Langjährige Forschungsarbeiten konnten die genetische, biochemische und physiologische Basis der Heterosis bis heute nicht klären. Das Promotionsprojekt ist Bestandteil eines längerfristigen Heterosisforschungsprojektes zur molekulargenetischen Charakterisierung der Ursachen von Heterosis. Ziel der Dissertation ist die Analyse von Chromosomenregionen bei der Maus, von denen ein Einfluß auf die Entstehung von Heterosis für Fruchtbarkeit ausgeht. Dabei wurde sowohl die Superdominanz- als auch die Dominanztheorie der Heterosis berücksichtigt. Es wurde eine reziproke Kreuzung der Inzuchtstämme C57BL/6J und Balb/cJ mit anschließendem F1-Interkross zur Erzeugung einer F2-Generation durchgeführt. Von den 948 weiblichen F2-Tieren sind Leistungsgruppen mit extrem hoher und niedriger Wurfgröße gebildet worden, um an diesen Tieren 56 Mikrosatelliten zu analysieren. Die Mikrosatelliten sind im Genom der Maus in einem durchschnittlichen Abstand von 32 cM lokalisiert. 12 von diesen Mikrosatelliten charakterisieren DNA-Loci mit Assoziationen zur Fruchtbarkeit. Entsprechend den Analysen nach der Superdominanztheorie der Heterosis konnte für sechs Mikrosatelliten eine signifikante Beziehung zwischen dem Heterozygotiegrad und der Heterosis für Wurfgröße nachgewiesen werden. Die Mikrosatelliten charakterisieren Regionen auf den Chromosomen 17 (18,2 - 22,3 cM), 18 (50 cM) und 19 (8 - 12 cM). Auf Chromosom 17 befinden sich in diesem Bereich die mit Fruchtbarkeit assoziierenden Gene Ped (Preimplantation embryonic development, 19,5 cM), Cyp21a1 (Cytochrome P450, 21, steroid 21 hydroxylase, 18,7 cM) und H2 (Histocompatibility-2, MHC, 23 cM). Nach dem Dominanzmodell zur Erklärung von Heterosis konnte für zwei Mikrosatelliten eine signifikante Beziehung zwischen dem Anteil Genotypen mit dominantem Leistungsallel und der Heterosis für Wurfgröße ermittelt werden. Die Mikrosatelliten sind auf Chromosom 5 (72 und 88 cM) lokalisiert. In diesen chromosomalen Regionen befinden sich die mit Fruchtbarkeit assoziierten DNA-Loci Pdgfa (Plateled derived growth faktor alpha, 77 cM) und Pmv12 (Polytropic murine leucemia virus-12, 88 cM). / Characterization of heterotic effects in litter size using DNA marker analyses in mice Long dated research could not explain the genetic, biochemical and physiological base of heterosis up to date. The dissertation project is a part of a long term heterosis research project concerning the molecular genetic characterization of the reasons of heterosis. The objective of the dissertation was to find out chromosomal regions of the mouse with a presumable influence on the rise of heterosis in fertility. Both the overdominance and the dominance theory of heterosis have been considered. A reciprocal cross of inbred strains C57BL/6J and Balb/cJ with following F1 intercross was accomplished to establish a F2 generation. From the 948 female F2 animals were formed performance groups with extreme high and low litter sizes to analyse 56 microsatellites on these animals. The microsatellites are located in an average distance of 32 cM in the mouse genome. Twelve of these microsatellites characterize DNA loci with associations to fertility. Corresponding to the analyses based on the overdominance theory of heterosis a significant correlation between the degree of heterozygosity and heterosis in litter size have been demonstrated for six microsatellites. The microsatellites characterize regions on the chromosomes 17 (18.2 - 22.3 cM), 18 (50 cM), and 19 (8 - 12 cM). The DNA loci Ped (Preimplantation embryonic development, 19,5 cM), Cyp21a1 (Cytochrome P450, 21, hydroxylase, 18,7 cM) and H2 (Histocompatibility-2, MHC, 23 cM) showing associations to fertility are located on chromosome 17 in these region. On the base of dominance theory as the reason of heterosis a significant relation between the portion of genotypes with dominant performance allel and heterosis in litter size have been found for two microsatellites. The microsatellites are located on chromosome 5 (72 and 88 cM). The DNA loci Pdgfa (Plateled derived growth factor alpha, 77 cM) and Pmv12 (Polytropic murine leucemia viruses 12, 88 cM) with known associations to fertility are located on these chromosomal regions.

