Integrase mutants defective for interaction with LEDGF/p75 are impaired in chromosome tethering and HIV-1 replication

The insertion of a DNA copy of its RNA genome into a chromosome of the host cell is mediated by the viral integrase with the help of mostly uncharacterized cellular cofactors. We have recently described that the transcriptional co-activator LEDGF/p75 strongly interacts with HIV-1 integrase. Here we show that interaction of HIV-1 integrase with LEDGF/p75 is important for viral replication. Using multiple approaches including two-hybrid interaction studies, random and directed mutagenesis, we could demonstrate that HIV-1 virus harboring a single mutation that disrupts integrase-LEDGF/p75 interaction, resulted in defective HIV-1 replication. Furthermore, we found that LEDGF/p75 tethers HIV-1 integrase to chromosomes and that this interaction may be important for the integration process and the replication of HIV-1.

Medidas de eficiência energética numa indústria cerâmica

O presente relatório resulta de um estágio realizado no âmbito da eficiência energética assente no programa Galp 20-20-20 que tem por génese uma parceria entre a Universidade de Aveiro e a empresa de coberturas cerâmicas, CS – Coelho da Silva S.A. A Fábrica alvo de estudo é uma consumidora intensiva de energia, despendeu no ano de 2013 cerca de 3.768 tep. Devido aos seus processos de cozedura e secagem, apresenta uma elevada dependência de Gás Natural, representando pouco mais de 78% do consumo global da fábrica. Deste consumo de energia térmica, 83% respeita ao forno e os restantes 17% encontram-se alocados ao secador, pelo que as medidas de eficiência energética presentes neste relatório centram-se da redução deste vetor energético. São então propostas três medidas para a redução da dependência deste vetor. A primeira, incide na recuperação de calor residual presente nos gases de exaustão através da instalação de um permutador de calor. Esta medida permite uma redução do consumo na ordem dos 10% e conta com um payback de 2,3 anos resultante de uma economia anual de 150.000 €. Para este estudo foi desenvolvido um modelo dinâmico em excel que permite a simulação de diversos cenários. São também propostas mais duas intervenções que incidem na alteração do circuito térmico. Estas medidas têm um impacte mais reduzido no que respeita ao percentual de redução energético, ambas com menos de 1% de redução do consumo global da fábrica. Contudo são medidas bastante interessantes dada a sua simplicidade e contam com poupanças anuais na ordem dos 6.000 € que resultam num payback inferior a 2 meses. Paralelamente executaram-se dois estudos para a iluminação, o primeiro sugere a instalação de um modelador de tensão que reduz a potência de iluminação em 36%, implicando uma redução da iluminância de cerca de 5%. A redução da potência resulta numa economia energética na ordem dos 0,4% da energia global da instalação. Este equipamento poderá ser adquirido por completo ou em renting. Ao optar pela compra integral, o investimento será apenas ressarcido em 2,8 anos resultante de uma poupança anula de perto de 6.500 €. Caso seja por renting este não tem qualquer custo adicional e as economias monetárias são partilhadas entre a empresa que fornece o equipamento e a CS-Coelho da Silva, S.A. Por fim é sugerida a substituição de parte da iluminação atual da fábrica por tecnologia LED, com esta medida reduz-se o consumo global em 0,76%. Esta medida gera uma economia monetária na ordem dos 11.500 € sendo ressarcida em 2,1 anos.

