Dissertations / Theses on the topic 'Direct sequencing'

We have designed an electrophoresis system that can purify plasmid DNA from a culture without centrifugation. This system is based on electrofiltration where bacterial cell lysates are loaded in one chamber and the purified plasmid DNA is recovered in an adjacent chamber. These two chambers are separated by a membrane made of regenerated cellulose, which allows plasmid DNA to migrate to the recovery chamber while retaining most contaminants in the loading chamber. Unfortunately, even with the optimization of the parameters involved in the electrofiltration, the only DNA that can pass through the middle membrane still has some contaminants, which prevent sequencing of the plasmid. Our results have shown that a pure plasmid cannot cross a membrane with pores small enough to prevent the migration of most of the contaminants. Only a plasmid complexed with some contaminants can cross a small pore membrane. In parallel, we have compared six direct sequencing methods that do not require any plasmid purification prior to the sequencing reaction. We compared the reliability, quality of sequences, time required, and cost of these six methods. We found that the best method was that of Zhang et al. (1999). This method is fast, reliable, produces good quality sequences and is inexpensive. The performance of this method is due to the amount of ABI's ready reaction mix used, the pre-sequencing heating step to lyse the cell, the large volume of the PCR sequencing reaction and the addition of BSA.

Objetivos: Analisar por meio da técnica de sequenciamento direto a presença de alterações moleculares nos genes SHH, SIX3, ZIC2 e TGIF1 em indivíduos com diagnóstico clínico de HPE. Analisar por meio da técnica de CGH-array a presença de alterações moleculares em indivíduos com diagnóstico clínico de HPE previamente submetidos à análise por sequenciamento direto. Local: Laboratório de Genética e Citogenética Humana HRAC/USP, Bauru-SP. Casuística e metodologia: Foram selecionados 50 indivíduos, de ambos os sexos com idades entre 03 meses a 50 anos com diagnóstico clínico para HPE. Todos foram analisados por meio da técnica de sequenciamento direto para os genes SHH e TGIF1 completamente e para os genes ZIC2 e SIX3 parcialmente. Dentre os indivíduos que não apresentaram alterações na técnica de sequenciamento oito indivíduos com fenótipo mais grave foram selecionados para a análise por CGH-array. Resultados e discussão: Foram analisados 50 indivíduos por meio da técnica de sequenciamento direto dos gene SHH e TGIF1, foram encontradas duas variantes patogênicas na análise do gene SHH, no caso 1 a variante p.24G>P foi identificada, e no caso 2 foi identificada a variante c.1031del C. No gene TGIF1 foram encontrados cinco polimorfismos já descritos na literatura. Foi identificada uma nova variante silenciosa no éxon 1 do gene ZIC2 p.Q46Q (c. 431 G>A) e um polimorfismo já descrito na literatura em dois indivíduos no gene SIX3. A análise por CGH-array revelou a presença de uma microdeleção no caso 37, de 1,5Mb no cromossomo 17p12 entre as posições genômicas 14,052,279-15,102,307. A mesma deleção foi encontrada na mãe, sendo que esta região nunca foi associada a HPE. Conclusão: A técnica de sequenciamento direto é uma ferramenta muito importante no diagnóstico molecular da HPE, a padronização do sequenciamento direto para os genes ZIC2 e SIX3 poderá auxiliar em diagnósticos mais precisos em estudos futuros dentro do HRAC/USP. O emprego de novas técnicas como CGH-array pode indicar novas relações entre regiões cromossômicas e os múltiplos fatores envolvidos na formação da HPE.
Objective: Analyze through direct sequencing technique the presence of molecular changes on the genes SHH, SIX3, ZIC2 and TGIF1 on individuals with clinical diagnosis of HPE. Analyze through array-CGH technique the presence of molecular changes on individuals with clinical diagnosis of HPE previously submitted to the direct sequencing analyzes. Local: Genetics and Human Cytogenetics Laboratory, HRAC/USP, Bauru-SP. Methods: Were selected 50 individuals from both genders with ages between 03 months and 50 years clinically diagnosed with HPE. Everyone was analyzed through the direct sequencing technique for the genes SHH and TGIF1 completely and for the genes ZIC2 and SIX3 partially. From those individuals which did not have shown changes on the direct sequencing technique, eight individuals with more severe phenotype were selected to the analysis through array-CGH. Results an Discussion: Were analyzed 50 individuals through the technique of direct sequencing of the genes SHH and TGIF1, were found two pathogenic variants in the analysis of SHH gene, in the case 1, the variant p.G24P was identified, and in the case 2 was identified the variant c.1031delC. On the TGIF1 gene were found five polymorphisms already described on the literature. Was identified a new silent variant on the exon 1 of the ZIC2 gene p. Q46Q(c.431G>A) and a polymorphism already described in the literature in two individuals on the gene SIX3. The analysis through array-CGH revealed the presence of one microdeletion in the case 37, of 1,5 Mb on the region 17p12 between the genomic positions 14,052,279-15,102,307. The same deletion was detected in the mother, though this region was never associated to the HPE. Conclusion: The direct sequencing technique is a very important tool for the molecular diagnosis of the HPE, and the direct sequencing standardization for the genes ZIC2 and SIX3 might help in more precise diagnostics on HRAC/USP future studies. The employ of new techniques such as array-CGH may indicate new relations between chromosomal regions and the multiple hit involved in the development of HPE.

Currently, a debate exists between the strengths and weaknesses of direct and inquiry instruction. Inquiry instruction is related to positive effect on learner motivation whereas supporters of direct instruction point to its ability to adequately support learners' working memories (Hmelo-Silver, Duncan, & Chinn, 2007; Kirschner, Sweller, & Clark, 2006; Kuhn, 2007; Sweller, 1988). This study examined the possibility of combining the best features of both inquiry and direct instruction by sequencing them together. A two-part lesson on electrical circuits was presented in three separate sequences of instruction to middle school students to determine if differences in student motivation and academic achievement emerge depending on whether a guided inquiry lab followed or preceded direct instruction. Results indicated equal levels of perceived competence by students across all instructional sequences and greater interest/enjoyment and perceived autonomy support when the instructional sequence began with a guided inquiry lesson. No significant differences in achievement were reported among the sequences.
ID: 030423060; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.A.)--University of Central Florida, 2011.; Includes bibliographical references (p. 42-48).
M.A.
Masters
Teaching, Learning and Leadership
Education
Applied Learning and Instrucation

The cereal seed plays a crucial role in society – both in the “food as medicine” paradigm, but also in food security. It is the starch and proteins present in the seed that lend it importance in these dissimilar anthropomorphic activities. This thesis investigation first characterized the post-translational processing of the potential diabetogen, wheat globulin-3. Globulin-3-like peptides were observed primarily in the embryo. These peptides varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting. Five major polypeptide spots were sequenced by mass spectrometry, allowing for the development of a model of the post-translational events contributing to the globulin-3 processing profile. Three separate investigations of starch granules from different cereal species were performed. In the first series of experiments, pathogen-susceptible maize kernels were injected with either conidia of the fungal pathogen Fusarium graminearum or sterile water controls. Proteins in the desiccated fungal remnants on the surface of the kernels as well as in the endosperm and embryo tissues of the control and infected kernels were isolated and these proteomes were sequenced using tandem mass spectrometry. Approximately 250 maize proteins were identified. These proteins were classified into functional categories. There was an increased representation of defense proteins in the both the embryo and endosperm tissues of infected maize samples. The proteome of the fungal remnants was composed of 18 proteins. Several of these proteins were categorized as being involved in the metabolism of plant-sourced molecules, or in stress response. The second series of experiments detail the investigation of commercially prepared rice and maize starches using tandem mass spectrometry. The majority of identified proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. Markers for seed maturity and for starch mobilization were also documented. Finally, the third series of experiments investigated the non-host proteomes present in commercially-prepared starches. Non-host proteins from a variety of species, including Homarus americanus were found in the starch samples. This documentation of H. americanus proteins in these starch samples may have food safety implications with regards to shellfish allergies.

