The objective of this dissertation is the isolation of DNA sequences that are differentially represented in males and females of the Oriental fruit fly Bactrocera dorsalis, specifically by initiating a molecular characterization of Y chromosome sequences in this species. Cytological observations have established the presence of a diminutive Y chromosome in B. dorsalis males. To isolate DNA sequences from the Y chromosome, a special method of genomic DNA isolation known as Representational Difference Analysis (RDA) was utilized to obtain DNA sequences unique to the B. dorsalis male genome. Genomic DNA from B. dorsalis males served as the "tester" DNA and female genomic DNA as the "driver" DNA. Six distinct RDA products were obtained following two complete rounds of DNA hybridization and difference enrichment via the Polymerase Chain Reaction (PCR). One ofthese products (RDA product 1) was used to isolate a genomic DNA clone (3.1a) from a B. dorsalis male genomic DNA minilibrary. This sequence shows similarity to the reverse transcriptase of R1 retrotransposable elements. The presence of R1 elements in the Tephritid insects has heretofore been undescribed, although these elements have been previously described in the genomes of other Dipteran species. Oligonucleotide primers for PCR were designed for the 3.1a clone. These primers consistently produce different amplification patterns in PCRs ofgenomic DNA from B. dorsalis males vs. females. Amplification using male genomic DNA produces 325 bp and 2.6 kb products while only a 2.6 kb product is obtained from female DNA. The amplification products obtained with these primers are also produced in PCRs of genomic DNA from B. dorsalis embryos and third instar larvae, suggesting the ability of this method to infer sex at pre-adult stages ofthe B. dorsalis life cycle. Similar amplification products have also been obtained in other Bactrocera species. Both the 325 bp male PCR product and the 2.6 kb products have regions of sequence similarity to R1 elements. The 2.6 kb product contains a putative 1.7 kb open reading frame (ORF) encoding 583 amino acids. Three amino acid motifs found in Drosophila R1 element reverse transcriptases are present in comparable locations within the hypothetical ORF product. Both of these sequences are also repetitively represented in the B. dorsalis male and female genomes. However, the 325 bp male product produces some bands that are male specific when used as a probe for Southern blots of B. dorsalis male and female genomic DNA. The amplification pattern produced by the 3.1a primers is consistent with what would be expected if the 2.6 kb and 325 bp PCR products originated from the B. dorsalis X and Y chromosomes, respectively. Thus, the cloned male-specific sequence recovered here is potentially useful both as a gateway into the relatively uncharacterized B. dorsalis Y chromosome and as a tool for the characterization of other aspects of the B. dorsalis genome.

The broad aim of this thesis is to generate fundamental knowledge about the structure and function of cyclotides, which are a topologically unique family of proteins. A long-term goal is to use the fundamental knowledge to assist in the development of drugs based on the stable cyclotide framework. Cyclotides are small proteins that are characterised by a cyclic cystine knot (CCK) motif, which is defined as a circular backbone combined with a cystine knot core. So far cyclotides have been found in plants of the Violaceae (violet) and Rubiaceae (coffee) plant families, and are believed to have a defence-related function. From an application perspective, the CCK framework has potential as a drug scaffold, being an ultra-stable alternative to linear peptide models. The reasons why cyclotides show promise as a drug template are three-fold – they have naturally high sequence diversity, suggesting that their framework can accommodate a range of epitopes; they are remarkably stable under various chemical, enzymatic and thermal conditions, which means that they have increased bioavailability; and they have a diverse range of bioactivities, supporting the notion that they can be used in a number of therapeutic applications. These three reasons are intimately linked to three core knowledge domains of cyclotide research, namely cyclotide sequences, structures and interactions. Thus, fundamental research into these three domains, as investigated in this thesis, is important as it may assist in the development of drugs based on the CCK scaffold. Chapter 1 of this thesis provides the background