Hundreds of thousands of putative small ORFs (smORFs) sequences are present in eukaryotic genomes, potentially coding for peptides less than 100 amino acids. smORFs have been deemed non-coding on the basis of their high numbers and their small size that makes it extremely challenging to assess their functionality both bioinformatically and biochemically. The recently developed Ribo-Seq technique, which is the deep sequencing of ribosome footprints, has generated significant controversy by showing extensive translation of smORFs outside of annotated protein coding regions, including putative non-coding RNAs.. Our lab adapted the Ribo-Seq technique by combining it with the polysome fractionation in order to assess smORF translation in Drosophila S2 cells. This thesis provides a high-throughput assessment of smORF translation in Drosophila melanogaster by firstly implementing complementary techniques such as transfection-tagging and Mass spectrometry methods in order to provide an independent corroboration of the S2 cell data (Chapter 3). Secondly, the in order to expand the catalogue of smORFs that are translated, I significantly improve upon the yield and sequencing efficiency of the Poly-Ribo-Seq protocol while adapting it to Drosophila embryos and then implementing it across embryogenesis divided in to Early, Mid and Late stages (Chapter 4). Currently, there is still a lot of debate in the field with regards to Ribo-Seq data analysis, and various computational metrics have been developed aimed at discerning 'real' translation events to background noise. Chapter 5 explores some of the metrics developed and establishes a translation cut-off suitable for designating small ORFs as translated. Altogether, the improvements introduced to the protocol and my data analysis shows the translation of 500 annotated smORFs, 500 smORFs in long non-coding RNAs and 5,000 uORFs, of which only one-third of each type of smORF has previous evidence of translation. These findings strengthen the establishment of smORFs as a distinct class of genes that significantly expand the protein coding complement of the genome.

One of the proteins that lie at the heart of the DNA Damage Response (DDR) is Topoisomerase II-binding protein I (TopBP1). TopBP1 was initially identified and has been extensively studied in the yeast model organisms. However, the lack of readily available tools, including genetically defined mutant cell lines, has rendered the characterisation of TopBP1 in higher eukaryotes more challenging. Sequence information obtained from the characterisation of the gallus gallus TopBP1 mRNA revealed a different splicing pattern at the 5'end to the one reported in the Genome Browser. Our assembled TopBP1 mRNA sequence containing a novel open reading frame (ORF) enabled the creation of a conditional knockout cell line of TopBP1 in DT40, which has been impossible with the use of the annotated cDNA sequence. Thus the avian TopBP1 ORF identified herein contained the necessary function(s) to sustain viability of DT40 cells in the absence of the endogenous protein. Additionally, the establishment of an isogenic set of stable cell lines from the chicken B cell line DT40 by targeted deletion of the TopBP1 alleles revealed a gene dosage-dependent reduction of the TopBP1 protein levels and functions. This work establishes a novel gene-dosage system that can be used for the knock in of point mutations within the endogenous TopBP1 locus. Using this system, a novel characterisation of knock-in point mutants of the ATR Activation Domain (AAD) of TopBP1 was carried out, providing in vivo evidence of its DDR function(s). Finally, a stably integrated overexpression system (SIOS) capable of producing increased amounts of a protein of interest has been established in DT40 cells. SIOS represents an easy to use versatile system for various experimental purposes in the field of DT40. The work presented in this thesis represents a novel characterisation of the avian TopBP1 mRNA and the TopBP1 protein and its functions. This is crucial to gain insight into the mechanistics of the DDR network and the genetic instability characterising cancer development.

Gene regulation id controlled by transcription factor proteins that bind to specific DNA sequences, known as transcription factor binding sites (TFBSs). Combinations of transcription factors working, co-operatively in cis-regulatory modules (CRMs), play a role in regulating gene expression. Current computational methods for TFBS prediction cannot distinguish between functional and non-functional sites, and predict very large numbers of false positives. The thesis focuses on the development of a novel computational model, based on artificial neural networks (ANNs), for the identification of functional TFBSs, and the CRMs within which they operate in the human genome. Datasets of 12,239 experimentally verified true positive (TP) TFBSs and 130,199 false positive (FP) TFBSs were extracted using a combination of position weight matrices from the JASPAR database and experimentally verified sites from the Encyclopedia of DNA elements (ENCODE). A number of machine learning alsgorithms were tested using a range of genetic information including gene expression, necleosome positioning, DNA methylation states and DNA entropy. The best model, that gave a mean area under the curve under a receiver operator characteristic curve of 0.800, was based on a feedforward ANN using backpropagation. This model was then used to predict functional TFBSs in a number of gene sets from the human genome. The predictions, combined with experimentally proven TFBSs from ENCODE, were used to investigate combinatorial patterns of TFBSs operating in CRMs. CRM patterns have been analysed in disease-associated genes located in linkage disequilibrium blocks containing SNPs obtained from Genome Wide Association Studies (GWAS). The potential for the model to make functional TFBS predictions to aid in the annotation of orphan genes of unknown function is discussed. In addition this thesis presents computational work on a number of smaller published studies.