Les deux virus d’ARN VIH et VHC attirent beaucoup d’attention de la santé publique parce qu’ils partagent des mêmes facteurs de risque de transmission par le sang et par des contacts sexuels. En plus, les maladies associées aux infections par le VIH et par le VHC sont des causes principales de mortalité et de morbidité globalement. Néanmoins, grâce à l’introduction des thérapies d’antirétroviraux pour le traitement de l’infection par le VIH et les antiviraux à action directe (AADs) pour le traitement de l’infection par le VHC, les patients infectés par ces virus constatent une amélioration significative de leur qualité de vie. Cependant, le taux de réplication élevé et l'absence de mécanisme de correction d'erreur de ces virus génèrent une population virale diversifiée appelée des quasi-espèces. Sous la pression sélective de traitement, les quasi-espèces virales sélectionnent des variants de résistance au médicament correspondant et rendent la thérapie inefficace, en particulier dans les cas où une surveillance appropriée du traitement n'est pas assurée. Afin de réserver un large choix de traitement antirétroviral tout au long de la vie chez les patients infectés par le VIH et, parallèlement, de réduire le coût de traitement des infections par le VIH et le VHC, il est fondamental de mener des recherches ciblées sur la détection, la surveillance et la transmission des mutations de résistance aux antiviraux. Dans cette thèse, nous avons utilisé les technologies de séquençage haut-débit (UDS) ou de séquençage de nouvelle génération (NGS) pour chercher des variants de résistance minoritaires (MiRVs) qui sont classiquement considérés comme représentant moins de 15%-25% de la population virale et ne sont pas trouvés par la technique de séquençage Sanger. La présence des MiRVs à baseline est responsable possiblement de l'échec de traitement et leur présence à l'échec peut limiter les options des traitements ultérieurs. Dans cette thèse, nous avons évalué la prévalence et l’impact clinique des MiRVs sur le gène de l’intégrase chez les patients infectés par le VIH qui ont été en échec d’un régime thérapeutique contenant un inhibiteur de l’intégrase. Nous avons également évalué l’impact des MiRVs chez les patients infectés par le VHC de génotype 3 et de génotype 4 et ayant échoué aux AADs. De plus, nous avons utilisé la technique UDS pour identifier et caractériser les chaines de transmission du VHC parmi une population clé d'hommes ayant des rapports sexuels avec des hommes, co-infectés par le VIH ou ayant un risque élevé d'acquisition du VIH. Nous avons également observé plusieurs cas d’infections mixtes de génotype du VHC dans cette population, probablement à cause du risque élevé de multiples expositions au VHC. Les questions sur l’avantage de l'UDS dans la recherche en virologie et l'applicabilité de cette technique en clinique ont été posées et vérifiées au travers de multiples types de projets dans cette thèse. L’UDS n’a pas été établi de manière concluante comme étant plus intéressant et bénéfique que la technique de séquençage de Sanger pour prévenir l’échec au traitement chez les patients infectés par le VIH ou le VHC et pour identifier les chaînes de transmission à grande échelle si le coût et le temps de l’expérience pour l’analyse des données sont pris en compte. Cependant, le développement dynamique des nouvelles technologies de l’UDS et les efforts sans cesse afin d’optimiser des procédures d'analyse montrent un rôle prometteur de l’UDS. Et l'applicabilité de l'UDS en pratique clinique devra encore être élucidée dans différents types de projets de recherche
The two RNA viruses HIV and HCV are getting a lot of public health concerns because both of them have overlapping risk factors for transmission through direct blood and sexual contacts. Furthermore, HIV and HCV infections are the leading cause of mortality and morbidity globally due to related diseases. However, with the introduction of antiretroviral therapy (ART) for the treatment of HIV infection and direct-acting antivirals (DAAs) for the treatment of HCV infection, patients infected by these viruses are witnessing significant improvement in their quality of life. However, the high replication rate and the lack of error correction mechanism of these viruses result in a diverse viral population referred to as quasispecies. Under drug- selective pressure, the viral quasispecies select resistance variants against corresponding drug and render the therapy ineffective especially in cases an appropriate treatment monitoring is not ensured.To reserve a wide range of possibilities for a life-long ART in HIV-infected patients and in parallel to reduce cost for treatment of both HIV and HCV infection, research focusing on detection, surveillance and transmission of resistance mutations is fundamental to prevent treatment failure on antivirals. In this PhD, we employed the ultra-deep sequencing (UDS) or next-generation sequencing (NGS) technologies to look for minority resistant variants (MiRVs) which are conventionally considered to represent less than 15%-25% of viral population and undetectable by Sanger sequencing. The presence of MiRVs at baseline is possibly responsible for the treatment failure and their presence at failure may limit options for subsequent therapies. In this PhD, we evaluated the prevalence and clinical impact of MiRVs on integrase gene in HIV-infected patients failing an integrase inhibitor containing regimen. We also evaluated the impact of MiRVs in HCV genotype 3 and genotype 4-infected patients failing DAAs. Furthermore, we used the UDS technique to identify and characterize the HCV transmission networks among a key population of men having sex with men either co-infected with HIV or at high risk of HIV acquisition. We also discovered several cases of mixed HCV genotype infections in this population probably for their high risk of multiple HCV exposures. The advantages of UDS in virology research and the applicability of this technique in clinic have been questioned and verified throughout multiple types of projects in this PhD. UDS has not been conclusively established to be more interesting and beneficial than Sanger sequencing in prevention of treatment failure in patients infected by HIV or HCV and in identifying the viral transmission networks at large scale if taking into account the experiment cost and time for data analysis. However, the dynamic development of UDS technologies and the continuing attempts in optimizing analysis procedures display a promising role of UDS. And the applicability of UDS in clinical practice still needs to be elucidated in different kinds of research projects

Aquatic ecotoxicology is the study of toxic chemicals and its effects on aquatic biological systems with the aim of minimising threats to human health and ensure self-sustained ecosystems. Freshwater bivalves are excellent sentinels for use in ecotoxilogical research due to their filter feeding properties, stationary lifestyle and inability to regulate body temperature. This project aimed to assess the feasibility and use of nanopore sequencing, a real-time single-molecule sequencing technology in comparative expression analysis by sequencing transcriptomic RNA from the freshwater mussel Anodonta anatina following exposure to copper. RNAs were extracted from 80 mg hepatopancreas tissue, followed by poly(A) RNA selection. Furthermore, the poly(A) RNA was used to construct a nanopore sequencing library. Sequencing a total amount of 560 ng poly(A) RNA over the course of two separate runs generated 239,448 reads, in which 75% of the reads were obtained during the first run (control) and 25% of the reads were obtained during the second run (case). The median read lengths ranged between 534-650 nucleotides, with a base call accuracy <90%. Due to the big differences in sequence data output between the two sequencing runs, the data was ineligible for comparative analysis. The findings conclude that nanopore sequencing is capable of generating longer read lengths when compared to other sequencing platforms. However, the technology is error-prone in terms of accurate base call identifications and relies on other platforms for error corrections. Future advances include de novo transcriptome assembly for efficient use of Anodonta anatina as a bioindicator in aquatic ecotoxicology.

Le diagnostic virologique est un sujet d’actualité particulièrement du fait des récentes épidémiesou pandémies telles que la pandémie d’influenza A(H1N1) en 2009 ou la diffusion du virus Zika dansles Amériques et la région du Pacifique entre 2014 et 2017, associée à des cas de microcéphalie et dessyndromes de Guillain Barré. Encore plus récemment, en août 2018, le ministre de la santé de laRépublique Démocratique du Congo annonçait la 10e épidémie de virus Ebola dans le pays et endécembre 2019, le coronavirus SARS-CoV-2 est à l’origine d’une pandémie au départ de la Chine. Avecle nombre croissant de migrants et de voyageurs favorisant la dissémination des maladies virales, leslaboratoires diagnostiques doivent être parés à la fois pour l’identification des virus communs maisaussi de ceux importés.Les techniques les plus anciennes de diagnostic virologique tendent à devenir obsolètes suite audéveloppement rapide des techniques moléculaires depuis les années 90. Cependant, nous utilisonstoujours un mélange de techniques moléculaires et non moléculaires au sein de notre laboratoire.Les objectifs de ce travail sont de passer en revue les différentes techniques communémentutilisées pour la détection directe des virus avec leurs avantages et leurs inconvénients et de fournirune réflexion sur la place de chaque technique, en 2020, dans un laboratoire diagnostique.Nous aborderons tout d’abord les cultures cellulaires et nous insisterons sur leur polyvalence quipermet parfois de mettre en évidence des micro-organismes que l’on ne suspectait pas. Nousillustrerons ce point par un article relatant la mise en évidence de Chlamydia trachomatis du serovar Lresponsables de la lymphogranulomatose vénérienne dans des prélèvements envoyés pour suspiciond’infection herpétique.Le travail se focalisera ensuite plus particulièrement sur le diagnostic des infections viralesrespiratoires. Nous verrons les principes des tests de détection antigéniques et discuterons de leurslimites en se basant sur un article qui traite du diagnostic des virus influenza A et B par 3 différentstests immunochromatographiques. Cet article montre que la sensibilité des tests varie en fonction dela charge virale dans le prélèvement ainsi que du sous-type de virus.Nous poursuivrons avec les tests d’amplification d’acides nucléiques (tests moléculaires) enexpliquant la technique de PCR (Polymerase Chain Reaction) et une technique d’amplificationisothermique (Nicking Enzyme Amplification Reaction - NEAR). Nous illustrerons par un article portantsur l’évaluation du test Alere i influenza A&B (technique NEAR) en comparaison du test Sofia influenzaA+B (immunochromatographie). Cet article montre un gain de sensibilité de l’Alere i par rapport auSofia pour le diagnostic de l’influenza A mais pas pour l’influenza B. Il constitue également un travailpréliminaire sur l’appréciation de l’utilité d’une technique PCR rapide dans la prise en charge despatients. La conclusion est qu’il pourrait y avoir un apport de ce type de technique pour la diminutiondes hospitalisations, de la prescription des examens complémentaires et des antibiotiques. Celapermettrait également une prescription plus adéquate de l’oseltamivir pour le traitement de la grippe.Le point important est que l’impact du résultat est d’autant plus grand qu’il est délivré précocementdans la prise en charge des patients, idéalement lorsqu’ils sont encore aux urgences.Suite au travail sur l’Alere i, nous avons entrepris d’évaluer un test PCR multiplex (FilmArrayRespiratory Panel) pour le diagnostic des virus afin de voir si la détection d’un plus grand nombre depathogènes pourrait avoir un impact plus grand sur la prise en charge des patients. Cette évaluation adonné lieu à deux articles. Le premier détaille les avantages et inconvénients des différents outils dediagnostic pour la détection des virus respiratoires et sert d’état des lieux sur les tests utilisésactuellement dans les laboratoires de virologie. Le deuxième article porte plus particulièrement surl’apport du FilmArray dans la prise en charge des patients. La conclusion est que ce n’est pas le résultatdu test qui a un impact sur cette prise en charge mais plutôt d’autres facteurs notamment l’âge ou desmarqueurs inflammatoires biologiques.Nous terminerons ce travail par un aperçu des techniques de séquençage qui seront sans aucundoute de plus en plus utilisées pour le diagnostic en virologie.
Doctorat en Sciences médicales (Médecine)
info:eu-repo/semantics/nonPublished