Large eddy simulation of shock boundary layer interaction control using micro-vortex generators

The performance of supersonic engine inlets and external aerodynamic surfaces can be critically affected by shock wave / boundary layer interactions (SBLIs), whose severe adverse pressure gradients can cause boundary layer separation. Currently such problems are avoided primarily through the use of boundary layer bleed/suction which can be a source of significant performance degradation. This study investigates a novel type of flow control device called micro-vortex generators (µVGs) which may offer similar control benefits without the bleed penalties. µVGs have the ability to alter the near-wall structure of compressible turbulent boundary layers to provide increased mixing of high speed fluid which improves the boundary layer health when subjected to flow disturbance. Due to their small size,µVGs are embedded in the boundary layer which provide reduced drag compared to the traditional vortex generators while they are cost-effective, physically robust and do not require a power source. To examine the potential of µVGs, a detailed experimental and computational study of micro-ramps in a supersonic boundary layer at Mach 3 subjected to an oblique shock was undertaken. The experiments employed a flat plate boundary layer with an impinging oblique shock with downstream total pressure measurements. The moderate Reynolds number of 3,800 based on displacement thickness allowed the computations to use Large Eddy Simulations without the subgrid stress model (LES-nSGS). The LES predictions indicated that the shock changes the structure of the turbulent eddies and the primary vortices generated from the micro-ramp. Furthermore, they generally reproduced the experimentally obtained mean velocity profiles, unlike similarly-resolved RANS computations. The experiments and the LES results indicate that the micro-ramps, whose height is h≈0.5δ, can significantly reduce boundary layer thickness and improve downstream boundary layer health as measured by the incompressible shape factor, H. Regions directly behind the ramp centerline tended to have increased boundary layer thickness indicating the significant three-dimensionality of the flow field. Compared to baseline sizes, smaller micro-ramps yielded improved total pressure recovery. Moving the smaller ramps closer to the shock interaction also reduced the displacement thickness and the separated area. This effect is attributed to decreased wave drag and the closer proximity of the vortex pairs to the wall. In the second part of the study, various types of µVGs are investigated including micro-ramps and micro-vanes. The results showed that vortices generated from µVGs can partially eliminate shock induced flow separation and can continue to entrain high momentum flux for boundary layer recovery downstream. The micro-ramps resulted in thinner downstream displacement thickness in comparison to the micro-vanes. However, the strength of the streamwise vorticity for the micro-ramps decayed faster due to dissipation especially after the shock interaction. In addition, the close spanwise distance between each vortex for the ramp geometry causes the vortex cores to move upwards from the wall due to induced upwash effects. Micro-vanes, on the other hand, yielded an increased spanwise spacing of the streamwise vortices at the point of formation. This resulted in streamwise vortices staying closer to the wall with less circulation decay, and the reduction in overall flow separation is attributed to these effects. Two hybrid concepts, named “thick-vane” and “split-ramp”, were also studied where the former is a vane with side supports and the latter has a uniform spacing along the centerline of the baseline ramp. These geometries behaved similar to the micro-vanes in terms of the streamwise vorticity and the ability to reduce flow separation, but are more physically robust than the thin vanes. Next, Mach number effect on flow past the micro-ramps (h~0.5δ) are examined in a supersonic boundary layer at M=1.4, 2.2 and 3.0, but with no shock waves present. The LES results indicate that micro-ramps have a greater impact at lower Mach number near the device but its influence decays faster than that for the higher Mach number cases. This may be due to the additional dissipation caused by the primary vortices with smaller effective diameter at the lower Mach number such that their coherency is easily lost causing the streamwise vorticity and the turbulent kinetic energy to decay quickly. The normal distance between the vortex core and the wall had similar growth indicating weak correlation with the Mach number; however, the spanwise distance between the two counter-rotating cores further increases with lower Mach number. Finally, various µVGs which include micro-ramp, split-ramp and a new hybrid concept “ramped-vane” are investigated under normal shock conditions at Mach number of 1.3. In particular, the ramped-vane was studied extensively by varying its size, interior spacing of the device and streamwise position respect to the shock. The ramped-vane provided increased vorticity compared to the micro-ramp and the split-ramp. This significantly reduced the separation length downstream of the device centerline where a larger ramped-vane with increased trailing edge gap yielded a fully attached flow at the centerline of separation region. The results from coarse-resolution LES studies show that the larger ramped-vane provided the most reductions in the turbulent kinetic energy and pressure fluctuation compared to other devices downstream of the shock. Additional benefits include negligible drag while the reductions in displacement thickness and shape factor were seen compared to other devices. Increased wall shear stress and pressure recovery were found with the larger ramped-vane in the baseline resolution LES studies which also gave decreased amplitudes of the pressure fluctuations downstream of the shock.