Este trabalho analisa filmes do cineasta Chris Marker que indagaram sobre a Unidade Popular do Chile nos anos 1970. Entre as produções abordadas, estão duas que foram remontadas a partir de documentários chilenos feitos durante o governo de Salvador Allende: La première année (1973) e On vous parle du Chili: ce que disait Allende (1973). Além disso, mais três filmes desse realizador se dedicaram ao tema: La Spirale (1976), Lambassade (1974) e O fundo do ar é vermelho (1977). Por meio do estudo dessa filmografia, identificaram-se quais foram as estratégias narrativas utilizadas e qual foi o discurso político sobre a chamada experiência chilena que essas estratégias elaboraram. A reflexão sobre o corpus permitiu verificar que o interesse por esse processo, visto desde a França, emergiu do espaço deixado pelas frequentes desilusões diante de uma referência política anterior, igualmente vinda da América Latina e que havia motivado os debates da esquerda francesa na década de 1960: a Revolução Cubana. Assim, optou-se por incluir também na pesquisa produções markerianas que nasceram do contato entre esse realizador e o Instituto Cubano del Arte e Industria Cinematográficos (ICAIC): Cuba si (1961), La bataille des dix millions (1970), On vous parle du Brésil: tortures (1969) e On vous parle du Brésil: Carlos Marighela (1970). A análise dos filmes elencados revela que, durante os anos da UP no poder, houve uma vontade de aproximação entre Chile e França que valorizava os caminhos empregados pela via chilena ao socialismo, apontando-os como possibilidades para uma Nova Esquerda europeia. Porém, com o golpe de Estado de 1973, essa postura passou por uma revisão, e a experiência chilena serviu à elaboração de leituras que apontassem um legado e lições que poderiam ser retirados da queda da Unidade Popular. Nesse sentido, as produções de Marker fazem uma denúncia das ações da direita que derrubaram Allende, bem como constroem uma visão autocrítica que indica os erros cometidos pela própria esquerda que possibilitaram essa derrota. Essa tentativa de atribuir um legado e lições buscava responder sobretudo aos anseios de uma esquerda francesa que dispunha de um programa comum de governo fortemente inspirado naquele da UP chilena, mas que deveria ser submetido a uma revisão após o fracasso de sua referência latinoamericana. Sob essa visão, esta tese propõe um estudo voltado à circulação de ideias culturais e políticas entre América Latina e França, que delega ao continente um papel central nessa relação durante os anos 1960 e 1970 e a Chris Marker a função de um mediador cinematográfico. / This work analyses the Chris Markers films that inquired about the Popular Unity in the 1970s. Among the productions approached, two of them have been reassembled from other Chilean documentaries that had been made during the Salvador Allendes government: La première année (1973) and On vous parle du Chili: ce que disait Allende (1973). Furthermore, three other films by Chris Marker have analysed this subject: La Spirale (1976), The embassy (1974) and The grin without a cat (1977). By studying his filmography, this research could identify the narratives strategies that had been used by the director, as well as the political discourse elaborated by them. The reflections about the corpus allowed verify how the interest for this Chilean process, viewed from France, has emerged from an empty space left by the usual delusion given by an earlier reference that had motivated the French left during the 1960s, also gone from Latin-American: the Cuban Revolution. So, we chose to also include in the research the Markers productions came from the relationship between this director and the Instituto Cubano del Arte e Industria Cinematográficos (ICAIC): Cuba si (1961), La bataille des dix millions (1970), On vous parle du Brésil: tortures (1969) and On vous parle du Brésil: Carlos Marighela (1970). The analysis of the films-selected shows that, during the UPs years in the power, there was a wish to approach Chile and France by valorising the Chileans way to the socialism and pointing it as a possibility to the European New Left. However, after the coup détat in 1973, this attempt has been revised and the Chilean experience has been used to elaborate lectures that pointed a legacy and lessons from the follow of Popular Unity. In this sense, the Markers productions denounce the rights actions to overthrow Allende, as well as built a self-criticism vision to indicate the lefts mistakes that had collaborated to the defeat. This wish to show a legacy and lessons has dialogued with the expectations of a Frenchs left that had have a common government programme tightly inspired in the Chileans UP ones, but that has required a revision after the failure of its Latin-American reference. From this point of view, this thesis proposes a study based on the circulation of cultural and political ideas between Latin American and France that delegated to this continent a central role in this relationship during the 1960s and the 1970s, and assigned to Chris Marker the function of a cinematographic mediator.

