Die transkranielle Magnetstimulation (TMS) ist ein nichtinvasives Hirnstimulationsverfahren, mit welchem sowohl die funktionelle Untersuchung umschriebener kortikaler Regionen als auch die Modulation der Erregbarkeit ebendieser sowie die Induktion neuroplastischer Phänomene möglich ist. Sie wurde in der Vergangenheit insbesondere bei der Erforschung des humanen zentralmotorischen Systems angewandt. Dabei zeigte sich, dass ein einzelner über dem primärmotorischen Areal (M1) applizierter TMS-Puls multiple deszendierende Erregungswellen im Kortikospinaltrakt induzieren kann. Von diesen Undulationen besitzt die D-Welle (direkte Welle) die kürzeste Latenz und sie rekurriert auf eine direkte Aktivierung kortikospinaler Neurone, wohingegen I-Wellen (indirekte Wellen) längere Latenzen besitzen und durch transsynaptische Aktivierung dieser Zellen entstehen. Bemerkenswert ist das periodische Auftreten der letztgenannten Erregungswellen mit einer Periodendauer von etwa 1,5 ms. Zwar sind die genauen Mechanismen noch unbekannt, welche der Entstehung dieser I-Wellen sowie dem Phänomen der I-Wellen-Fazilitierung, das sich in geeigneten TMS-Doppelpulsprotokollen offenbart, zugrunde liegen, jedoch existieren hierzu verschiedene Erklärungsmodelle. Im Mittelpunkt der vorliegenden Arbeit steht die repetitive Anwendung eines TMS-Doppelpulsprotokolls, bei dem das Interpulsintervall (IPI) im Bereich der I-Wellen-Periodizität liegt (iTMS) und das gleichsam durch eine Implementierung der I-Wellen-Fazilitierung in der repetitiven TMS charakterisiert ist. Da gezeigt werden konnte, dass iTMS mit einem IPI von 1,5 ms (iTMS_1,5ms) die kortikospinale Erregbarkeit signifikant intra- und postinterventionell zu steigern vermag, und die I-Wellen-Periodizität interindividuellen Schwankungen unterliegt, wurde in der hier vorgestellten Studie an Normalprobanden der Einfluss einer individuellen Anpassung des IPIs (resultierend in der iTMS_adj) auf die intrainterventionelle kortikospinale Erregbarkeit untersucht. In der Tat stellte sich heraus, dass die iTMS_adj der iTMS_1,5ms diesbezüglich überlegen ist. Dieses Ergebnis unterstreicht das Potential einer Individualisierung der interventionellen TMS für erregbarkeitsmodulierende Effekte und macht dasjenige der ohnehin auf physiologische Prozesse abgestimmten iTMS explizit, was insbesondere für klinische Anwendungen relevant sein mag.

Areas cultivated with eucalyptus forests have increased in Southern Brazil, and the effects on hydrosedimentological processes are not well known particularly at the watershed scale and in watersheds where land use has been changing. The Soil and Water Assessment Tool (SWAT) model has been applied in a few forested watersheds to evaluate and predict effects of commercial forest cultivation or land use change in hydrological processes, whilst erosion processes has not been the main goal. The present study aimed (i) to evaluate the effects of eucalyptus plantation on hydrosedimentological processes, and to evaluate the efficiency and limitations of the Soil and Water Assessment Tool (SWAT) model to simulate streamflow and sediment yield (daily and monthly) in nested eucalyptus watersheds and in two paired watersheds with eucalyptus and grassland, and (ii) to identify the contribution of the bank channel, unpaved roads and eucalyptus stands sediment-sources for two different size fractions of sediment (< 0.063 mm and 0.063-2 mm) deposited along the drainage network, and suspended sediment collected in the outlet of small watersheds. SWAT was used to evaluate hydrological and erosion processes for two nested forest watersheds (watershed-0.98 km2 and sub-watershed-0.39 km2) and two paired watersheds in Pampa Biome, one with grassland (1.10 km2) and other with eucalyptus (0.83 km2). Measured streamflow and sediment yield at the watersheds outlets was used to evaluate model sensitivity to selected model parameters, and for calibration from 2009 to 2013 for nested watersheds, and from 2009 to 2013 for paired watersheds. Time series plots and standard statistical measures were used to verify model predictions. For nested watersheds, predicted monthly streamflow was very good during calibration for the watershed and for the sub-watershed, respectively. Predicted monthly sediment yield was very good and satisfactory for the watershed, and unsatisfactory for the sub-watershed. Simulations for daily time-scale were satisfactory to predict streamflow and sediment yield for the watershed. For the sub-watershed, predicted streamflow was satisfactory , but sediment yield was unsatisfactory . For paired watersheds, predicted monthly streamflow was good during calibration for the grassland watershed and for the eucalyptus watershed. Predicted monthly sediment yield was satisfactory