information to define the molecules studied and to highlight their importance. Chapter 2 describes the main experimental techniques that were used in this thesis, including nuclear magnetic resonance spectroscopy and mass spectrometry. The development of the CCK technology may benefit from a thorough understanding of the natural diversity of cyclotide sequences and the significance of this diversity on activity. Chapter 3 reports on the discovery of cyclotides in Viola yedoensis, a Chinese violet that is interesting because it is widely used in Traditional Chinese Medicine to treat a number of illnesses including swelling and hepatitis. In this study, a total of eight cyclotides was characterised, including five novel sequences. Based on anti-HIV and haemolytic assays, a strong relationship between surface hydrophobicity and activity was established. The stability of cyclotides, which underpins their potential as a drug scaffold, is examined at a structural level in Chapter 4. The solution structure of varv F, a cyclotide from the European field pansy, Viola arvensis, was solved and compared to the crystal structure of the same peptide, confirming the core structural features of cyclotides responsible for their stability, including the topology of the cystine knot, which has previously attracted some debate. From a comparison of biophysical measurements of a representative group of five cyclotides, a conserved network of hydrogen bonds, which also stabilises the cyclotide framework, was defined. A subset of hydrogen bonds involving the highly conserved Glu in loop 1 of cyclotides was examined in more detail by solving the structure of kalata B12, the only naturally occurring cyclotide with an Asp instead of a Glu in loop 1. By comparison with the prototypical cyclotide kalata B1 and an Ala mutant E7A-kalata B1, it was shown that the highly conserved Glu is important for both stability and activity. Chapter 5 reports on studies that add to our understanding of the mechanism of action of cyclotides, which is believed to involve membrane interactions. Spin-label experiments were performed for two cyclotides, kalata B2 and cycloviolacin O2, which are representative cyclotides from the two cyclotide sub-families, Möbius and bracelet, respectively. This study showed that different cyclotides have different but very specific binding modes at the membrane surface. Currently, it is believed that for Möbius cyclotides at least (e.g. kalata B1 and kalata B2), self-association may lead to the formation of membrane pores. Oligomerisation of cyclotides was also studied in this chapter using NMR relaxation. A computer program, NMRdyn, was developed to extract microdynamic and self-association parameters from NMR relaxation data. This program was used to analyse 13C relaxation data on kalata B1, providing clues about the tetramer structure of kalata B1. Although the three areas of cyclotide research examined in this thesis – sequence, structure and interactions – are reported in separate sections, the areas are not independent of each other. For example, the mechanism of action of cyclotides, which is reported in Chapter 3, requires an understanding of cyclotide structures, which is reported in Chapter 4. Chapter 6 describes a database, CyBase, which integrates sequence/structure/activity data on cyclotides so that relationships between the three areas can be examined. The database also provides tools to assist in discovery and engineering of cyclic proteins. In summary, several key areas that are fundamental to our understanding of cyclotides have been investigated in this thesis, ranging from cyclotide sequence diversity to their mechanism of action. The work described in this thesis represents a significant advance in our current understanding of cyclotides by providing, for example, explanations to their observed structural stability and how they work through interactions with other biomolecules. The information presented in this thesis is potentially useful in facilitating the long-term goal of developing peptide therapeutics based on the stable cyclotide framework.

Thesis (Ph.D.)--McMaster University (Canada), 1995. / Source: Dissertation Abstracts International, Volume: 57-03, Section: B, page: 2045. Adviser: T. Jiang.

Thesis (Ph.D.)--Flinders University, School of Informatics and Engineering. / Typescript bound. Includes bibliographical references: (leaves 204-221) Also available online.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [119]-123).

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 152-153).

