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.

Late results after paediatric cardiac surgery

Background: Congenital heart defects include a wide range of inborn malformations. Depending on the defect, the life expectancy of a newborn with cardiac anomaly varies from a few days to a normal life span. In most instances surgery, is the only treatment available. The late results of surgery have not been comprehensively investigated. Aims: Mortality, morbidity and the life situation of all Finnish patients who had been operated on for congenital heart defect during childhood were investigated. Methods: Patient and surgical data were gathered from all hospitals that had performed heart surgeries on children. Late mortality and survival data were obtained from the population registry, and the causes of deaths from Statistics Finland. Morbidity of patients operated on during 1953-1989 was assessed by the usage of medicines. The pharmacotherapy data of patients and controls were obtained from the Social Insurance Institute. The life situation of patients was surveyed by mailed questionnaire. Survival, causes of deaths and life situation of patients were compared with those of the general population. Results: A total of 7240 cardiac operations were performed on 6461 children during the first 37 years of cardiac surgery (1953-1989). The number of procedures constantly rose during this period, and the increase continued in later years. The patient material varied over time, as more defects became surgically treatable. During 1953-1989 the operative mortality (death within 30 days of surgery) was 6.9%. In the 1990s a slight rise occurred in early mortality, as increasingly complicated patients were surgically treated. During 2000-2003 practically no defects were beyond the operative range. Thus, the operative mortality of 4.4% was excellent, decreasing even further to 2.0% in 2004-2007. The overall 45-year survival of patients operated on in 1953-1989 was 78%, and the corresponding figure for the general population was 93%. Survival depended on the defect, being worst among patients with univentricular heart. Late survival was also better during the 1990s and at the beginning of the 21st century. Of the 6028 early survivors, 592 died late (>30 days) after surgery. A total of 397 deaths (67%) were related and 185 (31%) unrelated to congenital heart defect. The cause of death was unknown in 10 cases. Of those 5774 patients who survived their first operation and had complete follow-up, 16% were operated on several times. Seventeen percent of patients used medicines for cardiac symptoms (heart failure, arrhythmia, hypertension and coronary disease). Patients risk of using cardiac medicines was 2.16 (Cl 1.97-2.37) times higher than that of controls. Patients also had more genetic syndromes and mental retardation and more often used medicines for asthma and epilepsy. Adult patients who had been operated on as children had coped surprisingly well with their defects. Their level of education was similar and their employment level even higher than expected, and they were living in a steady relationship as often as the general population. Conclusions: Cardiac surgery developed rapidly, and nowadays practically all defects can be treated. The overall survival of all operated patients was 78%, 16% less than that of the general population. However, it was significantly better than the anticipated natural survival. However, many patients had health problems; 16% needed reoperations and 17% cardiac medicines to maintain their condition. Most of the patients assessed their general health as good and lived a normal life.

Simplified Treatment of Childhood Acute Bone and Joint Infections

Acute childhood osteomyelitis (OM), septic arthritis (SA), and their combination osteomyelitis with adjacent septic arthritis (OM+SA), are treated with long courses of antimicrobials and immediate surgery. We conducted a prospective multi-center randomized trial among Finnish children at age 3 months to 15 years in 1983-2005. According to the two-by-two factorial study design, children with OM or OM+SA received 20 or 30 days of antimicrobials, whereas those with SA were treated for 10 or 30 days. In addition, the whole series was randomized to be treated with clindamycin or a first-generation cephalosporin. Cases were included only if the causative agent was isolated. The treatment was instituted intravenously, but only for the first 2-4 days. Percutaneous aspiration was done to obtain a representative sample for bacteriology, but all other surgical intervention was kept at a minimum. A total of 265 patients fulfilled our strict inclusion criteria and were analyzed; 106 children had OM, 134 SA, and 25 OM+SA. In the OM group, one child in the long and one child in the short-term treatment group developed sequelae. One child with SA twice developed a late re-infection of the same joint, but the causative agents differed. Regarding surgery, diagnostic arthrocentesis or corticotomy was the only surgical procedure performed in most cases. Routine arthrotomy was not required even in hip arthritis. Serum C-reactive protein (CRP) proved to be a reliable laboratory index in the diagnosis and monitoring of osteoarticular infections. The recovery rate was similar regardless of whether clindamycin or a first-generation cephalosporin was used. We conclude that a course of 20 days of these well-absorbing antimicrobials is sufficient for OM or OM+SA, and 10 days for SA in most cases beyond the neonatal age. A short intravenous phase of only 2-5 days often suffices. CRP gives valuable information in monitoring the course of illness. Besides diagnostic aspiration, surgery should be reserved for selected cases.

