Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.

Degradação de Ocratoxina a: estudo de processo e toxicidade

A ocratoxina A (OTA), micotoxina encontrada em diferentes níveis e em diversas matrizes, apresenta efeitos carcinogênicos, nefrotóxicos e teratogênicos. O desenvolvimento de métodos capazes de diminuir esta contaminação a níveis permitidos pela legislação é incentivado e os processos biológicos utilizados envolvem o uso de enzimas e/ou microrganismos para degradação da OTA e são preferenciais pela especificidade, bem como pelas condições brandas para a detoxificação. O objetivo do trabalho foi estudar a ação de carboxipeptidase A nos níveis e na toxicidade de OTA, visando aplicar a técnica para detoxificar farinhas de trigo. Primeiramente foi estimado o risco de exposição à ocratoxina A pelo consumo de farinhas de trigo. Para isso foram estabelecidas condições de determinação de OTA em farinhas de trigo, empregando técnicas de estatística multivariada para definir os principais interferentes na extração de OTA pelo método de QuEChERS e detecção em CLAE-FL. O método validado permitiu a avaliação da ocorrência natural em 20 amostras de farinha de trigo, estando estas contaminadas na faixa de 0,22 a 0,85 µg.kg-1 , apresentando um valor de ingestão diária de 0,08 ngOTA.dia-1 .kgmassacorpórea -1 e uma disponibilidade de 94,4%. Em seguida foi realizada a padronização da extração de carboxipeptidase A em biomassa de Rhizopus oryzae que consistiu em agitação ultrassônica durante 30 minutos numa potencia fixa de 150 W e 40 kHz e a triagem de agentes biológicos para degradação de OTA. Para o estudo da degradação in vitro de OTA, método de extração e detecção de OTA e OTα em CLAEFL foi validado e o processo de degradação foi realizado com Rhizopus oryzae e Trichoderma reesei, obtendo-se uma redução máxima de 63,5% e 57,7%, respectivamente. A degradação apresentou uma correlação alta (R>0,9) e significativa (p<0,05) com a produção de Otα, indicando que ocorreu a produção de enzimas capazes de hidrolisar a micotoxina, por exemplo, a carboxipeptidase A. O estudo da toxicidade de OTA e seu metabólito OTα foi realizado em neutrófilos humanos, onde foi observado a ausência de efeito tóxico de OTα. Também foi determinado o mecanismo de toxicidade de OTA pelo aumento de Ca2+ intracelular pela liberação a partir das reservas internas. Esta liberação, subsequentemente, provoca uma cascata de eventos, nomeadamente: a produção de espécies reativas, depleção de ATP, perda de ΔΨm, levando à morte por necrose. Para reduzir o risco de exposição à micotoxina pela ingestão de matéria prima contaminada, carboxipeptidase A extraída de diferentes fontes foi aplicada na hidrólise de OTA em farinha de trigo para posterior determinação do conteúdo residual de OTA e OTα, empregando método validado. O estudo mostrou uma redução de OTA entre 16,8 e 78,5% e produção de OTα entre 2 a 8,2 ng.g-1 . As carboxipeptidases mais promissoras para degradação foram as provenientes de Rhizopus e Trichoderma e a carboxipeptidase comercial. Ficou demonstrado que se pode recomendar a aplicação de enzimas proteolíticas, tipo carboxipeptidase, para reduzir o risco de exposição à micotoxina quando utilizada matéria prima contaminada, por exemplo, farinha de trigo para diferentes processos. A transformação de OTA para OTα e seus efeitos na redução da toxicidade da micotoxina corroboram com esta afirmação.

Relatório de Estágio

O presente relatório surge no âmbito da unidade curricular de Integração Curricular: Prática Educativa e Relatório de Estágio, inserida no plano de estudos do Mestrado em Ensino do 1.º e 2.º Ciclo do Ensino Básico. Este documento valida a obtenção do grau mestre, habilitando para a docência em 1.º e 2.º ciclo do Ensino Básico. Neste sentido, apresenta o percurso individual de formação, com opções fundamentadas e em contexto, articuladas entre os saberes teóricos e os saberes práticos construídos e aplicados ao longo da formação académica da mestranda. No desenvolvimento da Prática Educativa, a metodologia de investigação-ação foi a base de todo o trabalho desenvolvido pela professora estagiária. O seu processo cíclico faseado em quatro fases: observação, planificação, ação e reflexão, permitiu a construção de conhecimentos sólidos que sustentam a prática docente. A par desta metodologia, a supervisão pedagógica assenta num momento importante e insubstituível de aprendizagem na formação docente, uma vez que motiva à reflexão partilhada. A reflexão em colaboração com o par pedagógico, com os orientadores cooperantes e com os supervisores institucionais permite a partilha de saberes e de vivências com o objetivo de modificar e melhorar práticas de ensino. O rumo deste Relatório de Estágio desencadeia, ainda a reflexão crítica sobre todas instâncias da escola e da comunidade educativa, através da implementação de projetos contextualizados, que leva ao desenvolvimento de um processo ativo na construção pessoal, profissional e social do profissional de educação.

