Bayesian statistical analysis of bacterial diversity

Bacteria play an important role in many ecological systems. The molecular characterization of bacteria using either cultivation-dependent or cultivation-independent methods reveals the large scale of bacterial diversity in natural communities, and the vastness of subpopulations within a species or genus. Understanding how bacterial diversity varies across different environments and also within populations should provide insights into many important questions of bacterial evolution and population dynamics. This thesis presents novel statistical methods for analyzing bacterial diversity using widely employed molecular fingerprinting techniques. The first objective of this thesis was to develop Bayesian clustering models to identify bacterial population structures. Bacterial isolates were identified using multilous sequence typing (MLST), and Bayesian clustering models were used to explore the evolutionary relationships among isolates. Our method involves the inference of genetic population structures via an unsupervised clustering framework where the dependence between loci is represented using graphical models. The population dynamics that generate such a population stratification were investigated using a stochastic model, in which homologous recombination between subpopulations can be quantified within a gene flow network. The second part of the thesis focuses on cluster analysis of community compositional data produced by two different cultivation-independent analyses: terminal restriction fragment length polymorphism (T-RFLP) analysis, and fatty acid methyl ester (FAME) analysis. The cluster analysis aims to group bacterial communities that are similar in composition, which is an important step for understanding the overall influences of environmental and ecological perturbations on bacterial diversity. A common feature of T-RFLP and FAME data is zero-inflation, which indicates that the observation of a zero value is much more frequent than would be expected, for example, from a Poisson distribution in the discrete case, or a Gaussian distribution in the continuous case. We provided two strategies for modeling zero-inflation in the clustering framework, which were validated by both synthetic and empirical complex data sets. We show in the thesis that our model that takes into account dependencies between loci in MLST data can produce better clustering results than those methods which assume independent loci. Furthermore, computer algorithms that are efficient in analyzing large scale data were adopted for meeting the increasing computational need. Our method that detects homologous recombination in subpopulations may provide a theoretical criterion for defining bacterial species. The clustering of bacterial community data include T-RFLP and FAME provides an initial effort for discovering the evolutionary dynamics that structure and maintain bacterial diversity in the natural environment.

Statistical analysis of the associations of hereditary factors with the risk of Type 1 diabetes and Diabetic Nephropathy based on the familial data collected through ascertaiment from population-based registers

In genetic epidemiology, population-based disease registries are commonly used to collect genotype or other risk factor information concerning affected subjects and their relatives. This work presents two new approaches for the statistical inference of ascertained data: a conditional and full likelihood approaches for the disease with variable age at onset phenotype using familial data obtained from population-based registry of incident cases. The aim is to obtain statistically reliable estimates of the general population parameters. The statistical analysis of familial data with variable age at onset becomes more complicated when some of the study subjects are non-susceptible, that is to say these subjects never get the disease. A statistical model for a variable age at onset with long-term survivors is proposed for studies of familial aggregation, using latent variable approach, as well as for prospective studies of genetic association studies with candidate genes. In addition, we explore the possibility of a genetic explanation of the observed increase in the incidence of Type 1 diabetes (T1D) in Finland in recent decades and the hypothesis of non-Mendelian transmission of T1D associated genes. Both classical and Bayesian statistical inference were used in the modelling and estimation. Despite the fact that this work contains five studies with different statistical models, they all concern data obtained from nationwide registries of T1D and genetics of T1D. In the analyses of T1D data, non-Mendelian transmission of T1D susceptibility alleles was not observed. In addition, non-Mendelian transmission of T1D susceptibility genes did not make a plausible explanation for the increase in T1D incidence in Finland. Instead, the Human Leucocyte Antigen associations with T1D were confirmed in the population-based analysis, which combines T1D registry information, reference sample of healthy subjects and birth cohort information of the Finnish population. Finally, a substantial familial variation in the susceptibility of T1D nephropathy was observed. The presented studies show the benefits of sophisticated statistical modelling to explore risk factors for complex diseases.

