BACKGROUND:Neoadjuvant chemotherapy followed by cytoreduction surgery has been used where an accurate cytologic or pathologic diagnosis is usually required before the initiation of neoadjuvant chemotherapy. However, it is difficult to make definitive diagnosis of presence of cancer cells, particularly gynecologic versus non-gynecologic origin, from those ascites specimens due to the absence of specific biomarkers of gynecologic cancers. In the present study, we evaluated if, in addition to the routine morphologic diagnosis, the biomarker PAX8 could be useful in recognition of ovarian epithelial cancer cells prior to the neoadjuvant chemotherapy.METHODS:Two hundred and two cytology specimens including 120 pretreatment ovarian cancer samples, 60 benign controls, and 22 malignant non-gynecologic cases were studied. All cytology slides were morphologically reviewed in a blinded fashion without knowing corresponding pathology diagnosis, if present. A total of 168 cytology specimens with a cell block were stained with PAX8 and Calretinin. These included patients with potential for ovarian cancer neoadjuvant chemotherapy (n=96), metastatic cancers (n=22), and benign controls (n=50).RESULTS:Among the 96 ascitic samples prior to neoadjuvant chemotherapy, 76 (79%) showing morphologic features consistent with cancers of ovarian primary were all PAX+/Calretinin-. The remaining 20 (21%) cases were positive for adenocarcinoma, but morphologically unable to be further classified. Among the 22 metastatic cancers into the pelvis, one case with PAX8+/Calretinin- represented a renal cell carcinoma and the remaining 21 PAX8-/Calretinin- metastatic cancers were either breast metastasis (n=4) and the metastasis from gastrointestinal tract (n=17). Among the 50 benign control pelvic washing cases, 5 PAX8+/Calretinin-cases represented endosalpingiosis (n=4) and endometriosis (n=1), 25 PAX8-/Calretinin+cases showed reactive mesothelial cells, and the remaining 20 specimens with PAX8-/Calretinin- phenotype typically contained inflammatory or blood cells without noticeable diagnostic epithelia.CONCLUSIONS:PAX8 identifies all Mullerian derived benign or malignant epithelia. When combining with Calretinin, PAX8 is a sensitive marker to diagnose the carcinomas of ovarian origin, which will be ideal to be used for those patients with a possible advanced ovarian cancer prior to receiving neoadjuvant chemotherapy.

Neotyphodium species, endophytic fungi associated with cool-season grasses, enhance host fitness and stress tolerance, but also produce biologically active alkaloids including ergot alkaloids associated with fescue toxicosis in grazing animals. One approach to reduce fescue toxicosis is to manipulate genes in the ergot alkaloid pathway. The gene, dmaW, encoding the first pathway-specific step in ergot alkaloid biosynthesis, was cloned previously from Claviceps spp. and its function was demonstrated by expression in yeast. Putative homologs have been cloned from Neotyphodium coenophialum (from tall fescue) and Neotyphodium sp. Lp1 (from perennial ryegrass). In order to confirm the function of dmaW in ergot alkaloid production, dmaW in Neotyphodium sp. isolate Lp1 was knocked out by gene replacement. The dmaW knockout mutant produced no detectable ergovaline or simpler ergot alkaloids. Complementation with Claviceps fusiformis dmaW restored ergovaline production. These results confirmed that the cloned endophyte gene was dmaW, and represented the first genetic experiments to show the requirement of dmaW for ergot alkaloid biosynthesis. Neotyphodium coenophialum, endophyte of the grass tall fescue (Lolium arundinaceum) has two homologs of dmaW. Considering the possible field applications in future, the Cre/lox site-specific recombination system was chosen because of the potential to sequentially knock out both homologs and obtain marker-free dmaW mutants of N. coenophialum. One homolog, dmaW-2, was disrupted by marker exchange, and the marker was eliminated by Cre, thus demonstrating the application of Cre/lox system in N. coenophialum to eliminate a marker gene. The dmaW-2 knockout did not eliminate ergovaline production, indicating that the dmaW-1 was probably also active in N. coenophialum. A putative ergot alkaloid biosynthesis gene cluster was identified in Claviceps purpurea and C. fusiformis. C. purpurea and C. fusiformis produce different subsets of ergot alkaloids. Identification of nine common genes between them suggests the possible role of these genes in the early part of the ergot alkaloid biosynthetic pathway.

