Molecular reconstruction of a genetic code alteration

The genetic code establishes the rules that govern gene translation into proteins. It was established more than 3.5 billion years ago and it is one of the most conserved features of life. Despite this, several alterations to the standard genetic code have been discovered in both prokaryotes and eukaryotes, namely in the fungal CTG clade where a unique seryl transfer RNA (tRNACAG Ser) decodes leucine CUG codons as serine. This tRNACAG Ser appeared 272±25 million years ago through insertion of an adenosine in the middle position of the anticodon of a tRNACGA Ser gene, which changed its anticodon from 5´-CGA-3´ to 5´-CAG-3´. This most dramatic genetic event restructured the proteome of the CTG clade species, but it is not yet clear how and why such deleterious genetic event was selected and became fixed in those fungal genomes. In this study we have attempted to shed new light on the evolution of this fungal genetic code alteration by reconstructing its evolutionary pathway in vivo in the yeast Saccharomyces cerevisiae. For this, we have expressed wild type and mutant versions of the C. albicans tRNACGA Ser gene into S. cerevisiae and evaluated the impact of the mutant tRNACGA Ser on fitness, tRNA stability, translation efficiency and aminoacylation kinetics. Our data demonstrate that these mutants are expressed and misincorporate Ser at CUGs, but their expression is repressed through an unknown molecular mechanism. We further demonstrate, using in vivo forced evolution methodologies, that the tRNACAG Ser can be easily inactivated through natural mutations that prevent its recognition by the seryl-tRNA synthetase. The overall data show that repression of expression of the mistranslating tRNACAG Ser played a critical role on the evolution of CUG reassignment from Leu to Ser. In order to better understand the evolution of natural genetic code alterations, we have also engineered partial reassignment of various codons in yeast. The data confirmed that genetic code ambiguity affects fitness, induces protein aggregation, interferes with the cell cycle and results in nuclear and morphologic alterations, genome instability and gene expression deregulation. Interestingly, it also generates phenotypic variability and phenotypes that confer growth advantages in certain environmental conditions. This study provides strong evidence for direct and critical roles of the environment on the evolution of genetic code alterations.

Fito- e bacterioplâncton em albufeiras dominadas por cianobactérias no Algarve

Dissertação de mest., Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

Evolution, gene regulation and functional analysis of BMP2 in fish

Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor β (TGFβ) superfamily with a central role in bone formation and mineralization. BMP2, a founding member of this family, has demonstrated remarkable osteogenic properties and is clinically used to promote bone repair and fracture healing. Lack of basic data on factors regulating BMP2 expression and activity have hampered a better understanding of its role in bone formation and bone-related diseases. The objective of this work was to collect new functional data and determine spatiotemporal expression patterns in a fish system aiming towards a better understanding of BMP2 function and regulation. Transcriptional and post-transcriptional regulation of gilthead seabream BMP2 gene was inferred from luciferase reporter systems. Several bone- and cartilage-related transcription factors (e.g. RUNX3, MEF2c, SOX9 and ETS1) were found to regulate BMP2 transcription, while microRNA 20a was shown to affect stability of the BMP2 transcript and thus the mineralogenic capacity of fish bone-derived host cells. The regulation of BMP2 activity through an interaction with the matrix Gla protein (MGP) was investigated in vitro using BMP responsive elements (BRE) coupled to luciferase reporter gene. Although we demonstrated the functionality of the experimental system in a fish cell line and the activation of BMP signaling pathway by seabream BMP2, no conclusive evidence could be collected on a possible interaction beween MGP and BMP2. The evolutionary relationship among the members of BMP2/4/16 subfamily was inferred from taxonomic and phylogenetic analyses. BMP16 diverged prior to BMP2 and BMP4 and should be the result of an ancient genome duplication that occurred early in vertebrate evolution. Structural and functional data suggested that all three proteins are effectors of the BMP signaling pathway, but expression data revealed different spatiotemporal patterns in teleost fish suggesting distinct mechanisms of regulation. In this work, through the collection of novel data, we provide additional insight into the regulation, the structure and the phylogenetic relationship of BMP2 and its closely related family members.