The Feminist Transformation of Bioethics : An Analysis of Theoretical Perspectives and Practical Applications in Feminist Bioethics

The purpose of this study is to examine how transformation is defining feminist bioethics and to determine the nature of this transformation. Behind the quest for transformation is core feminism and its political implications, namely, that women and other marginalized groups have been given unequal consideration in society and the sciences and that this situation is unacceptable and should be remedied. The goal of the dissertation is to determine how feminist bioethicists integrate the transformation into their respective fields and how they apply the potential of feminism to bioethical theories and practice. On a theoretical level, feminist bioethicists wish to reveal how current ways of knowing are based on inequality. Feminists pay special attention especially to communal and political contexts and to the power relations endorsed by each community. In addition, feminist bioethicists endorse relational ethics, a relational account of the self in which the interconnectedness of persons is important. On the conceptual level, feminist bioethicists work with beliefs, concepts, and practices that give us our world. As an example, I examine how feminist bioethicists have criticized and redefined the concept of autonomy. Feminist bioethicists emphasize relational autonomy, which is based on the conviction that social relationships shape moral identities and values. On the practical level, I discuss stem cell research as a test case for feminist bioethics and its ability to employ its methodologies. Analyzing these perspectives allowed me first, to compare non-feminist and feminist accounts of stem cell ethics and, second, to analyze feminist perspectives on the novel biotechnology. Along with offering a critical evaluation of the stem cell debate, the study shows that sustainable stem cell policies should be grounded on empirical knowledge about how donors perceive stem cell research and the donation process. The study indicates that feminist bioethics should develop the use of empirical bioethics, which takes the nature of ethics seriously: ethical decisions are provisional and open for further consideration. In addition, the study shows that there is another area of development in feminist bioethics: the understanding of (moral) agency. I argue that agency should be understood to mean that actions create desires.

Molecular genetics and evolution of Puumala hantavirus

Puumala virus (PUUV) is the causative agent of nephropathia epidemica (NE), a mild form of hemorrhagic fever with renal syndrome. Finland has the highest documented incidence of NE with around 1000 cases diagnosed annually. PUUV is also found in other Scandinavian countries, Central Europe and the European part of Russia. PUUV belongs to the genus Hantavirus in the family Bunyaviridae. Hantaviruses are rodent-borne viruses each carried by a specific host that is persistently and asymptomatically infected by the virus. PUUV is carried by the bank voles (Myodes glareolus, previously known as Clethrionomys glareolus). Hantaviruses have co-evolved with their carrier rodents for millions of years and these host animals are the evolutionary scene of hantaviruses. In this study, PUUV sequences were recovered from bank voles captured in Denmark and Russian Karelia to study the evolution of PUUV in Scandinavia. Phylogenetic analysis of these strains showed a geographical clustering of genetic variants following the presumable migration pattern of bank voles during the recolonization of Scandinavia after the last ice age approximately 10 000 years ago. The currently known PUUV genome sequences were subjected to in-depth phylogenetic analyses and the results showed that genetic drift seems to be the major mechanism of PUUV evolution. In general, PUUV seems to evolve quite slowly following a molecular clock. We also found evidence for recombination in the evolution of some genetic lineages of PUUV. Viral microevolution was studied in controlled virus transmission in colonized bank voles and changes in quasispecies dynamics were recorded as the virus was transmitted from one animal to another. We witnessed PUUV evolution in vivo, as one synonymous mutation became repeatedly fixed in the viral genome during the experiment. The detailed knowledge on the PUUV diversity was used to establish new sensitive and specific detection methods for this virus. Direct viral invasion of the hypophysis was demonstrated for the first time in a lethal case of NE. PUUV detection was done by immunohistochemistry, in situ hybridization and RT-nested-PCR of the autopsy tissue samples.

The Reorganisation of Provincial Territories in Light of the Imperial Decision-Making Process : Later Roman Arabia and Tres Palaestinae as Case Studies