Die vorliegende Arbeit untersuchte die Bedeutung von Mikrosatelliteninstabilitäten (MSI) als Ausdruck einer Defizienz des MMR Systems im Prostatakarzinom. Neben der Bestimmung der Prävalenz von MSI lag das Hauptaugenmerk auf der Analyse von Korrelationen zwischen dem Auftreten von Mikrosatelliteninstabilitäten in Prostatakarzinomen und klinisch prognostischen Parametern. Von den insgesamt 153 untersuchten Prostatakarzinomen konnte in 24 Fällen (15,7%) Mikrosatelliteninstabilität nachgewiesen werden. 9 davon (5,9%) waren mit zwei oder mehr nachgewiesenen Instabilitäten definitionsgemäß hochinstabil (MSI H). Diese Prävalenz hochinstabiler Prostatakarzinome ist im Vergleich zu anderen MSI Studien niedrig, steht aber im Einklang mit konzeptionell vergleichbaren und validen Studienergebnissen. Eine statistisch signifikante Korrelation zwischen dem MSI Status und dem Alter der Patienten bei Diagnosestellung wurde beobachtet. Im untersuchten Patientenkollektiv traten hochinstabile Prostatakarzinome im Vergleich zu mikrosatellitenstabilen Karzinomen erst in einem deutlich höheren Lebensalter auf. Bezüglich der übrigen untersuchten Parameter zeigten die Analysen, dass hochinstabile Adenokarzinome der Prostata mit guter Differenzierung, niedrigeren Tumorstadien und fehlender Lymphknotenmetastasierung einhergehen. Den zweiten Schwerpunkt der Arbeit bildet die Detektion aberranter Expressionslevel der Spindelcheckpoint-Gene Bub1b und MAD2 und deren mögliche prognostische Bedeutung in Hinblick auf den klinischen Verlauf der Tumorerkrankung. Mittels quantitativer Expressionsanalysen wurden sowohl relative Über- als auch Unterexpressionen der Spindelcheckpoint-Gene Bub1b und MAD2 im Prostatakarzinom nachgewiesen. Im untersuchten Patientenkollektiv sind Überexpressionen dieser Gene vergleichsweise selten und scheinen somit für die Karzinomprogression keine bedeutende Rolle zu spielen. Hingegen weist eine Gruppe von Tumorproben insbesondere für Bub1b (19,1%), in geringerem Ausmaß auch für MAD2 (7,1%), vergleichsweise geringe Expressionslevel der untersuchten Spindelcheckpoint-Gene auf. Diese Prostatakarzinome mit reduzierten Expressionsleveln zeigen eine enge Assoziation mit verschiedenen biopathologischen Parametern. Prostatakarzinome mit reduzierter Bub1b Expression sind dabei in statistisch signifikantem Maße mit hohen Gleason-Scores, lokal fortgeschrittenen Tumorstadien und vermehrt lymphogener Metastasierung assoziiert. In Hinblick auf MAD2 sind mit der bislang untersuchten Patientenanzahl keine statistisch signifikanten Aussagen möglich. Jedoch fällt auch hier auf, dass untersuchte Prostatakarzinome mit reduzierter MAD2-Expression vergleichsweise schlecht differenzierte Karzinome in zum Großteil fortgeschritteneren Tumorstadien mit oftmals bereits nodaler Metastasierung sind. Die gezeigten Ergebnisse legen dem Spindelcheckpoint Gen Bub1b somit die Funktion eines Tumorsuppressors nahe. / The work at hand examined the significance of microsatellite instabilities (MSI) as an expression of a deficiency of the MMR-system in prostate carcinomas. Beside the determination of the prevalence of MSI the main focus was put on the analysis of correlations between the occurrence of microsatellite instabilities in prostate carcinomas and clinically prognostic parameters. From a total of 153 examined prostate carcinomas there were 24 cases (15.7%) in which instabilities of microsatellites could be proven. Nine of them (5.9%) had two or more detected instabilities and were consequently by definition highly instable (MSI-H). This prevalence of highly instable prostate carcinomas is low in comparison to other MSI-studies, it is, however, in accordance with conceptually comparable and valid study results. A statistically significant correlation between the MSI-status and the age of the patients at the time of the diagnosis was observed. Highly instable prostate carcinomas compared to microsatellite stable carcinomas occured only at a considerably higher age among the tested collective of patients. The analyses revealed in terms of the remaining tested parameters that highly instable prostate carcinomas are attended by well-differentiated carcinomas, lower tumour stages and absence of pathologic lymph nodes. The second focal point of this study depicts the detection of aberrant levels of expression of the spindle assembly checkpoint genes Bub1b and MAD2 and their possible prognostic relevance with regard to the clinical course of tumour disease. Via quantitative expression analyses both relative over- and underexpression of the spindle assembly checkpoint genes Bub1b and MAD2 in prostate