A hallmark of the cellular response to DNA double-strand breaks (DSBs) is histone H2AX phosphorylation by the protein kinase ATM. H2AX is unevenly distributed throughout chromatin and is rapidly phosphorylated to form γH2AX up to 2 megabases either side of DSBs. Studies in yeast systems have shown that while γH2A can spread in cis surrounding the break site, it can also spread in trans onto unbroken chromosomes located in close spatial proximity. Although the majority of data in the current literature presents the well characterised in cis spread of γH2AX, there are strong indications that it can also occur in trans in mammalian systems; analogous to the findings shown in yeast. This thesis lays out the steps taken to develop a novel system to address the spatial distribution of γH2AX around a nascent DSB. Since the first published live imaging experiments of the dynamics of chromatin by in vivo single particle tracking there has been extensive investigation into the regulation and biological function of movement of damaged DNA. In yeast, a relative consensus exists that DSB induction increases the movement of a DSB. In contrast to yeast however, data published of DSB movement in higher eukaryotes has been controversial, caused by conflicting results. Here, I developed a cell-based system, and utilised timelapse live cell imaging to show that a chromosomal locus containing a single endonuclease-induced DSB shows confined movement in comparison to an undamaged locus. Furthermore, this confined movement of a damaged locus is compounded by treatment with an ATM kinase inhibitor but not a DNA-PKcs kinase inhibitor, suggesting that the kinase activity of ATM and not the kinase activity of DNA-PKcs plays a significant role in the dynamics of DSBs.

乙酰膽鹼酯酶主要存在於神經肌肉接頭處和中樞神經系統的膽鹼能突觸處，是神經遞質傳遞過程中極其重要的膽鹼水解酶。研究表明，阿茲海默病人的大腦通常呈現出乙酰膽鹼酯酶的異常表達和分佈，並伴隨著β澱粉樣蛋白的沉澱。目前，乙酰膽鹼酯酶抑製劑是治療阿茲海默症的主要臨床藥物。 / 在本研究中，我們利用Bac-to-Bac 桿狀病毒表達系統分別使人類和雞泡魚的重組乙酰膽鹼酯酶基因在家蠶幼蟲裡得到了高效的表達。我們將乙酰膽鹼酯酶的cDNA序列克隆到pFastBac{U+2122} Dual質粒的多角體蛋白啟動子下游。為了易於監控蛋白表達水平，橙色熒光蛋白的cDNA序列也被克隆到同一個質粒的p10啟動子下游。此外，我們將多聚組氨酸標籤加在了乙酰膽鹼酯酶基因的碳端，從而使蛋白的純化效率得到了顯著提高。我們通過皮下注射含有乙酰膽鹼酯酶的重組bacmid對五齡期的家蠶幼蟲進行了病毒轉染。感染後約4-7天，重組乙酰膽鹼酯酶在蠶蟲內成功得到了表達。酶促反應動力學研究表明，重組乙酰膽鹼酯酶的活性與來自相同物種的天然乙酰膽鹼酯酶基本相似。這種高效率、低成本、高產量的蛋白表達方法可以為我們提供大量的重組乙酰膽鹼酯酶，用於體外篩選治療阿茲海默症的乙酰膽鹼酯酶抑製劑。 / 隨著對阿茲海默症分子學水平上的進一步了解，研究提出乙酰膽鹼酯酶可能通過外周陰離子位點誘導β澱粉樣多肽聚集, 從而形成澱粉樣纖維。因此，理想的乙酰膽鹼酯酶抑製劑應該既有抑制乙酰膽鹼酯酶的活性，又可以對抗β澱粉樣蛋白沉澱的毒性, 從而達到神經保護的作用。因此，我們採用AutoDock Vina軟件對ZINC數據庫內的天然化合物進行了兩輪虛擬篩選，篩選出的化合物理論上是可以同時作用於催化位點和外周陰離子位點。接下來，我們將對候選化合物進行體外驗證。 / Acetylcholinesterase (AChE: EC 3.1.1.7) is the acetylcholine-hydrolyzing enzyme that plays an essential role on cholinergic neurotransmission at the synapses of the brain and at the neuromuscular junctions. Abnormal expression and localization of AChE have been observed together with Aβ deposits in the brain of Alzheimer’s disease (AD) patient. Currently, AChE inhibitors are clinically used as drugs for AD treatment. / In this study, we demonstrated high-level expressions of functional recombinant human AChE and Tetraodon nigroviridis AChE using Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. The cDNA of AChE was cloned into the polyhedrin (PH) promoter of the plasmid pFastBac{U+2122} Dual. