Divergência interpopulacional e interespecífica e processo seletivo atuante nos genes ortólogos a fruitless e Odysseus em Drosophila mojavensis e D. arizonae

Pós-graduação em Biologia Animal - IBILCE

Análise de tripletos e de repetições em estruturas primárias de DNA

O desenvolvimento de equipamentos de descodificação massiva de genomas veio aumentar de uma forma brutal os dados disponíveis. No entanto, para desvendarmos informação relevante a partir da análise desses dados é necessário software cada vez mais específico, orientado para determinadas tarefas que auxiliem o investigador a obter conclusões o mais rápido possível. É nesse campo que a bioinformática surge, como aliado fundamental da biologia, uma vez que tira partido de métodos e infra-estruturas computacionais para desenvolver algoritmos e aplicações informáticas. Por outro lado, na maior parte das vezes, face a novas questões biológicas é necessário responder com novas soluções específicas, pelo que o desenvolvimento de aplicações se torna um desafio permanente para os engenheiros de software. Foi nesse contexto que surgiram os principais objectivos deste trabalho, centrados na análise de tripletos e de repetições em estruturas primárias de DNA. Para esse efeito, foram propostos novos métodos e novos algoritmos que permitirem o processamento e a obtenção de resultados sobre grandes volumes de dados. Ao nível da análise de tripletos de codões e de aminoácidos foi proposto um sistema concebido para duas vertentes: por um lado o processamento dos dados, por outro a disponibilização na Web dos dados processados, através de um mecanismo visual de composição de consultas. Relativamente à análise de repetições, foi proposto e desenvolvido um sistema para identificar padrões de nucleótidos e aminoácidos repetidos em sequências específicas, com particular aplicação em genes ortólogos. As soluções propostas foram posteriormente validadas através de casos de estudo que atestam a mais-valia do trabalho desenvolvido.

Identification of novel molecular determinants of tissue mineralization in fish

The identification of genes involved in signaling and regulatory pathways, and matrix formation is paramount to the better understanding of the complex mechanisms of bone formation and mineralization, and critical to the successful development of therapies for human skeletal disorders. To achieve this objective, in vitro cell systems derived from skeletal tissues and able to mineralize their extracellular matrix have been used to identify genes differentially expressed during mineralization and possibly new markers of bone and cartilage homeostasis. Using cell systems of fish origin and techniques such as suppression subtractive hybridization and microarray hybridization, three genes never associated with mechanisms of calcification were identified: the calcium binding protein S100-like, the short-chain dehydrogenase/reductase sdr-like and the betaine homocysteine S-methyltransferase bhmt3. Analysis of the spatial-temporal expression of these 3 genes by qPCR and in situ hybridization revealed: (1) the up-regulation of sdr-like transcript during in vitro mineralization of gilthead seabream cell lines and its specificity for calcified tissues and differentiating osteoblasts; (2) the up-regulation of S100-like and the down-regulation of bhmt3 during in vitro mineralization and the central role of both genes in cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish. While expression of S100-like and bhmt3 was restricted to calcified tissues, sdr-like transcript was also detected in soft tissues, in particular in tissues of the gastrointestinal tract. Functional analysis of gene promoters revealed the transcriptional regulation of the 3 genes by known regulators of osteoblast and chondrocyte differentiation/mineralization: RUNX2 and RAR (sdr-like), ETS1 (s100-like; bhmt3), SP1 and MEF2c (bhmt3). The evolutionary relationship of the different orthologs and paralogs identified within the scope of this work was also inferred from taxonomic and phylogenetic analyses and revealed novel protein subfamilies (S100-like and Sdr-like) and the explosive diversity of Bhmt family in particular fish groups (Neoteleostei). Altogether our results contribute with new data on SDR, S100 and BHMT proteins, evidencing for the first time the role for these three proteins in mechanisms of mineralization in fish and emphasized their potential as markers of mineralizing cartilage and bone in developing fish.