Dissection of the genetic background of childhood onset progressive myoclonic epilepsies

The progressive myoclonic epilepsies (PMEs) are a clinically and etiologically heterogeneous group of symptomatic epilepsies characterized by myoclonus, tonic-clonic seizures, psychomotor regression and ataxia. Different disorders have been classified as PMEs. Of these, the group of neuronal ceroid lipofuscinoses (NCLs) comprise an entity that has onset in childhood, being the most common cause of neurodegeneration in children. The primary aim of this thesis was to dissect the molecular genetic background of patients with childhood onset PME by studying candidate genes and attempting to identify novel PME-associated genes. Another specific aim was to study the primary protein properties of the most recently identified member of the NCL-causing proteins, MFSD8. To dissect the genetic background of a cohort of Turkish patients with childhood onset PME, a screen of the NCL-associated genes PPT1, TPP1, CLN3, CLN5, CLN6, MFSD8, CLN8 and CTSD was performed. Altogether 49 novel mutations were identified, which together with 56 mutations found by collaborators raised the total number of known NCL mutations to 364. Fourteen of the novel mutations affect the recently identified MFSD8 gene, which had originally been identified in a subset of mainly Turkish patients as the underlying cause of CLN7 disease. To investigate the distribution of MFSD8 defects, a total of 211 patients of different ethnic origins were evaluated for mutations in the gene. Altogether 45 patients from nine different countries were provided with a CLN7 molecular diagnosis, denoting the wide geographical occurrence of MFSD8 defects. The mutations are private with only one having been established by a founder-effect in the Roma population from the former Czechoslovakia. All mutations identified except one are associated with the typical clinical picture of variant late-infantile NCL. To address the trafficking properties of MFSD8, lysosomal targeting of the protein was confirmed in both neuronal and non-neuronal cells. The major determinant for this lysosomal sorting was identified to be an N-terminal dileucine based signal (9-EQEPLL-14), recognized by heterotetrameric AP-1 adaptor proteins, suggesting that MFSD8 takes the direct trafficking pathway en route to the lysosomes. Expression studies revealed the neurons as the primary cell-type and the hippocampus and cerebellar granular cell layer as the predominant regions in which MFSD8 is expressed. To identify novel genes associated with childhood onset PME, a single nucleotide polymorphism (SNP) genomewide scan was performed in three small families and 18 sporadic patients followed by homozygosity mapping to determine the candidate loci. One of the families and a sporadic patient were positive for mutations in PLA2G6, a gene that had previously been shown to cause infantile neuroaxonal dystrophy. Application of next-generation sequencing of candidate regions in the remaining two families led to identification of a homozygous missense mutation in USP19 for the first and TXNDC6 for the second family. Analysis of the 18 sporadic cases mapped the best candidate interval in a 1.5 Mb region on chromosome 7q21. Screening of the positional candidate KCTD7 revealed six mutations in seven unrelated families. All patients with mutations in KCTD7 were reported to have early onset PME, rapid disease progression leading to dementia and no pathologic hallmarks. The identification of KCTD7 mutations in nine patients and the clinical delineation of their phenotype establish KCTD7 as a gene for early onset PME. The findings presented in this thesis denote MFSD8 and KCTD7 as genes commonly associated with childhood onset symptomatic epilepsy. The disease-associated role of TXNDC6 awaits verification through identification of additional mutations in patients with similar phenotypes. Completion of the genetic spectrum underlying childhood onset PMEs and understanding of the gene products functions will comprise important steps towards understanding the underlying pathogenetic mechanisms, and will possibly shed light on the general processes of neurodegeneration and nervous system regulation, facilitating the diagnosis, classification and possibly treatment of the affected cases.