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP2) was cloned downstream to the p10 promoter of the same vector. We engineered a polyhistidine-tag (His-tag) tail to the C-terminal of each AChE gene to facilitate the purification. Transfection was carried out by subcutaneous injection of the recombinant bacmid DNA containing the AChE gene into the silkworm larvae of 5th instar. Approximately 4-7 days of post-infection, the recombinant AChE was expressed in the hemolymph of infected larvae. The kinetic studies showed that the biological activities of the recombinant AChEs were comparable to that of natural ones from other sources. This rapid, low-cost, and high yield production method could provide us sufficient amount of recombinant AChE for in vitro screening of AChE inhibitors for AD treatment. / Further advances in understanding the molecular basis of AD have proposed that AChE promote the assembly of Aβ peptide into amyloid fibrils through interaction at the peripheral anionic site of AChE. Consequently, new classes of AChE inhibitors are expected to be able to inhibit the active site of AChE and, at the same time, to protect neurons from Aβ toxicity. Therefore, we applied two rounds of virtual screening of ZINC database using AutoDock Vina to obtain new potential inhibitors which might be able to targeting both of the active and peripheral sites of AChE. The compounds with good performances in both of the two rounds of screening would be validated by the sequential in vitro tests. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Shuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-124). / Abstracts also in Chinese. / Acknowledgements --- p.I / Abstracts (English) --- p.II / Abstracts (Chinese) --- p.IV / Table of Contents --- p.VI / List of Abbreviations --- p.IX / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Acetylcholine mediated neutotransmission in nervous system --- p.1 / Chapter 1.2 --- Acetylcholineterase --- p.2 / Chapter 1.3 --- Comparison of AChE and BChE --- p.3 / Chapter 1.4 --- Molecular sturcture of AChE --- p.5 / Chapter 1.5 --- Molecular diversity of AChE --- p.7 / Chapter 1.5.1 --- Regulation at transcriptional level --- p.8 / Chapter 1.5.2 --- Regulation at post-transcriptional level --- p.11 / Chapter 1.5.3 --- Regulation at post-translational level --- p.12 / Chapter 1.6 --- Classic functions of AChE --- p.15 / Chapter 1.7 --- Non-classic functions of AChE --- p.18 / Chapter 1.8 --- Diseases associated with AChE --- p.19 / Chapter 1.8.1 --- Myasthenia gravis --- p.19 / Chapter 1.8.2 --- Alzheimer's disease --- p.20 / Chapter 1.8.2.1 --- Pathogenesis of AD --- p.20 / Chapter 1.8.2.2 --- Treatments for AD --- p.22 / Chapter 1.9 --- Silkworm larvae as biofactory for protein expression --- p.24 / Chapter 1.10 --- Traditional baculovirus expression system --- p.26 / Chapter 1.11 --- Bac-to-Bac baculovirus expression system --- p.29 / Chapter 1.12 --- Virtual screening with AutoDock Vina --- p.29 / Chapter 1.13 --- Project overview and the aim of study --- p.31 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.1.1 --- Chemicals and Reagents --- p.33 / Chapter 2.1.2 --- Primers --- p.35 / Chapter 2.1.3 --- Antibodies --- p.35 / Chapter 2.1.4 --- Silkworms --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Construction of the expression cassette --- p.36 / Chapter 2.2.1.1 --- Preparation of E.coli competent cells --- p.36 / Chapter 2.2.1.2 --- Transformation --- p.36 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.37 / Chapter 2.2.1.4 --- Gene clean --- p.37 / Chapter 2.2.1.5 --- Subcloning of target genes --- p.38 / Chapter 2.2.1.6 --- Plasmid DNA extraction --- p.40 / Chapter 2.2.1.7 --- Quantification of plasmid DNA by spectrophotometer --- p.41 / Chapter 2.2.1.8 --- Plasmid DNA sequencing --- p.41 / Chapter 2.2.2 --- Generation of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.1 --- Transposition --- p.42 / Chapter 2.2.2.2 --- White/Blue screening --- p.42 / Chapter 2.2.2.3 --- Extraction of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.4 --- Analysis of recombinant bacmid DNA by PCR --- p.44 / Chapter 2.2.