The dissertation examines Roman provincial administration and the phenomenon of territorial reorganisations of provinces during the Imperial period with special emphasis on the provinces of Arabia and Palaestina during the Later Roman period, i.e., from Diocletian (r. 284 305) to the accession of Phocas (602), in the light of imperial decision-making. Provinces were the basic unit of Roman rule, for centuries the only level of administration that existed between the emperor and the cities of the Empire. The significance of the territorial reorganisations that the provinces were subjected to during the Imperial period is thus of special interest. The approach to the phenomenon is threefold: firstly, attention is paid to the nature and constraints of the Roman system of provincial administration. Secondly, the phenomenon of territorial reorganisations is analysed on the macro-scale, and thirdly, a case study concerning the reorganisations of the provinces of Arabia and Palaestina is conducted. The study of the mechanisms of decision-making provides a foundation through which the collected data of all known major territorial reorganisations is interpreted. The data concerning reorganisations is also subjected to qualitative comparative analysis that provides a new perspective to the data in the form of statistical analysis that is sensitive to the complexities of individual cases. This analysis of imperial decision-making is based on a timeframe stretching from Augustus (r. 30 BC AD 14) to the accession of Phocas (602). The study identifies five distinct phases in the use of territorial reorganisations of the provinces. From Diocletian s reign there is a clear normative change that made territorial reorganisations a regular tool of administration for the decision-making elite for addressing a wide variety of qualitatively different concerns. From the beginning of the fifth century the use of territorial reorganisations rapidly diminishes. The two primary reasons for the decline in the use of reorganisations were the solidification of ecclesiastical power and interests connected to the extent of provinces, and the decline of the dioceses. The case study of Palaestina and Arabia identifies seven different territorial reorganisations from Diocletian to Phocas. Their existence not only testifies to wider imperial policies, but also shows sensitivity to local conditions and corresponds with the general picture of provincial reorganisations. The territorial reorganisations of the provinces reflect the proactive control of the Roman decision-making elite. The importance of reorganisations should be recognised more clearly as part of the normal imperial administration of the provinces and especially reflecting the functioning of dioceses.

Onnistuneen ikääntymisen ymmärrystä rakentamassa : Fysioterapeuttiopiskelijoiden oppiminen kestävää kehitystä edistävän kasvatuksen kehyksessä

The purpose of the present study was to investigate the possibilities and interconnec-tions that exist concerning the relationship between the University of Applied Sci-ences and the Learning by Developing action model (LbD), on the one hand, and education for sustainable development and high-quality learning as a part of profes-sional competence development on the other. The research and learning environment was the Coping at Home research project and its Caring TV project, which provided the context of the Physiotherapy for Elderly People professional study unit. The re-searcher was a teacher and an evaluator of her own students learning. The aims of the study were to monitor and evaluate learning at the individual and group level using tools of high-quality learning − improved concept maps − related to understanding the projects core concept of successful ageing. Conceptions were evaluated through aspects of sustainable development and a conceptual basis of physiotherapy. As edu-cational research this was a multi-method case study design experiment. The three research questions were as follows. 1. What kind of individual conceptions and conceptual structures do students build concerning the concept of successful ageing? How many and what kind of concepts and propositions do they have a) before the study unit, b) after the study unit, c) after the social-knowledge building? 2. What kind of social-knowledge building exists? a) What kind of social learn-ing process exists? b) What kind of socially created concepts, propositions and conceptual structures do the students possess after the project? c) What kind of meaning does the social-knowledge building have at an individual level? 3. How do physiotherapy competences develop according to the results of the first and second research questions? The subjects were 22 female, third-year Bachelor of Physiotherapy students in Laurea University of Applied Sciences in Finland. Individual learning was evaluated in 12 of the 22 students. The data was collected as a part of the learning exercises of the Physiotherapy for Elderly People study unit, with improved concept maps both at individual and group levels. The students were divided into two social-knowledge building groups: the first group had 15 members and second 7 members. Each group created a group-level concept map on the theme of successful ageing. These face-to-face interactions were recorded with CMapTools and videotaped. The data consists of both individually produced concept maps and group-produced concept maps of the two groups and the videotaped material of these processes. The data analysis was carried out at the intersection of various research traditions. Individually produced data was analysed based on content analysis. Group-produced data was analysed based on content analysis and dialogue analysis. The data was also analysed by simple statistical analysis. In the individually produced improved concept maps the students conceptions were comprehensive, and the first concept maps were found to have many concepts unrelated to each other. The conceptual structures were between spoke structures and chain structures. Only a few professional concepts were evident. In the second indi-vidual improved concept maps the conception was more professional than earlier, particulary from the functional point of view. The conceptual structures mostly re-sembled spoke structures. After the second individual concept mapping social map-ping interventions were made in the two groups. After this, multidisciplinary concrete links were established between all concepts in almost all individual concept maps, and the interconnectedness of the concepts in different subject areas was thus understood. The conceptual structures were mainly net structures. The concepts in these individual concept maps were also found to be more professional and concrete than in the previ-ous concept maps of these subjects. In addition, the wider context dependency of the concepts was recognized in many individual concept maps. This implies a conceptual framework for specialists. The social-knowledge building was similar to a social learning process. Both socio-cultural processes and cognitive processes were found to develop students conceptual awareness and the ability to engage in intentional learning. In the knowl-edge-building process two aspects were found: knowledge creation and pedagogical action. The discussion during the concept-mapping process was similar to a shared thinking process. In visualising the process with CMapTools, students easily comple-mented each others thoughts and words, as if mutually telepathic . Synthesizing, supporting, asking and answering, peer teaching and counselling, tutoring, evaluating and arguing took place, and students were very active, self-directed and creative. It took hundreds of conversations before a common understanding could be found. The use of concept mapping in particular was very effective. The concepts in these group-produced concept maps were found to be professional, and values of sustainable development were observed. The results show the importance of developing the contents and objectives of the European Qualification Framework as well as education for sustainable development, especially in terms of the need for knowledge creation, global responsibility and systemic, holistic and critical thinking in order to develop clinical practice. Keywords: education for sustainable development, learning, knowledge building, improved concept map, conceptual structure, competence, successful ageing

