Evidence-based perspective on CP rehabilitation : Reviews on physiotherapy, physiotherapy-related motor-based interventions and orthotic devices

This thesis utilises an evidence-based approach to critically evaluate and summarize effectiveness research on physiotherapy, physiotherapy-related motor-based interventions and orthotic devices in children and adolescents with cerebral palsy (CP). It aims to assess the methodological challenges of the systematic reviews and trials, to evaluate the effectiveness of interventions in current use, and to make suggestions for future trials Methods: Systematic reviews were searched from computerized bibliographic databases up to August 2007 for physiotherapy and physiotherapy-related interventions, and up to May 2003 for orthotic devices. Two reviewers independently identified, selected, and assessed the quality of the reviews using the Overview Quality Assessment Questionnaire complemented with decision rules. From a sample of 14 randomized controlled trials (RCT) published between January 1990 and June 2003 we analysed the methods of sampling, recruitment, and comparability of groups; defined the components of a complex intervention; identified outcome measures based on the International Classification of Functioning, Disability and Health (ICF); analysed the clinical interpretation of score changes; and analysed trial reporting using a modified 33-item CONSORT (Consolidated Standards of Reporting Trials) checklist. The effectiveness of physiotherapy and physiotherapy-related interventions in children with diagnosed CP was evaluated in a systematic review of randomised controlled trials that were searched from computerized databases from January 1990 up to February 2007. Two reviewers independently assessed the methodological quality, extracted the data, classified the outcomes using the ICF, and considered the level of evidence according to van Tulder et al. (2003). Results: We identified 21 reviews on physiotherapy and physiotherapy-related interventions and five on orthotic devices. These reviews summarized 23 or 5 randomised controlled trials and 104 or 27 observational studies, respectively. Only six reviews were of high quality. These found some evidence supporting strength training, constraint-induced movement therapy or hippotherapy, and insufficient evidence on comprehensive interventions. Based on the original studies included in the reviews on orthotic devices we found some short-term effects of lower limb casting on passive range of movement, and of ankle-foot orthoses on equinus walk. Long term effects of lower limb orthoses have not been studied. Evidence of upper limb casting or orthoses is conflicting. In the sample of 14 RCTs, most trials used simple randomisation, complemented with matching or stratification, but only three specified the concealed allocation. Numerous studies provided sufficient details on the components of a complex intervention, but the overlap of outcome measures across studies was poor and the clinical interpretation of observed score changes was mostly missing. Almost half (48%) of the applicable CONSORT-based items (range 28 32) were reported adequately. Most reporting inadequacies were in outcome measures, sample size determination, details of the sequence generation, allocation concealment and implementation of the randomization, success of assessor blinding, recruitment and follow-up dates, intention-to-treat analysis, precision of the effect size, co-interventions, and adverse events. The systematic review identified 22 trials on eight intervention categories. Four trials were of high quality. Moderate evidence of effectiveness was established for upper extremity treatments on attained goals, active supination and developmental status, and of constraint-induced therapy on the amount and quality of hand use and new emerging behaviours. Moderate evidence of ineffectiveness was found for strength training's effect on walking speed and stride length. Conflicting evidence was found for strength training's effect on gross motor function. For the other intervention categories the evidence was limited due to the low methodological quality and the statistically insignificant results of the studies. Conclusions: The high-quality reviews provide both supportive and insufficient evidence on some physiotherapy interventions. The poor quality of most reviews calls for caution, although most reviews drew no conclusions on effectiveness due to the poor quality of the primary studies. A considerable number of RCTs of good to fair methodological and reporting quality indicate that informative and well-reported RCTs on complex interventions in children and adolescents with CP are feasible. Nevertheless, methodological improvement is needed in certain areas of the trial design and performance, and the trial authors are encouraged to follow the CONSORT criteria. Based on RCTs we established moderate evidence for some effectiveness of upper extremity training. Due to limitations in methodological quality and variations in population, interventions and outcomes, mostly limited evidence on the effectiveness of most physiotherapy interventions is available to guide clinical practice. Well-designed trials are needed, especially for focused physiotherapy interventions.