Este trabalho apresenta uma técnica de diferenças finitas para resolver a equação constitutiva Fluido de Segunda Ordem para escoamentos tridimensionais com superfície livre. As equações governantes são resolvidas pelo método de diferenças finitas em uma malha deslocada 3D. A superfície livre é modelada por células marcadoras (Marker-and-Cell) e as condições de contorno a superfície livre são empregadas. O método numérico apresentado neste trabalho foi validado pela comparação entre as soluções numéricas obtidas para o escoamento em um tubo com a solução analítica correspondente para Fluidos de Segunda Ordem. Ao fazer refinamento de malha, a convergência do método numérico foi verificada. Resultados numéricos da simulação do problema do inchamento do extrudado para números de Deborah De \'< OU =\' 0:3 são apresentados / This work presents a finite difference method to simulate three-dimensional viscoelastic flow with free surfaces governed by the constitutive equation Second Order Fluid. The governing equations are solved by the finite difference method in a three-dimensional shifted mesh. The free surface of fluid is modeled by the Marker-and-Cell method which allows for the visualization and the location of the free surface of fluid. The full free surface stress conditions are employed. The numerical method developed in this work is validated by comparing the numerical and analytic solutions for the steady state flow of a Second Order Fluid in a pipe. By using mesh refinement convergence results are given. Numerical results of the simulation of the transient extrudate swell of a Second Order Fluid of the Deborah number De \'< OR =\' 0:3 are presented

Esta pesquisa busca, tendo como eixo principal Chris Marker e o filme Le fond de lair est rouge, fazer uma retrospectiva histórica dos principais debates na experiência do cinema-militante francês. Com um caráter introdutório e a partir daí construir alguns apontamentos iniciais para uma interpretação crítica do filme. Através desta aventura político-cinematográfica comentar um fragmento do que se oculta atrás das barricadas da memória. / This research aims, having as an leit-motif Chris Marker and the film Le fond de l\'air est rouge, making a historical retrospective of the major debates on the experience of French militant cinema, introductory in nature and serving as first notes to a critical interpretation of the film. Through this cinematics and politics adventure it trys to make a comment on a fragment of what is hidden behind the barricades of memory.

Background Prostate cancer (PCa) is the most frequently diagnosed malignant disease among adult males in the USA and the second leading cause of cancer deaths in men. Due to the lack of diagnostic tools that are able to differentiate highly malignant and aggressive cases from indolent tumors, overtreatment has become very common in the era of prostate specific antigen (PSA) screening. New diagnostic methods to determine biological status, malignancy, aggressiveness and extent of PCa are urgently needed. 1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy (1H HRMAS MRS) can be used to establish PCa metabolomic profiles while preserving tissue architecture for subsequent histopathological analysis. Immunohistochemistry (IHC), as opposed to conventional histopathology methods, has the potential to provide objective, more accurate and quantitative knowledge of tissue pathology. This diagnostic- accuracy study sought to evaluate a novel approach to quantitatively identify metabolomic markers of PCa by exploring the potential of PCa immunomarkers to quantify metabolomic profiles established by 1H HRMAS MRS. Material and Methods 1H HRMAS MRS was performed on tissue samples of 51 prostate cancer patients using a 14.1 Tesla NMR spectrometer (BRUKER Biospin, Billerica, MA) with a rotor synchronized CPMG pulse sequence. Spectral intensities of 36 regions of interest were measured as integrals of curve fittings with Lorentzian-Gaussian line shapes. Immunohistochemistry (IHC) was carried out following the spectroscopy scan, using three prostate immunomarkers to identify cancerous and benign glands: P504S (Alpha-methylacyl-CoA-racemace), CK903 (high-molecular weight cytokeratin) and p63. The immunostaining quality following 1H HRMAS MRS was evaluated and compared to unscanned sections of the same sample, to verify the stability and accessibility of the proposed immunomarkers. IHC images were automatically and quantitatively evaluated, using a quantitative image analysis program (QIAP), to determine the percentage of cancerous and benign epithelia in the tissue cross- sections. The results of the program were validated by a correlation with the results of a quantitative IHC review and quantitative conventional histopathology analysis performed by an experienced pathologist. Ultimately, spectral intensities and the cancer epithelium percentage, obtained from quantitative immunohistochemistry, were correlated in order to validate PCa metabolomic markers identified by 1H HRMAS MRS. Patient outcomes and incidence of recurrence were determined by retrospective review of medical records