Orientador: Cesar Martins / Banca: Luciana Bolsoni / Banca: Maeli Del Paiva / Resumo: O genoma dos organismos eucariotos apresenta-se organizado em seqüências simples e repetidas. As seqüências repetidas de DNA estão presentes em centenas a milhares de cópias dispersas ou agrupadas no genoma e localizam-se preferencialmente em regiões heterocromáticas, desempenhando papel relevante na organização do genoma desses organismos. Nesse sentido, a realização de estudos genéticos básicos sobre a organização genômica dessas seqüências repetidas é fundamental para uma melhor compreensão do seu papel biológico assim como o entendimento de sua dinâmica evolutiva entre os diversos grupos de vertebrados. Os Cichlidae constituem uma das mais especiosas famílias de peixes, com cerca de 3.000 espécies distribuídas pela América Central e do Sul, África, e sudeste da Índia. Este grupo passou por um rápido e extenso processo de radiação adaptativa ao longo dos tempos, constituindo-se em importantes entidades biológicas para a realização de estudos evolutivos. Dentre os Cichlidae, as espécies do gênero Cichla (tucunarés), com distribuição exclusiva na América do Sul, apresentam grande importância ecológica e econômica. No entanto, estudos genéticos envolvendo espécies desse gênero são ainda escassos. Assim, o presente trabalho teve por objetivo isolar e caracterizar seqüências repetidas de DNA no genoma de Cichla kelberi. Elementos repetidos de DNA foram isolados por PCR (elementos Rex1, Rex3, Rex6 e Tc1) e digestão enzimática (elemento Tuc), seqüenciados e mapeados cromossomicamente por FISH para o estudo de seu padrão de distribuição no genoma. O elemento Tuc apresentou elevada similaridade com seqüências do gene da transcriptase reversa de Oryzias melastigma, o que sugere tratar-se de um elemento retrotransponível. Análises comparativas do elemento Tuc a bancos de sequência mostraram alta similaridade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The genome of eucaryote organisms is organized into single and repetitive sequences. The repetitive DNA sequences are represented by hundreds to thousands of dispersed or tandem-arrayed copies preferentially localized on the heterochromatic regions, having important function on the genome organization of the organisms. Therefore, the development of basic genetic studies about the genome organization of these repetitive sequences are fundamental to a better comprehension of their biologic role and the understanding of their evolutionary dinamics. The Cichlidae are one of the most diverse fish families, having about 3.000 species distributed around Central and South America, Africa and Southeast India. This group underwent a large and rapid process of adaptative radiation, becoming an important biological model. Among the Cichlidae, the species of the genera Cichla (tucunarés), with exclusive distribution in South America, have a significative economic and ecologic importance. However genetic studies on species of this genera are scarce. Therefore, this work had the aim to isolate and characterize repetitive DNA sequences of the genome of Cichla kelberi. Repetitive DNA sequences were isolated using PCR (elements Rex1, Rex3, Rex6 and Tc1) and restriction digestion (element Tuc), sequenced and their genome distribution determined by FISH. The Tuc element showed high similarity to sequences of reverse transcriptase gene of the fish Oryzias melastigma, which suggests that such element correspond to an retrotransposon element. Comparative analysis of the Tuc element to DNA sequence data bank showed high similarity with repetitive sequences in the genome of several vertebrates, including fishes, amphibians and mammals. Results of FISH showed an accumulation of obtained elements preferentially in centromeres of all chromosomes of the complement, and few telomeric blocks in some... (Complete abstract click electronic access below) / Mestre

In this paper the non density of AP, the set of almost periodic functions in the sense of Bohr, in the space S of almost periodic functions in the sense of Stepanov is proven.

