Single-molecule imaging of transcription dynamics

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2019Transcription is the molecular process that synthesizes an RNA molecule that is complementary to the DNA template. In eukaryotes, transcription is catalyzed by one of three RNA polymerases that share structural features and subunits but transcribe different types of genes. RNA polymerase II (RNA Pol II) transcribes genes that code for proteins and several long non-coding genes. RNA Pol II transcription involves three main stages: initiation; transition into productive elongation; and termination. This thesis focuses on transcription termination and aims at studying its functional links with RNA processing and the formation of non-canonical nucleic acids structures, such as R-loops. R-loops are by-products of transcription that also have been found to play a role in transcription regulation, however, it is not well understood how they affect transcription termination and mRNA processing. To decipher how R-loop formation affect the process of transcription and RNA processing, we directly examined with single-molecule sensitivity the synthesis of transcripts upstream and downstream the cleavage and polyadenylation site (CPAS) in the nucleus of living human cells and calculated the transcription rate of RNA Pol II for that regions. By using two different RNA labeling methods, PP7 and λN22, we show that R-loops suppression impairs transcription termination. Further, we show that R-loop formation after the CPAS is not essential for an immediate transcription termination. A model for transcription termination, the torpedo model, suggests that exoribonuclease 2 (XRN2) digests the unprotected 5’ end of the RNA transcript until it collides with the RNA Pol II, leading to its dissociation from the template DNA and favoring transcription termination. Here, we provide evidence for the torpedo model by observing a non-immediate termination when XRN2 is depleted. Moreover, we show that splicing inhibition impairs transcript cleavage and transcription termination. Our data provide new insights in the role of R-loops in transcription termination and mRNA processing

Phylogenomics of pike cichlids (Cichlidae: Crenicichla): the rapid ecological speciation of an incipient species flock

© 2017 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2017 European Society For Evolutionary Biology The rapid rise of phenotypic and ecological diversity in independent lake-dwelling groups of cichlids is emblematic of the East African Great Lakes. In this study, we show that similar ecologically based diversification has occurred in pike cichlids (Crenicichla) throughout the Uruguay River drainage of South America. We collected genomic data from nearly 500 ultraconserved element (UCEs) loci and \u3e260 000 base pairs across 33 species, to obtain a phylogenetic hypothesis for the major species groups and to evaluate the relationships and genetic structure among five closely related, endemic, co-occurring species (the Uruguay River species flock; URSF). Additionally, we evaluated ecological divergence of the URSF based on body and lower pharyngeal jaw (LPJ) shape and gut contents. Across the genus, we recovered novel relationships among the species groups. We found strong support for the monophyly of the URSF; however, relationships among these species remain problematic, likely because of the rapid and recent evolution of this clade. Clustered co-ancestry analysis recovered most species as well delimited genetic groups. The URSF species exhibit species-specific body and LPJ shapes associated with specialized trophic roles. Collectively, our results suggest that the URSF consists of incipient species that arose via ecological speciation associated with the exploration of novel trophic roles

Determination of steroid estrogens in wastewater by high performance liquid chromatography-tandem mass spectrometry

This paper discusses the requirement for, and presents an analytical procedure for, the determination of four unconjugated steroid hormones and a conjugated steroid (estrone-3-sulfate) in wastewaters. The method quantifies the steroids by LC/MS/MS following solid phase extraction and a two stage clean-up procedure. Samples were extracted using C18 cartridges and eluates were then purified by gel permeation chromatography, followed by a further clean-up step on an aminopropyl cartridge. The limits of detection achieved were 0.2 ng l-1 for estriol, 17β-estradiol and 17α-ethinylestradiol, and 0.1 ng l-1 for estrone and the conjugate. The robustness of the method was demonstrated by achieving recoveries of >83% for all steroids in settled sewage and final effluent samples with relative standard deviations of 0.5 - 12%. The method was used to analyse a range of samples from a wastewater treatment works in south east England which demonstrated a >80% removal for estrone, estradiol and estriol with little impact on concentrations of ethinylestradiol or the conjugate