for the grassland watershed, and very good and satisfactory for eucalyptus watershed. Daily simulations were satisfactory to predict streamflow in both grassland and eucalyptus watersheds. The results suggest that the SWAT model is a promising tool to evaluate hydrological processes in Brazilian watersheds. However, more field work with continuous monitoring is required and empirical equations of SWAT must change to better represent the processes that predominate in each watershed and to represent the hydrological and erosion processes in sub-daily time-scale and, especially, for small watersheds where the time of concentration is less than one day. Sediment-sources was evaluated using geochemical properties, determined by inductively coupled plasma mass spectrometry and X-ray fluorescence analyses, and the data used to calculate proportional contributions of sediment. Source contributions were determined at points spatially-distributed along the main channel of the watershed. Source determination for in-stream sites was done using samples collected in one spot to evaluate source-contribution of area upstream of this site of interest to indicate how different sources dominate at different downstream locations. To examine whether different size-fractions shared similar origins, two size fractions of both source and suspended samples including fine (<0.063 mm) and coarse (0.063 2 mm) particles were analyzed. Fingerprinting approach indicated particle-size and location of sources within a watershed are major factors affecting the measured contribution of sources for coarse and fine sediments. The closer a sediment sampling site is to a potential source, the more likely this sediment-source will dominate the sampled material, especially for coarse particles. Based on the classification analysis, the relative contribution of each source to eroded sediments was bank channel, i.e. from the stream network. Modeling results and sediment-source identification can provide an improved understanding effect of land use or change in land use on sediment yield in watershed scale, which are useful and may be used as a management tool of natural resources. / Áreas cultivadas com florestas de eucalipto têm aumentado no Sul do Brasil e os efeitos sobre os processos hidrossedimentológicos são pouco conhecidos, especialmente em escala de bacia hidrográfica e para áreas em que ocorreu mudança no uso do solo. O modelo Soil and Water Assesment Tool (SWAT) tem sido utilizado em bacias hidrográficas florestais para avaliar e simular o efeito do cultivo de florestas comerciais ou mudança de uso do solo sobre os processos hidrológicos, mas os processos erosivos não têm sido avaliados. O presente estudo objetivou (i) avaliar os efeitos do plantio de eucalipto sobre os processos hidrossedimentológicos e a eficiência e as limitações do SWAT em simular o escoamento superficial e produção de sedimentos (diários e mensais) em bacias hidrográficas embutidas, ocupadas com eucalipto, e em duas bacias hidrográficas pareadas ocupadas principalmente com eucalipto e com campo natural, e (ii) identificar a contribuição das fontes margem da rede de drenagem, as estradas e os povoamentos de eucalipto na geração de duas diferentes frações de sedimentos (< 0,063 mm e 0,063-2 mm) depositados ao longo da rede de drenagem, e de sedimentos em suspensão coletados no exutório de bacias embutidas. O SWAT foi utilizado para avaliar os processos hidrológicos e de erosão em duas bacias florestais embutidas (bacia-0,94 km2 e sub-bacia-0,39 km2) e em duas bacias hidrográficas pareadas no Bioma Pampa, uma com campo natural (1,10 km2) e outra com eucalipto (0,83 km2). A vazão e a produção de sedimentos medidas nas seções de monitoramento das bacias hidrográficas foram utilizadas para avaliar a sensibilidade dos parâmetros selecionados do modelo e para calibração para as bacias embutidas (2009-2013), e para as bacias pareadas (2009-2013). As séries temporais e os parâmetros estatísticos foram utilizados para avaliar o potencial preditivo do modelo. Para as bacias embutidas, a representação da vazão mensal foi muito boa durante a calibração para a bacia e para a sub-bacia, respectivamente. A representação da produção de sedimentos mensal foi muito boa e satisfatória para a bacia, e insatisfatória para a sub-bacia. As simulações na escala diária foram satisfatórias para a representação da vazão e da produção de sedimentos para a bacia. Para a sub-bacia, a representação da vazão foi satisfatória, mas da produção de sedimentos foi insatisfatória. Para as bacias hidrográficas pareadas, a representação da vazão mensal foi boa durante a calibração para a bacia com pastagem e para a bacia com eucalipto. A representação da produção de sedimentos mensal foi satisfatória