Routine Measurement of Health-Related Quality of Life in Assessing Cost-Effectiveness in Secondary Health Care

Aims: The aims of this study were 1) to identify and describe health economic studies that have used quality-adjusted life years (QALYs) based on actual measurements of patients' health-related quality of life (HRQoL); 2) to test the feasibility of routine collection of health-related quality of life (HRQoL) data as an indicator of effectiveness of secondary health care; and 3) to establish and compare the cost-utility of three large-volume surgical procedures in a real-world setting in the Helsinki University Central Hospital, a large referral hospital providing secondary and tertiary health-care services for a population of approximately 1.4 million. Patients and methods: So as to identify studies that have used QALYs as an outcome measure, a systematic search of the literature was performed using the Medline, Embase, CINAHL, SCI and Cochrane Library electronic databases. Initial screening of the identified articles involved two reviewers independently reading the abstracts; the full-text articles were also evaluated independently by two reviewers, with a third reviewer used in cases where the two reviewers could not agree a consensus on which articles should be included. The feasibility of routinely evaluating the cost-effectiveness of secondary health care was tested by setting up a system for collecting HRQoL data on approximately 4 900 patients' HRQoL before and after operative treatments performed in the hospital. The HRQoL data used as an indicator of treatment effectiveness was combined with diagnostic and financial indicators routinely collected in the hospital. To compare the cost-effectiveness of three surgical interventions, 712 patients admitted for routine operative treatment completed the 15D HRQoL questionnaire before and also 3-12 months after the operation. QALYs were calculated using the obtained utility data and expected remaining life years of the patients. Direct hospital costs were obtained from the clinical patient administration database of the hospital and a cost-utility analysis was performed from the perspective of the provider of secondary health care services. Main results: The systematic review (Study I) showed that although QALYs gained are considered an important measure of the effectiveness of health care, the number of studies in which QALYs are based on actual measurements of patients' HRQoL is still fairly limited. Of the reviewed full-text articles, only 70 reported QALYs based on actual before after measurements using a valid HRQoL instrument. Collection of simple cost-effectiveness data in secondary health care is feasible and could easily be expanded and performed on a routine basis (Study II). It allows meaningful comparisons between various treatments and provides a means for allocating limited health care resources. The cost per QALY gained was 2 770 for cervical operations and 1 740 for lumbar operations. In cases where surgery was delayed the cost per QALY was doubled (Study III). The cost per QALY ranges between subgroups in cataract surgery (Study IV). The cost per QALY gained was 5 130 for patients having both eyes operated on and 8 210 for patients with only one eye operated on during the 6-month follow-up. In patients whose first eye had been operated on previous to the study period, the mean HRQoL deteriorated after surgery, thus precluding the establishment of the cost per QALY. In arthroplasty patients (Study V) the mean cost per QALY gained in a one-year period was 6 710 for primary hip replacement, 52 270 for revision hip replacement, and 14 000 for primary knee replacement. Conclusions: Although the importance of cost-utility analyses has during recent years been stressed, there are only a limited number of studies in which the evaluation is based on patients own assessment of the treatment effectiveness. Most of the cost-effectiveness and cost-utility analyses are based on modeling that employs expert opinion regarding the outcome of treatment, not on patient-derived assessments. Routine collection of effectiveness information from patients entering treatment in secondary health care turned out to be easy enough and did not, for instance, require additional personnel on the wards in which the study was executed. The mean patient response rate was more than 70 %, suggesting that patients were happy to participate and appreciated the fact that the hospital showed an interest in their well-being even after the actual treatment episode had ended. Spinal surgery leads to a statistically significant and clinically important improvement in HRQoL. The cost per QALY gained was reasonable, at less than half of that observed for instance for hip replacement surgery. However, prolonged waiting for an operation approximately doubled the cost per QALY gained from the surgical intervention. The mean utility gain following routine cataract surgery in a real world setting was relatively small and confined mostly to patients who had had both eyes operated on. The cost of cataract surgery per QALY gained was higher than previously reported and was associated with considerable degree of uncertainty. Hip and knee replacement both improve HRQoL. The cost per QALY gained from knee replacement is two-fold compared to hip replacement. Cost-utility results from the three studied specialties showed that there is great variation in the cost-utility of surgical interventions performed in a real-world setting even when only common, widely accepted interventions are considered. However, the cost per QALY of all the studied interventions, except for revision hip arthroplasty, was well below 50 000, this figure being sometimes cited in the literature as a threshold level for the cost-effectiveness of an intervention. Based on the present study it may be concluded that routine evaluation of the cost-utility of secondary health care is feasible and produces information essential for a rational and balanced allocation of scarce health care resources.