The epidermal growth factor receptor (EGFR) is a transmembrane receptor with tyrosine-kinase activity that can be activated by different ligands. This event triggers a cascade of signals downstream with effects on cell survival and proliferation. The mutational event of EGFR is able to induce the constitutional activation that can be effectively blocked by the administration of drugs with anti-receptor activity. The evaluation of EGFR mutational profile is a fundamental survey to select patients affected by advanced non-small cell lung cancer (NSCLC), which can undergo treatment with specific drugs. In fact, EGFR mutations are reported in a subpopulation of patients ( 8-20 %) with NSCLC, which grows in non-smokers, being female and belonging to Asian race. The most frequent mutations (90 % of the mutated samples) are located in Exon 19 (in-frame deletions: E746 - A750 ) and Exon 21 of the EGFR (point substitutions: L858R). Different technologies are used to evaluate the EGFR mutational status, including Sanger Sequencing, Pyrosequencing and Real Time PCR. Immunohistochemistry has also been proposed, even if not validated as a possible investigation complementary to those of molecular type. The purpose of the present work is to carry out a comparative analysis of these technologies (Sanger Sequencing, Pyrosequencing and Immunohistochemistry) in a series of patients with primary non-small cell lung cancer. The evaluation of the results took into account both the accuracy of the methods and other factors such as execution time, complexity of the procedure and cost/benefit ratio. Using tumor cell lines containing scalar dilutions of EGFR mutated alleles (1, 10, 20, 50%) we preliminarily stated that the Pyrosequencing in our hands, within a tumor population, is able to detect 10% of mutated alleles compared to 20% of mutated alleles detected by Sanger Sequencing. Then, we validated the diagnostic accuracy of these methods in our laboratory as part of two programs of quality controls developed at the national level on external occurences. The comparative research between Sanger Sequencing and Pirosequencing was carried out on 258 primary lung carcinomas, including 229 adenocarcinomas, using archival histological material formalin-fixed and paraffine-included which comprised materials of surgical origin both from therapeutic procedures (lung resections) and from diagnostic procedures (biopsies). The frequency of mutation detected by Pyrosequencing was 13,1% , higher than the one observed using Sanger Sequencing of 11,4%. Over the entire population of primary lung cancer with EGFR mutation (n=26), the gain in sensitivity of Pyrosequencing compared to Sanger Sequencing was 15,4% (4/26 cases). It is interesting to note that the cases identified as mutated by Pyrosequencing (not by Sanger Sequencing) came from samples characterized by reduced tumor cells (2 biopsies and 2 surgical microdissections). This finding is particularly significant taking into account the fact that EGFR mutation study is at present almost exclusively carried out on bioptic material taken from patients with lung cancer in advanced phase and thus inoperable. We then evaluated the diagnostic accuracy of immunohistochemistry in identifying typical mutations of EGFR. For this purpose, 73 adenocarcinomas with mutational profile related to Pyrosequencing [41 cases mutated for Exon 19 of EGFR (E746–A750del) and 31 mutated for Exon 21 (L858R)] were immunocolored using antibodies anti-E746-A750del for Exon 19 and anti-L858R for Exon 21. The diagnostic accuracy of immunohistochemistry was found to be between 80,3% (antibody anti-E746- A750del) and 90,7% ( antibody anti-L858R) with absolute specificity and sensitivity ranging from 67,7% to 69,0%. Overall, the immunohistochemical technique, even if limited to the identification of the two most frequent mutational events, turned out to be accurate enough and with much lower costs and execution times faster than molecular investigations. To sum up, our research proves both the suitability and the superiority of Pyrosequencing compared to Sanger Sequencing as for the ability to detect the presence of EGFR mutations in a consecutive series of primary non-small cell lung carcinomas. On the contrary, immunohistochemistry turned out to be a technique less sensitive in determining mutational arrangements of the same cases. Since this technique proved to get the same specificity and to be less expensive and faster than the first two, it is possible to use it in clinical diagnostics as preliminary screening investigation of the most frequent mutational events.

Aboveground and belowground ectomycorrhizal (ECM) communities associated with different age classes of the exotic plantation species Pinus radiata were investigated over the course of two years in the North Island of New Zealand. ECM species were identified with a combined approach of morphological and molecular (restriction fragment length polymorphism (RFLP) and DNA sequencing) analysis. ECM species richness and diversity of a nursery in Rotorua, and stands of different ages (1, 2, 8, 15 and 26 yrs of age at time of final assessment) in Kaingaroa Forest, were assessed above- and belowground; furthermore, the correlation between the above- and belowground ECM communities was assessed. It was found that the overall and stand specific species richness and diversity of ECM fungi associated with the exotic host tree in New Zealand were low compared to similar forests in the Northern Hemisphere but similar to other exotic plantations in the Southern Hemisphere. Over the course of this study, 18 ECM species were observed aboveground and 19 ECM species belowground. With the aid of molecular analysis the identities of Laccaria proxima and Inocybe sindonia were clarified. In the aboveground study, five species were found associated with P. radiata that were previously not reported with this host in New Zealand (Inocybe sindonia, Lactarius rufus, Lycoperdon gunii, Rhizopogon pseudoroseolus and Wilcoxina mikolae). Belowground, the species Psudotomentella sp., P. tristis, R. luteorubescens, Tomentella sp., Wilcoxina mikolae were found as new associates of P. radiata in New Zealand, additionally nine ECM types were found that could not be identified with molecular analysis. There was little correlation between the species fruiting and the species colonising root tips. Only seven species were found in common between the above- and belowground communities, furthermore the dominant species aboveground were not observed in the belowground ECM communities. The influence of host age on the above- and belowground ECM communities of different age classes of P. radiata plantations was investigated. The aboveground species richness increased from the nursery to the oldest age group investigated (26 yrs), while diversity increased to the 15 yr old age group and decreased slightly to the oldest stand. A clear sequence of ECM species changes was observed to be related to stand age with a growing complexity over the chronosequence. The belowground ECM communities showed a different picture and richness and diversity initially decreased from the nursery to the outplanting but increased thereafter. Belowground no change in ECM composition that was directly related to the age of the host was observed, but two distinct groups of ECM species were found – a 'young' and a 'plantation forest' group, with the respective discriminating species being Rhizopogon rubescens and Type unknown Basidiomycete/Amanita muscaria. Another aspect of the study was the fate of the nursery ECM species in the outplanting and the arrival of non-nursery species. The ECM communities of seedlings in the nursery were investigated in 2006 and these seedlings were followed up over eight assessments in the field for one year, furthermore data from the 1-, 2 and 8 yr old plantation stands was analysed. It was found that the nursery species do survive the first year of outplanting and are dominant in the first year. The first non-nursery species occurred six months after outplanting but was only in minor abundance. Nursery ECM were dominant for two years after the seedlings were planted, and were completely replaced after seven years. Rhizopogon rubescens was found to be the most persistent and dominant species in the outplanting, facilitating the successful establishment of the seedlings in the plantation forest.