Comparação filogenética de genomas de rizóbios por hibridização através de microarray

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de genômica comparativa de corynebacterium pseudotuberculosis linhagem 226 (biovar ovis)

Corynebacterium pseudotuberculosis é uma bactéria Gram-positiva, intracelular facultativa, nãoesporulante, não-capsulada e sem mobilidade, contudo possui fímbria, e pode assumir formas cocóides e filamentosas (pleomórfica), além disto, apresenta crescimento ótimo à 37°C. Este patógeno apresenta dois biovares: ovis que geralmente acomete pequenos ruminantes, e causa a doença linfadenite caseosa, e biovar equi, mais comum em equinos, bovinos, camelídeos, e bubalinos causando a Linfangite ulcerativa. A infecção por esta bactéria pode levar a condenação das carcaças e redução de lã (em ovinos e caprinos), leite e carne destes animais, e consequentemente a perdas econômicas para a indústria agropecuária mundial. Atualmente, ainda não existe uma vacina eficaz para estas doenças. A fim de obter um maior entendimento biológico entre as espécies o presente trabalho tem como objetivo principal analisar, por meio da genômica comparativa a linhagem C. pseudotuberculosis 226 biotipo ovis isolada de um caprino na Califórnia com outras linhagens do biovarar ovis e equi. Na análise de sintenia entre as linhagens foi possível identificar que a linhagem 226 apresenta alta conservação da ordem gênica entre as linhagens do biótipo ovis. Através de análises filogenômicas foi possível identificar que as linhagens I19 e 267 apresentaram maior e menor proximidade filogenética com a linhagem 226. A linhagem 1/06-A foi a que apresentou maior proximidade filogenômica entre as linhagens do biovar equi, quando comparadas a linhagem 226. Foram preditas 8 ilhas de patogenicidade, estando presente na ilha 1 os genes relacionados a virulência de C. pseudotuberculosis mais bem descritos na literatura. Não houveram regiões novas relacionadas a genes de virulência entre nenhuma das linhagens. Foram identificados 248 genes ortólogos entre as linhagens I19, 267 e 226 e 282 genes ortólogos entre as linhagens 258,1/06-A e 226. Com base nesse estudo é possível inferir que as linhagens do biovar ovis possuem um repertório gênico pouco variado e que as linhagens do biovar equi apresentam uma quantidade menor de genes compartilhados com a linhagem 226, corroborando com a diversidade gênica entre os biovares.

Factores transcripcionales de la clase DOF en Brachypodium distachyon: caracterización molecular de BdDOF24 durante la germinación de las semillas

