Thesis (Ph.D.)--University of California, San Francisco, 2004. / Bibliography: leaves 92-101. Also available online.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains ix, 146 p.; also includes graphics (some col.) Includes bibliographical references (p. 145-146). Available online via OhioLINK's ETD Center

Thesis (M.S.)--Brigham Young University. Dept. of Statistics, 2006. / Includes bibliographical references (p. 83-85).

Thesis (M.S.)--Montana State University--Bozeman, 2006. / Typescript. Chairperson, Graduate Committee: Christa Merzdorf. Includes bibliographical references (leaves 51-55).

Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Description based on contents viewed Feb. 12, 2009; title from PDF t.p. Includes bibliographical references (p. 40-41).

Orientador: Gonçalo Amarante Guimães Pereira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T09:23:56Z (GMT). No. of bitstreams: 1 Lepikson-Neto_Jorge_M.pdf: 1765356 bytes, checksum: c73289c953d58c6b221c2e0927805499 (MD5) Previous issue date: 2008 / Resumo: Com o intuito de obter informações relevantes para o melhoramento genético do eucalipto para a produção de biomassa, o presente trabalho buscou comparar a expressão dos genes relacionados com a formação e desenvolvimento da madeira em quatro diferentes espécies de eucalipto, tendo como objetivo identificar os padrões que as tornam mais aptas, bem como quais genes relacionados com determinadas características. Essa análise abre a possibilidade da identificação de genes chave que possam ser manipulados, através do melhoramento clássico ou da transgênia, para aumentar o conteúdo relativo de celulose das plantas, incrementando a sua eficiência para processos econômicos. 384 ESTs do banco de dados do Consórcio Genolyptus foram selecionadas para serem analisadas através da tecnologia de microarranjos de cDNA. Foram selecionadas ESTs de genes com funções conhecidas relacionadas com a formação da madeira, bem como de genes relacionados com o desenvolvimento do vegetal e de genes com função ainda desconhecida. Os dados obtidos foram cruzados com a biblioteca de ESTs do Consorcio Genolyptus (Northern Eletrônico), e foram feitos PCR em tempo real para os principais genes diferenciais nos microarranjos e para os genes da via de lignina e flavonóides. Os resultados mostraram que diferentes genes estão expressos nas espécies estudadas sendo um grande número deles ainda com função desconhecida no metabolismo do eucalipto. A maioria dos genes relacionados com a formação da parede celular não apresentou perfil de expressão diferencial nos microarranjos, sugerindo que as diferenças fenotípicas entre as madeiras das espécies estudadas podem estar sustentadas em vias alternativas, com fatores de elongação, cliclínas e outros genes desempenhando papel importante, bem como genes ainda não relacionados com o desenvolvimento da parede celular e ou ao desenvolvimento do vegetal. Os experimentos com PCR em tempo real mostraram que genes da via de flavonóides relacionados com a via de lignina e formação da madeira podem estar desempenhando papeis importantes no controle da formação da madeira da espécie. Esses resultados representam avanços significativos no entedimento da formação da madeira em eucalipto e servem como base para orientar futuras investigações no intuito de melhorar geneticamente esta espécie. / Abstract: In this work we intended to assemble relevant information for the genetic engineering of Eucalyptus for biomass production by comparing gene expression of genes related with the xylem and wood development of four different Eucalyptus species with distinct caractheristics, as a form to identify patterns and wich genes are possibly related to their differences. This analysis opens the possibilities to manipulate the specie and increase the overall celluloses content and its purpose for industrial production. 