Modern diagnosis of Epstein-Barr virus infections and post-transplant lymphoproliferative disease

Infection by Epstein-Barr virus (EBV) occurs in approximately 95% of the world s population. EBV was the first human virus implicated in oncogenesis. Characteristic for EBV primary infection are detectable IgM and IgG antibodies against viral capsid antigen (VCA). During convalescence the VCA IgM disappears while the VCA IgG persists for life. Reactivations of EBV occur both among immunocompromised and immunocompetent individuals. In serological diagnosis, measurement of avidity of VCA IgG separates primary from secondary infections. However, in serodiagnosis of mononucleosis it is quite common to encounter, paradoxically, VCA IgM together with high-avidity VCA IgG, indicating past immunity. We determined the etiology of this phenomenon and found that, among patients with cytomegalovirus (CMV) primary infection a large proportion (23%) showed antibody profiles of EBV reactivation. In contrast, EBV primary infection did not appear to induce immunoreactivation of CMV. EBV-associated post-transplant lymphoproliferative disease (PTLD) is a life threatening complication of allogeneic stem cell or solid organ transplantation. PTLD may present with a diverse spectrum of clinical symptoms and signs. Due to rapidity of PTLD progression especially after stem cell transplantation, the diagnosis must be obtained quickly. Pending timely detection, the evolution of the fatal disease may be halted by reduction of immunosuppression. A promising new PTLD treatment (also in Finland) is based on anti-CD-20 monoclonal antibodies. Diagnosis of PTLD has been demanding because of immunosuppression, blood transfusions and the latent nature of the virus. We set up in 1999 to our knowledge first in Finland for any microbial pathogen a real-time quantitative PCR (qPCR) for detection of EBV DNA in blood serum/plasma. In addition, we set up an in situ hybridisation assay for EBV RNA in tissue sections. In collaboration with a group of haematologists at Helsinki University Central Hospital we retrospectively determined the incidence of PTLD among 257 allogenic stem cell transplantations (SCT) performed during 1994-1999. Post-mortem analysis revealed 18 cases of PTLD. From a subset of PTLD cases (12/18) and a series of corresponding controls (36), consecutive samples of serum were studied by the new EBV-qPCR. All the PTLD patients were positive for EBV-DNA with progressively rising copy numbers. In most PTLD patients EBV DNA became detectable within 70 days of SCT. Of note, the appearance of EBV DNA preceded the PTLD symptoms (fever, lymphadenopathy, atypical lymphocytes). Among the SCT controls, EBV DNA occurred only sporadically, and the EBV-DNA levels remained relatively low. We concluded that EBV qPCR is a highly sensitive (100%) and specific (96%) new diagnostic approach. We also looked for and found risk factors for the development of PTLD. Together with a liver transplantation group at the Transplantation and Liver Surgery Clinic we wanted to clarify how often and how severely do EBV infections occur after liver transplantation. We studied by the EBV qPCR 1284 plasma samples obtained from 105 adult liver transplant recipients. EBV DNA was detected in 14 patients (13%) during the first 12 months. The peak viral loads of 13 asymptomatic patients were relatively low (<6600/ml), and EBV DNA subsided quickly from circulation. Fatal PTLD was diagnosed in one patient. Finally, we wanted to determine the number and clinical significance of EBV infections of various types occurring among a large, retrospective, nonselected cohort of allogenic SCT recipients. We analysed by EBV qPCR 5479 serum samples of 406 SCT recipients obtained during 1988-1999. EBV DNA was seen in 57 (14%) patients, of whom 22 (5%) showed progressively rising and ultimately high levels of EBV DNA (median 54 million /ml). Among the SCT survivors, EBV DNA was transiently detectable in 19 (5%) asymptomatic patients. Thereby, low-level EBV-DNA positivity in serum occurs relatively often after SCT and may subside without specific treatment. However, high molecular copy numbers (>50 000) are diagnostic for life-threatening EBV infection. We furthermore developed a mathematical algorithm for the prediction of development of life-threatening EBV infection.

Genetically Engineered Oncolytic Adenoviruses for the Treatment of Kidney and Breast Cancer

Metastatic kidney and breast cancer are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment are oncolytic adenoviruses, which have demonstrated excellent safety in many clinical trials. However, antitumor efficacy needs to be improved in order to make oncolytic viruses a viable treatment alternative. To be able to follow oncolytic virus replication in vivo, we set up a non-invasive imaging system based on coinjection of a replication deficient luciferase expressing virus and a replication competent virus. The system was validated in vitro and in vivo and used in other projects of the thesis. In another study we showed that capsid modifications on adenoviruses result in enhanced gene transfer and increased oncolytic effect on renal cancer cells in vitro. Moreover, capsid modified oncolytic adenoviruses demonstrated significantly improved antitumor efficacy in murine kidney cancer models. To transcriptionally target kidney cancer tissue we evaluated two hypoxia response elements for their usability as tissue specific promoters using a novel dual luciferase imaging system. Based on the results of the promoter evaluation and the studies on capsid modifications, we constructed a transcriptionally and transductionally targeted oncolytic adenovirus armed with an antiangiogenic transgene for enhanced renal cell cancer specificity and improved antitumor efficacy. This virus exhibited kidney cancer specific replication and significantly improved antitumor effect in a murine model of intraperitoneal disseminated renal cell cancer. Cancer stem cells are thought to be resistant to conventional cancer drugs and might play an important role in breast cancer relapse and the formation of metastasis. Therefore, we examined if capsid modified oncolytic adenoviruses are able to kill these cells proposed to be breast cancer initiating. Efficient oncolytic effect and significant antitumor efficacy on tumors established with breast cancer initiating cells was observed, suggesting that oncolytic adenoviruses might be able to prevent breast cancer relapse and could be used in the treatment of metastatic disease. In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of metastatic kidney and breast cancer.