five years after initial surgery. Categories of recurrence were correlated to spectral intensities to explore potential metabolomic markers of recurrence in the cohort. Results Immunostainings with P504S and CK903 showed excellent staining quality and accessibility following 1H HRMAS MRS, suggesting these markers to be suitable for the presented quantitative approach to determine metabolomics profiles of PCa. In contrast, the quality of p63 IHC was impaired after previously performed spectroscopy. IHC using the immunomarkers P504S and CK903 on adjacent slides was found to present a feasible quantitative diagnostic method to distinguish between benign and cancerous conditions in prostate tissue. The cancer epithelium percentage as determined by QIAP showed a significant correlation to the results of quantitative IHC analysis performed by a pathologist (p < 0.001), as well as to a quantitative conventional histopathology review (p = 0.001). The same was true for the benign epithelium percentage (p < 0.001 and p = 0.0183), validating the presented approach. Two metabolomic regions showed a significant correlation between relative spectral intensities and the cancer epithelium percentage as determined by QIAP: 3.22 ppm (p = 0.015) and 2.68 ppm (p = 0.0144). The metabolites corresponding to these regions, phosphocholine and citrate, could be identified as metabolomic markers of PCa in the present cohort. 45 patients were followed for more than 12 months. Of these, 97.8% were still alive five years after initial surgery. 11 patients (24.4%) experienced a recurrence during the follow- up time. The categories of recurrence showed a correlation to the spectral intensities of two regions, 2.33 – 2.3 ppm (p = 0.0403) and 1.28 ppm (p = 0.0144), corresponding to the metabolites phosphocreatine and lipids. Conclusion This study introduces a method that allows an observer-independent, quantitative analysis of IHC to help establish metabolomic profiles and identify metabolomic markers of PCa from spectral intensities obtained with 1H HRMAS NMR Spectroscopy. The immunomarkers P504S and CK903 have been found suitable IHC analysis following 1H HRMAS MRS. A prospective in vivo application of PCa metabolite profiles and metabolomic markers determined by the presented method could serve as highly sensitive, non- invasive diagnostic tool. This observer- independent, computer- automated, quantitative analysis could help to distinguish highly aggressive tumors from low-malignant conditions, avoid overtreatment and reduce risks and complications for cancer patients in the future. Further studies are needed to verify the identified PCa metabolomic markers and to establish clinical applicability. / Einführung Prostatakrebs ist eine häufigsten Krebserkrankungen in den USA und die zweithäufigste malignom- assoziierte Todesursache männlicher Patienten weltweit. Seit der Einführung des Prostata- spezifischen Antigen (PSA)- Screeningtests wird diese Krebsart in früheren Stadien diagnostiziert und therapiert, wodurch die Mortalitätsrate in den letzten Jahren deutlich reduziert werden konnte. Da moderne diagnostische Methoden bislang jedoch nicht ausreichend in der Lage sind, suffizient zwischen hochmalignen und weniger aggressiven Varianten dieses bösartigen Krebsleidens zu unterscheiden, werden häufig auch Patienten aggressiv therapiert, deren niedriggradiges Prostatakarzinom keine klinische Relevanz gehabt hätte. Es besteht daher ein großes wissenschaftliches Interesse an der Entwicklung neuer diagnostischer Methoden zur akkuraten Bestimmung von biologischem Status, Malignität, Aggressivität und Ausmaß einer Prostatakrebserkrankung. \\\\\\\"1H High Resolution Magic Angle Spinning Nuclear Magnetic Resonance Spectroscopy\\\\\\\" (1H HRMAS MRS) ist eine vielversprechende diagnostische Methode, welche es ermöglicht, metabolomische Profile von Prostatakrebs zu erstellen, ohne die Gewebsstruktur der analysierten Proben zu zerstören. Durch anschließende histopathologische Begutachtung lassen sich die erstellten Metabolitprofile validieren und evaluieren. Im Gegensatz zu konventionellen histopathologischen Methoden können durch immunhistochemische Verfahren dabei objektivere, akkuratere und quantifizierbare histopathologische Erkenntnisse gewonnen werden. Die vorliegende Studie präsentiert einen neuentwickelten diagnostischen Ansatz zur quantitativen Bestimmung von metabolomischen Markern von Prostatakrebs, basierend auf der Durchführung von 1H HRMAS NMR Spektroskopie und quantitativer Immunhistochemie. Material und Methoden Einundfünfzig Gewebsproben von Prostatakrebspatienten wurden mittels 1H HRMAS MRS an einem 14.1 T BRUKER NMR Spektrometer unter Einsatz einer CPMG-Pulssequenz untersucht. Spektrale Intensitäten in 36 Metabolitregionen wurden gemessen. Anschließend wurden die analysierten Gewebeproben mit drei Immunfärbemarkern für sowohl malignes (P504S, Alpha-methylacyl-CoA-racemase) als auch benignes (CK903, High-molecular weight cytokeratin, und p63) Prostatagewebe angefärbt und quantitativ mit Hilfe eines Bildanalyseprogramms (QIAP) ausgewertet. Die Anwendbarkeit und Auswertbarkeit der genannten Immunomarker