Macrophages are leukocytes of the innate immune system that display great phenotypic plasticity to mediate diverse functions. The ontogeny of tissue resident macrophages has been debated in recent decades. It is now recognised that tissue macrophages can be replenished from embryonically-derived precursors, and/or monocyte intermediates in a tissue specific manner. Interferon Regulatory Factor 5 (IRF5) is a transcription factor that promotes a pro-inflammatory phenotype in macrophages in vitro and in vivo. Indeed, IRF5 contributes to the pathogenesis of experimental inflammatory arthritis, lupus, and obesity via recruitment and activation of effector cells. Research described here as part of this thesis, involves the profiling of the intestinal Mononuclear Phagocyte system to investigate the role of IRF5 in the development of monocyte-derived macrophages in the Colonic Lamina Propria (cLP) which are exclusively replenished by adult Ly6C^hi monocytes. Using Mixed Bone Marrow Chimaeras (MBMCs) we showed that in shared environment Wild-Type (WT) cLP macrophages dominated IRF5-deficient (Irf5^-/-) cLP macrophages in both steady state and inflammation. The development of in vitro bone marrow derived macrophages, and the reconstitution of the haematopoietic compartment in bone marrow of MBMCs were not significantly affected by IRF5 deficiency. IRF5 promoted the accumulation of WT monocytes in the cLP of MBMCs in a process possibly dependent on the CCL2/CCR2 axis. Furthermore, IRF5 expression committed Ly6C^hi monocytes to a pro-inflammatory macrophage fate in the inflamed cLP, characterised by protein expression of the cytokines IL1β, and TNFα, and the expression of Ccl4 and Ccl8 transcripts, whilst loss of IRF5 favoured accumulation of CD11b⁺ IRF4-dependent Dendritic Cells. Of significance, IRF5 expression might have prevented further differentiation of inflammatory macrophages into tissue-resident macrophages, thus supporting an inflammatory state. Irf5-/- mice were protected from Helicobacter hepaticus + αIL10R colitis. Intriguingly, protection from colitis may also be conferred by the presence of Irf5-/- haematopoietic cells, evidenced by WT:Irf5-/- MBMCs . Modulation of IRF5 activity may therefore be a viable therapeutic strategy. RNA sequencing identified that C1q, Cd81, and Ccl8 were upregulated in WT macrophages from MBMC, which may prove therapeutic targets.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A implantação da cultura do feijão no sistema de plantio direto vem crescendo em todo território nacional, entretanto, o plantio direto exige uma adubação nitrogenada diferenciada, tornando-se necessário buscar mais informações a esse respeito. Nesse contexto, o trabalho foi desenvolvido em um LATOSSOLO VERMELHO Distrófico (LVd), em Selvíria - MS, com o objetivo de verificar o efeito da aplicação de diferentes níveis de nitrogênio em cobertura (0, 30, 60, 90, 120 e 150 kg ha-1), na cultura do feijão de inverno em sistema de plantio direto, após diferentes coberturas vegetais (milho, milheto, arroz, mucuna-preta, soja, Crotalaria juncea e milho + mucuna-preta). O delineamento experimental foi o de blocos casualizados em um esquema de parcelas subdivididas, com 4 repetições. Foram avaliadas: cobertura vegetal da cultura anterior, matéria seca das plantas, população de plantas, componentes produtivos (número de vagens e de grãos por planta, número de grãos por vagem e massa de 100 grãos), N total na parte aérea e nos grãos e, produtividade de grãos. Concluiu-se que A Crotalaria juncea e mucuna-preta proporcionaram a melhor cobertura do solo no período avaliado, com a Crotalaria juncea apresentando a maior quantidade de matéria seca (15.201 kg ha-1), o que é um fator importante no sistema de plantio direto; as diferentes coberturas vegetais não influenciaram significativamente a produtividade do feijoeiro cultivado em sucessão; a aplicação de doses crescentes de nitrogênio em cobertura aumentou a produtividade do feijoeiro, entretanto mesmo onde não houve aplicação do fertilizante a produtividade foi superior a 2000 kg ha-1, de forma que uma análise econômica passa a ser um fator importante para a tomada de decisão.
The no tillage system in common bean is improving in Brazil, however, there is controversy about sidedressing nitrogen fertilization, so is need more information about it. In this context, the experiment was carried out on a Typic Haplustox, in Selvíria, MS, Brazil, with the objective of evaluating the sidedressing nitrogen effects (0, 30, 60, 90, 120 and 150 kg ha-1), in common bean on no tillage system under different crop residues (corn, millet, rice, black velvet bean, soybean, crotalaria and corn + black velvet bean). The used design consisted of randomized blocks in a split-plot scheme and four replications. Crop residues, number of days to the bloom, dry matter weight, stand, number of pod and seeds per plant, number of seeds per pod, weight of 100 seeds, N in leaves and seeds and yield were evaluated. The results showed that: the Crotalaria juncea and black velvet bean provided the most crop residue, and the Crotalaria juncea with a highest dry matter weight (15.201 kg ha-1), it is a important factor on the no tillage system; there was not crop residues influence under common bean yield; the sidedressing nitrogen application increased the production, however where there was not N application the yield were higher to 2000 kg ha-1, with the economic analysis is the important to decision.

Orientador: Reginaldo Palazzo Junior
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Eletrica
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Resumo: A combinação das técnicas de espalhamento espectral, entrelaçamento e correção de erros provê aos sistemas de comunicações efetivos ganhos de processamento quando estes mesmos sistemas estão sujeitos à interferência intencional. Fundamentado nesta premissa, este trabalho apresenta a partir do modelo de um sistema de comunicações sob interferência intencional um pacote computacional para a realização de simulações e conseqüentemente possibilitando efetuar análises de desempenho e comparações das diferentes estratégias de combate à interferência intencional. Em particular serão analisados os efeitos da interferência intencional do tipo pulsada no sistema de comunicações usando a técnica de seqüência direta com relação ao espalhamento espectral, um entrelaçador do tipo bloco e um código cíclico
Mestrado
Mestre em Engenharia Elétrica

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O controle de pragas da soja representa até 36,50% do custo de produção. As lagartas desfolhadoras podem comprometer a produção de soja causando desfolhas de até 100%. A dificuldade de controle, associada ao aumento populacional e seriedade dos danos causados colocam a subfamília Plusiinae em destaque nacional, sendo mais abundante a espécie Pseudoplusia includens (Walker). Portanto, objetivou-se com este trabalho estudar a distribuição espacial da P. includens em dois sistemas de cultivo de soja, bem como, elaborar um plano de amostragem sequencial para a praga. Para monitoramento dos insetos foram realizadas coletas semanais através do método do pano de batida. Estudou-se a distribuição espacial do inseto, através de índices de dispersão e testes de ajuste às principais distribuições de probabilidade. Testaram-se os ajustes às distribuições de Poisson e binomial negativa. Os valores dos índices de dispersão, para lagartas de P. includens, em sistema de plantio convencional de soja, indicaram distribuição agregada para as duas categorias de lagartas (pequenas e grandes). Os resultados na área com sistema de plantio direto foram semelhantes. Desta forma, é possível afirmar que lagartas de P. includens apresentaram disposição agregada no campo independentemente do sistema de plantio. Lagartas de P. includens apresentam disposição agregada no campo independentemente do sistema de plantio. O ajuste do número de lagartas coletadas à distribuição de probabilidades binomial negativa possibilitou a elaboração de plano de amostragem sequencial para monitoramento da praga independentemente do sistema de plantio. O número mínimo de unidades amostrais necessário para tomada de decisão é quatro
The soybean pest control represents to 36.50% of the cost of production. The defoliating caterpillars may compromise the production of soy causing defoliation of up to 100%. The difficulty of control, with increased population and seriously damage the subfamily Plusiinae put into national prominence, being the most abundant species Pseudoplusia includens (Walker). Therefore, the aim of this work was to study the spatial distribution of P. includens in two systems of land use, as well as develop a sequential sampling plan for the pest. The insects were collected weekly by the ground cloth method. The spatial distribution of the insect was studied, using dispersion indices and testing the main probability distributions. Tested the adjustments to the distributions of Poisson and negative binomial. The values of aggregation index to P. includens larvae in conventionally tilled soybean indicated aggregated distribution for the two categories of larvae (small and large). The results in the area with no-tillage system were similar. Caterpillars of P. includens present aggregate provision in the field regardless of tillage system. Adjusting the number of caterpillars collected the negative binomial probability distribution enabled the development of sequential sampling plan for monitoring the pest regardless of tillage system. The minimum number of sample units required for decision making is four

Dissertação para obtenção do Grau de Mestre em Engenharia Geológica (Georrecursos)
Este trabalho apresenta uma metodologia para caracterizar espacialmente a contaminação em hidrocarbonetos totais do petróleo (Total Petroleum Hydrocarbons - TPH) no local de um antigo parque de combustíveis. A metodologia proposta foi desenvolvida com algoritmos de estimação e simulação geoestatística 3D e integra informação principal e secundária. Foram utilizados dados de 131 sondagens de uma anterior campanha de prospecção, para uma área de aproximadamente 14,5ha. As variáveis secundárias são apresentadas por classes(variável categórica) relativamente a três análises expeditas macroscópicas e sensoriais ao solo recolhido das sondagens. Ao todo foram analisados e codificados 820 troços, que constituem o comprimento total das sondagens. Dos 820 troços, 276 foram enviados para análise laboratorial tendo sido reportados os teores em TPH (informação principal). Comparativamente aos dados laboratoriais, os dados das análises sensoriais são muito expeditos e, por isso, mais abundantes; todavia têm um elevado grau de subjectividade na discriminação da contaminação. Pelo contrário, as determinações analíticas são menos expeditas e muito mais caras, mas os resultados são mais fiáveis do ponto de vista de identificar a contaminação. A metodologia proposta desenvolve-se em três etapas. Em primeiro lugar, procedeu-se à análise estatística preliminar para determinar qual das análises sensoriais e respectivas classes(cor, cheiro e reacção ao óleo) seriam mais adequadas para discriminar os teores em TPH. De seguida, fez-se a estimação 3D das classes da variável seleccionada por krigagem da indicatriz. Finalmente, na terceira etapa, faz-se a simulação dos teores em TPH. Na simulação utilizou-se uma variante inovadora do algoritmo de simulação sequencial directa com condicionamento a histogramas e médias locais. A metodologia proposta foi validada com um teste de validação cruzada, construído a partir de um subconjunto aleatório de 20% das amostras.