384 ESTs were selected from de Genolyptus database and analysed by microarryay cDNA experiments. Among the ESTs selected some were related to wood formation, cell wall assembly and some still had no general function known on the specie. The data from the microarray experiments were then crossed with the Genolyptus ESTs lybrary (Eletronic Northern) and Real-Time PCR were performed for the most relevant resultas as well as the genes from de lignin and flavonoid pathway. Results show that different genes are expressed among the xylem of the four species studied and most of them still have no related function to the metabolism of the plant. Most of the genes related to cell wall formation were not differentially expressed on the microarrays suggesting that the differences on the quality and structure of the wood among the four species might as well be resulted from the expression of alternative pathways and genes such as elongation factors, ciclins and others not yet related to the cell wall formation and wood development. Real-Time PCR experiments shown that genes from the flavonoid pathway related to lignin and wood formation might be playing a crucial role determining wicht pathway must be followed and therefore the type and quality of the wood on Eucalyptus. These results represent significant advances to our understanding on the formation of wood on Eucalyptus and will be valuable as a basis for future investigation aiming genetic engeneering of the specie. / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Orientador: Vera Lucia Gil da Silva Lopes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T00:02:33Z (GMT). No. of bitstreams: 1 Simioni_Milena_D.pdf: 16919327 bytes, checksum: fa845dbc925978762cb44d70536f9ec7 (MD5) Previous issue date: 2012 / Resumo: As fendas orofaciais típicas (FOT) são defeitos congênitos prevalentes que possuem múltiplas etiologias. Estudos populacionais apontaram diferentes genes relacionados às FOTs. Entretanto, em muitos casos, a etiologia permanece desconhecida. A investigação individualizada, utilizando em conjunto diferentes ferramentas laboratoriais, é uma abordagem mais complexa que pode contribuir na caracterização etiológica das FOTs. Assim, este estudo teve como objetivo investigar os fatores genéticos envolvidos na etiologia das FOTs em uma casuística composta por 23 indivíduos afetados, incluindo casos sindrômicos e não-sindrômicos, familiais e esporádicos. Participaram do grupo controle 20 indivíduos sem história familial de fenda em três gerações. Todos os indivíduos portadores de FOT foram previamente avaliados por um médico geneticista e por exame de cariótipo em linfócitos, sendo dois casos detectados com aberrações cromossômicas. A análise de alterações no número de cópias de segmentos de DNA (Copy Number Variation, CNVs) por hibridação genômica em arrays (aGH), baseada na comparação com grupo controle, apontou um novo gene de candidato para FOT, o gene TCEB3, e identificou uma duplicação no gene FGFR1. Em um paciente com fenda palatal submucosa e quadro sindrômico, a utilização da técnica de aGH, em conjunto à hibridação in situ fluorescente (FISH) e Multiplex Ligationdependent Probe Amplification (MLPA), possibilitou a caracterização da aberração cromossômica detectada pelo cariótipo. Em todos os portadores de FOT também foi realizado sequenciamento direto dos genes IRF6, FOXE1, GLI2, MSX2, SKI, SATB2, SPRY1, MSX1, FGF8, FGFR1; em um paciente específico, foi avaliado o gene P63. Alterações de sequencia inéditas foram encontradas nos genes FOXE1, MSX1, GLI2 e FGF8, assim como uma inserção no gene P63, cujos efeitos na proteína codificada deverão ser confirmados em estudos futuros. Os resultados deste trabalho exemplificam a diversidade de fatores genéticos envolvidos na etiologia das FOTs e o desenho de estudo utilizado mostrou a eficácia do uso concomitante de diferentes abordagens investigativas neste grupo de defeitos congênitos / Abstract: Typical oral cleft (TOC) is a prevalent and heterogeneous group of congenital defects with multiple etiologies, which remain unknown in several cases. Population studies detected several genes related to TOC. An individualized investigation, involving different laboratorial tools at same time, is an approach that can contribute on the etiological characterization of the TOC. The aim of this study was to investigate genetic factors involved on TOC in a sample composed by 23 individuals (syndromic and non-syndromic; familial and sporadic cases). The control group included 20 individuals without TOC in three generations. All patients were previously evaluated by clinical geneticists and performed