carcinomas were verified. Within the examined collective overexpression of those genes occur comparatively rarely, thus they seem not to play a decisive role for the tumour progression. On the other hand, a group of tumour samples especially for Bub1b (19.1%), to a minor degree also for MAD2 (7.1%), features comparatively low expression levels of the examined spindle assembly checkpoint genes. Those prostate carcinomas with reduced expression levels display a close association with different various biopathologic parameters. At the same time prostate carcinomas with reduced Bub1b-expression are associated at a statistically significant rate with high Gleason-scores, locally advanced tumour stages and increased nodal metastasis. With the so far examined patient collective statistically significant conclusions with regard to MAD2 are impossible. Here, however, it is also striking that examined prostate carcinomas with reduced MAD2-expression are comparatively poorly differentiated carcinomas at in large part advanced tumour stages with often already nodal metastasis. The shown results suggest that the spindle assembly checkpoint gene Bub1b holds the function of a tumour suppressor.

A identificação de leveduras por métodos moleculares e bioquímicos teve um grande impacto na sistemática e, devido aos resultados recentes este grupo sofreu alterações taxonômicas. A linhagem de levedura IZ-987 tem sido utilizada na fermentação de caldo de cana-de-açúcar para a obtenção de aguardente no Departamento de Agroindústria, Alimentos e Nutrição da ESALQ/USP. Esta linhagem, catalogada como Saccharomyces cerevisiae, não produz H2S durante o processo fermentativo e apresenta a capacidade de flocular em fermentação. A análise anterior de diversidade genética por marcadores de RAPD demonstrou existir um distanciamento elevado entre esta linhagem e S. cerevisiae. O presente trabalho teve então por objetivo avaliar a diversidade genética entre a linhagem IZ-987, e as linhagens PE-2 e IZ-259 utilizadas como leveduras padrões pertencentes à espécie Saccharomyces cerevisiae e a levedura CR-1 como padrão de Saccharomyces bayanus, por meio das características morfológicas, fisiológicas e bioquímicas, e também por meio de marcadores de RAPD, cariotipagem e análise da seqüência das regiões ITS1 e ITS2 (Internal Transcript Subunit). Os métodos mostraram-se eficientes na diferenciação das linhagens, pois as limitações de um método puderam ser complementadas por outro. A análise morfológica mostrou que a linhagem IZ-987 não apresenta similaridade com as linhagens padrões. Os testes fisiológicos e bioquímicos apresentaram maior similaridade entre as linhagens em estudo não sendo suficiente para identificar com segurança o gênero e a espécie. As análises de diversidade genética por marcadores de RAPD e cariotipagem demonstraram que existe um distanciamento elevado entre IZ-987 e os padrões de S. cerevisiae. A análise da seqüência das regiões ITS1 e ITS2 (incluindo a subunidade 5,8 S do RNA ribossomal) da linhagem IZ-987, mostrou similaridade (>96%) com a espécie Pichia anomala. / Identification of yeast by biochemical and molecular method has changed the systematic of this group. The strain IZ-987 has been used in Department of Agroindustry, Food and Nutrition, ESALQ/USP, Piracicaba/SP for alcohol production from sugarcane. This strain, which flocculates and is not able to produce H2S during fermentation, was previously identified by classical methodology as Saccharomyces cerevisiae. In previous work, RAPD analysis showed a low level of similarity between the IZ-987 and other of S. cerevisiae strains. The aim of the present work was to examines the genetic variability among the IZ-987 strain, S. cerevisae (IZ-259 and PE-2 strains) and S. bayanus (strain CR-1) by nutritional requirements, biochemical, physiological and morphological traits as well as RAPD markers. ITS (Internal Transcript Subunit) sequencing was used in further characterization of the IZ-987 strain. The methodologies used in the present analysis were able to distinguish the strains, since that the limitation observed in one was complemented by other technique. The morphological and molecular analysis distinguished the IZ-987 strain from those of Saccharomyces genera. However, physiological and biochemical evaluation show similarity among the evaluated strains. Therefore, the results were not able to define the specie or genera of the IZ-987 strain. The sequencing of ITS-1, ITS-2 and 5.8S rDNA of the IZ-987 strain and analysis by BLASTn (http://www.ncbi.nlm.nih.gov) showed similarity (>96%) of this strain with Pichia anomala.