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.1 --- Raising silkworm larvae --- p.45 / Chapter 2.2.3.2 --- Preparation of transfecting solution --- p.45 / Chapter 2.2.3.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.4 --- Collection of hemolymph after protein expression --- p.46 / Chapter 2.2.3.5 --- Oral infection of sikworm larvae --- p.46 / Chapter 2.2.4 --- Purification of AChE --- p.47 / Chapter 2.2.4.1 --- Nickel-chelating afinity chromatography --- p. 47 / Chapter 2.2.4.2 --- Determination of protein concenttration by BCA assay --- p.47 / Chapter 2.2.4.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.4.4 --- Western blot --- p.49 / Chapter 2.2.5 --- Kinetic analysis of AChE --- p.50 / Chapter 2.2.5.1 --- Ellman assay --- p.50 / Chapter 2.2.5.2 --- Curve fitting --- p.51 / Chapter 2.2.6 --- Virtual screening --- p.51 / Chapter Chapter 3 --- Expression of recombinant AChEs in silkworm larvae --- p.55 / Chapter 3.1 --- Construction of the expression cassette --- p.55 / Chapter 3.1.1 --- Human AChE and Tetraodon nigroviridis AChE --- p.55 / Chapter 3.1.2 --- Amplification of target genes from the parent vectors --- p.56 / Chapter 3.1.3 --- Insertion of target genes into pFastBac Dual --- p.58 / Chapter 3.2 --- Generation of recombinant bacmid DNA --- p.60 / Chapter 3.2.1 --- Phenotype verification --- p.60 / Chapter 3.2.2 --- PCR analysis of the recoombinant bacmid DNA --- p.64 / Chapter 3.3 --- Expression of AChE in silkworm larvae --- p.66 / Chapter 3.3.1 --- Raising silkworms --- p.66 / Chapter 3.3.2 --- High-level expression of AChE in silkworm larvae --- p.68 / Chapter 3.4 --- Oral infeciton --- p.72 / Chapter Chapter 4 --- Analysis of the recombinant AChEs --- p.73 / Chapter 4.1 --- Purification of recombinant AChEs by nickel-chelating affinity chromatography --- p.73 / Chapter 4.2 --- SDS-PAGE and western blot analysis of the recombinant AChEs --- p.76 / Chapter 4.3 --- Kinetic studies of recombinant AChEs --- p.79 / Chapter 4.4 --- Virtual screening --- p.84 / Chapter Chapter 5 --- Discussion and conclusion --- p.95 / Chapter 5.1 --- Demonstration of high-level expression of recombinant AChEs by Bac-to-Bac baculovirus expression system --- p.95 / Chapter 5.2 --- Oral infection of silkworm larvae --- p.98 / Chapter 5.3 --- Characterization of recombinant AChEs --- p.98 / Chapter 5.4 --- Discovery of new AChE inhibitors by virtual screening --- p.100 / Chapter 5.5 --- Future works --- p.101 / Chapter 5.6 --- Other applications --- p.102 / Chapter 5.7 --- Conclusion --- p.102 / References --- p.104 / Appendix I --- p.125 / Appendix II --- p.127 / Appendix III --- p.129