Variation in tracheid cross-sectional dimensions and wood viscoelasticity extent and control methods

Printing papers have been the main product of the Finnish paper industry. To improve properties and economy of printing papers, controlling of tracheid cross-sectional dimensions and wood viscoelasticity are examined in this study. Controlling is understood as any procedure which yields raw material classes with distinct properties and small internal variation. Tracheid cross-sectional dimensions, i.e., cell wall thickness and radial and tangential diameters can be controlled with methods such as sorting wood into pulpwood and sawmill chips, sorting of logs according to tree social status and fractionation of fibres. These control methods were analysed in this study with simulations, which were based on measured tracheid cross-sectional dimensions. A SilviScan device was used to measure the data set from five Norway spruce (Picea abies) and five Scots pine (Pinus sylvestris) trunks. The simulation results indicate that the sawmill chips and top pulpwood assortments have quite similar cross-sectional dimensions. Norway spruce and Scots pine are on average also relatively similar in their cross-sectional dimensions. The distributions of these species are somewhat different, but from a practical point of view, the differences are probably of minor importance. The controlling of tracheid cross-sectional dimensions can be done most efficiently with methods that can separate fibres into earlywood and latewood. Sorting of logs or partitioning of logs into juvenile and mature wood were markedly less efficient control methods than fractionation of fibres. Wood viscoelasticity affects energy consumption in mechanical pulping, and is thus an interesting control target when improving energy efficiency of the process. A literature study was made to evaluate the possibility of using viscoelasticity in controlling. The study indicates that there is considerable variation in viscoelastic properties within tree species, but unfortunately, the viscoelastic properties of important raw material lots such as top pulpwood or sawmill chips are not known. Viscoelastic properties of wood depend mainly on lignin, but also on microfibrillar angle, width of cellulose crystals and tracheid cross-sectional dimensions.

Microbial quality of hemp (Cannabis sativa L.) and flax (Linum usitatissimum L.) from plants to thermal insulation

Flax and hemp have traditionally been used mainly for textiles, but recently interest has also been focused on non-textile applications. Microbial quality throughout the whole processing chain of bast fibres has not previously been studied. This study concentrates on the microbial quality and possible microbial risks in the production chain of hemp and flax fibres and fibrous thermal insulations. In order to be able to utilize hemp and flax fibres, the bast fibres must be separated from the rest of the plant. Non-cellulosic components can be removed with various pretreatment processes, which are associated with a certain risk of microbial contamination. In this study enzymatic retting and steam explosion (STEX) were examined as pretreatment processes. On the basis of the results obtained in this study, the microbial contents on stalks of both plants studied increased at the end of the growing season and during the winter. However, by processing and mechanical separation it is possible to produce fibres containing less moulds and bacteria than the whole stem. Enzymatic treatment encouraged the growth of moulds in fibres. Steam explosion reduced the amount of moulds in fibres. Dry thermal treatment used in this study did not markedly reduce the amount of microbes. In this project an emission measurement chamber was developed which was suitable for measurements of emissions from both mat type and loose fill type insulations, and capable of interdisciplinary sampling. In this study, the highest amounts of fungal emissions were in the range of 10^3 10^5 cfu/m^3 from the flax and hemp insulations at 90% RH of air. The fungal emissions from stone wool, glass wool and recycled paper insulations were below 10^2 cfu/m^3 even at 90% RH. Equally low values were obtained from bast fibrous materials in lower humidities (at 30% and 80% RH of air). After drying of moulded insulations at 30% RH, the amounts of emitted moulds were in all cases higher compared to the emissions at 90% RH before drying. The most common fungi in bast fibres were Penicillium and Rhizopus. The widest variety of different fungi was in the untreated hemp and linseed fibres and in the commercial loose-fill flax insulation. Penicillium, Rhizopus and Paecilomyces were the most tolerant to steam explosion. According to the literature, the most common fungi in building materials and indoor air are Penicillium, Aspergillus and Cladosporium, which were all found in some of the bast fibre materials in this study. As organic materials, hemp and flax fibres contain high levels of nutrients for microbial growth. The amount of microbes can be controlled and somewhat decreased by the processing methods presented.