L'évolution des plantes à fleurs est remarquable par la vitesse et l'étendue de la diversification qui l'accompagne. La variabilité génétique qui découle de cette diversification pourrait provenir de nombreux processus. Parmi ceux-ci, les éléments transposables (ET) ont été considérés comme l'un des agents les plus importants. Les ET peuvent constituer de grandes proportions des génomes de plantes (>80%) et joueraient un rôle important dans l'établissement de la diversité génétique. Notre travail contribue à la compréhension du rôle des ET dans l'évolution des génomes d'espèces du genre Coffea, genre qui appartient à la famille des Rubiacées. Plus précisément, l'objectif de l'étude était d'étudier l'impact d'ET actifs sur le génome de certaines espèces du genre Coffea. Les données obtenues ont été divisées en deux parties qui composent les deux chapitres de la thèse. Dans le premier chapitre, dix rétrotransposons (LTR-RTs) ont été identifiés et annotés dans le génome de C. canephora. Le profil de leur polymorphisme d'insertion a été analysé à l'aide des approches IRAP et REMAP chez plusieurs génotypes des espèces C. canephora (18) et C. eugenioides ( 5), qui sont les espèces parentales de l'allotétraploïde, C. arabica (21). Les résultats soulignent la dynamique évolutive de ces éléments dans ces espèces, ainsi que leur transmission. Nos résultats montrent également des modifications de la structure du génome de l'hybride. Dans la seconde partie est constitué par une analyse complète de la répartition et de l'évolution d'un rétrotransposon particulier : Copia25. Ces analyses, menées in silico et in vitro, ont montré que ce LTR-RT est largement distribué au sein de la famille des Rubiacées et qu'il est également présent chez d'autres espèces proches ou bien plus éloignées phylogénétiquement (Asterides, Rosides ou Monocotylédones). Une situation particulière est constituée par la relation étroite qui existe entre les séquences de Copia25 identifiées dans le genre Musa, monocotylédone, et dans le genre Ixora, dicotylédones de la famille des Rubiacées. Nos résultats révèlent la complexité de la dynamique évolutive de Copia25 chez les angiospermes qui implique plusieurs processus incluant un mécanisme de conservation de la séquence, un « turnover » rapide, des pertes stochastiques et le transfert horizontal.L'article présenté dans le dernier chapitre a été accepté avec modifications par la revue « Plant Molecular Biology ». il est actuellement en cours de révision pour être renvoyé prochainement en vue de son acceptation définitive. / The evolutionary history of the flowering plants is remarkable for its rapid and extensive diversification. The background of the genetic variability for this diversification is originated by numerous processes, among them the transposable elements (TEs) have been considered as one of the most important agents. TEs may compose large amounts of plant genomes and might play important roles in the promotion of genetic diversity. Our study contributes for the understanding of TEs impact on the genome of Coffea species. Coffea is a genus that belongs to the Rubiaceae family. The goal of the study was to investigate the occurrence and diversity of active TEs in Coffea species and the data obtained were divided in two parts that compose the two chapters of the thesis. In the first chapter, ten retrotransposons with LTRs (LTR-RTs) were annotated in the C. canephora genome and had their insertion polymorphism profile analyzed, using the IRAP (inter-retrotransposon-amplified polymorphism) and REMAP (Retrotransposon-microsatellite amplified polymorphism) methods, in genotypes the progenitor species, C. canephora (18) and C. eugenioides (5), and the allotetraploid hybrid, C. arabica. The results outline the evolutionary dynamics of these elements in the species, as well as their inheritance. Our results also suggest the occurrence of genomic structural changes mediated by the LTR-RTs in the hybrid. And, the second part constitutes a wide analysis of the distribution and evolution of a particular LTR-RT, Copia25. In silico and in vitro analyses showed that Copia25 is widely distributed among the Rubiaceae family and that it is also present in other distantly related species belonging to asterids, rosids and monocots. A particular situation is the closer relationship between the Copia25 sequences of Musa, a monocot, and Ixora, a dicot species (Rubiaceae). Our results disclose the complexity of the evolutionary dynamics of Copia25 in angiosperm involving several processes including sequence conservation, rapid turnover, stochastic losses and horizontal transfer. The article presented in the last chapter was accepted for publication after modifications in “Plant Molecular Biology”, it is at the present under correction to be soon sent back for final approval.