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-09T15:48:50Z No. of bitstreams: 1 Milena da Silva Albuquerque.pdf: 915379 bytes, checksum: dd414d9f832cb823db0efc59b06ce1fb (MD5) / Made available in DSpace on 2017-02-09T15:48:50Z (GMT). No. of bitstreams: 1 Milena da Silva Albuquerque.pdf: 915379 bytes, checksum: dd414d9f832cb823db0efc59b06ce1fb (MD5) Previous issue date: 2014-12-17 / The objective of this work was to investigate the occurrence of encoding staphylococcal enterotoxin (sea, seb, sec and seg) genes and the toxin gene 1 of Toxic Shock Syndrome (tst) from Staphylococcus aureus, coming from mastitis cases bovine in the micro region of Garanhuns, State of Pernambuco. 93 isolates from Staphylococcus aureus were analyzed, which were obtained from milk samples from cows with clinical mastitis and subclinical from 17 properties in 11 municipalities of the Micro Region of Garanhuns, State of Pernambuco. For the molecular characterization of the species Staphylococcus aureus, one of the Polymerase Chain Reaction (PCR) was performed in order to identify the presence of the nuc gene, and to the molecular characterization of enterotoxins, and Staphylococcal toxin Toxic Shock Syndrome. Specific genes were identified: sea, seb, sec, seg and tst. 93 genes were analyzed and we observed the presence of enterotoxigenic gene in 20 (21.6%) samples, of which 11 (55.0%) were positive for tst gene, seven (35.0%) for the sec gene two (10.0%) for the seg gene. 20 isolates amplified segments to the presence of the sec, seg and tst genes. 16 of these (80.0%) were positive for only one gene, and four (20.0%) were positive for both genes (tst and sec). 17 properties were studied, of which seven (41.2%) had positive cultures for at least one of the genes sec, seg and tst. This was the first hit record of encoding gene of the toxin of toxic shock syndrome in mastitis cows milk samples in the state of Pernambuco. Since there was a variation in the distribution of sec, seg and tst genes in strains from different property, it can be inferred that there is genotypic variation in S. aureus strains that cause bovine mastitis. / Objetivou-se com este trabalho pesquisar a ocorrência de genes codificadores de enterotoxinas estafilocócicas (sea, seb, sec e seg) e do gene da toxina 1 da síndrome do choque tóxico (tst) a partir de isolados de Staphylococcus aureus procedentes de casos de mastite bovina na microrregião Garanhuns, estado de Pernambuco. Foram analisados 93 isolados de Staphylococcus aureus obtidos a partir de amostras de leite de vacas com mastite clínica e subclínica, provenientes de 17 propriedades localizadas em 11 municípios da microrregião Garanhuns, no estado de Pernambuco. Para a caracterização molecular da espécie Staphylococcus aureus, foi realizada uma Reação em Cadeia da Polimerase (PCR) visando a identificação pela presença do gene nuc, assim como para a caracterização molecular das Enterotoxinas Estafilocócicas e da toxina da Síndrome do Choque Tóxico. Foram identificados os genes específicos sea, seb, sec, seg e tst. Dos 93 isolados analisados, observou-se a presença de genes enterotoxigênicos em 20 (21,6%) amostras, das quais 11 (55,0%) foram positivas para o gene tst, sete (35,0%) para o gene sec, duas (10,0%) para o gene seg. Dentre os 20 isolados que amplificaram segmentos para a presença dos genes sec, seg e tst, 16 (80,0%) foram positivos apenas para um gene e quatro (20,0%) foram positivos para dois genes (sec e tst). Das 17 propriedades estudadas, sete (41,2%) apresentaram amostras positivas para pelo menos um dos genes sec, seg e tst. Este foi o primeiro registro de ocorrência do gene codificador da toxina da síndrome do choque tóxico em amostras de leite de vacas com mastite no estado de Pernambuco. Como houve uma variação na distribuição dos genes sec, seg e tst nas cepas procedentes de diferentes propriedades, pode-se inferir que há uma variação genotípica nas cepas de S. aureus que causam mastite bovina.