Asymmetrical flow field-flow fractionation in the study of water-soluble macromolecules

Asymmetrical flow field-flow fractionation (AsFlFFF) was constructed, and its applicability to industrial, biochemical, and pharmaceutical applications was studied. The effect of several parameters, such as pH, ionic strength, temperature and the reactants mixing ratios on the particle sizes, molar masses, and the formation of aggregates of macromolecules was determined by AsFlFFF. In the case of industrial application AsFlFFF proved to be a valuable tool in the characterization of the hydrodynamic particle sizes, molar masses and phase transition behavior of various poly(N-isopropylacrylamide) (PNIPAM) polymers as a function of viscosity and phase transition temperatures. The effect of sodium chloride salt and the molar ratio of cationic and anionic polyelectrolytes on the hydrodynamic particle sizes of poly (methacryloxyethyl trimethylammonium chloride) and poly (ethylene oxide)-block-poly (sodium methacrylate) and their complexes were studied. The particle sizes of PNIPAM polymers, and polyelectrolyte complexes measured by AsFlFFF were in agreement with those obtained by dynamic light scattering. The molar masses of PNIPAM polymers obtained by AsFlFFF and size exclusion chromatography agreed also well. In addition, AsFlFFF proved to be a practical technique in thermo responsive behavior studies of polymers at temperatures up to about 50 oC. The suitability of AsFlFFF for biological, biomedical, and pharmaceutical applications was proved, upon studying the lipid-protein/peptide interactions, and the stability of liposomes at different temperatures. AsFlFFF was applied to the studies on the hydrophobic and electrostatic interactions between cytochrome c (a basic peripheral protein) and anionic lipid, and oleic acid, and sodium dodecyl sulphate surfactant. A miniaturized AsFlFFF constructed in this study was exploited in the elucidation of the effect of copper (II), pH, ionic strength, and vortexing on the particle sizes of low-density lipoproteins.

Bayesian fisheries stock assessment: integrating and updating knowledge

In this thesis the use of the Bayesian approach to statistical inference in fisheries stock assessment is studied. The work was conducted in collaboration of the Finnish Game and Fisheries Research Institute by using the problem of monitoring and prediction of the juvenile salmon population in the River Tornionjoki as an example application. The River Tornionjoki is the largest salmon river flowing into the Baltic Sea. This thesis tackles the issues of model formulation and model checking as well as computational problems related to Bayesian modelling in the context of fisheries stock assessment. Each article of the thesis provides a novel method either for extracting information from data obtained via a particular type of sampling system or for integrating the information about the fish stock from multiple sources in terms of a population dynamics model. Mark-recapture and removal sampling schemes and a random catch sampling method are covered for the estimation of the population size. In addition, a method for estimating the stock composition of a salmon catch based on DNA samples is also presented. For most of the articles, Markov chain Monte Carlo (MCMC) simulation has been used as a tool to approximate the posterior distribution. Problems arising from the sampling method are also briefly discussed and potential solutions for these problems are proposed. Special emphasis in the discussion is given to the philosophical foundation of the Bayesian approach in the context of fisheries stock assessment. It is argued that the role of subjective prior knowledge needed in practically all parts of a Bayesian model should be recognized and consequently fully utilised in the process of model formulation.