para a bacia com pastagem, e muito boa e satisfatória para a bacia com eucalipto. As simulações diárias foram satisfatórias para a vazão em ambas as bacias pareadas. Os resultados sugerem que o modelo SWAT é uma ferramenta promissora para avaliar processos hidrológicos em bacias hidrográficas brasileiras. No entanto, fazem-se necessários mais tempo de monitoramento contínuo das variáveis hidrossedimentológicas e alteração de equações empíricas do SWAT para melhor representar processos erosivos que predominam em cada bacia e para representar os processos hidrológicos e erosivos em escala sub-diária, especialmente para pequenas bacias em que o tempo de concentração é menor do que um dia. As fontes de sedimentos foram avaliadas por meio do uso de propriedades geoquímicas, determinadas por análises de espectrometria de massas com plasma e de fluorescência de raios-X. A contribuição das fontes de sedimentos foi determinada em pontos espacialmente distribuídos ao longo do canal principal da bacia. Amostras de sedimento de um ponto de coleta foram utilizadas para avaliar a contribuição das fontes da área de contribuição à montante desse local de interesse e para indicar o a diferença da contribuição dessas fontes em diferentes localizações à jusante. Para avaliar se as diferentes frações de sedimentos tem origem semelhante, duas frações de tamanho de partículas das fontes e amostras de sedimento fino (< 0,063 mm) e grosso (0,063-2 mm) foram analisadas. A técnica fingerprinting indicou que o tamanho das partículas e localização das fontes dentro da bacia hidrográfica são os principais fatores que afetam a contribuição das fontes para a produção de sedimentos finos e grossos. As fontes mais próximas do local de amostragem são as potenciais fontes de sedimentos, sendo provável que essa fonte de sedimentos será dominante na amostra de sedimentos, principalmente para as frações grossas. Com base na análise de classificação, a contribuição relativa de cada fonte indicou que a principal fonte de sedimentos foi a margem do canal da rede de drenagem. Os resultados da modelagem e identificação das fontes de sedimentos contribuem para uma melhor compreensão do efeito do uso do solo ou da alteração no uso sobre a produção de sedimentos na escala de bacia hidrográfica, que são úteis e podem ser utilizados como ferramenta de gestão dos recursos naturais.

Made available in DSpace on 2016-06-02T20:06:10Z (GMT). No. of bitstreams: 1 6128.pdf: 965623 bytes, checksum: 08d957ba051c6348918f8348a857eff7 (MD5) Previous issue date: 2014-04-23 / Financiadora de Estudos e Projetos / The modelling of paired counts data is a topic that has been frequently discussed in several threads of research. In particular, we can cite bivariate counts, such as the analysis of sports scores. As a result, in this work we present the bivariate Poisson distribution to modelling positively correlated scores. The possible independence between counts is also addressed through the double Poisson model, which arises as a special case of the bivariate Poisson model. The main characteristics and properties of these models are presented and a simulation study is conducted to evaluate the behavior of the estimates for different sample sizes. Considering the possibility of modeling parameters by insertion of predictor variables, we present the structure of the bivariate Poisson regression model as a general case as well as the structure of an effects model for application in sports data. Particularly, in this work we will consider applications to Brazilian Championship Serie A 2012 data, in which the effects will be estimated by double Poisson and bivariate Poisson models. Once obtained the fits, the probabilities of scores occurence are estimated and then we obtain forecasts for the outcomes. In order to obtain more accurate forecasts, we present the weighted likelihood method from which it will be possible to quantify the relevance of the data according to the time they were observed. / A modelagem de dados provenientes de contagens pareadas e um típico que vem sendo frequentemente abordado em diversos segmentos de pesquisa. Em particular, podemos citar os casos em que as contagens de interesse são bivariadas, como por exemplo na analise de placares esportivos. Em virtude disso, neste trabalho apresentamos a distribuição Poisson bivariada para os casos em que as contagens de interesse sao positivamente correlacionadas. A possível independencia entre as contagens tambem e abordada por meio do modelo Poisson duplo, que surge como caso particular do modelo Poisson bivariado. As principais características e propriedades desses modelos são apresentadas e um estudo de simulação é realizado, visando avaliar o comportamento das estimativas para diferentes tamanhos