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Osteogenesis Imperfecta (OI) is a genetic desease characterized by patient s bone fragility and deformity, in which severity ranges from a barely detectable connective tissue disorder to lethality in the perinatal period. The diversity of clinical variability in patients is caused by the different location or type of mutations in one of the ten genes related with the disease. This wide clinical variability difficults the perfect clinical diagnoses, due to that the use of molecular biology techniques becomes necessary to obtain a correct diagnoses and for genotype: phenotype correlation. One of the relevant genes associated with recessive forms of OI is the LEPRE-1 gene, responsible for encoding the prolyl 3 hidroxylase 1 protein. This protein and two others are components of the complex responsible for pro-collagen alfa 1 chains 3 prolyl hydroxylation. The target of this research was to analyze the LEPRE-1 gene in eight non consanguineous patients clinically diagnosed as severe Osteogenesis Imperfecta suggestive of autossomic recessive heritage by DNA sequencing of exons 1, 3, 5, 6 and 14 of the gene. In addition, the data obtained was used to analyze the efficiency of the SSCP technique by comparing the results between screening for mutations methodologies and gene sequencing methodologies. On exon 6, for instance, a mutation in one patient was found: a heterozygose base change (c.1087A>G / p.Lys363Glu), consequently, lysine was produced instead of glutamic acid. On the other exons, there was no mutation found on the patients chosen. All the results obtained in this research were compatible with datas generated by SSCP and suggest high efficient of SSCP technique for LEPRE-1 gene to recessive cases of Osteogenesis Imperfecta
A Osteogênese Imperfeita é uma doença genética caracterizada por fragilidade e deformidade esquelética, onde o quadro clínico pode variar desde simples deformidades ósseas à forma letal perinatal. A alta variabilidade clínica apresentada pelos indivíduos afetados ocorre devido ao tipo e à localização da mutação em um dos dez genes relacionados a doença. A grande heterogeneidade genética existente exige a utilização de técnicas da biologia molecular para o diagnóstico e compreensão das correlações genótipo: fenótipo da doença. Um dos genes relevantes associados com as formas recessivas da Osteogênese Imperfeita é o gene LEPRE-1 codificador da proteína prolil 3 hidroxilase 1. Esta é uma das três proteínas componentes do complexo responsável pela prolil 3 - hidroxilação das cadeias de pró-colágeno alfa 1 formadoras da molécula do colágeno tipo I, expresso, predominantemente em ossos, tendões e pele. Este projeto de pesquisa teve como objetivo analizar o gene LEPRE-1 em oito pacientes não consanguineos com Osteogênese Imperfeita tipo grave sugestivos de herança autossômica recessiva por meio do sequenciamento direto dos exons 1, 3, 5, 6 e 14 do gene. Além disso, o resultado gerado foi utilizado para avaliar a eficiência da técnica de triagem de mutações por Polimorfismo Conformacional de Fita Simples (SSCP) por meio da comparação de resultados entre as metodologias de triagem de mutações e sequenciamento direto do gene. Foi identificada, no exon 6, uma mutação de troca de nucleotídeos em heterozigose, c.1087A>G / p.Lys363Glu, levando a produção do aminoácido ácido glutâmico ao invés da lisina em um dos pacientes avaliados. Não foram encontradas mutações em nenhum dos pacientes para os demais exons analisados. Estes resultados corroboram dados gerados por meio de SSCP, e sugerem grande eficiência da técnica de triagem para o gene LEPRE-1 para formas recessivas de Osteogênese Imperfeita

Mestrado em Engenharia Geológica
Os modelos de subsuperfície inferidos a partir de dados de reflexão sísmica permitem avaliar espacialmente as propriedades petro-elásticas de reservatório (por exemplo, densidade, impedâncias acústica e elástica) que são essenciais para uma boa caracterização e modelação de reservatórios de hidrocarbonetos. Os modelos invertidos gerados a partir de metodologias geoestatísticas e da integração de dados de logs de poços com dados de reflexão sísmica, dentro de uma grelha de reservatório, permitem ainda avaliar e quantificar, localmente, a incerteza espacial associada. O principal objetivo desta dissertação de mestrado consiste na comparação e discussão dos diversos resultados obtidos através da aplicação de diferentes metodologias de inversão sísmica geoestatística com multi-distribuições e modelos de continuidade espacial locais, de forma a gerar modelos próximos da realidade, em condições de não-estacionaridade, que permitam uma melhor caracterização da geologia de subsuperfície. Dentro das várias aproximações de inversão geoestatística existentes, para a elaboração desta tese foram utilizadas a Inversão Estocástica Global e a Inversão Elástica Global recorrendo ao algoritmo de simulação sequencial direta para a geração dos modelos elásticos de sub-superfície. Esta tese baseia-se na realização de 6 ensaios distintos, para cada uma das metodologias de inversão referidas, segundo multi-distribuições e modelos de continuidade locais (zonalidades) previamente definidas a um conjunto de dados sintéticos altamente não estacionários. No conjunto de dados disponíveis foram integrados dados de impedâncias (acústica e elástica) de 32 poços, bem como dados de reflexão sísmica post-stack. Os resultados mostram que a divisão da malha do reservatório em diferentes zonas tem um grande impacto na convergência dos diferentes métodos de inversão geoestatística utilizados no âmbito desta tese.
The subsurface models inferred from seismic reflection data allow the spatial evaluation of the petro-elastic properties of the reservoir (e.g., density, elastic and acoustic impedances) that are essential for a good characterization and modeling of hydrocarbon reservoirs. The generated inverted models from geostatistical methodologies and the integration of well log data with seismic reflection data, within a reservoir grid, allow to assess and quantify, locally, the associated spatial uncertainty. The main objective of this MSc dissertation is the comparison and discussion of the results obtained from applying different geostatistical seismic inversion methodologies, with local multi-distributions and spatial continuity models, to generate models closer to reality, under non-stationarity conditions, that allow a better characterization of the subsurface geology. From the several existing geostatistical inversion approaches, for the realization of this thesis the Global Stochastic Inversion and the Global Elastic Inversion were selected, using the direct sequential simulation algorithm to generate the subsurface elastic models. This thesis is based on the realization of 6 different trials for each of the inversion methodologies, under local multi-distributions and continuity models (zonality) predefined in a highly non-stationary synthetic dataset. In the available dataset impedance (acoustic and elastic) data from 32 wells and post-stack seismic reflection data were integrated. The results show that the division of reservoir grid into different zones has high impact on the convergence of the different inversion geostatistical methods used within this thesis.

Yams are an important food source grown throughout the Pacific but the international movement of the crop is restricted due to a lack of understanding of the viruses infecting these plants. This study focused on identifying and characterising badnaviruses infecting yams and the subsequent development of sensitive and reliable diagnostic tests. These tests can be used in virus-screening programs to enable the safe international movement of virus-free yam germplasm thus contributing to food and nutritional security of Pacific Island Countries.

碩士
國立臺灣大學
化學研究所
84
Breast cancer is the most common malignancy cancer of women in Western countries. The mortality rate have held almost steady over the past 20 years, even though the number of new cases has grown. From 1970 to 1990, breast cancer is an important cancer of women in Taiwan and incidence of age in breast cancer trend to early onset. At molecular level, various genetic alteration have been implicated in the development of breast cancer. Loss of heterozygosity(LOH) at five different regions of human genome,including chromosome 1q, 3p, 11p, 13q and 17p, has been observed in a high percentage of primary breast cancer. Because the extremely high frequency(60%) of p53 mutation in primary breast cancer of Western countries, we will focus on mutations of the p53 tumor suppressor gene in breast cancer of Taiwan. First, we use immunohistochemical analysis to determine whether p53 gene expression in 171 breast cancer specimen of Taiwan can be detected. Second,the validity of p53 immunopositivity as an indicator for p53 mutation was verified using two molecular assays of p53 mutation: Single Strand Conformation Polymorphism(SSCP) and direct DNA sequencing. We have found p53 mutations in 26.9% of the breast cancer specimen in Taiwan. In addition, the mutation patterns of p53 gene mainly locate at exon 7∼8 and G to A (22%) transition is the major type of nucleotide changes. But in Western countries, the mutation patterns of p53 gene major locate at exon 5∼6 and C to G (20%) transversion is the major type of nucleotide changes. The spectrum of mutation in breast cancer of Taiwan differs from that of Western countries, further indicating that unknown carcinogens involve in the formation of breast cancer in Taiwan.

碩士
國立成功大學
藥理學研究所
85
p53是一種癌症抑制基因(tumor supressor gene)，在人類常見的癌症當 中，可發現p53基因的等位基因缺失(allelic deletion)或點突變(point mutation)所造成的功能喪失。當DNA受到了損傷之後，p53蛋白的量在細 胞內急遽增加，隨之使細胞週期停留在G1期；細胞若包含突變的p53或缺 乏p53則不能誘導此種細胞週期停止。此外，突變的p53基因使細胞進行計 畫性細胞自殺(apoptosis)的能力降低，因此癌細胞內的p53基因若突變則 其對化學療法或放射線療法將具有抵抗性。在與其他種致癌基因比較之下 ，p53基因的突變或缺失是在乳房癌中最常見的基因變化。因此，若能建 立p53基因與乳房癌惡化程度之間的關係，將有助於診斷乳房癌或者評估 其在治療後復發的可能性。本實驗的目的在於偵測不同病期的乳房癌中其 p53基因突變的情形，藉此來推斷乳房癌的預後(prognosis)。我們首先利 用去氧核醣核酸單股結構多型性(single-stand conformation polymorphism)，偵測涵蓋了p53之exons 5~9的部分，篩選出具有突變的 片段，再據此進行直接循環去氧核糖核酸定序(direct-cyclic DNA sequencing)以建立p53基因在乳房癌中突變的圖譜。經篩選及定序之後， 我們發現了三個病人在exon 5的位置產生了突變。編號33的病人，其p53 基因的第175個密碼子(codon)由CGC變為TGC，即胺基酸由原本的arginine 變為cysteine。編號36的病人其p53基因第175個密碼子由原本的CGC變為 CAC，即胺基酸由正常的arginine變為histidine。編號54的病人p53基因 的第138個密碼子則由原本的GCC變為CCC，即胺基酸由正常的alanine變為 proline，而以上三者均屬於錯譯突變(missense mutation)，並且其第一 個等位基因發生突變後伴隨著第二個等位基因的缺失，此現象稱之為loss of heterozygosity，是p53基因常被發現的現象之一。若想根據以上三個 p53突變之病人，來找出其病程與預後之關係，則尚無法確切地下結論。 其原因為此三人目前仍繼續在門診，其中雖有一人有癌症轉移之現象，但 代表性不夠，故仍需進一步的觀察。 p53 is a tumor supressor gene whose inactivation, either by allelic deletion or point mutation, is one of the most common genetic changes in human malignancies. The P53 protein level is strongly increased after experimental DNA damage and this is followed by a specific arrest of the cell cycle in G1 phase. Cells containing mutated p53 or lacking P53 are unable to induce this cell cycle arrest; furthermore, these cells would have a reduced capacity to induce apoptosis after DNA damage incurred by ionizing radiotherapy or some therapeutic drugs, suggesting that cancer cells containing mutated p53 are more resistant to radiotherapy or chemotherapy. Compared with the other oncogenes, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer. Therefore, p53 gene mutations coupled with the disease status could provide an invaluable tumor marker for diagnosis and/or prognosis of breast neoplasm. This research project aims at detecting p53 gene mutations from breast tumor samples of various neoplastic stages and with these information to infer the prognosis of breast cancer. We first obtained genomic DNA from archival and frozen-breast cancer tissues, then amplified exons 5,6,7,8 and 9 of p53 gene by the method of polymerase chain reaction (PCR). We used single-strand conformation polymorphism (SSCP) as a means to prescreen mutations in some specific exon fragments. Eleven variant fragments was identified by their band shifting following polyacrylamide gel electrophoresis (PAGE). Those variants are subjected to direct-cyclic sequencing so as to establish the mutation spectrum of p53 gene. Out of 11 SSCP variants, 3 showed nucleotide mutations upon sequencing. In patient number 33, codon 175 was changed from CGC to TGC, and patient number 36 has the same mutation site in codon 175: CGC ->CAC. Patient number 54 has mutation in codon 138: GCC->CCC. In agreement with other studies, these point mutations are believed to lead to changes in the conformation of p53 protein and loss of its normal functions.