New Perspectives on the Roman Coinage on the Eastern Limes in the Late Republican and Roman Imperial Period

The Ph.D. thesis discusses the monetary development in Roman Syria and Judaea in the Late Republican and the Early Imperial Period, from a numismatic, archaeological and historical point of view. In effect, the work focuses on the 1st century B.C. to the 1st century A.D., that is, the assumed time of introduction of Roman denarii to the region. The work benefits from the silver coin hoards of Khirbet Qumran recently published by the author. Though discovered as early as 1955 at Qumran, where the famous Dead Sea Scrolls had been found prior to that in 1947, most hoards remained unpublished until 2007. A second important source utilized is the so-called Tax Law from Palmyra in Syria. Its significance lies in the fact that Palmyra used to be one of the most important cities on the Silk Road, along which luxury goods were transported into the Roman Empire and Rome itself. During the research conducted, studies of the provincial coinage of Judaea (A.D. 6-66) shed new light on the authority of the Roman governors in economic and monetary matters in eastern Mediterranean regions. Furthermore, a new suggestion as to the length of the mandate period of Pontius Pilate is made. The extent of Emperor Augustus monetary reforms as well as the military history of Judaea are discussed in the light of new analytical studies, which show that the production of Roman base metal coins appears to have been a highly controlled process, contrary to popular opinion. Statistical calculations related to the coin alloy revealed striking similarities with Roman and other local metalwork found in Israel; a fact previously unknown. Results indicate that both Roman and local metalwork consisted of outstandingly systematized practises and may have exploited the same metal sources. Information: Kenneth Lönnqvist (*25.7.1962) has studied at the University of Helsinki since 1981. Furthermore, Lönnqvist has lived in the Mediterranean countries and the Near East, and made research there at various scientific institutions and universities for ca. 7 years. Contact and sales of thesis: kenneth.lonnqvist@helsinki.fi

Death, Destruction and Commemoration : Tracing ritual activities in Finnish Late Iron Age cemeteries (AD 550-1150)

The thesis is connected with death, memory and ancestor commemoration during the Merovingian Period, the Viking Age and the beginning of the Crusade Period (AD 550-1150) in Finland. During this time, cremation was the dominant burial rite. It was not until the end of the Viking Age that inhumation became more common but both cremations and inhumations are performed even at the same sites throughout the time. Three different burial types 1) cremation cemeteries below level ground, 2) inhumation burials and 3) water burials are discussed in five articles. I consider these burial forms from three different viewpoints; collectivity-individuality, visibility-invisibility and cremation-inhumation. The thesis also discusses the topics of memory, memorialisation and monument re-use, which have been neglected subjects in Finnish archaeology until now. Both cremation cemeteries below level ground and inhumation burials have been re-used during their time of usage, and on most occasions are situated in a landscape that is overlaid by other monuments as well. The main questions of the thesis are: What kinds of ritual behaviour can we detect in the burials during the period (AD 550-1150)? How did people perceive the moraine hills that functioned as burial places? What kind of re-use can be detected in the Iron Age cemeteries? Why have ancient sites and artefacts been re-used? This thesis shows that it is possible to claim that both artefact and site re-use is a much more widespread phenomenon than has previously been thought in Finnish archaeology. It is also a conscious and deliberate behaviour that can be related to an ancestor cult and commemoration of the dead. The funerary rituals during this time period show great variation and complex, both regionally and nationally. Not only have the dead been buried using elaborate rituals, they have also been mourned and commemorated in intricate ways that proves that death was not an end product, but the start of something new.