amostrais. Considerando a possibilidade de se modelar os parâmetros por meio da inserçao de variáveis preditoras, apresentamos a estrutura do modelo de regressão Poisson bivariado como caso geral, bem como a estrutura de um modelo de efeitos para aplicação a dados esportivos. Particularmente, neste trabalho vamos considerar aplicações aos dados da Serie A do Campeonato Brasileiro de 2012, na qual os efeitos serão estimados por meio dos modelos Poisson duplo e Poisson bivariado. Uma vez obtidos os ajustes, estimam-se as probabilidades de ocorrência dos placares e, a partir destas, obtemos previsões para as partidas de interesse. Com o intuito de se obter previsões mais acuradas para as partidas, apresentamos o metodo da verossimilhança ponderada, a partir do qual seria possível quantificar a relevância dos dados em função do tempo em que estes foram observados.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Chapter I - Toxoplasmosis is a zoonosis caused by the intracellular parasite Toxoplasma gondii, which infects a range of hosts, including about one-third of the world\'s human population. One of the most severe manifestations of this infection in humans corresponds to congenital toxoplasmosis, which occurs when there is placental transmission of the parasite to the fetus in cases of primary maternal infection during pregnancy. Congenital infection may cause abortions or fetal losses, as well as severe ocular and/or cerebral sequelae in newborns. The serological screening of pregnant women and newborns is mainly based on the detection of IgG and IgM antibodies to T. gondii and constitutes an important measure to be adopted in programs to control this infection, despite the limitations in the antibody detection and interpretation of results. Chapter II - A total of 173 pairs of serum samples from mothers with suspected primary toxoplasmosis during pregnancy and their newborns, obtained from the Department of Pediatrics and Neonatology of Clinical Hospital of the Federal University of Uberlândia (HC-UFU) from 2006 to June 2014, was analyzed by ELISA for the detection of IgG, IgM and IgA anti-T. gondii, and the results were correlated with clinical data obtained from research in the clinical records of each patient. It was concluded that (i) prenatal serological screening is very important for the identification of pregnant women exposed to toxoplasmosis during pregnancy; (ii) maternal treatment reduces congenital transmission of T. gondii; (iii) neonatal serologic screening, associated with analysis of clinical parameters, allows the identification of vertically infected newborns, mainly through simultaneous detection of IgM and IgA antibodies; and (iv) serological follow-up of newborns is important in clarifying doubtful situations, especially in cases of asymptomatic newborns that present suggestive serology of congenital infection. Chapter III - Different antigenic fractions derived from soluble antigen of tachyzoites of T. gondii (STAg) were obtained from sequential precipitation with increasing concentrations of ammonium sulfate and used in immunoblotting technique to detect IgG antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) present in paired serum samples from mothers with presumptive serology of recent toxoplasmosis during pregnancy and their newborns. It was concluded that the use of antigenic fractions obtained from STAg precipitation in the diagnosis of human toxoplasmosis proved to be interesting to detect IgG and its subclasses, allowing differentiation between positive and negative samples, but it was not a good alternative for the diagnosis of congenital toxoplasmosis, presenting results considered inferior to the STAg, due to the lower frequency of recognized antigenic bands and the absence of differential recognition of antigens by sera of newborns. Chapter IV - The amino acid sequences of sixteen immunodominant antigens of T. gondii were used to perform B cell linear epitope prediction using a software-based approach. A total of 22 epitopes of antigens from surface (SRS), rhoptries (ROP), micronemes (MIC) and dense granules (GRA) of T. gondii were identified, and 15 residues from their amino acid sequences were used to synthesize peptides chemically linked to bovine serum albumin backbone, and the diagnostic performance of these synthetic peptides was evaluated in ELISA to detect specific IgG antibodies in sera of patients with suspected acute toxoplasmosis (G1) or chronic (G2). It was concluded that synthetic peptides designed from B cell linear epitope prediction constitute promising antigens in serological assays to diagnose toxoplasmosis and differentiate acute from chronic phases of toxoplasmosis, representing an alternative to the use of native or recombinant