FAM49 - A novel regulator of the protrusive behaviour and motility of cells

Most eukaryotic cell motility relies on plasma membrane protrusions, which depend on the actin cytoskeleton and its tight regulation. The SCAR/WAVE complex, a pentameric assembly comprising SCAR/WAVE, Nap1, CYFIP/Pir121, Abi and HSPC300, is a key driver of actin-based protrusions such as pseudopods. SCAR/WAVE is thought to activate the Arp2/3 complex, a crucial actin nucleator, after being itself activated by upstream signals such as active Rac1. Despite recent progress on the study of the SCAR/WAVE complex, its regulation is still incompletely understood, with Nap1’s role being particularly enigmatic. Upon screening for potential Nap1 binding partners in the social amoeba Dictyostelium discoideum – a well established model organism in the study of the actin cytoskeleton and cell motility – we found FAM49, a ~36 kDa protein of unknown function which is highly conserved in Metazoa (animals) and evolutionarily closer species such as D. discoideum. Interestingly, D. discoideum’s FAM49 and its homologs contain a DUF1394 domain, which is also predicted in CYFIP/Pir121 proteins and most likely involved in their direct binding to active Rac1, which in turn contributes to SCAR/WAVE’s activation. FAM49’s unknown role, apparent high degree of conservation and potential connections to SCAR/WAVE and Rac1 persuaded us to start investigating its function and biological relevance in D. discoideum, leading to the work presented in this thesis. Several pieces of our data collectively support a function for FAM49 in modulating the protrusive behaviour, and ultimately motility, of D. discoideum cells, as well as a regulatory link between FAM49 and Rac1. FAM49’s involvement in protrusion regulation was first hinted at by our observation that GFP-tagged FAM49 is enriched in pseudopods. The possibility of a link with Rac1 was then strengthened by two additional observations: first, pseudopodial GFP-FAM49 is substantially co-enriched with active Rac, both showing fairly comparable spatio-temporal accumulation dynamics; second, when dominant-active (G12V) Rac1 is expressed in cells, it triggers the recruitment and persistent accumulation of GFP-FAM49 at the plasma membrane, where both become highly co-enriched. We subsequently determined that fam49 KO cells differ from wild-type cells in the way they protrude and move, as assessed in under-agarose chemotaxis assays. In particular, our data indicate that fam49 KO cells tend to display a lower degree of global protrusive activity, their protrusions extend more slowly and are less discrete, and the cells end up moving at lower speeds and with higher directional persistence. This phenotype was substantially rescued by FAM49 re-expression. While re-expressing FAM49 in fam49 KO cells we generated putative FAM49 overexpressor cells; compared to wild-type cells, they displayed atypically thin pseudopods and what seemed to be an excessively dynamic, and perhaps less coordinated, protrusive behaviour. Additional data in our study suggest that pseudopods made by fam49 KO cells are still driven by SCAR/WAVE, which is clearly not being replaced by WASP (as is now known to be the case in D. discoideum cells lacking a functional SCAR/WAVE complex). Nonetheless, the peculiar dynamics of those pseudopods imply that SCAR/WAVE’s activity is regulated differently when FAM49 is lost, though it remains to be determined how. This thesis is the first report of a dedicated study on FAM49 and lays the foundation for future research on it.

Metoda projektów w uczeniu się i nauczaniu fizyki w gimnazjum

Wydział Studiów Edukacyjnych: Zakład Pedeutologii

Ruchy gałek ocznych i gesty jako korelaty przetwarzania języka w tłumaczeniu konsekutywnym i symultanicznym