Approaching Sacred Pregnancy : The Cult of the Visitation and Narrative Altarpieces in Late Fifteenth-Century Florence

The study focuses on the Visitation as a narrative subject of altarpieces in late fifteenth-century Florence. Although the Visitation was a well-known story in both verbal and visual representations since the early medieval period, it became a popular subject of altarpieces only towards the end of the fifteenth century. In this study, the first part provides an overview of the complex religious and historical background to an emerging cult of the Visitation. Devotional practices focusing on the Visitation belong in a context of late medieval Marian devotion and in 1389 a new feast of the Visitation was introduced into the liturgical calendar of the Catholic Church. Because of the ongoing schism within the Catholic Church, the feast was not unanimously accepted across Western Europe until the later part of the fifteenth century. Contrary to a widely disseminated view, the feast of the Visitation cannot be associated with Franciscan spirituality, but was rather a clearly defined Dominican project that primarily emphasised the importance of peace and unity within the Christian Church. Simultaneously with the gradual acceptance of the new feast, visual representations of the Visitation began to appear at the centre of altarpieces. The Visitation exemplifies an increasing preference for narrative subjects within the genre of the altarpiece. The second part of the study presents an analysis of the concept of the narrative altarpiece and highlights the complexities involved in combining a narrative content with the traditional devotional function of the altarpiece. In detailed case studies some prominent art works produced in Florence between 1490 and 1503 are discussed within a framework of contextual analysis, narrative theory and iconography. Altarpieces by Domenico Ghirlandaio, Piero di Cosimo and Mariotto Albertinelli represent visual manifestations of a cult of the Visitation with roots in late medieval devotional practices. At the same time, the altarpieces highlight the multiple functions of altarpieces in a culture where art works responded to a variety of social and religious needs. Building on earlier studies, each case study presents new insights and evidence not considered in previous art historical research.

Childhood and adolescent antecedents of work stress and early atherosclerosis

Work stress is after musculoskeletal disorders the second most common work-related health problem in the European Union, affecting 28% of EU employees. Furthermore, a 50% excessive cardiovascular disease risk among employees with work stress is reported. High job demands combined with low job control according to the Job Demands-Job Control model, or high effort combined with low rewards according to Effort-Reward Imbalance model, are likely to produce work stress in the majority of employees. Atherosclerotic wall thickening is a validated marker of an increased risk of cardiovascular disease. This study examined the role of childhood and adolescent factors as antecedents of work stress and early atherosclerosis, and in the relationship between them. The Cardiovascular Risk in Young Finns Study, (the CRYF project) started in 1980 when the participants were at the age of three to 18 years. Follow-ups have been conducted every three years until 1992, after that in 1997 and 2001, and the latest is ongoing in 2008. The participants parents reported their socioeconomic position in 1980 and 1983, and their life satisfaction in 1983. Biological risk factors were measured in 1980 and 2001. Type A behaviour was reported in 1986, 1989 and 2001. In the 2001 follow-up when the participants were aged 24 to 39, work stress was assessed from responses to questionnaires on job demands-job control and effort-reward imbalance, and education. Ultrasound measurement of the carotid intima-media thickness (CIMT) was used to assess atherosclerosis. There were 755, 746, 1014 and 494 participants in studies I-IV, respectively. The results showed that low parental socioeconomic position and parental life dissatisfaction during childhood and adolescence predicted higher levels of job strain 18 years later, and that education mediated the relationship between parental socioeconomic position and job strain. Childhood and adolescent family factors were not related to the effort-reward imbalance. Parental life satisfaction was associated with high rewards at work among the men, and high parental socioeconomic position was associated with high reward among the women. Among the men, the eagerness-energy component of Type A behaviour across different developmental periods predicted increased CIMT. Among the women, hard-driving component of Type A behaviour predicted decreased CIMT. Low leadership characteristic in adolescence and early adulthood was associated with both high job strain and increased CIMT, and attenuated the relationship between job strain and CIMT to non-significance in men. The current findings add to the literature on the relationship between job strain and health literature in adopting a developmental perspective. The results imply that work stress does not completely originate from work. There are childhood and adolescent environmental and dispositional effects on work stress and CIMT several years later, and these partly seem to operate through educational attainment.