antigens. / Capítulo I - Toxoplasmose é uma zoonose causada pelo parasita intracelular Toxoplasma gondii, que infecta diferentes hospedeiros, incluindo cerca de um terço da população humana mundial. Uma das manifestações mais graves da infecção por este parasita em humanos corresponde à toxoplasmose congênita, quando há transmissão placentária do parasita para o feto durante infecção materna primária na gestação. A infecção congênita pode causar abortos ou perdas fetais, além de sequelas graves em recém-nascidos (RNs), principalmente oculares e/ou cerebrais. A triagem sorológica de gestantes e RNs, baseada principalmente na detecção de anticorpos IgG e IgM anti-T. gondii constitui-se em importante medida a ser adotada em programas de controle desta infecção, apesar das limitações na detecção dos anticorpos e na interpretação dos resultados. Capítulo II - Um total de 173 pares de amostras de soros de mães com suspeita de toxoplasmose primária na gestação e seus respectivos RNs, provenientes do Setor de Pediatria e Neonatologia do Hospital de Clínicas da Universidade Federal de Uberlândia (HC-UFU) no período de 2006 a junho de 2014, foi analisado por ELISA para a detecção de anticorpos IgG, IgM e IgA anti-T. gondii, e os resultados obtidos foram correlacionados com dados clínicos obtidos a partir de pesquisa nos prontuários clínicos de cada paciente. Concluiu-se que (i) a triagem sorológica pré-natal é de grande relevância na identificação de gestantes expostas à toxoplasmose durante a gestação; (ii) o tratamento materno reduz a transmissão congênita de T. gondii; (iii) a triagem sorológica neonatal, aliada à análise de parâmetros clínicos permite identificar RNs verticalmente infectados, principalmente por meio da detecção conjunta de anticorpos IgM e IgA; e (iv) o acompanhamento sorológico de RNs é importante no esclarecimento de situações duvidosas, principalmente em casos de RNs assintomáticos, mas com sorologia sugestiva de infecção congênita. Capítulo III - Diferentes frações antigênicas derivadas do antígeno solúvel de taquizoítas de T. gondii (STAg) foram obtidas a partir de precipitação sequencial em concentrações crescentes de sulfato de amônio e utilizadas na técnica de immunoblotting para a detecção de anticorpos IgG total e suas subclasses (IgG1, IgG2, IgG3 e IgG4) presentes em amostras pareadas de soros de mães com sorologia presuntiva de toxoplasmose recente na gestação e seus respectivos RNs. Concluiu-se que a utilização destas frações antigênicas de STAg no diagnóstico da toxoplasmose humana se mostrou interessante na detecção de anticorpos IgG e suas subclasses, permitindo diferenciação entre amostras positivas e negativas, mas não se mostrou uma boa alternativa no diagnóstico da toxoplasmose congênita, com resultados inferiores aos do STAg, em função da menor frequência de bandas antigênicas reconhecidas e da ausência de reconhecimento diferencial de antígenos pelos soros dos RNs, em relação aos soros maternos. Capítulo IV - As sequências de aminoácidos de 16 antígenos imunodominantes de T. gondii foram usadas para a predição de epítopos lineares de células B usando ferramentas de bioinformática. Um total de 22 epítopos de antígenos de superfície (SRS), roptrias (ROP), micronemas (MIC) e grânulos densos (GRA) de T. gondii foram identificados e utilizados para a síntese de peptídeos com 15 resíduos de aminoácidos, os quais foram quimicamente conjugados ao esqueleto proteico da albumina sérica bovina (BSA), e o desempenho diagnóstico destes peptídeos sintéticos foi avaliado por ELISA para a detecção de anticorpos IgG em soros de pacientes com suspeita de toxoplasmose aguda (G1) ou crônica (G2). Concluiu-se que peptídeos sintéticos obtidos a partir da predição de epítopos lineares de células B constituem antígenos promissores em ensaios sorológicos para o diagnóstico da toxoplasmose e para a diferenciação das fases aguda e crônica da infecção, representando uma alternativa ao uso de antígenos nativos ou recombinantes. / Doutor em Imunologia e Parasitologia Aplicadas

Diploma thesis "Health care expenditures- future prospects and current situation" is focused on three areas linked to health care expenditures in EU and USA. First area, health expenditures financing, is analyzed in first chapter of the thesis. In the chapter, the development of modern health systems from its beginnings to 2014 is described. Second chapter is based on statistical hypothesis testing- paired two-sample t-tests. Firstly, share of health expenditures on GDP in 2000 and 2014 is tested. Secondly, share of public financing on health expenditures in 2000 and 2014 in tested. Third chapter explores influence of demography factors on health care expenditures and tests structure of health care expenditures in 2004 and 2013.