Os agrupamentos de ferro-enxofre (Fe-S) são grupos prostéticos necessários para a manutenção da vida, pois estão envolvidos em diversos processos incluindo a transferência de elétrons, reações metabólicas, sinalização e regulação da expressão gênica. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a síntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Neste trabalho foi realizada uma análise transcricional dos genes NFS1, ISA1 e ISU1 de E. grandis após diferentes estúmlos por meio de PCR quantitativa (qRT-PCR) e microarranjos. Após o tratamento de plântulas de E. grandis com frio, foram realizados experimentos de qRT-PCR. Os resultados foram normalizados com os genes constitutivos codificadores da histona H2B e da ribonucleoproteína L23A. Considerando tal normalização, ISU1 aumentou sua expressão em 0,6 e 1,7 vezes, NFS1 apresentou um aumento de 6 e 8 vezes, enquanto ISA1 apresentou um aumento de 69 a 114 vezes em relação à condição controle. Utilizando-se a técnica de microarranjos, foi analisada a diferença de expressão entre folhas e xilema de árvores maduras de E. grandis. O gene NFS1 apresentou maior expressão nas folhas do que em xilema, porém os genes ISA1 e ISU1 apresentaram um padrão de expressão equivalente entre os dois tipos de tecidos. Esses resultados sugerem que (i) os genes NFS1 e ISA1 podem estar relacionados à resposta celular ao estresse causado por frio; e que (ii) os aumentos na expressão devem-se, provavelmente, ao metabolismo de enxofre e à indução de enzimas antioxidantes. Foi também realizado um experimento de curva de tempo com a submissão de plântulas de E. grandis ao resfriamento, objetivando-se verificar em que momento esses genes começam a ter suas expressões aumentadas. O gene ISU1 apresentou maior expressão gênica nas primeiras duas horas de tratamento, caindo drasticamente logo após este período. O gene ISA1, que havia apresentado a maior expressão relativa no experimento anterior, não apresentou diferença significativa no padrão de expressão durante as 16 horas de resfriamento, assim como o gene NFS1. Esses resultados indicam que as proteínas Fe-S, frente ao resfriamento, estão possivelmente envolvidas na recuperação das plantas após tal estresse. / Iron-sulfur (Fe-S) clusters are prosthetic groups required for the maintenance of life because they are involved in various vital processes, including electron transfers, metabolic reactions, signaling and regulation of gene expression. Plants perform photosynthesis and respiration, two processes that require Fe-S proteins, and are the only organisms in which the synthesis of these proteins is compartmentalized. Several factors and stresses affect the development of plants including low temperature, which is a productivity-limiting factor and restricts plants to certain geographical distributions, including Eucalyptus grandis, a species with significant economic importance. The aim of this study is to perform an analysis of E. grandis NFS1 and ISA1 gene expressions after different stimuli through quantitative PCR (qRT-PCR) and microarrays. qRT-PCR experiment were conducted on plants submitted to a cold treatment. The results were normalized with the housekeeping genes encoding histone H2B and ribonucleoprotein L23A. Considering such normalizations, ISU1 increased the expression 0.6 and 1-fold, NFS1 showed a 6 and 8-fold increase in comparison with the control condition, while ISA1 gene increased 69 and 114-fold. Using microarrays, the difference in expression between leaves and xylem of E. grandis was analyzed. The NFS1 gene showed higher expression in leaves than in xylem, but the ISA1and ISU1 showed equivalent pattern of expression in both types of tissues. These results suggest that (i) NFS1 and ISA1 genes are related to the cellular response to the stress caused by chilling, and that (ii) the increased expression should be probably due to the metabolism of sulfur and to the induction of antioxidative enzymes. A time-course experiment was also conducted during the cold stress of E. grandis plants to look at which moment these genes begin to increase their expressions. The ISU1 gene showed higher expression in the first 2 hours of treatment, and than decreased severally after this period. The ISA1 gene, which had shown the highest expression in the previous experiment, did not show significant differences in the pattern of expression during the 16 hours of chilling treatment, as well as the NFS1 gene. These results indicate that Fe-S proteins, in response to low temperature, are possibly involved in the recovery of the plants after this stress.