Algorithms for 13C metabolic flux analysis

The metabolism of an organism consists of a network of biochemical reactions that transform small molecules, or metabolites, into others in order to produce energy and building blocks for essential macromolecules. The goal of metabolic flux analysis is to uncover the rates, or the fluxes, of those biochemical reactions. In a steady state, the sum of the fluxes that produce an internal metabolite is equal to the sum of the fluxes that consume the same molecule. Thus the steady state imposes linear balance constraints to the fluxes. In general, the balance constraints imposed by the steady state are not sufficient to uncover all the fluxes of a metabolic network. The fluxes through cycles and alternative pathways between the same source and target metabolites remain unknown. More information about the fluxes can be obtained from isotopic labelling experiments, where a cell population is fed with labelled nutrients, such as glucose that contains 13C atoms. Labels are then transferred by biochemical reactions to other metabolites. The relative abundances of different labelling patterns in internal metabolites depend on the fluxes of pathways producing them. Thus, the relative abundances of different labelling patterns contain information about the fluxes that cannot be uncovered from the balance constraints derived from the steady state. The field of research that estimates the fluxes utilizing the measured constraints to the relative abundances of different labelling patterns induced by 13C labelled nutrients is called 13C metabolic flux analysis. There exist two approaches of 13C metabolic flux analysis. In the optimization approach, a non-linear optimization task, where candidate fluxes are iteratively generated until they fit to the measured abundances of different labelling patterns, is constructed. In the direct approach, linear balance constraints given by the steady state are augmented with linear constraints derived from the abundances of different labelling patterns of metabolites. Thus, mathematically involved non-linear optimization methods that can get stuck to the local optima can be avoided. On the other hand, the direct approach may require more measurement data than the optimization approach to obtain the same flux information. Furthermore, the optimization framework can easily be applied regardless of the labelling measurement technology and with all network topologies. In this thesis we present a formal computational framework for direct 13C metabolic flux analysis. The aim of our study is to construct as many linear constraints to the fluxes from the 13C labelling measurements using only computational methods that avoid non-linear techniques and are independent from the type of measurement data, the labelling of external nutrients and the topology of the metabolic network. The presented framework is the first representative of the direct approach for 13C metabolic flux analysis that is free from restricting assumptions made about these parameters.In our framework, measurement data is first propagated from the measured metabolites to other metabolites. The propagation is facilitated by the flow analysis of metabolite fragments in the network. Then new linear constraints to the fluxes are derived from the propagated data by applying the techniques of linear algebra.Based on the results of the fragment flow analysis, we also present an experiment planning method that selects sets of metabolites whose relative abundances of different labelling patterns are most useful for 13C metabolic flux analysis. Furthermore, we give computational tools to process raw 13C labelling data produced by tandem mass spectrometry to a form suitable for 13C metabolic flux analysis.

Computational Methods for Locating and Analyzing Conserved Gene Regulatory DNA Elements

This thesis presents methods for locating and analyzing cis-regulatory DNA elements involved with the regulation of gene expression in multicellular organisms. The regulation of gene expression is carried out by the combined effort of several transcription factor proteins collectively binding the DNA on the cis-regulatory elements. Only sparse knowledge of the 'genetic code' of these elements exists today. An automatic tool for discovery of putative cis-regulatory elements could help their experimental analysis, which would result in a more detailed view of the cis-regulatory element structure and function. We have developed a computational model for the evolutionary conservation of cis-regulatory elements. The elements are modeled as evolutionarily conserved clusters of sequence-specific transcription factor binding sites. We give an efficient dynamic programming algorithm that locates the putative cis-regulatory elements and scores them according to the conservation model. A notable proportion of the high-scoring DNA sequences show transcriptional enhancer activity in transgenic mouse embryos. The conservation model includes four parameters whose optimal values are estimated with simulated annealing. With good parameter values the model discriminates well between the DNA sequences with evolutionarily conserved cis-regulatory elements and the DNA sequences that have evolved neutrally. In further inquiry, the set of highest scoring putative cis-regulatory elements were found to be sensitive to small variations in the parameter values. The statistical significance of the putative cis-regulatory elements is estimated with the Two Component Extreme Value Distribution. The p-values grade the conservation of the cis-regulatory elements above the neutral expectation. The parameter values for the distribution are estimated by simulating the neutral DNA evolution. The conservation of the transcription factor binding sites can be used in the upstream analysis of regulatory interactions. This approach may provide mechanistic insight to the transcription level data from, e.g., microarray experiments. Here we give a method to predict shared transcriptional regulators for a set of co-expressed genes. The EEL (Enhancer Element Locator) software implements the method for locating putative cis-regulatory elements. The software facilitates both interactive use and distributed batch processing. We have used it to analyze the non-coding regions around all human genes with respect to the orthologous regions in various other species including mouse. The data from these genome-wide analyzes is stored in a relational database which is used in the publicly available web services for upstream analysis and visualization of the putative cis-regulatory elements in the human genome.

Dental Service Utilization, Dental Health Production and Equity in Dental Care: the Finnish Experience