karyotype test that showed chromosomal aberrations in two cases. Copy number variation (CNV) investigation by genomic hibridization in arrays (aGH), based in a comparative to control group data, detected a new candidate gene to TOC (TCEB3), and identified a duplication affecting FGFR1 gene. In one patient with syndromic form of submucous cleft palate, the use of aGH technique, together with Fluorescent in situ hibridization (FISH) and Multiplex ligation-dependent probe amplification (MLPA), characterized the chromosomal aberration previously detected by karyotype. Direct sequencing of IRF6, FOXE1, GLI2, MSX2, SKI, SATB2, SPRY1, MSX1, FGF8 and FGFR1 genes was performed in all individuals; in a specific case, P63 gene was investigated. New sequence alterations were found in FOXE1, MSX1, GLI2 e FGF8 genes, as well as an insertion in P63 gene, which effects will be verified in futures studies. In conclusion, results here described reflect the diversity of genetic factors involved in the etiology of TOC and the type of study show the efficiency of the use of different techniques in the etiological investigation of this congenital defect / Doutorado / Ciencias Biomedicas / Doutor em Ciências Médicas

The expression of genetic information, in all organisms, might be characterized as in a constant state of flux with only a fraction of the gene within a genome being expressed at any given time. The genes’ expression pattern reflects the response of cells to stimuli that control growth, development and signal environmental changes. Understanding genes’ expression at the level of transcription and/or other stages of gene regulation at the mRNA level (half life of mRNA, RNA production from primary transcript) might reveal insights into the genes expression mechanisms that control these changes. With the DNA microarray technology researchers are now able to determine, in a single experiment, the gene expression profiles of hundreds to tens of thousands of genes in tissue, tumors, cells or biological fluids. Accordingly, and since the patterns of gene expression are strongly functionally correlated, microarrays might provide unprecedented information both on basic research (e.g. expression profiles of different tissues) and on applied research (e.g. human diseases, drug and hormone action etc). While the simultaneous measurement of thousands of gene expression levels potentially serves as source of profound knowledge, genes quantification (i.e. extraction of the genes expression levels) is confounded by various types of noise originating both from the microarray experimental procedure (e.g. sample preparation) and the probabilistic characteristics of the microarray detection process (e.g. scanning errors). The “noisy” nature of the measured gene expression levels obscures some of the important characteristics of the biological processes of interest. The latter, as a direct effect, renders the extraction of biological meaningful conclusions through microarray experiments difficult and affects the accuracy of the biological inference. Thus, as a major challenge in DNA microarray analysis, and especially for the accurate extraction of genes expression levels, might be considered the effective separation of “true” gene expression values from noise. Noise reduction is an essential process, which has to be incorporated into the microarray image analysis pipeline in order to minimize the “errors” that propagate throughout the microarray analysis pipeline and, consequently, affect the extracted gene expression levels. A possible solution, as proposed in previous studies, for addressing microarray image noise is image enhancement. Results of these studies have indicated a superior quality of the enhanced images, without however examining whether enhancement leads to more accurate spot segmentation or reduces the variability of the extracted gene expression levels. As foresaid, noise also complicates the extraction of meaningful biological conclusions. While more advanced methods have been introduced [28-32] that attempt to prevent the noisy set of genes from being grouped, there is a lack of consensus among experts on the selection of a single method for determining meaningful clusters of genes. The latter, directly