Finnish Homicides and Mental Disorders: An investigation of offense and offender characteristics

Although the majority of people with mental illness are not violent, scientific studies over the last decades show that certain psychiatric disorders increase the risk of violent behavior, including homicide. This thesis examined crime scene behaviors and offender background characteristics among mentally ill Finnish homicide offenders. Previously, homicide crime scene behaviors have been investigated in relation to offender demographic characteristics, whereas this study compares the behaviors of offenders with various mental illnesses. The study design was a retrospective chart review of the forensic psychiatric statements of Finnish homicide offenders. The work consists of four substudies. The aims of the study were as follows: To describe differences in the childhood and family backgrounds as well as in the adolescent and adult adjustment of Finnish homicide offenders belonging to different diagnostic categories (schizophrenia, personality disorder, alcoholism, drug addiction or no diagnosis). Further, the study examined associations between the crime scene behaviors and mental status of these offenders. Also, the distinguishing characteristics between two groups of offenders with schizophrenia were examined: early starters, who present antisocial behavior before the onset of schizophrenia, and late starters, who first offend after the onset of mental disorder. Finally, it was investigated how the use of excessive violence is associated with clinical and circumstantial variables as well as offender background characteristics among homicide offenders with schizophrenia. The main findings of the study can be summarized as follows. First, offenders with personality disorder or drug addiction had experienced multiple difficulties in their early environments: both family and individual problems were typical. Offenders with schizophrenia were relatively well-adjusted in childhood compared to the other groups. However, in adolescence and adulthood, social isolation, withdrawal and other difficulties attributable to these offenders illness became evident. In several aspects, offenders with alcohol dependency resembled offenders with no diagnosis in that these offenders had less problematic backgrounds compared to other groups. Second, the results showed that crime scene behaviors, victim gender and the victim-offender relationship differ between the groups. In particular, offenders with a diagnosis of schizophrenia or drug addiction have some unique features in their crime scene behaviors and choice of victims. Offenders with schizophrenia were more likely to kill a blood relative, to use a sharp weapon and to injure the victim s face. Drug addiction was associated with stealing from the victim and trying to cover up the body. Third, the results suggest that the offense characteristics of early- and late-start offenders with schizophrenia differ only modestly. However, several significant differences between the groups were found in characteristics of offenders: early starters had experienced a multitude of problems in their childhood surroundings and also later in life. Fourth, violent acts where the offender did not commit the offense alone or had previous homicidal history were predictive of excessive violence among offenders with schizophrenia. Positive psychotic symptoms did not predict the use of excessive violence. Nearly one third of the cases in the sample involved multiple and severe violence, including features such as sadism, mutilation, sexual components or extreme stabbing. In sum, mentally disordered homicide offenders are heterogeneous in their offense characteristics as well as their background characteristics. Empirically based information on how the offender s mental state is associated with specific crime scene behaviors can be utilized within the police force in developing methods of prioritizing suspects in unsolved homicide cases. Also, these results emphasise the importance of early interventions for problem families and children at risk of antisocial behavior. They may also contribute to the development of effective treatment for violent offenders.

Self-rating scales in the assessment of current and childhood symptoms of attention deficit hyperactivity disorder in adults