Identification of the cellular and molecular mechanism by which CD2AP, a regulator of neuronal intracellular transport contributes to the development of Alzheimer’s disease

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências,2014

Phylogenetic insights of Avena genus based on genomic analysis of 45S rDNA molecular organization

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências, 2014

Processamento e interpretação de dados aerogeofísicos magnetométricos e gamaespectrométricos no estudo do controle das mineralizações auríferas na região do Complexo Bossoroca, São Sepé, RS

Sete ocorrências auriferas estão inseridas na Seqüência Campestre do Complexo Bossoroca, no Escudo Sul-rio-grandense, municípios de São Sepé, no Estado do Rio Grande do Sul. Nessa região, foi realizado um aerolevantamento geofisico no qual foram coletados dados magnetométricos e aerogamaespectrométricos (K, U, Th e contagem total), em convênio finnado entre órgãos federais brasileiros (CPRM, DNPM e CNEN) e a empresa canadense Texas Instruments no período 1972-1973. Nessa dissertação são apresentados as interpretações e resultados dos dados e a metodologia utilizada para a interpretação das principais estruturas magnéticas e radiométricas visando aplicação na exploração mineral de ouro. Esse tipo de processamento, correlacionado aos dados geológicos, é uma prática usual de campanhas de prospecção mineral desde 1940 e é considerada como uma ferramenta de grande potencial para esse propósito. O processamento de dados magnetométricos forneceu mapas de primeira e segunda derivadas verticais, sinal analítico, gradiente horizontal, integral vertical, continuação para cima (1000, 2000 e 3000 metros) e seus principais lineamentos do campo magnético residual. Os dados foram tratados e posterionnente transfonnados em mapas de contorno e imagens pseudocoloridos e em tons de cinza com relevo sombreado (iluminação N, NE e SE) para realçar os principais lineamentos. Os mapas produzidos a partir dos dados aerogamaespectrométricos foram os de contagem total, K, U e Th, suas razões Th/K, U/K e UITh; mapas temários nos padrões RGB e CMY, potássio e urânio anômalos e parfunetro F, também foram transfonnados em mapas de contorno, pseudocolridos e tons de cinza com relevo sombreado (iluminação a N, NE e SE) e um mapa geológico interpretativo da região do Complexo Bossoroca As imagens magnetométricas mostraram-se importantes na caracterização estrutural regional, possibilitando a identificação de lineamentos NE-SW e NW-SE, não mapeados anterionnente e que podem estar relacionadas diretamente às ocorrências auriferas da região do Complexo Bossoroca. A aplicação das técnicas de processamento e interpretação de estruturas magnéticas por meio dos filtros como gradiente horizontal, primeira e segunda derivada, sinal analítico, continuação para cima, possibilitaram a determinação de descontinnidades fisicas que podem ser traduzidas em estruturas geológicas. O processamento de dados aerogamaespectrométricos possibilitou a caracterização de unidades geológicas regionais, a identificação de possíveis trends e zonas anômalas em potássio, relacionadas com as principais ocorrências auriferas da região. As imagens ternárias nos padrões RGB e CMY pennitiram a distinção e a caracterização das grandes unidades geológicas. Pode-se individualizar três regiões principais dentro da Seqüência Campestre, não mapeadas anterionnente, onde se encontram as ocorrências auriferas.

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.

Processamento e interpretação de dados 2D e 3D de GPR :aplicações no imageamento de feições kársticas e estruturas de dissolução no campo de petróleo de Fazenda Belém-CE