Submitted by Pablo Gonzalez Pagotto null (pgp_2008@hotmail.com) on 2016-09-28T16:16:44Z No. of bitstreams: 1 final.pdf: 1327249 bytes, checksum: 26bc65e3566fd3813a93f271a02744c1 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-09-30T14:43:09Z (GMT) No. of bitstreams: 1 pagotto_pg_me_sjrp.pdf: 1327249 bytes, checksum: 26bc65e3566fd3813a93f271a02744c1 (MD5) / Made available in DSpace on 2016-09-30T14:43:09Z (GMT). No. of bitstreams: 1 pagotto_pg_me_sjrp.pdf: 1327249 bytes, checksum: 26bc65e3566fd3813a93f271a02744c1 (MD5) Previous issue date: 2016-08-30 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo principal deste trabalho é apresentar um estudo sobre Homologia de Espaços Fibrados, baseado no livro Elements of Homotopy Theory de G.W.Whitehead. O conceito de fibração apareceu em torno de 1930 e pode ser visto como uma extensão da teoria de fibrados. Existe uma sequência exata longa que relaciona os grupos de homotopia dos espaços base, total e da fibra de uma fibração. Porém, relacionar os grupos de homologia desses espaços é uma tarefa mais complicada. O caso geral é feito utilizando sequências espectrais. Porém, há casos particulares em que podemos obter relações sem utilizar a maquinaria das sequências espectrais. / The main goal of this work is to present a study on Homology of Fibre Spaces, based on the book of G.W. Whitehead: ``Elements of Homotopy Theory''. The concept of fibration appeared around 1930 and can be seen as an extension of the theory of bundles. There is a long exact sequence that relates the homotopy groups of the total, base and fiber spaces of a fibration. However, relating the homology groups of such spaces is more complicated. The general case is obtained using spectral sequences. Nevertheless there are particular cases where one can obtain such relations without the need of the machinery of spectral sequences. / FAPESP: 2013/22249-0

A localização cromossômica do DNA ribossômico (rDNA) foi estudada em cromossomos politênicos e em tecidos diplóides de quatro espécies de sciarídeos: Trichosia pubescens; Rhynchosciara americana; R. milleri e Schwenkfeldina sp.. Resultados de hibridação em cromossomos mitóticos mostraram a existência de um único locus de rDNA; entretanto, sondas ribossomais hibridaram em mais de uma região dos cromossomos politênicos em todas as espécies analisadas devido à adesão de micronucléolos em regiões específicas dos cromossomos. Os micronucléolos são estruturas arredondadas que contêm, provavelmente, DNA extracromossômico transcricionalmente ativo. Em T. pubescens, o rDNA está predominantemente localizado nas secções cromossômicas X-10 e X-8. Em R. americana o rDNA está freqüentemente associado à heterocromatina centromérica dos cromossomos X, C, B e A, e também às secções X-1 e B-13. Sondas ribossômicas em R. milleri hibridaram, em alta freqüência, em regiões teloméricas e pericêntricas de cromossomos politênicos. Schwenkfeldina sp. apresenta uma distribuição incomum do rDNA em núcleos politênicos, caracterizada pela adesão de micronucléolos em muitas regiões cromossômicas. Os resultados mostraram que os micronucléolos estão preferencialmente associados à heterocromatina intercalar ou terminal de todos os sciarídeos analisados e, dependendo da espécie, estão aderidos a um número pequeno (Trichosia), moderado (Rhynchosciara) e grande (Schwenkfeldina sp.) de sítios em cromossomos politênicos. Este trabalho também descreve a caracterização de seqüências presentes nas extremidades cromossômicas de R. americana, que se iniciou através da triagem de uma microbiblioteca plasmidial, feita a partir de uma extremidade microdissecada B-1. Uma repetição do tipo satélite foi identificada e sua composição de bases, estrutura genômica e localização cromossômica são semelhantes às repetições teloméricas complexas de Nematocera que já foram descritas. Contudo, dados obtidos em outras espécies de Rhynchosciara, assim como a localização desse satélite e da transcriptase reversa, sugerem que o elemento repetitivo caracterizado neste trabalho não atinge as extremidades dos cromossomos. A caracterização de seqüências terminais e subterminais presentes nos cromossomos de R. americana foi continuada através da triagem de uma biblioteca de DNA desse díptero clonada em fagos Dash. Escolhemos como sonda para a triagem o clone pRaM47.33, representativo do elemento repetitivo M22, caracterizado em R. americana. Foram