Objective: Attention deficit hyperactivity disorder (ADHD) is a life-long condition, but because of its historical status as a self-remitting disorder of childhood, empirically validated and reliable methods for the assessment of adults are scarce. In this study, the validity and reliability of the Wender Utah Rating Scale (WURS) and the Adult Problem Questionnaire (APQ), which survey childhood and current symptoms of ADHD, respectively, were studied in a Finnish sample. Methods: The self-rating scales were administered to adults with an ADHD diagnosis (n = 38), healthy control participants (n = 41), and adults diagnosed with dyslexia (n = 37). Items of the self-rating scales were subjected to factor analyses, after which the reliability and discriminatory power of the subscales, derived from the factors, were examined. The effects of group and gender on the subscales of both rating scales were studied. Additionally, the effect of age on the subscales of the WURS was investigated. Finally, the diagnostic accuracy of the total scores was studied. Results: On the basis of the factor analyses, a four-factor structure for the WURS and five-factor structure for the APQ had the best fit to the data. All of the subscales of the APQ and three of the WURS achieved sufficient reliability. The ADHD group had the highest scores on all of the subscales of the APQ, whereas two of the subscales of the WURS did not statistically differ between the ADHD and the Dyslexia group. None of the subscales of the WURS or the APQ was associated with the participant's gender. However, one subscale of the WURS describing dysthymia was positively correlated with the participant's age. With the WURS, the probability of a correct positive classification was .59 in the current sample and .21 when the relatively low prevalence of adult ADHD was taken into account. The probabilities of correct positive classifications with the APQ were .71 and .23, respectively. Conclusions: The WURS and the APQ can provide accurate and reliable information of childhood and adult ADHD symptoms, given some important constraints. Classifications made on the basis of the total scores are reliable predictors of ADHD diagnosis only in populations with a high proportion of ADHD and a low proportion of other similar disorders. The subscale scores can provide detailed information of an individual's symptoms if the characteristics and limitations of each domain are taken into account. Improvements are suggested for two subscales of the WURS.

Lapsi, lapsuus ja perhe varhaiskasvatusasiakirjoissa 1967-1999

The aim of this study is to describe and interpret discourses in Finnish national early childhood education and care (ECEC) documents concerning the child, childhood and family, including preschool education of six-year-old children. The study begins with preparation phase of the Act on Children s Day Care (1967) and concludes with the definition of ECEC policy (1999). The research data consists of committee memoranda and national ECEC curriculum guidelines. The total number of documents studied was 20, comprising some 1700 pages. The research data was examined with qualitative text analysis and employed a discursive approach. A semiotic square (Greimas rectangle) served as a tool for clarifying the discourses and constructions reflected in the research data. The theoretical framework of the study consists of the theories of childhood and family studies. The main concepts from childhood studies used in this study were childhood as a cultural construct and child-centred pedagogy in ECEC. The theoretical approaches from family studies used were the formation of modern and late-modern parenthood and family, as well as the concept of familism. Two main discursive lines were constructed from the ECEC documents. The notion of universalistic childhood suggests that early education and care aim to create the same good childhood for all children, regardless of their family background or living area. The second discursive line followed in the documents is the familistic discourse. This discourse contains emphasis on the priority of parental care. The construct of the competent child was found in the research data as early as in the mid-1970s. On the other hand, the construct of the weak family is distinguishable throughout almost the entire research period. This raises the question of whether Finnish ECEC system has been developed for the competent and self-sufficient child of a weak family which needs constant support and guidance of welfare experts. According to the study, it appears that within the Finnish ECEC system the relatively heavy emphasis on social work rather than on early education has been legitimised by the construct of the weak family. This study also shows that a more thorough analysis should be given to what we mean when we say that the main task of ECEC system is to support families in the upbringing of their children. The study was completed during the period when historical decisions concerning the administration in Finland were in the making (i.e. the potential transfer of ECEC services from the Ministry of Social Affairs and Health to the Ministry of Education). Also, over the past decade, a major reformation of the Act on Children s Daycare has been on the agenda, but no concrete measures have been implemented. Based on the findings of this study, we can ask for what kind of child and family we are preparing the ECEc reforms of the new millennium. Key words: ECEC policy Finland, childhood, family, familism, discourse analysis, semiotic square

Mitochondrial Malfunction in Tumor Formation and Neuromuscular Diseases : Consequences of defects in fumarate hydratase and in the respiratory chain