O silenciamento gênico tem sido utilizado extensivamente para facilitar a investigação de eventos da biologia vegetal. Ele pode ser induzido de diferentes modos, sendo que o passo chave para sua ocorrência é a presença de RNA fita dupla (dsRNA). A fim de avaliar um método rápido de obtenção de silenciamento para análise funcional em larga escala, foi inoculado dsRNA ou siRNA diretamente em folhas de plantas de arroz e de Nicotiana benthamiana. Os dsRNAs de seqüências do gene pds e do gene codificante da ferritina foram obtidos através da síntese in vivo e in vitro e o siRNA de PDS de arroz, derivado somente de síntese in vitro. O gene pds codifica a enzima fitoeno desaturase (PDS), uma enzima chave na biossíntese de carotenóides, que protege a clorofila das plantas contra o foto-branqueamento. O silenciamento deste gene mostra um fenótipo visível, claramente demonstrado através do uso de um inibidor químico da síntese desta enzima. No presente estudo, plantas de arroz e de N. benthamiana inoculadas com dsRNAs derivados do gene pds não apresentaram mudanças fenotípicas. Para avaliação de um outro método foi adaptado um vetor de silenciamento estável, no qual foram inseridos fragmentos de cDNA nas orientações senso e antisenso do gene de ferritina de arroz. O papel da ferritina, segundo experimentação in vitro, pode estar ligada à tolerância de certos cultivares de arroz às altas concentrações de ferro. O aprimoramento das metodologias de silenciamento e de transformação poderá resultar em uma ferramenta mais útil para a análise funcional em larga escala de genes de arroz. / Gene silencing has been used extensively to facilitate the investigation of different events in plant biology. It can be induced through different ways. The key step for its occurrence is the presence of double strand RNA (dsRNA). In order to evaluate a fast method to obtain silencing for functional analysis in a wide scale, either dsRNAs or siRNAs were directly inoculated in the leaves of rice and N. benthamiana plants. The dsRNAs the sequences of the pds gene and of the coding region of the ferritin gene were obtained through in vivo or in vitro synthesis, the siRNA PDS of rice were derived from synthesis in vitro. The pds gene codifies for the enzyme phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids, which protects the clorophil from the plants against photo-bleaching. The silencing of this gene shows a visible phenotype, clearly demonstrated through a chemical inhibition of PDS synthesis. However, no phenotypical changes were observed in either rice or N. benthamiana plants inoculated with dsRNA of the pds gene. In the present study, it was also adapted a vector for stable transformation, in which were inserted cDNA fragments of both, sense and antisense, orientations of the rice ferritin gene. In vitro experiments show that ferritin could be connected to the tolerance to high concentrations of iron of certain cultivars of rice. The improvement of silencing and transformation methodologies could result in a more useful tool for the functional analysis of rice genes in a wide scale.

A utilização massiva de fertilizantes químicos na agricultura tem efeitos perniciosos ao ambiente. As bactérias do gênero Azospirillum são amplamente estudadas, pois são capazes de promover o crescimento vegetal. Essa característica lhes confere potencial para serem utilizadas na agricultura como uma alternativa ecologicamente compatível. Embora a espécie Azospirillum amazonense seja menos conhecida, o estudo de sua biologia molecular poderia contribuir para a elucidação dos mecanismos envolvidos na promoção do crescimento vegetal. Na primeira parte deste trabalho, foram descritas ferramentas genéticas que podem facilitar o estudo da biologia molecular de A. amazonense. Métodos de conjugação e eletroporação foram otimizados utilizando vetores com origens de replicação de amplo espectro (pVS1 e pBBR1). Além disso, mutantes para o gene glnK foram gerados utilizando o sistema do vetor pK19MOBSACB. Finalmente, um protocolo de análise de promotores baseado na expressão de proteínas fluorescentes foi desenvolvido para permitir estudos de regulação gênica. Na segunda parte do trabalho, uma análise abrangente das características do draft do genoma de A. amazonense foi realizada. Essa espécie apresenta um repertório versátil de genes, crucial para seu modo de vida na rizosfera. Genes putativos relacionados com metabolismo de nitrogênio e de carbono, produção de energia, produção de fitormônio, transporte, quorum sensing, resistência a antibióticos, síntese de bacteriofitocromo, quimiotaxia e motilidade foram identificados. Os genes da fixação do nitrogênio e da nitrilase poderiam estar diretamente relacionados com a promoção do crescimento vegetal. A identificação de genes da RubisCO sugere que A. amazonense seja capaz de fixar carbono, característica do seu metabolismo antes desconhecida. Outro aspecto relevante é que alguns genes de A. amazonense, como os da nitrogenase e da RubisCO, são mais próximos filogeneticamente aos genes de membros da ordem Rhizobiales do que dos de espécies do mesmo gênero. / The massive use of chemical fertilizers in agriculture has harmful effects to the environment. Bacteria from the Azospirillum genus are widely studied, since they are able to promote plant growth. This feature gives them the potential to be used in agriculture as an ecologically compatible alternative. Although the Azospirillum amazonense is a lesserknown species, the study of its molecular biology could