gn glycoprotein consists of an external domain, a transmembrane domain and a C-terminal cytoplasmic domain. In addition, the S segment of some hantaviruses, including Tula and Puumala virus, have an open reading frame (ORF) encoding a nonstructural potein NSs that can function as a weak interferon antagonist. The mechanisms of hantavirus-induced pathogenesis are not fully understood but it is known that both hemorrhagic fever with renal syndrome (HFRS) and hantavirus (cardio) pulmonary syndrome (HCPS) share various features such as increased capillary permeability, thrombocytopenia and upregulation of TNF-. Several hantaviruses have been reported to induce programmed cell death (apoptosis), such as TULV-infected Vero E6 cells which is known to be defective in interferon signaling. Recently reports describing properties of the hantavirus Gn cytoplasmic tail (Gn-CT) have appeared. The Gn-CT of hantaviruses contains animmunoreceptor tyrosine-based activation motif (ITAM) which directs receptor signaling in immune and endothelial cells; and contain highly conserved classical zinc finger domains which may have a role in the interaction with N protein. More functions of Gn protein have been discovered, but much still remains unknown. Our aim was to study the functions of Gn protein from several aspects: synthesis, degradation and interaction with N protein. Gn protein was reported to inhibit interferon induction and amplication. For this reason, we also carried out projects studying the mechanisms of IFN induction and evasion by hantavirus. We first showed degradation and aggresome formation of the Gn-CT of the apathogenic TULV. It was reported earlier that the degradation of Gn-CT is related to the pathogenicity of hantavirus. We found that the Gn-CT of the apathogenic hantaviruses (TULV, Prospect Hill virus) was degraded through the ubiquitin-proteasome pathway, and TULV Gn-CT formed aggresomes upon treatment with proteasomal inhibitor. Thus the results suggest that degradation and aggregation of the Gn-CT may be a general property of most hantaviruses, unrelated to pathogenicity. Second, we investigated the interaction of TULV N protein and the TULV Gn-CT. The Gn protein is located on the Golgi membrane and its interaction with N protein has been thought to determine the cargo of the hantaviral ribonucleoprotein which is an important step in virus assembly, but direct evidence has not been reported. We found that TULV Gn-CT fused with GST tag expressed in bacteria can pull-down the N protein expressed in mammalian cells; a mutagenesis assay was carried out, in which we found that the zinc finger motif in Gn-CT and RNA-binding motif in N protein are indispensable for the interaction. For the study of mechanisms of IFN induction and evasion by Old World hantavirus, we found that Old World hantaviruses do not produce detectable amounts of dsRNA in infected cells and the 5 -termini of their genomic RNAs are monophosphorylated. DsRNA and tri-phosphorylated RNA are considered to be critical activators of innate immnity response by interacting with PRRs (pattern recognition receptors). We examined systematically the 5´-termini of hantavirus genomic RNAs and the dsRNA production by different species of hantaviruses. We found that no detectable dsRNA was produced in cells infected by the two groups of the old world hantaviruses: Seoul, Dobrava, Saaremaa, Puumala and Tula. We also found that the genomic RNAs of these Old World hantaviruses carry 5´-monophosphate and are unable to trigger interferon induction. The antiviral response is mainly mediated by alpha/beta interferon. Recently the glycoproteins of the pathogenic hantaviruses Sin Nombre and New York-1 viruses were reported to regulate cellular interferon. We found that Gn-CT can inhibit the induction of IFN activation through Toll-like receptor (TLR) and retinoic acid-inducible gene I-like RNA helicases (RLH) pathway and that the inhibition target lies at the level of TANK-binding kinase 1 (TBK-1)/ IKK epislon complex and myeloid differentiation primary response gene (88) (MyD88) / interferon regulatory factor 7 (IRF-7) complex.