La semilla es el órgano que garantiza la propagación y continuidad evolutiva de las plantas espermatofitas y constituye un elemento indispensable en la alimentación humana y animal. La semilla de cereales acumula en el endospermo durante la maduración, mayoritariamente, almidón y proteínas de reserva. Estas reservas son hidrolizadas en la germinación por hidrolasas sintetizadas en la aleurona en respuesta a giberelinas (GA), siendo la principal fuente de energía hasta que la plántula emergente es fotosintéticamente activa. Ambas fases del desarrollo de la semilla, están reguladas por una red de factores de transcripción (TF) que unen motivos conservados en cis- en los promotores de sus genes diana. Los TFs son proteínas que han desempeñado un papel central en la evolución y en el proceso de domesticación, siendo uno de los principales mecanismos de regulación génica; en torno al 7% de los genes de plantas codifican TFs. Atendiendo al motivo de unión a DNA, éstos, se han clasificado en familias. La familia DOF (DNA binding with One Finger) participa en procesos vitales exclusivos de plantas superiores y sus ancestros cercanos (algas, musgos y helechos). En las semillas de las Triticeae (subfamilia Pooideae), se han identificado varias proteínas DOF que desempeñan un papel fundamental en la regulación de la expresión génica. Brachypodium distachyon es la primera especie de la subfamilia Pooideae cuyo genoma (272 Mbp) ha sido secuenciado. Su pequeño tamaño, ciclo de vida corto, y la posibilidad de ser transformado por Agrobacterium tumefaciens (plásmido Ti), hacen que sea el sistema modelo para el estudio de cereales de la tribu Triticeae con gran importancia agronómica mundial, como son el trigo y la cebada. En este trabajo, se han identificado 27 genes Dof en el genoma de B. distachyon y se han establecido las relaciones evolutivas entre estos genes Dof y los de cebada (subfamilia Pooideae) y de arroz (subfamilia Oryzoideae), construyendo un árbol filogenético en base al alineamiento múltiple del dominio DOF. La cebada contiene 26 genes Dof y en arroz se han anotado 30. El análisis filogenético establece cuatro grupos de genes ortólogos (MCOGs: Major Clusters of Orthologous Genes), que están validados por motivos conservados adicionales, además del dominio DOF, entre las secuencias de las proteínas de un mismo MCOG. El estudio global de expresión en diferentes órganos establece un grupo de nueve genes BdDof expresados abundantemente y/o preferencialmente en semillas. El estudio detallado de expresión de estos genes durante la maduración y germinación muestra que BdDof24, ortólogo putativo a BPBF-HvDOF24 de cebada, es el gen más abundante en las semillas en germinación de B. distachyon. La regulación transcripcional de los genes que codifican hidrolasas en la aleurona de las semillas de cereales durante la post‐germinación ha puesto de manifiesto la existencia en sus promotores de un motivo tripartito en cis- conservado GARC (GA-Responsive Complex), que unen TFs de la clase MYB-R2R3, DOF y MYBR1-SHAQKYF. En esta tesis, se ha caracterizado el gen BdCathB de Brachypodium que codifica una proteasa tipo catepsina B y es ortólogo a los genes Al21 de trigo y HvCathB de cebada, así como los TFs responsables de su regulación transcripcional BdDOF24 y BdGAMYB (ortólogo a HvGAMYB). El análisis in silico del promotor BdCathB ha identificado un motivo GARC conservado, en posición y secuencia, con sus ortólogos en trigo y cebada. La expresión de BdCathB se induce durante la germinación, así como la de los genes BdDof24 y BdGamyb. Además, los TFs BdDOF24 y BdGAMYB interaccionan en el sistema de dos híbridos de levadura e in planta en experimentos de complementación bimolecular fluorescente. En capas de aleurona de cebada, BdGAMYB activa el promotor BdCathB, mientras que BdDOF24 lo reprime; este resultado es similar al obtenido con los TFs ortólogos de cebada BPBF-HvDOF24 y HvGAMYB. Sin embargo, cuando las células de aleurona se transforman simultáneamente con los dos TFs, BdDOF24 tiene un efecto aditivo sobre la trans-activación mediada por BdGAMYB, mientras que su ortólogo BPBF-HvDOF24 produce el efecto contrario, revirtiendo el efecto de HvGAMYB sobre el promotor BdCathB. Las diferencias entre las secuencias deducidas de las proteínas BdDOF24 y BPBF-HvDOF24 podrían explicar las funciones opuestas que desempeñan en su interacción con GAMYB. Resultados preliminares con líneas de inserción de T-DNA y de sobre-expresión estable de BdGamyb, apoyan los resultados obtenidos en expresión transitoria. Además las líneas homocigotas knock-out para el gen BdGamyb presentan alteraciones en anteras y polen y no producen semillas viables. ABSTRACT The seed is the plant organ of the spermatophytes responsible for the dispersion and survival in the course of evolution. In addition, it constitutes one of the most importan elements of human food and animal feed. The main reserves accumulated in the endosperm of cereal seeds through the maturation phase of development are starch and proteins. Its degradation by hydrolases synthetized in aleurone cells in response to GA upon germination provides energy, carbon and nitrogen to the emerging seedling before it acquires complete photosynthetic capacity. Both phases of seed development are controlled by a network of transcription factors (TFs) that interact with specific cis- elements in the promoters of their target genes. TFs are proteins that have played a central role during evolution and domestication, being one of the most important regulatory mechanisms of gene expression. Around 7% of genes in plant genomes encode TFs. Based on the DNA binding motif, TFs are classified into families. The DOF (DNA binding with One Finger) family is involved in specific processes of plants and its ancestors (algae, mosses and ferns). Several DOF proteins have been described to play important roles in the regulation of genes in seeds of the Triticeae tribe (Pooideae subfamily). Brachypodium distachyon is the first member of the Pooideae subfamily to be sequenced. Its small size and compact structured genome (272 Mbp), the short life cycle, small plant size and the possibility of being transformed with Agrobacterium tumefaciens (Ti-plasmid) make Brachypodium the model system for comparative studies within cereals of the Triticeae tribe that have big economic value such as wheat and barley. In this study, 27 Dof genes have been identified in the genome of B. distachyon and the evolutionary relationships among these Dof genes and those frome barley (Pooideae subfamily) and those from rice (Oryzoideae subfamily) have been established by building a phylogenetic tree based on the multiple alignment of the DOF DNA binding domains. The barley genome (Hordeum vulgare) contains 26 Dof genes and in rice (Oryza sativa) 30 genes have been annotated. The phylogenetic analysis establishes four Major Clusters of Orthologous Genes (MCOGs) that are supported by additional conserved motives out of the DOF domain, between proteins of the same MCOG. The global expression study of BdDof genes in different organs and tissues classifies BdDof genes into two groups; nine of the 27 BdDof genes are abundantly or preferentially expressed in seeds. A more detailed expression analysis of these genes during seed maturation and germination shows that BdDof24, orholog to barley BPBF-HvDof24, is the most abundantly expressed gene in germinating seeds. Transcriptional regulation studies of genes that encode hydrolases in aleurone cells during post-germination of cereal seeds, have identified in their promoters a tripartite conserved cis- motif GARC (GA-Responsive Complex) that binds TFs of the MYB-R2R3, DOF and MYBR1-SHAQKYF families. In this thesis, the characterization of the BdCathB gene, encoding a Cathepsin B-like protease and that is ortholog to the wheat Al21 and the barley HvCathB genes, has been done and its transcriptional regulation by the TFs BdDOF24 and BdGAMYB (ortholog to HvGAMYB) studied. The in silico analysis of the BdCathB promoter sequence has identified a GARC motif. BdCathB expression is induced upon germination, as well as, those of BdDof24 and BdGamyb genes. Moreover, BdDOF24 and BdGAMYB interact in yeast (Yeast 2 Hybrid System, Y2HS) and in planta (Bimolecular Fluorecence Complementation, BiFC). In transient assays in aleurone cells, BdGAMYB activates the BdCathB promoter, whereas BdDOF24 is a transcriptional repressor, this result is similar to that obtained with the barley orthologous genes BPBF-HvDOF24 and HvGAMYB. However, when aleurone cells are simultaneously transformed with both TFs, BdDOF24 has an additive effect to the trans-activation mediated by BdGAMYB, while its ortholog BPBF-HvDOF24 produces an opposite effect by reducing the HvGAMYB activation of the BdCathB promoter. The differences among the deduced protein sequences between BdDOF24 and BPBF-HvDOF24 could explain their opposite functions in the interaction with GAMYB protein. Preliminary results of T-DNA insertion (K.O.) and stable over-expression lines of BdGamyb support the data obtained in transient expression assays. In addition, the BdGamyb homozygous T-DNA insertion (K.O.) lines have anther and pollen alterations and they do not produce viable seeds.