Prognóstico de falhas baseado em redes neurais com estados de eco

Tese (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Mecânica, 2015.

Protocolo de roteamento para redes oportunistas utilizando otimização por enxame de partículas

In this research work, a new routing protocol for Opportunistic Networks is presented. The proposed protocol is called PSONET (PSO for Opportunistic Networks) since the proposal uses a hybrid system composed of a Particle Swarm Optimization algorithm (PSO). The main motivation for using the PSO is to take advantage of its search based on individuals and their learning adaptation. The PSONET uses the Particle Swarm Optimization technique to drive the network traffic through of a good subset of forwarders messages. The PSONET analyzes network communication conditions, detecting whether each node has sparse or dense connections and thus make better decisions about routing messages. The PSONET protocol is compared with the Epidemic and PROPHET protocols in three different scenarios of mobility: a mobility model based in activities, which simulates the everyday life of people in their work activities, leisure and rest; a mobility model based on a community of people, which simulates a group of people in their communities, which eventually will contact other people who may or may not be part of your community, to exchange information; and a random mobility pattern, which simulates a scenario divided into communities where people choose a destination at random, and based on the restriction map, move to this destination using the shortest path. The simulation results, obtained through The ONE simulator, show that in scenarios where the mobility model based on a community of people and also where the mobility model is random, the PSONET protocol achieves a higher messages delivery rate and a lower replication messages compared with the Epidemic and PROPHET protocols.

Dielectric studies of Liquid Crystal Nanocomposites and Nanomaterial systems.

Liquid crystals (LCs) have revolutionized the display and communication technologies. Doping of LCs with inorganic nanoparticles such as carbon nanotubes, gold nanoparticles and ferroelectric nanoparticles have garnered the interest of research community as they aid in improving the electro-optic performance. In this thesis, we examine a hybrid nanocomposite comprising of 5CB liquid crystal and block copolymer functionalized barium titanate ferroelectric nanoparticles. This hybrid system exhibits a giant soft-memory effect. Here, spontaneous polarization of ferroelectric nanoparticles couples synergistically with the radially aligned BCP chains to create nanoscopic domains that can be rotated electromechanically and locked in space even after the removal of the applied electric field. The resulting non-volatile memory is several times larger than the non-functionalized sample and provides an insight into the role of non-covalent polymer functionalization. We also present the latest results from the dielectric and spectroscopic study of field assisted alignment of gold nanorods.

Optical Enhancement of Exoskeleton-Based Estimation of Glenohumeral Angles

In Robot-Assisted Rehabilitation (RAR) the accurate estimation of the patient limb joint angles is critical for assessing therapy efficacy. In RAR, the use of classic motion capture systems (MOCAPs) (e.g., optical and electromagnetic) to estimate the Glenohumeral (GH) joint angles is hindered by the exoskeleton body, which causes occlusions and magnetic disturbances. Moreover, the exoskeleton posture does not accurately reflect limb posture, as their kinematic models differ. To address the said limitations in posture estimation, we propose installing the cameras of an optical marker-based MOCAP in the rehabilitation exoskeleton. Then, the GH joint angles are estimated by combining the estimated marker poses and exoskeleton Forward Kinematics. Such hybrid system prevents problems related to marker occlusions, reduced camera detection volume, and imprecise joint angle estimation due to the kinematic mismatch of the patient and exoskeleton models. This paper presents the formulation, simulation, and accuracy quantification of the proposed method with simulated human movements. In addition, a sensitivity analysis of the method accuracy to marker position estimation errors, due to system calibration errors and marker drifts, has been carried out. The results show that, even with significant errors in the marker position estimation, method accuracy is adequate for RAR.