O Diabetes Mellitus (DM) é uma doença metabólica que implica em altas incidências de mortalidade e morbidade. A hiperglicemia crônica é responsável pelo surgimento de inúmeras complicações em longo prazo nestes pacientes. Atualmente, é recomendado por diretrizes internacionais que pacientes com DM sejam monitorados e manejados em seu tratamento a partir dos níveis de hemoglobina glicada (A1C). A A1C é formada por reações não enzimáticas de glicação na hemoglobina, refletindo a glicemia dos últimos 120 dias. A A1C possui forte associação com os desfechos clínicos no DM e apresenta uma excelente padronização de seus métodos analíticos. Contudo, diversas situações clínicas podem interferir falsamente em seus níveis e prejudicar a interpretação de seus resultados, como em anemias (carenciais ou hemolíticas), hemoglobinopatias, gravidez, doença renal crônica, etc. Por outro lado, a albumina glicada (AG) é uma frutosamina formada por glicações na albumina e reflete uma glicemia média de cerca de 2 a 3 semanas. A AG não é influenciada pela concentração de outras proteínas no plasma e também não sofre interferência pelas condições que afetam a A1C. Este marcador tem sido fortemente avaliado como uma ferramenta alternativa para a A1C, a partir da análise de seus níveis por um novo método enzimático descrito em 2002. Estudos tem demonstrado uma forte associação entre estes dois marcadores e grande semelhança em predizer as complicações do DM. Entretanto, a AG se mostra melhor para avaliar flutuações nos níveis de glicose e a resposta ao tratamento terapêutico. Neste trabalho, foi avaliado o desempenho analítico de dois kits enzimáticos de AG e realizado uma comparação entre os métodos, encontrando excelentes resultados. Ainda, foi determinado o intervalo de referência para os níveis de AG em brasileiros saudáveis. A forte correlação encontrada entre AG e A1C demonstra que a AG pode ser um teste útil para o controle glicêmico no DM, principalmente quando a A1C não é recomendada. / Diabetes Mellitus is a metabolic disease with high incidence rates of mortality and morbidity. Chronic hyperglycemia is responsible for several long-term complications in these patients. Currently, international guidelines recommend that glycemic monitoring in DM should be performed by glycated hemoglobin (A1C) levels, to provide a correct clinical conduction. A1C is relative to non-enzymatic glycation reactions in hemoglobin and reflects the glucose levels from the last 120 days. It is well established the great association between A1C and clinical outcomes in DM, besides, its analytical methods present an excellent standardization. However, some conditions may influence and imply misinterpretation in A1C results, such as anemia, hemoglobinophaties, pregnancy, chronic renal disease, etc. On the other hand, glycated albumin (GA) is a fructosamine produced by glycation reactions in albumin and it reflects a mean glycemia at around 2 to 3 weeks. GA is not influenced by the concentrations of other plasma proteins, as well as by those conditions that interfere in A1C. GA has been strongly evaluated as an alternative marker to A1C, through its quantitative measurement by an enzymatic methodology described in 2002. Recent studies have demonstrated a high association between GA and A1C and a great similarity between these tests in predicting DM future complications. Nevertheless, GA has showed be better to assess the glucose fluctuations in blood and the response to treatment. This study evaluated the analytical performance of two GA enzymatic kits and also executed a methods comparison, and found excellent results. Also, we established the reference range for GA levels in healthy Brazilians. The high correlation found between GA and A1C indicates that GA could be a useful test for glycemic control in DM, especially when A1C is unreliable.