This thesis is grounded on four articles. Article I generally examines the factors affecting dental service utilization. Article II studies the factors associated with sector-specific utilization among young adults entitled to age-based subsidized dental care. Article III explores the determinants of dental ill-health as measured by the occurrence of caries and the relationship between dental ill-health and dental care use. Article IV measures and explains income-related inequality in utilization. Data employed were from the 1996 Finnish Health Care Survey (I, II, IV) and the 1997 follow-up study included in the longitudinal study of the Northern Finland 1966 Birth Cohort (III). Utilization is considered as a multi-stage decision-making process and measured as the number of visits to the dentist. Modified count data models and concentration and horizontal equity indices were applied. Dentist s recall appeared very efficient at stimulating individuals to seek care. Dental pain, recall, and the low number of missing teeth positively affected utilization. Public subvention for dental care did not seem to statistically increase utilization. Among young adults, a perception of insufficient public service availability and recall were positively associated with the choice of a private dentist, whereas income and dentist density were positively associated with the number of visits to private dentists. Among cohort females, factors increasing caries were body mass index and intake of alcohol, sugar, and soft drinks and those reducing caries were birth weight and adolescent school achievement. Among cohort males, caries was positively related to the metropolitan residence and negatively related to healthy diet and education. Smoking increased caries, whereas regular teeth brushing, regular dental attendance and dental care use decreased caries. We found equity in young adults utilization but pro-rich inequity in the total number of visits to all dentists and in the probability of visiting a dentist for the whole sample. We observed inequity in the total number of visits to the dentist and in the probability of visiting a dentist, being pro-poor for public care but pro-rich for private care. The findings suggest that to enhance equal access to and use of dental care across population and income groups, attention should focus on supply factors and incentives to encourage people to contact dentists more often. Lowering co-payments and service fees and improving public availability would likely increase service use in both sectors. To attain favorable oral health, appropriate policies aimed at improving dental health education and reducing the detrimental effects of common risk factors on dental health should be strengthened. Providing equal access with respect to need for all people ought to take account of the segmentation of the service system, with its two parallel delivery systems and different supplier incentives to patients and dentists.

Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus Replicase Proteins

Viruses are submicroscopic, infectious agents that are obligate intracellular parasites. They adopt various types of strategies for their parasitic replication and proliferation in infected cells. The nucleic acid genome of a virus contains information that redirects molecular machinery of the cell to the replication and production of new virions. Viruses that replicate in the cytoplasm and are unable to use the nuclear transcription machinery of the host cell have developed their own transcription and capping systems. This thesis describes replication strategies of two distantly related viruses, hepatitis E virus (HEV) and Semliki Forest virus (SFV), which belong to the alphavirus-like superfamily of positive-strand RNA viruses. We have demonstrated that HEV and SFV share a unique cap formation pathway specific for alphavirus-like superfamily. The capping enzyme first acts as a methyltransferase, catalyzing the transfer of a methyl group from S-adenosylmethionine to GTP to yield m7GTP. It then transfers the methylated guanosine to the end of viral mRNA. Both reactions are virus-specific and differ from those described for the host cell. Therefore, these capping reactions offer attractive targets for the development of antiviral drugs. Additionally, it has been shown that replication of SFV and HEV takes place in association with cellular membranes. The origin of these membranes and the intracellular localization of the components of the replication complex were studied by modern microscopy techniques. It was demonstrated that SFV replicates in cytoplasmic membranes that are derived from endosomes and lysosomes. According to our studies, site for HEV replication seems to be the intermediate compartment which mediates the traffic between endoplasmic reticulum and the Golgi complex. As a result of this work, a unique mechanism of cap formation for hepatitis E virus replicase has been characterized. It represents a novel target for the development of specific inhibitors against viral replication.

Mediastinitis after cardiac surgery

Mediastinitis as a complication after cardiac surgery is rare but disastrous increasing the hospital stay, hospital costs, morbidity and mortality. It occurs in 1-3 % of patients after median sternotomy. The purpose of this study was to find out the risk factors and also to investigate new ways to prevent mediastinitis. First, we assessed operating room air contamination monitoring by comparing the bacteriological technique with continuous particle counting in low level contamination achieved by ultra clean garment options in 66 coronary artery bypass grafting operations. Second, we examined surgical glove perforations and the changes in bacterial flora of surgeons' fingertips in 116 open-heart operations. Third, the effect of gentamicin-collagen sponge on preventing surgical site infections (SSI) was studied in randomized controlled study with 557 participants. Finally, incidence, outcome, and risk factors of mediastinitis were studied in over 10,000 patients. With the alternative garment and textile system (cotton group and clean air suit group), the air counts fell from 25 to 7 colony-forming units/m3 (P<0.01). The contamination of the sternal wound was reduced by 46% and that of the leg wound by >90%. In only 17% operations both gloves were found unpunctured. Frequency of glove perforations and bacteria counts of hands were found to increase with operation time. With local gentamicin prophylaxis slightly less SSIs (4.0 vs. 5.9%) and mediastinitis (1.1 vs. 1.9%) occurred. We identified 120/10713 cases of postoperative mediastinitis (1.1%). During the study period, the patient population grew significantly older, the proportion of women and patients with ASA score >3 increased significantly. In multivariate logistic regression analysis, the only significant predictor for mediastinitis was obesity. Continuous particle monitoring is a good intraoperative method to control the air contamination related to the theatre staff behavior during individual operation. When a glove puncture is detected, both gloves are to be changed. Before donning a new pair of gloves, the renewed disinfection of hands will help to keep their bacterial counts lower even towards the end of long operation. Gentamicin-collagen sponge may have beneficial effects on the prevention of SSI, but further research is needed. Mediastinitis is not diminishing. Larger populations at risk, for example proportions of overweight patients, reinforce the importance of surveillance and pose a challenge in focusing preventive measures.