Ferrous iron sensing and responding in Pseudomonas aeruginosa

Controlling iron distribution is important for all organisms, and is key in bacterial pathogenesis. It has long been understood that cystic fibrosis (CF) patient sputum contains elevated iron concentrations. However, anaerobic bacteria have been isolated from CF sputum and hypoxic zones in sputum have been measured. Because ferrous iron [Fe(II)] is stable in reducing, acidic conditions, it could exist in the CF lung. I show that a two-component system, BqsRS, specifically responds to Fe(II) in the CF pathogen, Pseudomonas aeruginosa. Concurrently, a clinical study found that Fe(II) is present in CF sputum at all stages of lung function decline. Fe(II), not Fe(III) correlates with patients in the most severe disease state. Furthermore, transcripts of the newly identified BqsRS were detected in sputum. Two component systems are the main method bacteria interact with their extracellular environment. A typical two-component system contains a sensor histidine kinase, which upon activation phosphorylates a response regulator that then acts as a transcription factor to elicit a cellular response to stimuli. To explore the mechanism of BqsRS, I describe the Fe(II)-sensing RExxE motif in the sensor BqsS and determine the consensus DNA sequence BqsR binds. With the BqsR binding sequence, I identify novel regulon members through bioinformatic and molecular biology techniques. From the predicted function of new BqsR regulon members, I find that Fe(II) elicits a response that globally protects the cells against cationic stressors, including clinically relevant antibiotics. Subsequently, I use BqsR as a case study to determine if promoter outputs can accurately be predicted based only on a deep understanding of a transcriptional activator’s operator or if a broader regulatory context is required for accurate predictions at all genomic loci. This work highlights the importance of Fe(II) as a (micro)environmental factor, even in conditions typically thought of as aerobic. Since the presence of Fe(II) can alter P. aeruginosa’s antibiotic susceptibility, combining the current strategy of targeting Fe(III) with a new approach targeting Fe(II) may help eradicate infections in the CF lung in the future.

Estudo da imobilização das proteases aspárticas da flor de Cynara cardunculus L. Reologia da coalhada

Mestrado em Biotecnologia, Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2005

A review of maize-beans and maize-cowpea intercrop systems in the semiarid Northeast Brazil.

Fifty-one experiments on maize -beans and 34 on maize-cowpea intercropping systems conducted mostly in semiarid Northeast Brazil were analysed to get an understanding of the performance of these intercrops in terms of their productivity as well as stability. Both the intercrop systems produced higher yields over their respective sole crops under a wide range of agroclimates; the average advantage with maize-beans was 32%, while that from maize-cowpea was 41%. The optimum row proportion for maize-beans was one maize: three beans, requiring 59% of sole crop maize population and 75% sole bean population. In maize-cowpea, alternate rows or one maize: two cowpea arrangement with about 50% of sole maize density and 100% of sole cowpea population seemed to be optimum. The intercrops failed less frequently compared to sole crops to meet specified incomes or yields. Sorghum seemed to be a good alternative to the traditional cereal because of its improved and consistent performance. Future research needs are discussed for further yield improvement in these two intercrop system.s

Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output

The response regulator RpaB (regulator of phycobilisome associated B), part of an essential two-component system conserved in cyanobacteria that responds to multiple environmental signals, has recently been implicated in the control of cell dimensions and of circadian rhythms of gene expression in the model cyanobacterium Synechococcus elongatus PCC 7942. However, little is known of the molecular mechanisms that underlie RpaB functions. In this study we show that the regulation of phenotypes by RpaB is intimately connected with the activity of RpaA (regulator of phycobilisome associated A), the master regulator of circadian transcription patterns. RpaB affects RpaA activity both through control of gene expression, a function requiring an intact effector domain, and via altering RpaA phosphorylation, a function mediated through the N-terminal receiver domain of RpaB. Thus, both phosphorylation cross-talk and coregulation of target genes play a role in the genetic interactions between the RpaA and RpaB pathways. In addition, RpaB∼P levels appear critical for survival under light:dark cycles, conditions in which RpaB phosphorylation is environmentally driven independent of the circadian clock. We propose that the complex regulatory interactions between the essential and environmentally sensitive NblS-RpaB system and the SasA-RpaA clock output system integrate relevant extra- and intracellular signals to the circadian clock.

Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells -- and only those cells -- to be labeled with azides. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affnity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins -- including proteins with previously unknown expression patterns -- expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.

Transcription-Coupled DNA Supercoiling in Escherichia Coli: Mechanisms and Biological Functions

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.

On the Wick product and Wong-Zakai approximations.

This thesis is a compilation of 6 papers that the author has written together with Alberto Lanconelli (chapters 3, 5 and 8) and Hyun-Jung Kim (ch 7). The logic thread that link all these chapters together is the interest to analyze and approximate the solutions of certain stochastic differential equations using the so called Wick product as the basic tool. In the first chapter we present arguably the most important achievement of this thesis; namely the generalization to multiple dimensions of a Wick-Wong-Zakai approximation theorem proposed by Hu and Oksendal. By exploiting the relationship between the Wick product and the Malliavin derivative we propose an original reduction method which allows us to approximate semi-linear systems of stochastic differential equations of the Itô type. Furthermore in chapter 4 we present a non-trivial extension of the aforementioned results to the case in which the system of stochastic differential equations are driven by a multi-dimensional fraction Brownian motion with Hurst parameter bigger than 1/2. In chapter 5 we employ our approach and present a “short time” approximation for the solution of the Zakai equation from non-linear filtering theory and provide an estimation of the speed of convergence. In chapters 6 and 7 we study some properties of the unique mild solution for the Stochastic Heat Equation driven by spatial white noise of the Wick-Skorohod type. In particular by means of our reduction method we obtain an alternative derivation of the Feynman-Kac representation for the solution, we find its optimal Hölder regularity in time and space and present a Feynman-Kac-type closed form for its spatial derivative. Chapter 8 treats a somewhat different topic; in particular we investigate some probabilistic aspects of the unique global strong solution of a two dimensional system of semi-linear stochastic differential equations describing a predator-prey model perturbed by Gaussian noise.

Decoding Agc1 deficiency: bioinformatics approaches to unveil the functional implications of Slc25a12 on the transcriptome and epigenome

In the brain, mutations in SLC25A12 gene encoding AGC1 cause an ultra-rare genetic disease reported as a developmental and epileptic encephalopathy associated with global cerebral hypomyelination. Symptoms of the disease include diffused hypomyelination, arrested psychomotor development, severe hypotonia, seizures and are common to other neurological and developmental disorders. Amongst the biological components believed to be most affected by AGC1 deficiency are oligodendrocytes, glial cells responsible for myelination. Recent studies (Poeta et al, 2022) have also shown how altered levels of transcription factors and epigenetic modifications greatly affect proliferation and differentiation in oligodendrocyte precursor cells (OPCs). In this study we explore the transcriptomic landscape of Agc1 in two different system models: OPCs silenced for Agc1 and iPSCs from human patients differentiated to neural progenitors. Analyses range from differential expression analysis, alternative splicing, master regulator analysis. ATAC-seq results on OPCs were integrated with results from RNA-Seq to assess the activity of a TF based on the accessibility data from its putative targets, which allows to integrate RNA-Seq data to infer their role as either activators or repressors. All the findings for this model were also integrated with early data from iPSCs RNA-seq results, looking for possible commonalities between the two different system models, among which we find a downregulation in genes encoding for SREBP, a transcription factor regulating fatty acids biosynthesis, a key process for myelination which could explain the hypomyelinated state of patients. We also find that in both systems cells tend to form more neurites, likely losing their ability to differentiate, considering their progenitor state. We also report several alterations in the chromatin state of cells lacking Agc1, which confirms the hypothesis for which Agc1 is not a disease restricted only to metabolic alterations in the cells, but there is a profound shift of the regulatory state of these cells.