affects the biological inference, since different number of clusters are produced when different clustering techniques or either different parameters in the clustering algorithms are utilized. Thus, it turns up that it is not only important to assess the performance of each analysis stage independently (i.e. whether the techniques employed in the microarray analysis pipeline provide accurate extracted gene expression levels or the clustering techniques group biologically related genes) but it is also necessary to ensure an acceptable performance of all steps, as a whole, in terms of biologically meaningful information. This thesis has been carried out towards the development of a complete microarray image processing and analysis framework in order to improve the extraction and, consequently, the quantification of gene expression levels on spotted complementary DNA (cDNA) microarray images. The aims of the present thesis are: a) to model and address the effects of cDNA microarray image noise in such a way that it will increase the accuracy of the extracted gene expression levels, b) to investigate the impact of noise and facilitate genes expression data analysis in order to allow biologists to develop an integrated understanding of the process being studied, c) to introduce a semi-supervised biologically informed criterion for the detection of meaningful biological clusters of genes that answer specific biological questions, d) to investigate the performance and the impact of various state-of-art and novel cDNA microarray image segmentation techniques in the quantification of genes expression levels For exploring all of these aspects, a complete and robust framework of microarray image processing and analysis techniques was designed, built and implemented. The framework incorporated in the microarray analysis pipeline a novel combination of image processing and analysis techniques originating from the comprehensive quantitative investigation of the impact of noise on spot segmentation, intensity extraction and data mining. Additionally, novel formulations of known image segmentation techniques have been introduced, implemented and evaluated in the task of microarray image segmentation. The usefulness of the proposed methods has been validated experimentally on both simulated and real cDNA microarray images. / Η έκφραση της γενετικής πληροφορίας, σε όλους τους οργανισμούς, χαρακτηρίζεται από μια σταθερή κατάσταση «ροής» στην οποία όμως μόνο ένα μέρος του γονιδίου μέσα στο γονιδίωμα (genome) εκφράζεται ανά χρονική στιγμή. Το γονιδιακό μοτίβο έκφρασης (gene expression pattern or gene expression profile) θα μπορούσαμε να πούμε ότι αντανακλά την αντίδραση των κυττάρων στα διάφορα εξωτερικά ερεθίσματα. Για να μπορέσουν να απαντηθούν ερωτήματα σχετικά με τους μηχανισμούς που επηρεάζουν και μεταβάλλουν τη γονιδιακή έκφραση ανάλογα με το εξωτερικό ερέθισμα είναι απαραίτητη η μελέτη της γονιδιακής έκφρασης σε μεταγραφικό επίπεδο (transcription level) ή/και άλλα στάδια (παράγοντες) που ρυθμίζουν τη γονιδιακή έκφραση (gene regulation) σε επίπεδο mRNA. Με τη χρήση της τεχνολογίας των μικροσυστοιχιών, οι ερευνητές έχουν πλέον τη δυνατότητα να μελετήσουν ταυτόχρονα την γονιδιακή έκφραση δεκάδων ή και εκατοντάδων χιλιάδων γονιδίων σε ιστούς, κύτταρα όγκους κλπ με τη χρήση ενός και μόνο πειράματος. Κατά συνέπεια, και από τη στιγμή που τα γονιδιακά μοτίβα έκφρασης συσχετίζονται έντονα λειτουργικά (functionally correlated), η τεχνολογία των μικροσυστοιχιών παρέχει ανεκτίμητης αξίας πληροφορίες που μπορούν να δώσουν ώθηση τόσο στην ανάπτυξη της βασικής έρευνας π.χ. μελέτη των γονιδιακών προφίλ έκφρασης διαφορετικών ιστών όσο και στην ανάπτυξη της εφαρμοσμένης έρευνας π.χ. μελέτη ασθενειών, δράση φαρμάκων και ορμονών κλπ. Παρά τη δυνατότητα που παρέχει η τεχνολογία των μικροσυστοιχιών για την ταυτόχρονη μέτρηση των επιπέδων έκφρασης χιλιάδων γονιδίων, η ποσοτικοποίηση της γονιδιακής έκφρασης (δηλ. η εξαγωγή των επιπέδων έκφρασης των γονιδίων), επηρεάζεται από τους διάφορους τύπους θορύβου που υπεισέρχονται τόσο κατά την πειραματική διαδικασία κατασκευής των μικροσυστοιχιών (π.χ. προετοιμασία δειγμάτων) όσο και από τα πιθανοκρατικά χαρακτηριστικά που διέπουν τη διαδικασία ανίχνευσης (microarray scanning procedure) των μικροσυστοιχιών (π.