nach Spektroskopie wurde evaluiert und mit der Färbungsqualität von nicht- gescannten Schnitten verglichen. Die Resultate der automatischen Auswertung durch QIAP konnten durch einen erfahrenen Pathologen in einer quantitativen Analyse der Immunfärbungen sowie konventioneller histologischer Färbungen derselben Gewebsproben validiert werden. Die spektralen Intensitäten aus den Messungen mit 1H HRMAS MRS wurden mit den korrespondierenden Ergebnissen der quantitativen Auswertung der Immunfärbungen korreliert, um metabolomische Marker von Prostatakrebs zu identifizieren. Der klinische Verlauf und die Rezidivrate der Patienten wurden 5 Jahre nach der initialen Prostatektomie retrospektiv bestimmt. Rezidivkategorien wurden erstellt und mit den bestimmten spektralen Intensitäten korreliert, um metabolomische Marker für das Auftreten von Prostatakrebsrezidiven zu identifizieren. Ergebnisse Die Immunfärbungen mit P504S und CK903 zeigten exzellente Qualität und Auswertbarkeit nach vorheriger 1H HRMAS MRS. Beide Marker eigneten sich zur Durchführung von quantitativer Immunhistochemie an spektroskopierten Gewebeproben. Im Gegensatz dazu war die Qualität der Immunfärbungen mit p63 nach Spektroskopie vermindert. Quantitative Immunfärbungen unter Einsatz der Immunmarker P504S und CK903 stellten eine praktikable diagnostische Methode dar, um zwischen malignen und benignem Prostatagewebe zu unterscheiden. Der Anteil von bösartig verändertem Prostatagewebe, bestimmt durch QIAP, korrelierte signifikant mit den Ergebnissen der quantitativen Analyse der Immunfärbungen durch den Pathologen (p < 0.001), sowie mit der quantitativen Auswertung der konventionellen histopathologischen Färbung (p = 0.001). Ebenso ließ sich die Bestimmung des Anteils von benignem Gewebe mit QIAP zu den Ergebnissen der pathologischen Analyse korrelieren (p < 0.001 und p = 0.0183). Für zwei metabolomische Regionen konnte ein signifikante Korrelation zwischen relativen spektralen Intensitäten, bestimmt mit 1H HRMAS NMR Spektroskopie, und dem Anteil von malignem Epithelium in derselben Gewebeprobe, ermittelt durch QIAP, festgestellt werden: 3.22 ppm (p = 0.015) und 2.68 ppm (p = 0.0144). Die zu diesen Regionen korrespondierenden Metaboliten, Phosphocholin und Zitrat, konnten als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Die retrospektiven Analyse der klinischen Daten der Patienten fünf Jahre nach Prostatektomie ergab eine Überlebensrate von 97.8%. Elf dieser Patienten (24.4%) erlitten ein Rezidiv ihrer Erkrankung. Die bestimmten Rezidivkategorien korrelierten signifikant mit zwei metabolomischen Regionen (2.33 – 2.3 ppm, p = 0.0403 und 1.28 ppm, p = 0.0144), welche zu den Metaboliten Phosphokreatin und Lipiden korrespondierten. Schlussfolgerung Die vorliegende Studie präsentiert einen diagnostischen Ansatz zur objektiven und quantitativen Bestimmung metabolomischer Marker von Prostatakrebs unter Verwendung von 1H HRMAS MRS und Immunhistochemie. P504S und CK903 eignen sich als Immunmarker für quantitative Immunfärbungen nach vorheriger Durchführung von 1H HRMAS MRS. Die Metaboliten Phosphocholin und Zitrat konnten in der vorliegenden Patientenkohorte als potentielle metabolomische Marker für Prostatakrebs identifiziert werden. Eine mögliche in vivo Anwendung der gefundenen metabolomischen Marker könnte als hochsensitives, objektives und nicht- invasives diagnostisches Werkzeug der Prostatakrebsdiagnostik dienen. Der vorliegende untersucherunabhängige, automatisierte und quantitative diagnostischer Ansatz hat das Potential, zwischen hochmalignen und weniger aggressiven Krebsfällen zu unterscheiden und somit unnötige Risiken und Komplikationen für Prostatakrebspatienten zu reduzieren. Weitere Untersuchungen sind notwendig, um die identifizierten metabolomischen Marker zu verifizieren und eine klinische Anwendung zu etablieren.

The presented diploma thesis deals with the description of verbal epistemic parentheses such as (ich) glaub(e) or (ich) mein(e) in German and myslím in Czech. These types usually do not belong among the means of expressing epistemic modality and are often neglected in current dictionaries and grammars, although they have a different meaning than the verb they originated from. The diploma thesis analyzes parentheses (ich) glaub(e) in German and myslím in Czech via spoken language corpora. The German data were acquired from FOLK (Forschungs- und Lehrkorpus gesprochenes Deutsch) which is a part of IDS Mannheim corpora. The Czech data come from ORAL v1 (Český národní korpus). The overall aim was to describe their syntactic behaviour, their semantics, and in some cases their prosodic features. The theoretical part focuses on distinguishing between discourse markers and particles. Such distinction forms a foundation for the analysis. The differentiation between grammaticalization and pragmaticalization is also commented on. Both parentheses were examined with relation to their syntactic position, syntactic integration, and their potential function as fillers. Based on the analysis, both constructions evince an advanced level of particularization and have features of particles and/or discourse markers....