Lung segmentation in CT scans of patients with lower respiratory tract infection: influence of respiratory physiotherapy

The goal of the study was to assess the effects of a respiratory physiotherapy intervention in the tracheobronchial tree (TBT) volume and lung function in patients with lower respiratory tract infection (LRTI). In this framework, a method for TBT segmentation in Computer Tomography (CT) images of patients with LRTI was implemented, allowing to obtain volumetric data and study their correlation with the lung function (spirometry) parameterspublishe

Caseinolytic activity of fruit extract from Opuntia ficus-indica on bovine, caprine, and ovine Sodium Caseinates

The rates and extents of hydrolysis of RS- and â-caseins from bovine, caprine, and ovine sodium caseinates produced by an enzymatic extract of the fruit of Opuntia ficusindica, (L.) Miller were evaluated and compared with those produced by a commercial animal rennet. A mechanistic model based on a pseudo-first-order enzymatic reaction, in the presence of first-order deactivation of the enzyme, was postulated and successfully fitted to the experimental data. The animal rennet exhibited higher enzymatic efficiency than the fruit extract, irrespective of the source (i.e., bovine, caprine, or ovine) and the type (i.e., RS- or â-casein) of substrate. The enzymatic efficiency (kcat/Km) for RS-casein ranged from 72 to 220 and from 43 to 65 L g-1 h-1, and for â-casein from 242 to 742 and from 55 to 164 L g-1 h-1, for the animal rennet and the enzymatic extract of O. ficus-indica, respectively. Finally, it was observed that â-casein from caprine and ovine caseinates was degraded by O. ficus-indica faster than its RS counterpart, but the reverse was observed for bovine caseinate

Spiders (Arachnida: Araneae) associated with two degraded forests in the humid Chaco of Corrientes, Argentina.

El importante avance de la degradacion de bosques en la Provincia Biogeografica del Chaco, Argentina, conlleva una perdida importante de su poco conocida biodiversidad. Se realizo un estudio de la fauna de aranas en dos bosques de la provincia de Corrientes, Argentina, correspondientes al Distrito Oriental Humedo del Chaco, en las localidades de Laguna Brava y El Perichon. Se realizaron muestreos estacionales entre los anos 2001 y 2002. Las aranas recolectadas (2 067 individuos de 33 familias y 226 especies/ morfoespecies) fueron obtenidas por golpeteo del estrato arbustivo y tamizado de hojarasca. Las familias Araneidae, Anyphaenidae, Salticidae y Theridiidae fueron las mas abundantes en los dos bosques. El gremio de aranas constructoras de telas orbiculares presento el mayor numero de individuos (n=382) y el de cazadoras al acecho la mayor riqueza (S=56). En Laguna Brava se observo mayor abundancia en el verano (n=287) y primavera (n=273) y en el Perichon en invierno (n=315). La riqueza especifica y el valor de los indices de diversidad, fueron mayores en Laguna Brava (S=134, H'=4.23, E=0.86, D=0.023) que en El Perichon (S=127, H'=4.08, E=0.84, D=0.029). La similitud entre ambos bosques fue MH=0.611.The advancing degradation of the forest in the biogeographic Chaco province (Argentina) produces an important loss of its little known biodiversity. We studied the spider biodiversity in two forests of Corrientes, Argentina’s “Distrito Oriental Húmedo del Chaco”: Laguna Brava and El Perichón. Seasonal samplings of foliage and fallen leaves between 2001 and 2002 produced 2 067 individuals from 33 families and 226 species/ morphospecies). The families Araneidae, Anyphaenidae, Salticidae and Theridiidae were the most abundant in both forests. The “orb weavers” guild had the highest number of specimens (n=382) and “stalkers” the highest richness (S=56). In Brava, highest abundance was in the summer (n=287) and spring (n=273), in Perichón, it was in winter (n=315). The specific richness and the diversity indexes were higher in Brava (S=134, H´=4.23, E=0.86, D=0.023) than in Perichón (S=127, H´=4.08, E=0.84, D=0.029). The similarity value between both forests was MH=0.611.Centro de Estudios Parasitológicos y de Vectore