Aritmetiikasta algebraan : Muutoksia osaamisessa peruskoulun päättöluokalla 20 vuoden aikana

From Arithmetic to Algebra. Changes in the skills in comprehensive school over 20 years. In recent decades we have emphasized the understanding of calculation in mathematics teaching. Many studies have found that better understanding helps to apply skills in new conditions and that the ability to think on an abstract level increases the transfer to new contexts. In my research I take into consideration competence as a matrix where content is in a horizontal line and levels of thinking are in a vertical line. The know-how is intellectual and strategic flexibility and understanding. The resources and limitations of memory have their effects on learning in different ways in different phases. Therefore both flexible conceptual thinking and automatization must be considered in learning. The research questions that I examine are what kind of changes have occurred in mathematical skills in comprehensive school over the last 20 years and what kind of conceptual thinking is demonstrated by students in this decade. The study consists of two parts. The first part is a statistical analysis of the mathematical skills and their changes over the last 20 years in comprehensive school. In the test the pupils did not use calculators. The second part is a qualitative analysis of the conceptual thinking of pupils in comprehensive school in this decade. The study shows significant differences in algebra and in some parts of arithmetic. The largest differences were detected in the calculation skills of fractions. In the 1980s two out of three pupils were able to complete tasks with fractions, but in the 2000s only one out of three pupils were able to do the same tasks. Also remarkable is that out of the students who could complete the tasks with fractions, only one out of three pupils was on the conceptual level in his/her thinking. This means that about 10% of pupils are able to understand the algebraic expression, which has the same isomorphic structure as the arithmetical expression. This finding is important because the ability to think innovatively is created when learning the basic concepts. Keywords: arithmetic, algebra, competence

Kivaa ja hyödyllistä, mutta jo se riittää : Yläasteen kielikylpyoppilaiden käsityksiä ruotsinkielisestä kielikylpyopetuksesta Helsingissä

The aim of this study was to find out how immersion students experience immersion education, how they feel about the implementation of immersion education methods and what role immersion plays in immersion students’ lives outside the school context. In addition, the influence of sex, grade level, school and type of immersion education on students’ perceptions was studied. The population included all students at the lower secondary level in Helsinki who participated in Swedish immersion education during 2002–2003. The sample consisted of 128 students who represented two different forms of immersion: 47% of the students had previously participated in early total immersion while 53 % of the students had taken part in early partial immersion. The data were gathered through a questionnaire and interviews. All 128 students answered the questionnaire, and 10 students were chosen to focus interviews through purposive sampling. In addition, students’ parents were invited to fill in a questionnaire where students’ background information was requested. The data were collected during the spring of 2003. Principal Component Analysis and one-way variance analysis were used as statistical analysis methods. Also frequencies, average, correlations and cross tabs were studied. In the PCA a right-angled varimax-rotation was performed separately to every thematic entity that arose from the theoretical background. Sum variables were formed from the Principal Components by summing up all the items that received over .400 charges for the specific Principal Component. Significance testing of the mean was performed with F and t-tests. Results indicate that immersion students in lower secondary school experience immersion quite diversely as a whole. Students are satisfied with the fact that they are in the immersion class but not with the amount of teaching in Swedish. Students feel it is very important and useful to learn Swedish bearing in mind their future studies and working life. The students estimate their language skills to be very high. Yet they prefer using Finnish during classes. The fact that teachers use Swedish does not considerably affect how well the students learn the factual content in various subjects, especially if the student knows Swedish well. Theoretical subjects seemed to cause most problems. Swedish played only a very small part in students’ lives outside the school context and it was used merely when travelling abroad and in different kinds of guiding situations. Unless the students were talked to in Swedish, they kept on speaking Finnish. When asked about students’ experiences no statistically significant differences between sexes were found in this study. On the contrary, in some cases their grade level but especially their school and form of immersion had clear statistically significant differences on students’ perceptions.