The mitochondrion is an organelle of outmost importance, and the mitochondrial network performs an array of functions that go well beyond ATP synthesis. Defects in mitochondrial performance lead to diseases, often affecting nervous system and muscle. Although many of these mitochondrial diseases have been linked to defects in specific genes, the molecular mechanisms underlying the pathologies remain unclear. The work in this thesis aims to determine how defects in mitochondria are communicated within - and interpreted by - the cells, and how this contributes to disease phenotypes. Fumarate hydratase (FH) is an enzyme of the citrate cycle. Recessive defects in FH lead to infantile mitochondrial encephalopathies, while dominant mutations predispose to tumor formation. Defects in succinate dehydrogenase (SDH), the enzyme that precedes FH in the citrate cycle, have also been described. Mutations in SDH subunits SDHB, SDHC and SDHD are associated with tumor predisposition, while mutations in SDHA lead to a characteristic mitochondrial encephalopathy of childhood. Thus, the citrate cycle, via FH and SDH, seems to have essential roles in mitochondrial function, as well as in the regulation of processes such as cell proliferation, differentiation or death. Tumor predisposition is not a typical feature of mitochondrial energy deficiency diseases. However, defects in citrate cycle enzymes also affect mitochondrial energy metabolism. It is therefore necessary to distinguish what is specific for defects in citrate cycle, and thus possibly associated with the tumor phenotype, from the generic consequences of defects in mitochondrial aerobic metabolism. We used primary fibroblasts from patients with recessive FH defects to study the cellular consequences of FH-deficiency (FH-). Similarly to the tumors observed in FH- patients, these fibroblasts have very low FH activity. The use of primary cells has the advantage that they are diploid, in contrast with the aneuploid tumor cells, thereby enabling the study of the early consequences of FH- in diploid background, before tumorigenesis and aneuploidy. To distinguish the specific consequences of FH- from typical consequences of defects in mitochondrial aerobic metabolism, we used primary fibroblasts from patients with MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) and from patients with NARP (neuropathy, ataxia and retinitis pigmentosa). These diseases also affect mitochondrial aerobic metabolism but are not known to predispose to tumor formation. To study in vivo the systemic consequences of defects in mitochondrial aerobic metabolism, we used a transgenic mouse model of late-onset mitochondrial myopathy. The mouse contains a transgene with an in-frame duplication of a segment of Twinkle, the mitochondrial replicative helicase, whose defects underlie the human disease progressive external ophthalmoplegia. This mouse model replicates the phenotype in the patients, particularly neuronal degeneration, mitochondrial myopathy, and subtle decrease of respiratory chain activity associated with mtDNA deletions. Due to the accumulation of mtDNA deletions, the mouse was named deletor. We first studied the consequences of FH- and of respiratory chain defects for energy metabolism in primary fibroblasts. To further characterize the effects of FH- and respiratory chain malfunction in primary fibroblasts at transcriptional level, we used expression microarrays. In order to understand the in vivo consequences of respiratory chain defects in vivo, we also studied the transcriptional consequences of Twinkle defects in deletor mice skeletal muscle, cerebellum and hippocampus. Fumarate accumulated in the FH- homozygous cells, but not in the compound heterozygous lines. However, virtually all FH- lines lacked cytoplasmic FH. Induction of glycolysis was common to FH-, MELAS and NARP fibroblasts. In deletor muscle glycolysis seemed to be upregulated. This was in contrast with deletor cerebellum and hippocampus, where mitochondrial biogenesis was in progress. Despite sharing a glycolytic pattern in energy metabolism, FH- and respiratory chain defects led to opposite consequences in redox environment. FH- was associated with reduced redox environment, while MELAS and NARP displayed evidences of oxidative stress. The deletor cerebellum had transcriptional induction of antioxidant defenses, suggesting increased production of reactive oxygen species. Since the fibroblasts do not represent the tissues where the tumors appear in FH- patients, we compared the fibroblast array data with the data from FH- leiomyomas and normal myometrium. This allowed the determination of the pathways and networks affected by FH-deficiency in primary cells that are also relevant for myoma formation. A key pathway regulating smooth muscle differentiation, SRF (serum response factor)-FOS-JUNB, was found to be downregulated in FH- cells and in myomas. While in the deletor mouse many pathways were affected in a tissue-specific basis, like FGF21 induction in the deletor muscle, others were systemic, such as the downregulation of ALAS2-linked heme synthesis in all deletor tissues analyzed. However, interestingly, even a tissue-specific response of FGF21 excretion could elicit a global starvation response. The work presented in this thesis has contributed to a better understanding of mitochondrial stress signalling and of pathways interpreting and transducing it to human pathology.