La demenza è una sindrome clinica caratterizzata da declino cognitivo le cui principali manifestazioni comprendono la perdita di memoria, deficit del linguaggio, deterioramento delle capacità di pianificazione e di giudizio, cambiamenti di comportamento e, non di rado, compromissione delle funzioni motorie ed infine dell'autonomia. Oggigiorno, si stima che circa 36 milioni di persone in tutto il mondo siano affette da demenza, stime che, secondo le ultime predizioni, è destinata a crescere fino a 115 milioni nel 2050. Considerando non solo la prevalenza bensì anche l'impatto sociale ed economico che questa malattia comporta, negli ultimi anni la ricerca scientifica sta investendo molte risorse in studi finalizzati all'identificazione delle cause e dei meccanismi patologici che sottendono a questa malattia. Lo scopo ultimo è quello di riuscire ad identificare dei trattamenti terapeutici efficaci che vadano a sostituire le cure palliative, le uniche disponibili oggi per questa malattia. Esistono diverse forme di demenza e, tra esse, la Malattia di Alzheimer (MA) è la più frequente, seguita dalla Demenza Vascolare (DV) e dalla Degenerazione Frontotemporale Lobare (FTLD), la quale, rappresenta il sottotipo più comune dopo la MA negli individui al di sotto dei 65 anni. Tutti e tre questi disordini possono presentare una componente genetica e, malgrado i notevoli investimenti e progressi degli ultimi anni per chiarirne le basi molecolari, la conoscenza dei meccanismi patologici che li determinano è ancora limitata. Tre forme di demenza, che possono avere una componente genetica, sono state incluse in questo studio: la Malattia di Alzheimer, la Degenerazione Frontotemporale Lobare e l'Arteriopatia Cerebrale Autosomica Dominante con Infarti Sottocorticali e Leucoencefalopatia (CADASIL), quest'ultima rappresentante un sottotipo di DV. I principali obiettivi di questo studio hanno compreso l'analisi mutazionale dei geni associati a queste malattie con il duplice intento di definirne le frequenze mutazionali in una casistica italiana e di valutare l'esistenza di eventuali correlazioni genotipo-fenotipo. Lo scopo finale era quello di definire degli algoritmi diagnostici utili ai medici specialisti nella selezione dei pazienti da sottoporre ad indagine genetica. Sono state allestite due coorti: una comprendente pazienti con diagnosi di MA, FTLD o disordini correlati, e l'altra, pazienti con CADASIL. L'analisi mutazionale è stata eseguita mediante due principali approcci: la Cromatografia Liquida Denaturante (DHPLC) ed il sequenziamento diretto. Sono stati analizzati i seguenti geni: proteina Precursore dell'Amiloide (APP), Presenilina1 (PSEN1) e Presenilina2 (PSEN2) per la Malattia di Alzheimer; Progranulina (PGRN), Proteina Tau Associata ai Microtubuli (MAPT), Proteina Contenente Valosina (VCP), Proteina di Modificazione della Cromatina 2B (CHMP2B), Proteina Legante TAR DNA (TARDBP) per la Degenerazione Frontotemporale Lobare ed il gene NOTCH3 per il CADASIL. L'analisi mutazionale dei geni associati a MA e FTLD ha portato all'identificazione di 8 mutazioni: 3 nuove variazioni, PSEN1 p.Ile437Asn, PSEN2 p.Thr18Met e PGRN p.His400ThrfsX12 e, cinque sostituzioni già descritte, PSEN2 p.Arg71Trp e p.Met174Val, PGRN p.Phe86SerfsX170 e p.Thr272SerfsX10 e MAPT c.IVS10+16C>T. I risultati delle analisi molecolari e delle simulazioni al computer depongono a favore di una natura patogenetica per le variazioni dei geni Presenilina, malgrado il ruolo di alcune di esse sia dibattuto in letteratura. Le sostituzioni identificate nei geni PGRN e MAPT sono mutazioni il cui meccanismo patologico è già stato descritto. Il fenotipo clinico riscontrato nei pazienti con le mutazioni identificate non era univoco e, specialmente le mutazioni nella PGRN erano associate ad una notevole variabilità delle manifestazioni cliniche, anche all'interno di una stessa famiglia. Inoltre, l'identificazione di una famiglia con diagnosi di FTD-MND in linkage al cromosoma 9 sottolinea ulteriormente l'eterogeneità genetica delle FTLD. L'analisi mutazionale del gene NOTCH3 ha portato all'identificazione di 21 diverse mutazioni, incluse 7 nuove variazioni, distribuite non uniformemente lungo il gene. Si è osservata una clusterizzazione delle mutazioni in aree geografiche diverse: la maggior parte identificate in pazienti provenienti dal nord-est Italia, alcune nel nord-ovest ed altre nell'Italia centrale. Nell'ipotesi che questa regionalizzazione rispecchiasse un effetto fondatore, si sono studiati gli aplotipi associati a queste mutazioni: i risultati, sebbene compatibili, non hanno permesso di confermare l'effetto fondatore in quanto la maggior parte di queste variazioni cadeva nell'aplotipo più frequente. I risultati di questo studio, complessivamente sono stati utili per definire degli algoritmi diagnostici che potrebbero aiutare i clinici ad identificare i pazienti, suggestivi di una base molecolare, da sottoporre ad analisi genetica.
Dementia is a clinical syndrome associated with progressive deterioration of intellectual functions including loss of memory, difficulties with language, simple calculations, planning and judgment, and motor skills with eventually loss of autonomy. Currently there are an estimated 36 million people worldwide with dementia and this figure is set to increase to more than 115 million people by 2050. Given the prevalence of dementia and the associated significant financial and human costs, in recent years there has been a huge burst of studies aimed to identify the causes of this disorder and its underlain pathological mechanisms, in order to define therapeutic treatments to replace the nowadays available palliative cares. Among different subtypes of dementia, Alzheimer's Disease (AD) is the most frequent form, followed by Vascular Dementia and Frontotemporal Lobar Degeneration (FTLD) which represents the second most common form in people younger than 65 years. All three of these diseases may have a genetic component and, despite considerable progress and efforts made in recent years to clarify their molecular basis, little is known about the pathological mechanisms determining these diseases. Three forms of dementia, which may have a genetic component, were included in this study: Alzheimer's Disease, Frontotemporal Lobar Degeneration and Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leuokoencephalopathy (CADASIL) which is a subtype of vascular dementia. The main objectives of this study included mutational analysis of the genes associated with these diseases with the dual purpose of defining their mutational frequencies in an Italian series and to evaluate the presence of possible genotype-phenotype correlations. The secondary purpose is to be able to draw diagnostic algorithms useful to clinicians in selecting which patients submit to genetic testing. Two clinical cohorts were set up; one consisting in patients with diagnosis of AD, FTLD or related disorders, and the other made up of patients with CADASIL. The mutational analysis was performed by two main techniques: Denaturing High Performance Liquid Chromatography and direct sequencing. The analyzed genes encompassed: Amyloid Precursor Protein (APP), Presenilin1 (PSEN1) and Presenilin2 (PSEN2) for Alzheimer's Disease; Progranulin (PGRN), Microtubule Associated Protein Tau (MAPT), Valosin Containing Protein (VCP), Charged Multivesicular Body Protein 2B (CHMP2B), TAR DNA Binding Protein (TARDBP) for Frontotemporal Lobar Degeneration and NOTCH3 for CADASIL. The mutational analysis of AD- and FTLD-associated genes led to the identification of 8 mutations: three novel variations, PSEN1 p.Ile437Asn, PSEN2 p.Thr18Met and PGRN p.His400ThrfsX12 and five already described substitutions, PSEN2 p.Arg71Trp and p.Met174Val, PGRN p.Phe86SerfsX170 and p.Thr272SerfsX10 and MAPT c.IVS10+16C>T. The molecular data and in silico analyses performed in this study argue in favour of pathogenetic nature for Presenilins variations even though the role of some of them is debated in literature. The substitution identified in PGRN and MAPT are mutations whose pathogenic mechanism has already been described. The clinical phenotype associated to identified mutations was not unique and, especially mutations in the PGRN gene showed a marked variability in clinical presentations, even within the same family. Furthermore, the identification of a FTD-MND family linked to a locus on chromosome 9 further emphasizes the genetic heterogeneity of FTLD. The mutational screening of NOTCH3 gene led to the identification of 21 different mutations, including 7 novel variations, distributed unevenly along the gene. A geographical clustering was observed with mutations identified only in patients living in North-East Italy, a few in North-West and other in Central Italy. Haplotype analysis was performed to assess a possible founder effected underlying this regionalization but, although consistent, it was not confirmed as the majority of mutations was associated with the most common haplotype. The results of this study together were useful to define diagnostic algorithms that could help clinicians to identify patients suggestive of a molecular basis of disease to address to genetic testing.