Microarrays in molecular profiling of cancer - focus on head and neck squamous cell carcinoma

Microarrays have a wide range of applications in the biomedical field. From the beginning, arrays have mostly been utilized in cancer research, including classification of tumors into different subgroups and identification of clinical associations. In the microarray format, a collection of small features, such as different oligonucleotides, is attached to a solid support. The advantage of microarray technology is the ability to simultaneously measure changes in the levels of multiple biomolecules. Because many diseases, including cancer, are complex, involving an interplay between various genes and environmental factors, the detection of only a single marker molecule is usually insufficient for determining disease status. Thus, a technique that simultaneously collects information on multiple molecules allows better insights into a complex disease. Since microarrays can be custom-manufactured or obtained from a number of commercial providers, understanding data quality and comparability between different platforms is important to enable the use of the technology to areas beyond basic research. When standardized, integrated array data could ultimately help to offer a complete profile of the disease, illuminating mechanisms and genes behind disorders as well as facilitating disease diagnostics. In the first part of this work, we aimed to elucidate the comparability of gene expression measurements from different oligonucleotide and cDNA microarray platforms. We compared three different gene expression microarrays; one was a commercial oligonucleotide microarray and the others commercial and custom-made cDNA microarrays. The filtered gene expression data from the commercial platforms correlated better across experiments (r=0.78-0.86) than the expression data between the custom-made and either of the two commercial platforms (r=0.62-0.76). Although the results from different platforms correlated reasonably well, combining and comparing the measurements were not straightforward. The clone errors on the custom-made array and annotation and technical differences between the platforms introduced variability in the data. In conclusion, the different gene expression microarray platforms provided results sufficiently concordant for the research setting, but the variability represents a challenge for developing diagnostic applications for the microarrays. In the second part of the work, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 38 laryngeal and oral tongue squamous cell carcinoma cell lines and primary tumors. Our aim was to pinpoint genes for which expression was impacted by changes in copy number. The data revealed that especially amplifications had a clear impact on gene expression. Across the genome, 14-32% of genes in the highly amplified regions (copy number ratio >2.5) had associated overexpression. The impact of decreased copy number on gene underexpression was less clear. Using statistical analysis across the samples, we systematically identified hundreds of genes for which an increased copy number was associated with increased expression. For example, our data implied that FADD and PPFIA1 were frequently overexpressed at the 11q13 amplicon in HNSCC. The 11q13 amplicon, including known oncogenes such as CCND1 and CTTN, is well-characterized in different type of cancers, but the roles of FADD and PPFIA1 remain obscure. Taken together, the integrated microarray analysis revealed a number of known as well as novel target genes in altered regions in HNSCC. The identified genes provide a basis for functional validation and may eventually lead to the identification of novel candidates for targeted therapy in HNSCC.

Tracking and identifying wastewater toxicants and assessing their biodegradation products

Screening of wastewater effluents from municipal and industrial wastewater treatment plants with biotests showed that the treated wastewater effluents possess only minor acute toxic properties towards whole organisms (e.g. bacteria, algae, daphnia), if any. In vitro tests (sub-mitochondrial membranes and fish hepatocytes) were generally more susceptible to the effluents. Most of the effluents indicated the presence of hormonally active compounds, as the production of vitellogenin, an egg yolk precursor protein, was induced in fish hepatocytes exposed to wastewater. In addition, indications of slight genotoxic potential was found in one effluent concentrate with a recombinant bacteria test. Reverse electron transport (RET) of mitochondrial membranes was used as a model test to conduct effluent assessment followed by toxicant characterisations and identifications. Using a modified U.S. EPA Toxicity Identification Evaluation Phase I scheme and additional case-specific methods, the main compound in a pulp and paper mill effluent causing RET inhibition was characterised to be an organic, relatively hydrophilic high molecular weight (HMW) compound. The toxicant could be verified as HMW lignin by structural analyses using nuclear magnetic resonance. In the confirmation step commercial and in-house extracted lignin products were used. The possible toxicity related structures were characterised by statistical analysis of the chemical breakdown structures of laboratory-scale pulping and bleaching effluents and the toxicities of these effluents. Finally, the biological degradation of the identified toxicant and other wastewater constituents was evaluated using bioassays in combination with chemical analyses. Biological methods have not been used routinely in establishing effluent discharge limits in Finland. However, the biological effects observed in this study could not have been predicted using only routine physical and chemical effluent monitoring parameters. Therefore chemical parameters cannot be considered to be sufficient in controlling effluent discharges especially in case of unknown, possibly bioaccumulative, compounds that may be present in small concentrations and may cause chronic effects.