analisados cerca de 12kb de um único inserto de fago, que continha, alem das repetições M22, uma nova repetição de 16pb, organizada em tandem e que denominamos de M16. Resultados de hibridações in situ revelaram a presença da repetição M16 nas 5 extremidades cromossômicas não-telocêntricas de R. americana. Essa repetição também foi utilizada como sonda em uma outra triagem da mesma biblioteca genômica, o que permitiu a seleção e análise de aproximadamente 50kb de DNA cromossômico terminal de R. americana. Encontramos também, ao longo dessas 50kb de DNA analisado, repetições de 414pb anteriormente caracterizadas em R. americana; parte de seqüências do transposon Ramar1 e do retrotransposon RaTART . Além disso, foram observadas também seqüências que não apresentam semelhança significativa com seqüências depositadas no banco de dados GenBank, e que tampouco apresentam motivos repetitivos. Os resultados obtidos apontam para a possibilidade de que a região telomérica de R. americana seja composta por mais de um tipo de elemento repetitivo. / The chromosomal localization of ribosomal DNA (rDNA) was studied in polytene and diploid tissues of four sciarid species, Trichosia pubescens, Rhynchosciara americana, R. milleri and Schwenkfeldina sp. While hybridization to mitotic chromosomes showed the existence of a single rDNA locus, ribosomal probes hybridized to more than one polytene chromosome region in all the species analyzed as a result of micronucleolar attachment to specific chromosome sites. Micronucleoli are small, round bodies containing transcriptionally active, probably extrachromosomal rDNA. In T. pubescens the rDNA is predominantly localized in chromosome sections X-10 and X-8. In R. americana the rDNA is frequently found associated with centromeric heterochromatin of the chromosomes X, C, B and A, and also with sections X-1 and B-13. Ribosomal probes in R. milleri hybridized with high frequency to pericentric and telomeric regions of its polytene complement. Schwfenkfeldina sp. displays a remarkably unusual distribution of rDNA in polytene nuclei, characterized by the attachment of micronucleoli to many chromosome regions. The results showed that micronucleoli preferentially associate with intercalary or terminal heterochromatin of all sciarid flies analyzed and, depending on the species, are attached to a few (Trichosia), moderate (Rhynchosciara) or a large (Schwenkfeldina sp.) number of polytene chromosome sites. This work also describes the characterization of chromosome end sequences of Rhynchosciara americana, initiated with the screening of a plasmid microlibrary made from a microdissected polytene chromosome end. We report the identification and sequencing of an R. americana satellite displaying base composition, genomic structure and chromosomal localization similar to the complex telomeric repeats of Nematocera that have previously been characterized. However, data obtained in other Rhynchosciara species, as well as distinct chromosomal localization of satellite and reverse transcriptase loci in R. americana, suggest that the repetitive element characterized does not reach the very end of the chromosome. The characterization of chromosome end sequences of Rhynchosciara americana continued with the screening of a phage library made with its genomic DNA. We choose pRaM47.33, a clone whose insert is a repetitive microsatellite characterized in the subtelomeric region of R. americana chromosomes, as a probe for the screening. We analyzed 12kb of a single phage insert, composed of M22 tandem arrays and a new microsatellite which was 16pb long, arranged in tandem (named M16). In situ hybridization showed the presence of M16 repeats in the five telomeric termini of R. americana chromosomes. The M16 repeat was used as a probe in another screen of the same phage library, which allowed us to analyze approximately 50kb of terminal DNA. We find that repetitive sequences, such as the 414pb repeat previously characterized in R. americana and stretches of Ramar1 and RaTART mobile elements, also characterized in R. americana, compose the subtelomeric region of R. americana chromosomes. Additionally, we find sequences that do not match sequences in the GenBank database and do not present repetitive motifs. Our results suggest that the telomeric regions of R. americana chromosomes are composed of more than one type of repetitive sequence.