My diploma thesis deals with genetic variability of entomopathogenic fungi of the Isaria genus encountered in the Czech Republic. Individual representative of the genus can be found in soil where they attack all developmental stages of insects, giving preference to larvae and pupae. The Isaria fungi find application first and foremost where plants have to be provided biological protection. In case of mitosporic fungi is the precise identification very difficult, taxonomy is often unclear in many genera, including the genus Paecilomyces/Isaria to demonstrate their polyphyletic nature. The fungi are classified primarily with reliance on morphological studies. The most common markers used to identify fungi are the shapes and sizes of their conidia and the biological properties (germination of spores, tests of biological efficiency). Identification made in consideration of the morphological markers is inaccurate and very variable. To overcome those accuracies, there are very useful molecular DNA markers, which can be relevant in ecology, biology and in fungi genetics. This paper relies on applying the ITS region (Internal Transcribed Spacer) as a molecular marker. ITS regions are partial constituent rDNA carrying no code - that is why the regions are likely to accumulate evolutionary changes in the DNA sequence, which makes them suitable for extensive use in taxonomic analyses of many organisms. The study results in a phylogenetic trees constructed by comparing different sequences of ITS regions obtained from the samples of entomopathogenic fungi of the Isaria genus gathered in the Czech Republic during the monitoring stage 2013 to 2014. Thereunder detection of Isaria sp. occurring in the Czech Republic.

Made available in DSpace on 2015-03-26T13:51:52Z (GMT). No. of bitstreams: 1 texto completo.pdf: 800491 bytes, checksum: 274cc18672a32ed45926e416e0de5305 (MD5) Previous issue date: 2011-02-15 / The homologue recombination in Kluyveromyces lactis is not the preferential way used as repair mechanism of DNA double strand, desirable in proteins expression construction dependent on gene-specific integration. In order to obtain K. lactis strains to recombinant protein expression efficient in homologue recombination way, KU80 gene was interrupted. The perfect running of non-homologue ends junctions (NHEJ) way depends on that gene and is responsible by exogenous DNA random integration in the host genome. KU80 gene deletion was made by Split-Marker. Two fragments were generated by PCR fusion, each one with a flanker sequence of KU80 coding region (5 and 3 ends) and part of geneticin resistance gene (KanMX). Deletion cassette resulting from in vivo recombination of two fragments had KanMX gene flanked by KU80 coding regions end sand was used for KU80 deletion by homologue recombination in the genome in two K. lactis strains, JA6 and HP108. The 3.7 Kb fragment obtained by PCR amplification with external primers to KU80 coding region confirmed deletion cassette integration in the target gene. Integration efficiency with Split-Marker resulting fragments was 100 %. pKLAC1 and pKLAC1/cStp vectors with streptavidin affinity domain by biotin were used to determine homologue recombination efficiency of KU80 (JA6&#916;KU80 and HP108&#916;KU80) mutant strains. Pectin lyase coding gene (plg1) from Penicillium griseoroseum was cloned in those vectors to evaluate the capacity of K. lactis strains to produce and secrete the recombinant protein. The transformation efficiency (transformants/&#956;g of DNA) of mutants JA6&#916;KU80 and HP108&#916;KU80 with pKLAC1/Plg1 and pKLAC1/cStp-Plg1 vectors was higher than to parental strains JA6 and HP108. Target gene integration efficiency was 100 % in most strains, except to strains JA6/Plg1and HP108&#916;KU80/Plg1 that showed an integration efficiency of 80 and 70 %, respectively. Although the high efficiency of specific-gene integration there was no pectin lyase (PL) or cStp-Plg1 secretion as expected for pKLAC1 vector. PL intracellular activity was significant when compared with parental strain HP108/Plg1, that presented specific activity of 9,525 U.mg-1 protein. / A recombinação homóloga em Kluyveromyces lactis não é a via preferencial usada como mecanismo de reparo de quebra de fita dupla de DNA, o que pode ser indesejado em construções de expressão de proteínas dependentes de integração gene-específico. Para obter linhagens de K. lactis hospedeiras para a expressão de proteínas recombinantes eficientes na via de recombinação homóloga realizou-se a mutação do gene KU80. Este gene é essencial para perfeito funcionamento da via de junções de extremidades não-homólogas (NHEJ), responsável pela integração aleatória do DNA exógeno no genoma hospedeiro. A deleção do gene KU80 foi feita utilizando a técnica Split-Marker. Dois fragmentos foram obtidos por fusão por PCR, cada um contendo uma sequência flanqueadora da