Early Childhood Caries and a Community Trial of its Prevention in Tehran, Iran

Varhaislapsuuden karies ja sen ehkäisy kehittyvän terveydenhuollon maassa Varhaislapsuuden karies on merkittävä kansanterveysongelma varsinkin lapsirikkaissa maissa ja väestöissä. Karieksen hoitaminen vie paljon voimavaroja ja aiheuttaa mittavia taloudellisia seuraamuksia. Karies voi ilmaantua lapselle jo vauvaikäisenä, pian ensimmäisten maitohampaiden puhjettua suuhun. Alle 3-vuotiaiden karieksesta on kuitenkin niukasti tilastotietoja. Maailman terveysjärjestökin suosittaa tietojen keräämistä vasta 3-vuotiaiden ikäryhmästä. Heistä kariesta sairastaa Suomessa 16 %, Yhdysvalloissa 25 %, Englannissa 30 %, Iranissa 46 % ja Saudi-Arabiassa 61 %. Tämä väitöstutkimus selvitti karieksen esiintymistä ja sen vaaratekijöitä 1─3-vuotiailla Teheranissa. Lisäksi tutkimus arvioi perusterveydenhuoltoon sisällytetyn karieksen ehkäisyn tuloksellisuutta. Tutkimuskohteiksi arvottiin Teheranista 18 neuvolaa. Jokaisessa oltiin 4 päivää, jolloin kaikkia rokotuksiin tulleita 1─3-vuotiaita äiteineen pyydettiin osallistumaan tutkimukseen. Kahta lukuun ottamatta kaikki äidit suostuivat, ja aineistoon tuli kaikkiaan 504 lasta äiteineen. Kaikki 1-vuotiaat, 242 lasta äiteineen, valittiin karieksen ehkäisykokeiluun. Sitä varten neuvolat jaettiin kolmeen ryhmään, joista kaksi (A ja B) oli koeryhmiä ja yksi (C) oli vertailuryhmä. Tutkimus alkoi äidin haastattelulla. Siinä selvitettiin perheen koulutus- ja tulotaso sekä lapsen ruokinnasta imetyksen kesto, yösyötöt ja päiväaikaan nautitut makeat. Vielä kysyttiin lapsen ja äidin suuhygieniatavoista ja äidin kokemuksista lapsen suun puhdistamisessa. Sitten hammaslääkäri tutki lapsen suun ja kirjasi karieksen ja hammasplakin esiintymät. Suun tutkimuksen jälkeen äiti ja lapsi siirtyivät rokotushuoneeseen. Koeryhmissä (A ja B) äidit saivat terveydenhoitajalta suunterveyttä koskevan esitteen ja kehotuksen lukea se huolellisesti. Lisäksi ryhmässä A terveydenhoitaja kertoi suun ja hampaiden terveydenhoidosta saman esitteen avulla, ja neuvolan henkilökunta muistutti suunhoidon tärkeydestä puhelimitse kahdesti seuraavan puolen vuoden kuluessa. Vertailuryhmässä äideille ei annettu suunhoidon ohjeita. Kaikissa ryhmissä äitejä muistutettiin seuraavan rokotuskerran ajankohdasta, muttei mainittu tulevaa toista hammastarkastusta. Varhaislapsuuden kariesta sairasti ikäryhmästä riippuen 3─26 % tutkituista 1─3-vuotiaista, ja 65─76 %:lla oli hammasplakkia. Äideistä 68 % harjasi hampaansa päivittäin ja 39 % puhdisti lapsensa suun päivittäin. Mitä useammin äiti harjasi omat hampaansa, sitä paremmin hän huolehti lapsen suun puhtaudesta. Rintaruokinta oli yleistä eikä lisännyt kariesvaaraa. Yöllä pullomaitoa saavilla karies oli 5 kertaa yleisempää kuin muilla. Neuvolassa saatu ohjeistus ehkäisi selvästi karieksen syntyä puolen vuoden kokeessa.