Caracterização molecular dos genes PSO2 e PSO4 de Saccharomyces cerevisiae envolvidos na reparação do DNA : análise funcional e identificação de interações proteína-proteína

O alelo mutante termo-condicionalmente letal pso4-1 do gene PRP19, que codifica uma proteína associada ao spliceossoma, permitiu investigar a influência deste gene no processamento de pré-mRNA, reparação do DNA e esporulação. Fenótipos relacionados a genes portadores de introns foram correlacionados à temperatura. Sistemas repórteres para processamento de pré-mRNA e RT-PCR mostraram que eficiência de processamento de mRNA no mutante pso4-1 é inversamente correlacionada com a temperatura de crescimento. Uma mutação pontual, substituindo uma leucina por uma serina foi identificada dentro da região codificadora N-terminal do alelo Pso4-1 e afeta as propriedades bioquímicas de Pso4-1p. Entre 24 clones isolados utilizando o sistema doishíbridos, 7 foram identificados como partes dos genes RAD2, RLF2 e DBR1. RAD2 codifica uma endonuclease indispensável para a via reparação por excisão de nucleotídeos (NER), RLF2 codifica a subunidade maior do fator de montagem da cromatina I, cuja deleção resulta em sinsibilidade à radiação UVC, enquanto que DBR1 codifica uma enzima que atua sobre substratos de RNA na forma lariat, degradando estruturas lariat em introns durante o processamento de mRNA. A caracterização dos fenótipos após tratamentos com mutágenos em linhagens mutantes simples e duplos de rad2∆, rlf2∆ e pso4-1 mostraram sensibilidade aumentada para para mutantes rad2∆/pso4-1 e rlf2∆/pso4-1, sugerindo uma interferência funcional destas proteínas no processo dereparação do DNA em Saccharomyces cerevisiae. O mecanismo exato de reparação de pontes inter-cadeia (ICL) em S. cerevisiae não é ainda totalmente conhecido. Identificando novos fenótipos e isolando proteínas potencialmente capazes de interagir com Pso2p através da técnica do sistema dois-híbridos, foi possível extender a caracterização deste gene específica para reparação de pontes intercadeia. Tratamentos com acetaldeído (ACA), um metabólito natural da via glicolítica, foi mais tóxico a linhagem mutante pso2 em comparação com a linhagem selvagem e também capaz de induzir a expressão da fusão contendo o promotor de PSO2 à lacZ (PSO2-lacZ) por um fator comparável à tratamentos com outros agentes indutores de ICLs, indicando que o metabólito natural ACA pode causar danos do tipo ICL em S. cerevisiae. A utilização do sistema dois-híbridos permitiu isolar partes de proteínas codificadas por nove diferentes genes, entre eles a proteína quinase Pak1p, um supressor de mutações termosensíveis da DNA Polimerase alfa. Pak1p interage com a extremidade C-terminal conservada de Pso2p, uma região da proteína recentemente nomeada β-CASP entre v ortólogos conhecidos do gene PSO2. A integridade do domínio β-CASP é essencial para a reparaçãode DNA proficiente como demonstrado em ensaios de complementação com mutantes pso2 ∆. Comparação da sobrevivência após tratamento com agentes mutagênicos de simples mutantes pso2 ∆ e pak1 ∆ assim com o dulpo mutante pso2 ∆/pak1 ∆ revelaram que o gene PAK1 é necessário para reparação do DNA proficiente como na linhagem selvagem. A interação epistática dos dois alelos mutantes na linhagem duplo mutante sugere que Pak1p atua na mesma via de reparação a qual PSO2 pertence e que PAK1 constitui um novo locus envolvido na reparação do DNA em S. cerevisiae.