região codificante do gene KU80 (extremidades 5 e 3 ) e uma parte do gene de resistência a geneticina (KanMX). O cassete de deleção resultante da recombinação in vivo dos dois fragmentos gerados continha o gene KanMX flanqueado pelas extremidades da região codificante do gene KU80, e foi utilizado para deleção do gene KU80 por recombinação homóloga no genoma de duas linhagens de K. lactis, JA6 e HP108. O fragmento de 3,7 Kb obtido por amplificação por PCR com oligonucleotídeos externos à região codificante do gene KU80 confirmou a integração do cassete de deleção no gene alvo. A eficiência de integração com os fragmentos resultantes do Split-Marker foi de 100 %. Os vetores pKLAC1, e pKLAC1/cStp contendo o domínio de afinidade da estreptavidina pela biotina, foram usados para determinar a eficiência de recombinação homóloga das linhagens mutantes KU80 (JA6&#916;KU80 e HP108&#916;KU80). O gene da pectina liase (plg1) de Penicillium griseoroseum foi clonado nesses vetores para avaliar a capacidade de linhagens de K. lactis em produzir e secretar a proteína recombinante. A eficiência de transformação (transformantes/&#956;g de DNA) dos mutantes JA6&#916;KU80 e HP108&#916;KU80 com os vetores pKLAC1/Plg1 e pKLAC1/cStp-Plg1, foi superior à das linhagens parentais JA6 e HP108. A eficiência de integração no gene alvo foi de 100% para maioria das linhagens, com exceção das linhagens JA6/Plg1e HP108&#916;KU80/Plg1 que apresentaram, respectivamente, eficiência de 80 e 70 % de integração geneespecífico. Apesar da eficiência de integração por recombinação homóloga, não houve secreção de PL e da fusão cStp-Plg1 como esperado ao se utilizar o vetor pKLAC1. A atividade intracelular de pectina liase (PL) só foi significativa em relação à parental para a cepa HP108/Plg1, que demonstrou atividade específica de 9,525 U.mg-1 proteína.

Made available in DSpace on 2015-03-26T13:51:58Z (GMT). No. of bitstreams: 1 texto completo.pdf: 952076 bytes, checksum: 7fbcb43414ecacd3439620826b6172cd (MD5) Previous issue date: 2012-07-20 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Colletotrichum lindemuthianum is the causal agent of common bean anthracnose. Genes that encode cell wall-degrading enzymes are essential for the development of this disease. The pectinases are characterized as the most important group of cell wall- degrading enzymes produced by phytopathogen fungi. The gene coding for pectate lyase, pecCl1, was previously identified in a suppressive subtractive library of bean infected with C. lindemuthianum. Isolation of the gene pecCl1 made it possible to obtain mutants and to analyze the regulation of this gene during development of anthracnose, determining whether the pectate lyase is a pathogenic factor. Thus, the aim of our study was structurally and functionally characterize the gene encoding pectate lyase in C. lindemuthianum. Initially, was performed the structural analysis of the gene pecCl1. The complete nucleotide sequence of the gene pecCl1 was deposited in Genbank with accession number JX270683. The analysis of the promoter region revealed some putative cis-elements and potential binding motifs of transcription factors involved in the regulation of pectate lyase gene expression. The deduced amino acid sequence of pecCl1 showed sequence identity with the pectate lyase F of Colletotrichum higginsianum and the pectate lyase C of Glomerella graminicola M1.001. Furthermore, it was found putative conserved domain pfam03211 of the pectate lyases superfamily. The gene pecCl1 is represented by a single copy in the C. lindemuthianum genome. However, into the genome of Colletotrichum graminicola, three sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, and into the genome of C. higginsianum seven sequences encoding pectate lyase showed sequence identity with the gene pecCl1 of C. lindemuthianum, indicating that the C. lindemuthianum genome can possess other genes encoding pectate lyase. Phylogenetic analysis of pectate lyase amino acid sequences of filamentous fungi exhibited the formation of two distinct groups which are grouped on the basis of members of the pectate lyases multigene family. The Split-Marker technique was effective in C. lindemuthianum pecCl1 gene inactivation, allowing the study of pecCl1 function in a mutant by specific integrations and without ectopic integrations. The pecCl1 gene inactivation did not lead to complete loss of the pectate lyase activity, and consequently only decreased anthracnose symptoms in its host, which is consistent with the presence of other genes coding pectate lyase, allowing greater flexibility in pathogen aggressiveness. The analysis of differential expression of gene pecCl1 by qPCR was performed at different stages of bean infection and were observed expression levels of pecCl1 at all stages of development of the fungus in the plant, but a significant increase was observed five days