The main topic of the thesis is the temporal dimension of aesthetic experience in its relation to memory and imagination. The initial approach to the topic is the concept of relationship between time and narrative of Paul Ricoeur, primarily contained in his three volume book entitled Time and Narrative. Gradually, the specificity of the time experience will be investigated, as well as operations of memory and imagination in narrative fiction. Attention is focused on a narrative as means to designate a coherent whole, respectively a meaningful concatenation of events. The main relationship explored is the interaction between the world of work and the lived world. The functionality of theoretical views is presented on the work of French director Chris Marker, especially on his two films La Jetée (1962) and Sans Soleil (1982).

O cerrado brasileiro é um dos biomas de maior riqueza e endemismo de plantas, mas com alto índice de desmatamento nas últimas décadas, o que resultou na fragmentação dos habtats e na ameaça de extinção de centenas de espécies vegetais. Dentre estas espécies ameaçadas está Anacardium humile, conhecida como cajuzinho-do-campo, uma planta caméfita de ampla distribuição pelo país, servindo de alimento para o homem e para alguns animais e apresentando propriedades medicinais. Estudos sobre a estrutura de populações de Anacardium humile são escassos na literatura, e seu entendimento é fundamental para a preservação e conservação da espécie. Dessa forma, o presente trabalho teve dois objetivos: 1) em duas fitofisionomias de cerrado distintas, estimar a abundância de ramets da espécie, seu padrão de distribuição espacial em macro e microescala, e a influencia da porcentagem de abertura do dossel na determinação deste padrão; 2) a construção de uma biblioteca genômica enriquecida de microssatélites para a espécie e o desenho de primers a partir desta biblioteca, a fim de isolar locos com potencial para uso como marcadores genéticos. Com relação ao primeiro objetivo, três parcelas de 0,5 ha, divididas em 200 subparcelas, foram instaladas (duas num fragmento de cerrado típico à cerradão e uma num fragmento de cerrado aberto), e todos os ramets da espécie foram amostrados por contagem, sendo discriminados os com e sem ataque de Contarinia sp. (Diptera: Cecidomyiidae). Fotografias foram tiradas do centro de cada parcela para determinar a porcentagem de abertura do dossel. A abundância de ramets foi maior nas áreas mais abertas, mas a incidência de ataque de Contarinia sp foi maior no fragmento mais fechado. As análises de macroescala (Índice de Dispersão) e de microescala (Autocorrelação Espacial) mostraram que os ramets da espécie apresentam padrão agregado em ambas as áreas, mas que essa agregação, em microescala, não está relacionada à porcentagem de abertura do dossel, embora haja diferenças significativas para este ultimo fator entre os grids, indicando que o padrão é devido à sua forma de vida. Já em relação ao segundo objetivo, a construção da biblioteca genômica enriquecida de microssatélites resultou em 180 clones, dos quais 84 foram seqüenciados, sendo detectadas 23 sequências contendo microssatélites, o que representa um enriquecimento da biblioteca de 27,38%. Foram desenhados 15 pares de primers dentre os 34 microssatélites obtidos, mas apenas 7 pares amplificaram. Destes, cinco pares foram visualizados em acrilamida, e um loco polimórfico foi observado. O número de clones obtidos está dentro do observado em estudos com outras espécies da família Anacardiaceae, mostrando a eficiência do protocolo. Problemas com a otimização de reagentes podem ter impedido a amplificação de alguns primers, uma vez que os procedimentos para o desenho dos mesmos foram adequados. Os resultados de polimorfismo são preliminares, devido ao número baixo de indivíduos avaliados. Ao menos um loco apresenta potencial como marcador genético. / Brazilian cerrado is one of the richest and plants endemism biomes, but with a high deforestation in the last decades, resulted in habitats fragmentation and extinction threat of hundreds of plant species. Among these threatened species is Anacardium humile, known as cajuzinho-do-campo, a camephyth plant with a wide distribution through the country, which serves as aliment to man and some animals and presents some medical properties. Studies about Anacardium humiles populations structure are very scarce in the literature, and its understanding is fundamental to the preservation and conservation of the species. Thus, the present work had two objectives: 1) in two distincts cerrado plant physiognomies, estimate the abundance of species ramets, its spatial distribution pattern in macro and microscale, and the influence of canopy openness percentage in the determination of this pattern; 2) construction of a genomic enriched library with microsatellites to the species and primers design from this library, to isolate loci with potential to be used as genetic markers. In relation to the first objective, three 0,5 ha quadrats, divided in 200 contiguous quadrats