Assessment of neuroleptic-induced movement disorders in a naturalistic schizophrenia population

The prevalence and assessment of neuroleptic-induced movement disorders (NIMDs) in a naturalistic schizophrenia population that uses conventional neuroleptics were studied. We recruited 99 chronic schizophrenic institutionalized adult patients from a state nursing home in central Estonia. The total prevalence of NIMDs according to the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) was 61.6%, and 22.2% had more than one NIMD. We explored the reliability and validity of different instruments for measuring these disorders. First, we compared DSM-IV with the established observer rating scales of Barnes Akathisia Rating Scale (BARS), Simpson-Angus Scale (SAS) (for neuroleptic-induced parkinsonism, NIP) and Abnormal Involuntary Movement Scale (AIMS) (for tardive dyskinesia), all three of which have been used for diagnosing NIMD. We found a good overlap of cases for neuroleptic-induced akathisia (NIA) and tardive dyskinesia (TD) but somewhat poorer overlap for NIP, for which we suggest raising the commonly used threshold value of 0.3 to 0.65. Second, we compared the established observer rating scales with an objective motor measurement, namely controlled rest lower limb activity measured by actometry. Actometry supported the validity of BARS and SAS, but it could not be used alone in this naturalistic population with several co-existing NIMDs. It could not differentiate the disorders from each other. Quantitative actometry may be useful in measuring changes in NIA and NIP severity, in situations where the diagnosis has been made using another method. Third, after the relative failure of quantitative actometry to show diagnostic power in a naturalistic population, we explored descriptive ways of analysing actometric data, and demonstrated diagnostic power pooled NIA and pseudoakathisia (PsA) in our population. A subjective question concerning movement problems was able to discriminate NIA patients from all other subjects. Answers to this question were not selective for other NIMDs. Chronic schizophrenia populations are common worldwide, NIMD affected two-thirds of our study population. Prevention, diagnosis and treatment of NIMDs warrant more attention, especially in countries where typical antipsychotics are frequently used. Our study supported the validity and reliability of DSM-IV diagnostic criteria for NIMD in comparison with established rating scales and actometry. SAS can be used with minor modifications for screening purposes. Controlled rest lower limb actometry was not diagnostically specific in our naturalistic population with several co-morbid NIMDs, but it may be sensitive in measuring changes in NIMDs.

Juvenile rheumatoid knee : Management of knee growth disturbances in children with juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is associated with growth disturbances, especially leg length discrepancy (LLD) and knee valgus deformity (KVD). Studies have demonstrated growth plate stimulation with chronic arthritis. In the context of surgical treatment of LLD or KVD of a growing knee, the less invasive procedures, which allow immediate mobilisation, are preferred. Establishment of the skeletal age and the correction potential in the knees of rheumatic children is difficult due to rheumatic changes. In this present work, an analysis of the efficacy, safety and long-term results of temporary epiphyseal arrests performed in Rheumatism Foundation Hospital (Heinola, Finland). The distribution of diagnoses among children (n=71) with JIA and LLD (68 knees) was consistent with the normal oligoarthritis-predominated population of children with JIA. A higher male:female ratio (1:1.7 vs. 1:2.4 in population-based studies (PBS)) and earlier mean onset age (4 vs. 7 years in PBSs) were, however, distinct features in the study population. In most cases the correction was reliable and temporary arrest produced a mean correction of 1mm per month. The time of arrest required, however, varied significantly, probably due to the effect of underlying diseases and medication, and the age of the child. All complications encountered (10%) were minor. The correction achieved persisted in long-term follow-up. KVD (n=112, 177 knees) was associated with a high proportion of polyarthritic disease subtype (45% vs. 12-31% in PBSs), and the male:female distribution was grossly female-dominated (1:4.9 vs. 1:2.4 in PBSs). The early mean onset age (3 vs. 7 years in PBSs) was also notable in this cohort. Successful correction was achieved in 2/3 cases and the mean angular correction was 0.7 degrees per month. The required time of arrest, however, varied considerably. In 13% of knees the paucity of follow-up visits resulted in over-correction to varus. The complication rate (3%) in the knees operated for KVD was considerably lower compared to ten per cent in the management of LLD. Most of the complications related to epiphyseal stapling were reversible. However, the risk of premature closure of growth plates does exist. The number of over-corrections was notably high, with 13% knees turning to varus. The correction achieved persisted in long-term follow-up.