after infection, in the onset of necrotrophic stage. At this stage, secondary hyphae cause extensive degradation of plant cell wall through the secretion of wide range of depolymerases, among these, the pectate lyase. Thus, the pectate lyase encoded by the gene pecCl1 is important to aggressiveness of C. lindemuthianum. The analysis of pectate lyases in C. lindemuthianum can not only assist in understanding the disease, but may also lead to discovery of one more target for disease control. / Colletotrichum lindemuthianum é o agente causal da antracnose do feijoeiro comum. Genes que codificam enzimas que degradam a parede celular são essenciais para o desenvolvimento dessa doença. As pectinases são caracterizadas como o grupo de enzimas que hidrolisam a parede celular mais importante produzidas por fungos fitopatogênicos. O gene pecCl1, que codifica pectato liase, foi previamente identificado em uma biblioteca subtrativa supressiva de feijoeiro infectado com C. lindemuthianum. O isolamento do gene tornou possível a obtenção de mutantes e análise da regulação deste gene durante o desenvolvimento da antracnose, visando determinar se a pectato liase é um fator de patogenicidade. Desta forma, o objetivo do nosso trabalho foi caracterizar estruturalmente e funcionalmente o gene que codifica pectato liase em C. lindemuthianum. Inicialmente, foi realizada a análise estrutural do gene pecCl1. A sequência completa de nucleotídeos do gene pecCl1 foi deposita no Genbank com número de acesso JX270683. A análise da região promotora revelou alguns possíveis cis-elementos e sítios de ligação a fatores de transcrição envolvidos na regulação da expressão gênica da pectato liase. A sequência de aminoácidos deduzida de pecCl1 apresentou identidade de sequências com a pectato liase F de Colletotrichum higginsianum e a pectato liase C de Glomerella graminicola M1.001. Além disso, detectou-se um possível domínio conservado pfam03211 da superfamília de pectato liases. O gene pecCl1 encontra-se representado por uma cópia única no genoma de C. lindemuthianum. No entanto, no genoma de Colletotrichum graminicola, três sequências que codificam pectato liase apresentaram identidade de sequências com o gene pecCl1 de C. lindemuthianum, e no genoma de C. higginsianum sete sequências que codificam pectato liase apresentaram identidade de sequências com o gene pecCl1 de C. lindemuthianum, indicando que o genoma de C. lindemuthianum pode possuir além do gene pecCl1 outros genes que codificam pectato liase. A análise filogenética de sequências de aminoácidos de pectato liases de fungos filamentosos mostrou a formação de dois grupos distintos, que se agruparam com base nos membros da família multigênica de pectato liases. A técnica de Split-Marker mostrou-se eficiente na inativação do gene pecCl1 de C. lindemuthianum, possibilitando o estudo da função do gene pecCl1, em um mutante com integração específica e livre de integrações ectópicas. A inativação do gene pecCl1 não levou a perda completa da atividade de pectato liase, e consequentemente, somente diminuiu os sintomas de antracnose em seu hospedeiro, o que é consistente com a presença de outros genes que codificam pectato liase no fungo, permitindo ao patógeno uma maior flexibilidade em sua agressividade. Foi realizada a análise da expressão diferencial do gene pecCl1 por qPCR nos diferentes estágios de infecção no feijoeiro e foram observados transcritos de pecCl1 em todas as fases de desenvolvimento do fungo na planta, mas houve um aumento significativo destes transcritos cinco dias após a infecção, no início da fase necrotrófica do fungo. Nesta fase, as hifas secundárias causam degradação extensiva da parede celular vegetal por meio da secreção de vasta gama de despolimerases, dentre estas, a pectato liase. Portanto, a pectato liase codificada pelo gene pecCl1 é importante para agressividade de C. lindemuthianum. A análise de pectato liases poderá não somente auxiliar na compreensão da antracnose em feijoeiro comum, mas também poderá levar a descoberta de mais um alvo para o controle dessa doença.

Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include immunoglobulin heavy chain or T-cell receptor gene rearrangements, recurrent cytogenetic abnormalities and mutations in important hematological genes. Whereas in the majority of adult acute lymphoblastic leukemia patients a suitable MRD target can be identified, in adult acute myeloid leukemia patients well-characterized targets are found in only half of cases. The identification of new specific molecular markers of leukemic blasts for MRD assessment, particularly in AML patients, is therefore highly desirable. Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker. Using a combination of cytogenetic (chromosome banding, chromosome microdissection), molecular cytogenetic (mFISH, mBAND) and molecular biological (next- generation sequencing, long-range...