Ranunculus trichophyllus agg. (thread-leaved water crowfoot) represents a taxonomically challenging group of aquatic plants in which the presence of several significantly different genotypes and the genome size variation have been recently revealed. The results of previous studies suggest that cryptic taxa occur in this group, being so far overlooked due to considerable morphological reduction and extensivephenotypic plasticity. In this thesis, the variation and genetic relationships of four morphologically similar homophyllous water-crowfoot species was critically assessed in the area of Central Europe, using a combination of modern biosystematic methods (flow cytometry, direct DNA sequencing, morphometric analyses), specially focusing on the complex of R. trichophyllus.. The genome size analysis via flow cytometry was confirmed as a suitable method for determining the studied species; further, several hybrid combinations were revealed using this approach. However, recent interspecific hybridization is rather infrequent in the interest group. The results of DNA analyses indicate an importance of hybridization events in the evolution of sect. Batrachium: all the polyploid taxa studied are probably of allopolyploid origin. Two cryptic taxa within the traditionally recognized species R. trichophyllus have...

abstract: ABSTRACT Whole genome sequencing (WGS) and whole exome sequencing (WES) are two comprehensive genomic tests which use next-generation sequencing technology to sequence most of the 3.2 billion base pairs in a human genome (WGS) or many of the estimated 22,000 protein-coding genes in the genome (WES). The promises offered from WGS/WES are: to identify suspected yet unidentified genetic diseases, to characterize the genomic mutations in a tumor to identify targeted therapeutic agents and, to predict future diseases with the hope of promoting disease prevention strategies and/or offering early treatment. Promises notwithstanding, sequencing a human genome presents several interrelated challenges: how to adequately analyze, interpret, store, reanalyze and apply an unprecedented amount of genomic data (with uncertain clinical utility) to patient care? In addition, genomic data has the potential to become integral for improving the medical care of an individual and their family, years after a genome is sequenced. Current informed consent protocols do not adequately address the unique challenges and complexities inherent to the process of WGS/WES. This dissertation constructs a novel informed consent process for individuals considering WGS/WES, capable of fulfilling both legal and ethical requirements of medical consent while addressing the intricacies of WGS/WES, ultimately resulting in a more effective consenting experience. To better understand components of an effective consenting experience, the first part of this dissertation traces the historical origin of the informed consent process to identify the motivations, rationales and institutional commitments that sustain our current consenting protocols for genetic testing. After understanding the underlying commitments that shape our current informed consent protocols, I discuss the effectiveness of the informed consent process from an ethical and legal standpoint. I illustrate how WGS/WES introduces new complexities to the informed consent process and assess whether informed consent protocols proposed for WGS/WES address these complexities. The last section of this dissertation describes a novel informed consent process for WGS/WES, constructed from the original ethical intent of informed consent, analysis of existing informed consent protocols, and my own observations as a genetic counselor for what constitutes an effective consenting experience.
Dissertation/Thesis
Ph.D. Biology 2013

This study aimed to optimise Oenococcus oeni for more efficient malolactic fermentation in wine with multiple stressors. First, a previously evolved ethanol tolerant strain, A90, was characterised for resistance to combined pH and ethanol stress in both MRSAJ and Red Fermented Chemically Defined Grape Juice Medium (RFCDGJM). A90 showed a similar viability in RFCDGJM compared to its parent, SB3, indicating the need for further improvement. With the success of the previous proof-of-concept directed evolution (DE) in O. oeni, a new DE was carried out to determine 1) if DE can be applied to further improve A90 in a wine-like environment using combinations of stressors to generate more superior strains with better general stress resistance; 2) how much further can A90 be developed, and how stable the new phenotype would be; 3) possible new patterns of stress response through study of the genetic basis for the superior phenotype. A continuous culture of A90 was established in a bioreactor and grown in a wine-like environment for approximately 350 generations with increasing ethanol and sulfur dioxide (SO₂), and decreasing pH over time. Samples of the population in the bioreactor were collected at three significant times during the DE to screen for improved isolates based on L-malic acid consumption and growth. Three strains, namely 1-161, 2-49 and 3-83, outperformed from a total of 378 isolates. With a view to applying these strains to the industry, in-depth physiological characterisations were undertaken. Aspects examined included tolerance to various oenologically related stressors such as ethanol, pH, SO₂ and medium chain fatty acids, as well as phenotype stability and fermentation ability under more realistic winemaking conditions, i.e. un-filtered wine and winery scale fermentation. Overall, 2-49 and 3-83 constantly displayed better growth and malolactic activity than the parent strain A90 in either lab-scale or winery-scale trials. Finally, whole genome sequencing of strains SB3, A90, 2-49 and 3-83 and genetic characterisation were utilised to investigate changes during DE in O. oeni. A total of 19 single nucleotide polymorphisms (SNPs) were found in 2-49 and 3-83 strains compared to A90. The SNPs identified may affect cell envelope and fatty acids biosynthesis, DNA translation and homeostasis of internal pH, leading to the improved performance of DE strains. Sequences were also compared to the available sequence for commercial strain VP41. Several mutations were identified in stress response genes, indicating VP41 and SB3-related strains might have different responses to stressors. SNPs in the predicted mleA promoter sequence may suggest a new mechanism of MLF activation. Additionally, Nucleotide BLAST was used to analyse the presence of genes with oenological traits in SB3-related strains. Genes associated with the release of desirable aromas were found, whilst genes involved in the formation of biogenic amines were absent. This study expands the knowledge regarding optimisation of O. oeni, and may be helpful for the further improvement of food-related microbes with enhanced performance.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2017.