Estudo de genes expressos diferencialmente durante o processo de estrobilização in vitro de Mesocestoides corti

Os cestódeos são agentes etiológicos de doenças parasíticas em humanos e em animais domesticados. Estamos utilizando Mesocestoides corti como um sistema modelo para estudar a biologia do desenvolvimento dos cestódeos, particularmente a transição da fase larval para a fase adulta segmentada. Com o propósito de isolar seqüências diferencialmente expressas durante o processo de segmentação, aplicamos a metodologia de análise das diferenças de representações de cDNA (cDNA RDA) utilizando RNA total extraído de larvas e vermes segmentados em cultivos in vitro de M. corti. Duas bibliotecas de cDNA subtraídas, enriquecidas com seqüências diferencialmente expressas das formas larvais ou segmentadas, foram construídas usando uma razão de driver:tester de 100:1 e 800:1 no 1º e 2º ciclos de subtração, respectivamente. A eficiência de subtração foi avaliada com experimentos de hibridização, utilizando as seqüências subtraídas como sondas contra os produtos de cDNA amplificados por PCR, com análise de macroarranjos e com confirmação individual, em experimentos de Northern virtual, de clones selecionados. Uma estratégia de RT-PCR em tempo real para confirmação dos resultados está sendo otimizada e resultados preliminares são apresentados. Após o seqüenciamento de 1036 clones de cDNA independentes e adoção de uma estratégia de seqüenciamento de alta qualidade, foram identificadas 190 seqüências, preferencialmente expressas em tetratirídeos (49) ou em vermes segmentados (141). Entre os genes identificados, 71 foram funcionalmente anotados, incluindo seqüências relacionadas a reguladores de estrutura de cromatina e controle de transcrição, cujos ortólogos estão implicados em processos de desenvolvimento em Drosophila e em vertebrados.

Análise comparativa dos genes de reparo do DNA em Gluconacetobacter diazotrophicus e Gluconobacter oxydans