Joint Regression and Association Models for Repeated Categorical Responses

The focus of this study is on statistical analysis of categorical responses, where the response values are dependent of each other. The most typical example of this kind of dependence is when repeated responses have been obtained from the same study unit. For example, in Paper I, the response of interest is the pneumococcal nasopharengyal carriage (yes/no) on 329 children. For each child, the carriage is measured nine times during the first 18 months of life, and thus repeated respones on each child cannot be assumed independent of each other. In the case of the above example, the interest typically lies in the carriage prevalence, and whether different risk factors affect the prevalence. Regression analysis is the established method for studying the effects of risk factors. In order to make correct inferences from the regression model, the associations between repeated responses need to be taken into account. The analysis of repeated categorical responses typically focus on regression modelling. However, further insights can also be gained by investigating the structure of the association. The central theme in this study is on the development of joint regression and association models. The analysis of repeated, or otherwise clustered, categorical responses is computationally difficult. Likelihood-based inference is often feasible only when the number of repeated responses for each study unit is small. In Paper IV, an algorithm is presented, which substantially facilitates maximum likelihood fitting, especially when the number of repeated responses increase. In addition, a notable result arising from this work is the freely available software for likelihood-based estimation of clustered categorical responses.

Ecological impacts of Phlebiopsis gigantea biocontrol treatment against Heterobasidion spp. as revealed by fungal community profiling and population analyses

Wood decay fungi belonging to the species complex Heterobasidion annosum sensu lato are among the most common and economically important species causing root rot and stem decay in conifers of the northern temperate regions. New infections by these pathogens can be suppressed by tree stump treatments using chemical or biological control agents. In Finland, the corticiaceous fungus Phlebiopsis gigantea has been formulated into a commercial biocontrol agent called Rotstop (Verdera Ltd.). This thesis addresses the ecological impacts of Rotstop biocontrol treatment on the mycoflora of conifer stumps. Locally, fungal communities within Rotstop-treated and untreated stumps were analyzed using a novel method based on DGGE profiling of small subunit ribosomal DNA fragments amplified directly from wood samples. Population analyses for P. gigantea and H. annosum s.l. were conducted to evaluate possible risks associated with local and/or global distribution of the Rotstop strain. Based on molecular community profiling by DGGE, we detected a few individual wood-inhabiting fungal species (OTUs) that seemed to have suffered or benefited from the Rotstop biocontrol treatment. The DGGE analyses also revealed fungal diversity not retrieved by cultivation and some fungal sequence types untypical for decomposing conifer wood. However, statistical analysis of DGGE community profiles obtained from Rotstop-treated and untreated conifer stumps revealed that the Rotstop treatment had not caused a statistically significant reduction in the species diversity of wood-inhabiting fungi within our experimental forest plots. Locally, ISSR genotyping of cultured P. gigantea strains showed that the Rotstop biocontrol strain was capable of surviving up to six years within treated Norway spruce stumps, while in Scots pine stumps it was sooner replaced by successor fungal species. In addition, the spread of resident P. gigantea strains into Rotstop-treated forest stands seemed effective in preventing the formation of genetically monomorphic populations in the short run. On a global scale, we detected a considerable level of genetic differentiation between the interfertile European and North American populations of P. gigantea. These results strongly suggest that local biocontrol strains should be used in order to prevent global spread of P. gigantea and hybrid formation between geographically isolated populations. The population analysis for H. annosum s.l. revealed a collection of Chinese fungal strains that showed a high degree of laboratory fertility with three different allopatric H. annosum s.l. taxa. However, based on the molecular markers, the Chinese strains could be clearly affiliated with the H. parviporum taxonomical cluster, which thus appears to have a continuous distribution range from Europe through southern Siberia to northern China. Keywords: Rotstop, wood decay, DGGE, ISSR fingerprinting, ribosomal DNA

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.