χ. λάθη ανίχνευσης). Η «θορυβώδης» φύση των γονιδίων και κατά συνέπεια των μετρούμενων γονιδιακών εκφράσεων «κρύβει» (obscure) μερικά από τα πιο σημαντικά χαρακτηριστικά των βιολογικών διαδικασιών ενδιαφέροντος και καθιστά δύσκολη την εξαγωγή χρήσιμων βιολογικών συμπερασμάτων. Από τα παραπάνω διαφαίνεται ότι η μείωση του θορύβου είναι μια πολύ σημαντική διαδικασία η οποία θα πρέπει να ενσωματωθεί στην αλγοριθμική μεθοδολογία που μέχρι στιγμής χρησιμοποιείται για την εξαγωγή των γονιδιακών εκφράσεων από τις εικόνες μικροσυστοιχιών. Με αυτό τον τρόπο θα ελαχιστοποιηθούν τα πιθανά «λάθη» τα οποία μεταφέρονται (propagate) κατά τη διαδικασία εξαγωγής των εντάσεων (μέσω της χρησιμοποιούμενης αλγοριθμικής μεθοδολογίας) και τελικά επηρεάζουν την «ακριβή» εξαγωγή των γονιδιακών εκφράσεων. ‘Ως πιθανή λύση για την αντιμετώπιση του θορύβου στις εικόνες μικροσυστοιχιών, έχει προταθεί στη διεθνή βιβλιογραφία η χρήση τεχνικών αναβάθμισης εικόνας. Τα αποτελέσματα αυτών των επιστημονικών εργασιών συμπεραίνουν ότι με τη χρήση τεχνικών αναβάθμισης η ποιότητα των επεξεργασμένων εικόνων είναι σαφώς καλύτερη. Ωστόσο, καμία από αυτές τις εργασίες δεν μελετάει εάν οι τεχνικές αναβάθμισης οδηγούν στον ακριβέστερο προσδιορισμό των παρυφών των κουκίδων (spot) από τις οποίες εξάγονται οι γονιδιακές εκφράσεις ή εάν βοηθάνε στη μείωση της μεταβλητότητας (variability) των εξαγόμενων γονιδιακών εκφράσεων. Επιπρόσθετα, όπως έχει ήδη προαναφερθεί, ο θόρυβος παρεμποδίζει την εξαγωγή χρήσιμων βιολογικών συμπερασμάτων. Παρά το μεγάλο πλήθος εξελιγμένων μεθόδων που έχουν προταθεί στη διεθνή βιβλιογραφία για την αποτροπή της ομαδοποίησης γονιδίων που χαρακτηρίζονται ως «θορυβώδη», δεν έχει καθοριστεί ακόμα (από τους ειδικούς) μια ενιαία μέθοδος που να βρίσκει και να ομαδοποιεί γονίδια τα οποία θα παρέχουν βιολογικά χρήσιμες πληροφορίες. Αποτέλεσμα αυτής της «ασυμφωνίας» μεταξύ των ειδικών αποτελεί η εξαγωγή διαφορετικών βιολογικών συμπερασμάτων ανάλογα α) με τον αριθμό των δημιουργούμενων γονιδιακών ομάδων (που εξαρτάται άμεσα από τη διαφορετική μέθοδο ομαδοποίησης (clustering)) και β) με τις διαφοροποιήσεις που μπορεί να έχουμε στις παραμέτρους των διαφόρων μεθόδων ομαδοποίησης. H παρούσα διατριβή στοχεύει στη δημιουργία ενός ολοκληρωμένου πλαισίου για την επεξεργασία και ανάλυση εικόνων μικροσυστοιχιών με σκοπό την βελτιστοποίηση της εξαγωγής και κατά συνέπεια της ποσοτικοποίησης των γονιδιακών εντάσεων από εικόνες μικροσυστοιχιών κουκίδων (spotted cDNA microarray images). Οι στόχοι της παρούσας διατριβής συνοψίζονται ως εξής: α) μοντελοποίηση και περιορισμός των επιδράσεων του θορύβου σε εικόνες μικροσυστοιχιών κουκίδων κατά τέτοιο τρόπο ώστε να αυξηθεί η ακρίβεια των εξαγόμενων γονιδιακών εκφράσεων, β) μελέτη της επίδρασης του θορύβου και βελτιστοποίηση των μεθόδων ανάλυσης των γονιδιακών εκφράσεων με σκοπό τη διευκόλυνση των βιολόγων στην εξαγωγής βιολογικών συμπερασμάτων και την καλύτερη κατανόηση της βιολογικής διεργασίας που μελετάται, γ) εισαγωγή ενός ημιεποπτευόμενου (semi-supervised) κριτηρίου που στηριζόμενο σε βιολογικές πληροφορίες θα αποσκοπεί στην ανεύρεση βιολογικά σημαντικών ομάδων γονιδίων τα οποία ταυτόχρονα θα απαντούν σε συγκεκριμένα βιολογικά ερωτήματα ,δ) μελέτη της επίδρασης και της απόδοσης διαφόρων τεχνικών κατάτμησης εικόνων μικροσυστοιχιών κουκίδων, τόσο ανωτάτου επιπέδου (state-of-art) όσο και νέων, στην ποσοτικοποίηση γονιδιακών εκφράσεων. Για την πραγματοποίηση των παραπάνω στόχων σχεδιάστηκε και κατασκευάστηκε μια πλήρως δομημένη μεθοδολογία (a complete and robust framework) που περιελάμβανε αλγοριθμους επεξεργασίας και ανάλυσης εικόνας κουκίδων μικροσυστοιχιών Η προτεινόμενη μεθοδολογία ενσωμάτωσε στην ήδη υπάρχουσα αλγοριθμική μεθοδολογία (microarray analysis pipeline) έναν πρωτότυπο συνδυασμό τεχνικών επεξεργασίας και ανάλυσης εικόνας βασισμένο στην εις βάθος ποσοτική έρευνα της επίδρασης του θορύβου στην κατάτμηση κουκίδων (spot segmentation), στην εξαγωγή εντάσεων και στην εξόρυξη δεδομένων (data mining). Επιπρόσθετα, κατά την παρούσα διατριβή προτάθηκαν, κατασκευάστηκαν και αξιολογήθηκαν νέες τεχνικές κατάτμησης εικόνας από μικροσυστοιχές κουκίδων. Η χρησιμότητα των προτεινόμενων μεθοδολογιών αξιολογήθηκε τόσο σε εικονικές (simulated) όσο και σε πραγματικές εικόνες μικροσυστοιχιών κουκίδων.