The aetiological diagnosis of recessive non-syndromic hearing loss poses a challenge owing to marked heterogeneity and the lack of identifying clinical features. The finding that up to 50% of recessive non-syndromal genetic hearing loss among Caucasians was due to mutations in GJB2, the gene encoding Connexin 26 (Cx26) was a breakthrough, whose value as a diagnostic tool has been limited by the significant variation in the prevalence of deafness genes and loci among population groups. The significant association of the GJB6-D13S1830 deletion among individuals with one mutant GJB2 allele highlighted the need to explore population specific genetic mutations for NSHL. Although data from Sub-Saharan Africa is limited, reported studies found a high prevalence of R143W GJB2 mutation among Ghanaian, the 35delG mutation in 5 out of 139 Sudanese and a low prevalence of GJB2 variations among 385 Kenyan deaf children. The mutation spectrum of Waardenburg Syndrome (WS) in Africans has not been documented. During a visit to a School for the Deaf in the Limpopo Province of South Africa in 1997, it was noted that a high number of students came from Nzhelele sub-district. All had childhood onset hearing loss with no associated anomalies or disorders. The question arose as to whether there was a high-risk area for deafness in the Limpopo Province and what the aetiology of this hearing loss was.The main aim of this study was to investigate the role of GJB2, the GJB6-D13S1830 deletion, and the four common mitochondrial mutations, A1555G, A3243G, A7511C and A7445G, in the African hearing-impaired population of Limpopo province in South Africa, and to identify the mutation spectrum of the deafness genes found. The type and degree of hearing loss in this hearing impaired population would also be assessed. Secondly, this study sought to identify the mutations in a sibling pair with 2 clinical WS and to use the findings in a future study to establish the mutation spectrum of WS in the African population of the Limpopo province and of South Africa in general. The study was designed as a two phase study, in which phase 1 was used for hypothesis formulation and phase 2 was for hypothesis testing. While phase 1 was a descriptive retrospective case study, phase 2 was a combination of sample survey and prospective descriptive case study. In phase 1, demographic data of 361 students in two schools of the deaf in the Limpopo province was analyzed for evidence of areas of high risk populations for deafness in the province. In phase 2, a group of 182 individuals with genetic non-syndromic hearing loss (NSHL) and two siblings with clinical WS from two schools for the Deaf in the Limpopo Province of South Africa were investigated. A thorough clinical examination, audiological evaluation and urinalysis were done. Mutational screening was carried out in all 184 subjects using genomic DNA using single-strand conformation polymorphism (SSCP), multiplex polymerase chain reaction (PCR), and direct sequencing for GJB2, and Restriction Fragment-Length Polymorphism (PCR–RFLP) analysis for GJB6, and SSCP, hetero-duplex analysis, and direct sequencing of the first 8 exons of PAX3 and all of MITF for Waarenburg syndrome. Data analysis was by geographical mapping, frequency tables, tests of association with calculation of odds ratios, and binary logistic regression analysis using STATA and GIS mapping systems. The results indicate that there seem to be areas of genuine populations at risk for hearing loss in the Limpopo province of South Africa, namely Mutale and parts of Makhado and Thulamela municipalities. In Thulamela (NP343) wards 11-15, 26-30 and 31-35, and in Mutale (NP 344) wards 6-10, together accounted for 67 (18%) of participants in phase 1, and 33 (18%) of the participants in phase 2 of the study. Mutale municipality in the Vhembe 3 district gave with a projected prevalence of at least 13.14 deaf children per 100,000 African population attending the local school for the deaf. The observed hearing loss is a genetic, non-syndromic form, which is mainly severe and severe to profound, although without any clear defining configuration or shape. It is a stable, non-progressive and prelingual form of hearing loss, implying that this may be a recessive form of deafness. No identifiable environmental confounding factors or associations were identified. The deafness is not linked the common known auditory gene mutations in GJB2, the GJB6-D13S1830 deletion, or the common mitochondrial mutations A1555G, A3243G, A7511C and A7445G. Severe and profound levels of hearing loss were found in 22.8% and 75% of the cohort respectively, with the majority exhibiting flat (70.1%) or sloping (23.4%) audiograms that were commonly symmetrical (81.5%). However, as indicated, there was no clear pattern in the audiological findings overall. None of the 184 hearing impaired individuals exhibited any of the reported disease causing mutations of GJB2, including 35delG. There was, however, a high prevalence of two variants, the C>T variant at position g.3318-15 and the C>T variant at position g.3318-34, occurring in 21.4% and 46.2% of the deaf cohort respectively. The same variants were found to occur in 35% and 42.6% of a normal hearing control group (n = 63) respectively, indicating that these variations are polymorphisms. In three subjects (1.63% of the cohort), a T>A homozygous variation at position g.3318-6 was detected. Its significance in the causation of NSSNHL is yet to be determined. The GJB6-D13S1830 deletion was not detected in any of the participants. None of the four mitochondrial mutations screened for were found. 4 These results indicate that GJB2 is not a significant deafness gene in the African population of the Limpopo Province of South Africa and that significant genes for non-syndromic recessive hearing loss in this population are yet to be found. The geographical clustering of deafness found in this study, combined with the lack of identifiable common associated clinical features among the subjects of this study (excluding the WS sibling pair), suggests that these subjects have a genetic recessive non-syndromal type of hearing loss. In the context of historical and cultural evidence of consanguinity in this population, a founder effect cannot be ruled out. A rare mutation, R223X, previously identified only once out of 470 WS patients, was identified in the PAX3 gene among the WS sibling pair. A novel silent change GGG>GGT at amino acid 293, was also identified. These identical findings document, for the first time, a molecular defect in WS in an African sibling pair, and confirm WS Type I in this family, which could be found in other WS type I South Africans in the Limpopo Province of South Africa. The current study demonstrated that parents of genetically hearing impaired children in these areas are able to detect hearing loss at an early age, with over 60% suspecting their children’s hearing loss below 6 months of age. A child-centered management model encompassing all the areas relevant to childhood deafness/hearing impairment, which takes into consideration the prevailing logistical and financial constraints of the available healthcare system, is proposed. The implementation of this model requires a paradigm shift from the current fragmented model of service delivery to a cohesive patient-centered approach, based on concrete data from appropriate community based research, in which all the relevant parties communicate and share resources. 5 It would achieve the goals of early detection and intervention, as well as inclusive education for all. The relevant health and education policies are already in place and the posts funded. Equitable implementation of these policies would require appropriate community based research, as well as improved communication and consultation between the various stakeholders to ensure an efficient and affordable quality healthcare service for all hearing impaired South Africans.

Text in English, Tswana and siSwati
This dissertation examines the multiplicity of social positions within which African women in the postcolonial era find themselves. It focuses on how the dramatic dialogue depicts the positions of women in Zakes Mda’sThe Nun’s Romantic Story, And the Girls in their Sunday Dresses and You Fool, How can the Sky Fall. The study is intended to explore the dramatic dialogue in these plays and to show whether there is any evidence of change in women’s positions. It seeks to demonstrate the extent to which the positions of women have changed and also how the dramatic dialogue in the selected plays of Zakes Mda indicates the change in women’s positions.
Thutopatlisiso eno e tlhatlhoba maemo a loago a mantsintsi a basadi ba maAforika ba ba tshelang mo motlheng wa morago ga puso ya bokoloniale ba iphitlhelang ba le mo go ona. E tota ka moo puisano ya terama e bontshang maemo a basadi ka gona mo The Nun’s Romantic Story, And the Girls in their Sunday Dresses le You Fool, How can the Sky Fall tsa ga Zakes Mda. Maikaelelo a thutopatlisiso ke go sekaseka puisano ya terama mo metshamekong eno go bontsha gore a go na le bosupi bope jwa diphetogo mo maemong a basadi. E batla go bontsha ka moo maemo a basadi a fetogileng ka gona le ka moo puisano ya terama mo metshamekong e e tlhophilweng ya ga Zakes Mda e bontshang diphetogo mo maemong a basadi ka gona.
Ledisetheshini ihlolisisa tikhundlanyenti tetenhlalo bomake base-Afrika labatitfola bakuto ngemuva kwesikhatsi sembuso webukolonali (umbusobucalu). Igcile ekutsini inkhulumomphendvulwane emidlalweni yaZakes Mda itikhombisa kanjani letikhundla tabomake; i-The Nun’s Romantic Story [Indzaba yelutsandvo yemasisitela], ne-Girls in their Sunday Dresses [Emantfombatana etingutjeni tawo teLisontfo] ne-You Fool [Wena Silima], How can the Sky Fall [Singawa kanjani Sibhakabhaka]. Lolucwaningo lwentelwe kuhlolisisa inkhulumomphendvulwane kulemidlalo kanye nekukhombisa kutsi ingabe bukhona yini bufakazi bengucuko etikhundleni tabomake. Ifuna kukhombisa kutsi tikhundla tabomake tigucuke kangakanani kanye nekutsi inkhulumomphendvulwane emidlalweni lekhetsiwe yaZakes Mda ikukhombisa kanjani kugucuka kwetikhundla tabomake.
English Studies
M.A.(Theory of Literature)

O presente trabalho tem como principal objetivo apresentar e testar uma metodologia que permita a integração de perfis sísmicos de reflexão (informação secundária ou soft) e dados de sondagens e poços (informação principal ou hard) na modelação estocástica da morfologia de horizontes geológicos. Divide-se em duas etapas principais: primeiro são geradas superfícies representativas dos horizontes, só condicionais aos dados da sísmica e, posteriormente, faz-se o condicionamento destas superfícies aos dados de sondagens e poços. Para a geração das superfícies primárias, só condicionais aos dados da sísmica, testaram-se duas abordagens de simulação condicional: i) condicionamento a leis de distribuição regionais de cotas; ii) condicionamento a matrizes locais de desníveis. Para efetuar o condicionamento das superfícies aos dados de sondagens e poços, calcularam-se coeficientes de correlação locais, entre as superfícies primárias e os dados das sondagens, estimaram-se por krigagem estes coeficientes de correlação locais para toda a área de estudo e, deste modo, co-simularam-se as superfícies, com condicionamento aos dados das sondagens e poços às superfícies previamente simuladas conforme os coeficientes de correlação locais. A metodologia proposta foi testada numa zona da Bacia Cenozóica do Baixo Tejo. Foram utilizados perfis sísmicos de reflexão, provenientes de campanhas de prospeção petrolífera, realizadas na bacia durante o séc. XX, poços profundos e sondagens. Os resultados mostram que as muitas contradições entre a informação sísmica e os dados dos poços e sondagens são resolvidas pela metodologia proposta, prevalecendo a informação principal em detrimento da informação secundária quando existem as duas em simultâneo, mas mantendo as tendências regionais capturadas pela sísmica.

Esta dissertação apresenta uma metodologia original para simular a morfologia e as propriedades petrofísicas de reservatórios de hidrocarbonetos em sistemas de canais turbidíticos. Estes sistemas são constituídos por complexos, ou seja, conjuntos de canais de arquitetura meandriforme, e são considerados importantes alvos para a indústria petrolífera. A simulação da morfologia divide-se em duas partes, primeiramente é simulada a trajetória do complexo e depois são simuladas as trajetórias dos canais propriamente ditos condicionadas à trajetória do complexo. O algoritmo de simulação utiliza as classes de ângulos azimutais de linhas poligonais de treino como uma variável aleatória. As trajetórias são simuladas também como linhas poligonais, condicionais a estatísticas multiponto das trajetórias de treino e a pontos de controlo. As estatísticas multiponto são organizadas em árvore, que guarda sequências de classes de orientação que ocorrem na trajetória de treino e as respetivas probabilidades de ocorrência. Para avaliar as propriedades petrofísicas, o modelo morfológico das trajetórias é convertido para uma malha de blocos de alta resolução, identificando-se, em cada bloco, a fácies preponderante de acordo com um modelo conceptual de zonamento da secção dos canais. A conversão prioriza os canais mais recentes (do topo) sobre os mais antigos (da base). A cada fácies é associada uma lei de distribuição da porosidade e permeabilidade, assim são geradas imagens destas propriedades petrofísicas por Simulação Sequencial Direta com histogramas locais. Finalmente, o número de blocos da malha é reduzido por upscaling e as simulações são ordenadas para poderem ser utilizadas nos simuladores de fluxo. Para ilustrar a metodologia, utilizaram-se imagens de sísmica 3D de um reservatório turbidítico na Bacia do Baixo Congo para extrair leis de distribuição das dimensões dos canais e trajetórias de treino. Os resultados representam corretamente a arquitetura complexa destes sistemas.