In Fazenda Belém oil field (Potiguar Basin, Ceará State, Brazil) occur frequently sinkholes and sudden terrain collapses associated to an unconsolidated sedimentary cap covering the Jandaíra karst. This research was carried out in order to understand the mechanisms of generation of these collapses. The main tool used was Ground Penetrating Radar (GPR). This work is developed twofold: one aspect concerns methodology improvements in GPR data processing whilst another aspect concerns the geological study of the Jandaíra karst. This second aspect was strongly supported both by the analysis of outcropping karst structures (in another regions of Potiguar Basin) and by the interpretation of radargrams from the subsurface karst in Fazenda Belém. It was designed and tested an adequate flux to process GPR data which was adapted from an usual flux to process seismic data. The changes were introduced to take into account important differences between GPR and Reflection Seismic methods, in particular: poor coupling between source and ground, mixed phase of the wavelet, low signal-to-noise ratio, monochannel acquisition, and high influence of wave propagation effects, notably dispersion. High frequency components of the GPR pulse suffer more pronounced effects of attenuation than low frequency components resulting in resolution losses in radargrams. In Fazenda Belém, there is a stronger need of an suitable flux to process GPR data because both the presence of a very high level of aerial events and the complexity of the imaged subsurface karst structures. The key point of the processing flux was an improvement in the correction of the attenuation effects on the GPR pulse based on their influence on the amplitude and phase spectra of GPR signals. In low and moderate losses dielectric media the propagated signal suffers significant changes only in its amplitude spectrum; that is, the phase spectrum of the propagated signal remains practically unaltered for the usual travel time ranges. Based on this fact, it is shown using real data that the judicious application of the well known tools of time gain and spectral balancing can efficiently correct the attenuation effects. The proposed approach can be applied in heterogeneous media and it does not require the precise knowledge of the attenuation parameters of the media. As an additional benefit, the judicious application of spectral balancing promotes a partial deconvolution of the data without changing its phase. In other words, the spectral balancing acts in a similar way to a zero phase deconvolution. In GPR data the resolution increase obtained with spectral balancing is greater than those obtained with spike and predictive deconvolutions. The evolution of the Jandaíra karst in Potiguar Basin is associated to at least three events of subaerial exposition of the carbonatic plataform during the Turonian, Santonian, and Campanian. In Fazenda Belém region, during the mid Miocene, the Jandaíra karst was covered by continental siliciclastic sediments. These sediments partially filled the void space associated to the dissolution structures and fractures. Therefore, the development of the karst in this region was attenuated in comparison to other places in Potiguar Basin where this karst is exposed. In Fazenda Belém, the generation of sinkholes and terrain collapses are controlled mainly by: (i) the presence of an unconsolidated sedimentary cap which is thick enough to cover completely the karst but with sediment volume lower than the available space associated to the dissolution structures in the karst; (ii) the existence of important structural of SW-NE and NW-SE alignments which promote a localized increase in the hydraulic connectivity allowing the channeling of underground water, thus facilitating the carbonatic dissolution; and (iii) the existence of a hydraulic barrier to the groundwater flow, associated to the Açu-4 Unity. The terrain collapse mechanisms in Fazenda Belém occur according to the following temporal evolution. The meteoric water infiltrates through the unconsolidated sedimentary cap and promotes its remobilization to the void space associated with the dissolution structures in Jandaíra Formation. This remobilization is initiated at the base of the sedimentary cap where the flow increases its abrasion due to a change from laminar to turbulent flow regime when the underground water flow reaches the open karst structures. The remobilized sediments progressively fill from bottom to top the void karst space. So, the void space is continuously migrated upwards ultimately reaching the surface and causing the sudden observed terrain collapses. This phenomenon is particularly active during the raining season, when the water table that normally is located in the karst may be temporarily located in the unconsolidated sedimentary cap

Estruturação do Eumenine Mastroparano por dinâmica molecular em misturas de TFE-água

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudos estruturais da Purina Nucleosídeo Fosforilase do Mycobacterium tuberculosis complexada com ligantes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Interação do peptídeo MP-I com micela de SDS em solução aquosa: um estudo por dinâmica molecular

Pós-graduação em Biofísica Molecular - IBILCE

Caracterização molecular de UsnRNAs em trypanosoma cruzi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Simulação de poli(etileno glicol) em água por dinâmica molecular

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Caracterização funcional de eIF5A: análise genética e molecular utilizando o modelo de Saccharomyces cerevisiae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)