This study assessed the impact of the Texas Counselors' Network (TCN) on students of counselors who attended TCN workshops. TCN is a professional organization created in 1996 for the professional development of counselors. TCN impacts primary and secondary school students by providing counselors with selected skills to assist them in helping students. In theory, TCN thus impacts these students by improving overall skills of participating counselors. This study assessed the progress of students before and after implementation of TCN. Students' progress was considered in four areas: Texas Accountability Assessment Scores (TAAS), attendance rates, dropout rates, and high school student enrollment in technical programs. The current study compared student performance in the above four areas during a two to three year period prior to the establishment of TCN, with the initial six years of TCN existence. This study examined data attained through the Texas Educational Agency (TEA) Website using their Academic Excellence Indicator System (AEIS) for four regional TCN groups. The study used a paired t-test to compare the performance of students before versus after counselor participation in network workshops. The findings indicated that overall, counselor participation in TCN could have a significant effect on student performance. In fact, eight tests were run and all were found significant at the .05 alpha level.

Theoretical part of this diploma thesis gives a brief characteristic of human mobile elements (transposons), which represents nearly 50% of human genome. It provides basic transposon clasification and describes types of transposons present in hunam genome, as well as mobilization, activation and regulation mechanisms. The work also deals with the domestication of transposons, describes the ways in which TE contribute to DNA damage and summarizes the diseases caused by mutagenic activity of transposons in the human genome. Conclusion of theoretical part describes next-generation sequencing technologies (NGS). As practical part, data from RNA-seq experimet were analyzed in order to compare differen transposon activity in normal and cancer cells from prostate and colorectal tissues. As like as publicly available sophisticated tools (TopHat), new scripts were created to analyze these data. The results show that cancer cells exhibit overexpression of transposons. This corresponds with the published results and suggests a connection of transposon activation with cancer development.

An influence of variations in human genome is perceptible at a first glance on human itself to see differences between the individuals and entire populations. Also, behavior or probability of certain diseases are influenced in large way by differences at genome's level. This work presents methods for detecting variations in the human genome that were developed after an arose of the second-generation sequencing technologies. A new tool that combines read pair and split read methods, with information about a depth of coverage was also designed and implemented. The tool was tested on simulated and real data and compared with a reference outputs.

The relations between environmental resources and biodiversity are crucial in the proper management and conservation of grasslands. Three nature reserves were chosen around the Gauteng Province, namely Roodeplaat Nature Reserve (RNR) in Tshwane, Suikerbosrand Nature Reserve (SNR) in Heidelberg, and Abe Bailey Nature Reserve (ANR) in Carletonville. We selected three sites within RNR, SNR and ANR, and paired these with adjacent sites in private farming/grazing areas adjacent to the nature reserves. At each site, species composition, species richness and plant diversity were determined with the use of 50m x 20m Modified-Whittaker plots (MWP), making a total of eighteen plots (MWP) at the nine paired sites. Two paired sites had high Shannon-Wiener Index (H’) average values at the adjacent grazing area as compared to the nature reserve area at ANR. Roodeplaat Nature Reserve (RNR) and Suikerbosrand Nature Reserve (SNR) had two paired sites with high H’ average values in the nature reserve as compared to the adjacent grazing area. The vegetation structure was similar for the three study locations, consisting of graminoids, herbs and isolated patches of shrubs. The species composition showed similarities between ANR and SNR sites, while RNR showed different species composition. SNR soils had the highest organic carbon (OC), total Carbon (C), total Nitrogen (N), Calcium (Ca), Potassium (K), Magnesium (Mg) and Sodium (Na) as compared to both ANR and RNR. Species richness had a significantly positive relationship with Organic Carbon and Total Nitrogen. Species diversity difference was detected between the nature reserves and adjacent grazing areas and the difference are likely due to a number of factors including soil properties, land disturbance and land use and management. More research is necessary to further understand the aspects impacting species richness, species diversity and species composition in grasslands. / School of Agriculture and Life Sciences / M. Sc.

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.