Gluconacetobacter diazotrophicus é uma alfa-proteobactéria Gram-negativa, tolerante a meios ácidos, fixadora de nitrogênio atmosférico e foi a primeira bactéria diazotrófica endofítica isolada da cana-de-açúcar. Por sua vez, Gluconobacter oxydans, também alfa-proteobactéria Gram-negativa, possui a capacidade de oxidar incompletamente alcoóis e carboidratos. Ambas de interesse biotecnológico e industrial, essas bactérias tiveram seus genomas seqüenciados completamente em 2007. Desta forma, foi de interesse desse trabalho analisar e comparar os genes de reparo do DNA devido sua importância na manutenção da integridade genômica. Sendo assim, as vias de reparo presentes nos dois organismos foram identificadas, utilizando como base uma terceira alfa-proteobactéria, a Caulobacter crescentus, cujos genes de reparo foram descritos por um trabalho anterior e também os genes bem estabelecidos para o reparo do DNA em Escherichia coli. Para esse estudo, um banco de dados contendo ortólogos para os genes de reparo de DNA encontrados nos organismos foi criado e análises comparativas por similaridade usando o pacote Blast e o software Clustal foram feitas. Este estudo demonstrou que as principais vias de reparo ao DNA reparos por excisão, reparo direto, reparo recombinacional e reparo pelo sistema SOS estão presentes nos organismos analisados, demonstrando, na maioria das vezes, boa similaridade com E. coli. Interessantemente, foram encontradas duplicações gênicas nos quais uma das cópias estava presente no cromossomo e a outra, no plasmídeo, como no caso de UvrD, DnaE e Ssb, possivelmente caracterizando eventos de transferência lateral. Por fim, uma grande novidade foi a identificação de ortólogos para RecB em G. diazotrophicus e G. oxydans e de ortólogos duplicados de RecD em G. diazotrophicus. Até o momento, não havia sido relatada a presença de membros da via de iniciação RecBCD do reparo recombinacional em alfaproteobactérias

Error, Bias, and Long-Branch Attraction in Data for Two Chloroplast Photosystem Genes in Seed Plants

Sequences of two chloroplast photosystem genes, psaA and psbB, together comprising about 3,500 bp, were obtained for all five major groups of extant seed plants and several outgroups among other vascular plants. Strongly supported, but significantly conflicting, phylogenetic signals were obtained in parsimony analyses from partitions of the data into first and second codon positions versus third positions. In the former, both genes agreed on a monophyletic gymnosperms, with Gnetales closely related to certain conifers. In the latter, Gnetales are inferred to be the sister group of all other seed plants, with gymnosperms paraphyletic. None of the data supported the modern ‘‘anthophyte hypothesis,’’ which places Gnetales as the sister group of flowering plants. A series of simulation studies were undertaken to examine the error rate for parsimony inference. Three kinds of errors were examined: random error, systematic bias (both properties of finite data sets), and statistical inconsistency owing to long-branch attraction (an asymptotic property). Parsimony reconstructions were extremely biased for third-position data for psbB. Regardless of the true underlying tree, a tree in which Gnetales are sister to all other seed plants was likely to be reconstructed for these data. None of the combinations of genes or partitions permits the anthophyte tree to be reconstructed with high probability. Simulations of progressively larger data sets indicate the existence of long-branch attraction (statistical inconsistency) for third-position psbB data if either the anthophyte tree or the gymnosperm tree is correct. This is also true for the anthophyte tree using either psaA third positions or psbB first and second positions. A factor contributing to bias and inconsistency is extremely short branches at the base of the seed plant radiation, coupled with extremely high rates in Gnetales and nonseed plant outgroups. M. J. Sanderson,* M. F. Wojciechowski,*† J.-M. Hu,* T. Sher Khan,* and S. G. Brady

Enhanced human bone marrow stromal cell affinity for modified poly(L-lactide) surfaces by the upregulation of adhesion molecular genes

To enhance and regulate cell affinity for poly (l-lactic acid) (PLLA) based materials, two hydrophilic ligands, poly (ethylene glycol) (PEG) and poly (l-lysine) (PLL), were used to develop triblock copolymers: methoxy-terminated poly (ethylene glycol)-block-poly (l-lactide)-block-poly (l-lysine) (MPEG-b-PLLA-b-PLL) in order to regulate protein absorption and cell adhesion. Bone marrow stromal cells (BMSCs) were cultured on different composition of MPEG-b-PLLA-b-PLL copolymer films to determine the effect of modified polymer surfaces on BMSC attachment. To understand the molecular mechanism governing the initial cell adhesion on difference polymer surfaces, the mRNA expression of 84 human extracellular matrix (ECM) and adhesion molecules was analysed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). It was found that down regulation of adhesion molecules was responsible for the impaired BMSC attachment on PLLA surface. MPEG-b-PLLA-b-PLL copolymer films improved significantly the cell adhesion and cytoskeleton expression by upregulation of relevant molecule genes significantly. Six adhesion genes (CDH1, ITGL, NCAM1, SGCE, COL16A1, and LAMA3) were most significantly influenced by the modified PLLA surfaces. In summary, polymer surfaces altered adhesion molecule gene expression of BMSCs, which consequently regulated cell initial attachment on modified PLLA surfaces.

A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.