of 25 m2, were installed (two in a typical cerrado to cerradão fragment and one in an open cerrado fragment), and all species ramets were sampled by count, being differentiated in with and without Contarinia sp attack ((Diptera: Cecidomyiidae). Photographies of the center of each parcel were taken to determinate the percentage of canopys openness. The abundance of ramets were higher in the most opened areas, but the incidence of Contarinia sp attack were higher in the closest fragment. Macroscale (Dispersion Index) and microscale analysis (Spatial Autocorrelation) showed that species ramets present an aggregated pattern in both areas, but this aggregation is not related to the percentage of canopys openness, although there are significant differences to this last factor among the grids, indicating that pattern is due to its way of life. In relation to the second objective, the construction of genomic library enriched with microsatellites resulted in 180 clones, which 84 were sequenced, being detected 23 sequences containing microsatellites, representing a librarys enrichment of 27,38%. 15 primers pairs were designed among the 34 obtained microsatellites, but only 7 pairs amplified. From these, five pairs were visualized in acrylamide, and one polymorphic loco was observed. The number of obtained clones is in accordance with the observed in studies with another species from Anacardiaceae family, showing protocols efficiency. Problems with the optimization of reagents may have impeded the amplification of some primers, once that procedures to their design were suitable. Polymorphism results are preliminaries, due to low number of evaluated individuals. At least one loco presents potential as genetic marker.

Student Number : 0201444H - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Due to the paucity of sensitive mutation markers available for studying mycobacterial species it was decided to explore the suitability of mycobacteriophage L5 as an analogous mutation detection system to phage Lambda in E. coli. The system relies on the detection of an increased production of clear plaque mutants (CPM) arising from turbid plaques, in response to DNA damage. A number of L5 phage experimental tools were developed and optimized, including a lysogen-based CPM confirmation assay. The mutant induction system was applied to wild type M. smegmatis mc2155 and its recA mutant, dinP mutant as well as an M. smegmatis(L5) lysogen. The lysogen system proved to be insensitive with respect to mutant induction since elevated CPM frequencies could not be detected. Interestingly, the wild type M. smegmatis mc2155 system demonstrated slightly elevated CPM frequencies in response to transfection of untreated L5 on UV irradiated host cells. This result suggests that a host SOS mutagenic system is able to act on normal, undamaged DNA bases. The involvement of the SOS response in untargeted mutagenesis was confirmed by the abrogation of increased CPM frequency, in an M. smegmatis recA mutant. This data supports suggestions that RecA is responsible for the control of the SOS response. The M. smegmatis dinP mutant system showed a decrease in CPM frequency which supports evidence that this gene does have mutator polymerase activity, as is in seen E. coli dinP homologues.

The Crimson-spotted rainbowfish (Melanotaenia fluviatilis) from the Murray-Darling basin of Australia is a common indicator species in Australian ecotoxicology. Biochemical changes have been investigated in this species, but not molecular markers of ecotoxicological interest. In this study genes of M. fluviatilis were isolated using a cDNA library and sequences analysed. Of 345 randomly selected clones, 94 shared similarity with 26 different genes in other organisms in public databases. Amongst these, reproductive genes coding for vitellogenin, retinol binding protein, sialyltransferase and zona pellucida protein were considered of interest in ecotoxicology. The vitellogenin gene was selected for study as it has been widely used as a molecular marker of exposure to 17â-estradiol (E2) in teleosts. Gene expression was examined via northern blot, RT-PCR and Real-Time PCR relative to the housekeeping gene (18S rRNA). The expression of vitellogenin mRNA was observed a t 12 hours post-exposure, peaked at 48 hours according to northern blot analysis; and cleared within 4 days, partly consistent with RT-PCR. However, Real-time PCR yielded an inconclusive result, probably due to differences between pooled and individual samples. Vitellogenin in blood plasma was confirmed by western blot, found to be significantly increased and retained in the plasma in fish treated with E2 compared to controls. It was concluded that vitellogenin mRNA is a molecular marker of exposure to 17â-estradiol in the rainbowfish, and could potentially be used as a marker of exposure to environmental estrogenic chemicals. Further investigations of the expression of genes in the cDNA library, could establish other molecular markers of ecotoxicological interest in M. fluviatilis.