contribute to a better understanding of the mechanisms implicated in plant growth. In the first part of this study, genetic tools that can support the study of the molecular biology of A. amazonense were described. Conjugation and electrotransformation methods were established utilizing vectors with broad host-replication origins (pVS1 and pBBR1). Furthermore, glnK-specific A. amazonense mutants were generated utilizing the pK19MOBSACB vector system. Finally, a promoter analysis protocol based on fluorescent protein expression was optimized to aid genetic regulation studies on this bacterium. In the second part of this study a comprehensive analysis of the genomic features of this species was presented. The species A. amazonense presents a versatile repertoire of genes crucial for its plant-associated lifestyle. Genes of A. amazonense related to nitrogen/carbon metabolism, energy production, phytohormone production, transport, quorum sensing, antibiotic resistance, chemotaxis/motility and bacteriophytochrome biosynthesis were identified. Noteworthy genes were the nitrogen fixation genes and the nitrilase gene, which could be directly implicated in plant growth promotion, and the carbon fixation genes, which had previously been poorly investigated in this genus. One important finding was that some A. amazonense genes, like the nitrogenase genes and RubisCO genes, were closer phylogenetically to genes from Rhizobiales members than to those from species of its own order.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Ciências Médicas, Programa de Pós-Graduação em Ciências Médicas, 2015. / A Síndrome de Robinow se caracteriza por dismorfias faciais associadas a encurtamento mesomélico e genitália hipoplásica. Defeitos de segmentação costovertebral que incluem fusões de costelas também podem ser observados e permitem distinguir os pacientes com a forma autossômica recessiva da síndrome daqueles com a forma autossômica dominante. A forma recessiva é causada por mutações no gene ROR2 (related orphan receptor 2), sendo que apenas dezenove diferentes mutações foram descritas em pacientes com a síndrome. O objetivo desse trabalho foi identificar novas mutações no gene ROR2 em pacientes com a Síndrome de Robinow autossômica recessiva. Foram selecionados onze pacientes originários do Brasil, Estados Unidos, Índia e Turquia com características clínicas da forma recessiva da síndrome. O sequenciamento foi realizado a partir de amostras de sangue e saliva por sequenciamento Sanger, ou por sequenciamento de nova geração na plataforma Ion PGM. Em um paciente a mutação foi detectada por MLPA. Identificamos 13 diferentes mutações sendo que 10 ainda não haviam sido descritas na literatura. A comparação dos dados clínicos dos pacientes estudados com as frequências dos sinais clínicos proposta por Mazzeu e cols. (2007) revelou frequência semelhante para a maioria dos sinais clínicos com exceção da fusão de costelas, ausente no paciente 5, a língua bífida presente em todos os 11 pacientes, o lábio superior fino que teve uma frequência de 72% e anteriormente era de 26% e a má-oclusão dentária que teve uma frequência de 60% enquanto que a frequência descrita anteriormente era de 93%. Assim, a descrição clínica detalhada de pacientes com diagnóstico confirmado molecularmente contribuiu para a definição das frequências dos principais sinais clínicos nessa forma da síndrome. Observamos também uma maior gravidade das manifestações esqueléticas nos pacientes com mutações que alteram o quadro de leitura mostrando assim que pode haver correlação entre o tipo de mutação e o fenótipo dos afetados. O conjunto de novas mutações detectadas no trabalho colaboram para um melhor entendimento da fisiopatologia da síndrome. / Robinow syndrome is characterized by facial dysmorphisms, mesomelic limb shortening and hypoplastic genitalia. Costovertebral segmentation defects including rib fusions may be present and allow the distinction between the autosomal recessive and the autosomal dominant forms. Recessive form is caused by mutations in ROR2 gene (related orphan receptor 2) and only 19 different mutations have been described in the literature in patients with the syndrome. The main goal of this work was to identify new mutations in ROR2 in patients with RRS. Eleven patients with clinical signs of RRS from Brazil, United States, India and Turkey have been selected. Sequencing was performed from either blood or saliva samples by Sanger sequencing or Next generation sequencing using Ion PGM platform. In one patient the mutation was detected by MLPA. We identified 13 different mutations, 10 not previously described in the literature. Comparison of clinical data from our patients and the frequencies of clinical signs proposed by Mazzeu e cols., (2007) revealed similar frequencies for most clinical signs except for rib fusions, absent in patient 5; bifid tongue, present in all 11 patients; thin upper lip that had a frequency of 72% compared to 26% in the previous report and dental malocclusion that showed a frequency of 60% and the previous frequency was 93%. Therefore, a detailed clinical description of patients with a molecularly confirmed diagnosis contributed to the definition of frequencies of the main clinical signs of the syndrome. We also observed a more severe skeletal phenotype in patients with frameshift mutations showing that genotype and phenotype may correlate. The group of new mutations detected in the present work contribute to a better understanding of the physiopathology of the syndrome.