Wydział Anglistyki

THERE GOES THE ELECTORAL NEIGHBORHOOD: LOCAL NETWORKS, ATTRIBUTION OF RESPONSIBILITY, AND THE (UN) FAIRNESS OF ELECTIONS

During the last two decades there have been but a handful of recorded cases of electoral fraud in Latin America. However, survey research consistently shows that often citizens do not trust the integrity of the electoral process. This dissertation addresses the puzzle by explaining the mismatch between how elections are conducted and how the process is perceived. My theoretical contribution provides a double-folded argument. First, voters’ trust in their community members (“the local experience”) impacts their level of confidence in the electoral process. Since voters often find their peers working at polling stations, negative opinions about them translate into negative opinions about the election. Second, perceptions of unfairness of the system (“the global effect”) negatively impact the way people perceive the transparency of the electoral process. When the political system fails to account for social injustice, citizens lose faith in the mechanism designed to elect representatives -and ultimately a set of policies. The fact that certain groups are systematically disregarded by the system triggers the notion that the electoral process is flawed. This is motivated by either egotropic or sociotropic considerations. To test these hypotheses, I employ a survey conducted in Costa Rica, El Salvador, Honduras, and Guatemala during May/June 2014, which includes a population-based experiment. I show that Voters who trust their peers consistently have higher confidence in the electoral process. Whereas respondents who were primed about social unfairness (treatment) expressed less confidence in the quality of the election. Finally, I find that the local experience is predominant over the global effect. The treatment has a statistically significant effect only for respondents who trust their community. Attribution of responsibility for voters who are skeptics of their peers is clear and simple, leaving no room for a more diffuse mechanism, the unfairness of the political system. Finally, now I extend analysis to the Latin America region. Using data from LAPOP that comprises four waves of surveys in 22 countries, I confirm the influence of the “local experience” and the “global effect” as determinants of the level of confidence in the electoral process.

Designing Hydrogels that Transform their Shape in Response to Molecular Cues

There has been considerable interest in developing shape-changing soft materials for potential applications in drug delivery, microfluidics and biosensing. These shape- changing materials are inspired by the morphological changes exhibited by plants in nature, such as the Venus flytrap. One specific class of shape-change is that from a flat sheet to a folded structure (e.g., a tube). Such “self-folding” materials are usually composed of polymer hydrogels, and these typically fold in response to external stimuli such as pH and temperature. In order to develop these hydrogels for the previously described applications, it is necessary to expand the range of triggers. The focus of this dissertation is the advancement of shape-changing polymer hydrogels that are sensitive to uncommon cues such as specific biomolecules (enzymes), the substrates for such enzymes, or specific multivalent cations. First, we describe a hybrid gel that responds to the presence of low concentrations of a class of enzymes known as matrix metalloproteinases (MMPs). The hybrid gel was created by utilizing photolithographic techniques to combine two or more gels with distinct chemical composition into the same material. Certain portions of the hybrid gel are composed of a biopolymer derivative with crosslinkable groups. The hybrid gel is flat in water; however, in the presence of MMPs, the regions containing the biopolymer are degraded and the flat sheet folds to form a 3D structure. We demonstrate that hydrogels with different patterns can transform into different 3D structures such as tubes, helices and pancakes. Furthermore, this shape change can be made to occur at physiological concentrations of enzymes. Next, we report a gel with two layers that undergoes a shape change in the presence of glucose. The enzyme glucose oxidase (GOx) is immobilized in one of the layers. GOx catalyzes the conversion of glucose to gluconic acid. The production of gluconic acid decreases the local pH. The decrease in local pH causes one of the layers to swell. As a result, the flat sheet folds to form a tube. The tube unfolds to form a flat sheet when it is transferred to a solution with no glucose present. Therefore, this biomolecule- triggered shape transformation is reversible, meaning the glucose sensing gel is reusable. Furthermore, this shape change only occurs in the presence of glucose and it does not occur in the presence of other small sugars such as fructose. In our final study, we report the shape change of a gel with two layers in the presence of multivalent ions such as Ca2+ and Sr2+. The gel consists of a passive layer and an active layer. The passive layer is composed of dimethylyacrylamide (DMAA), which does not interact with multivalent ions. The active layer consists of DMAA and the biopolymer alginate. In the presence of Ca2+ ions, the alginate chains crosslink and the active layer shrinks. As a result, the gel converts from a flat sheet to a folded tube. What is particularly unusual is the direction of folding. In most cases, when flat rectangular gels fold, they do so about their short-side. However, our gels typically fold about their long-side. We hypothesize that non-homogeneous swelling determines the folding axis.

Mechanistic Dissection of the Cop9 Signalosome’s Deneddylation Activity on Cullin-RING Ligases

We set out to understand the precise mechanisms that regulate the activation and deactivation of Cullin-RING Ligases (CRLs). While a great deal of work has already gone into identifying the players involved in these pathways and the cellular consequences associated with the loss of each, the biochemical mechanisms regulating these steps have remained elusive. In this work we sought to gain a better understanding of the mechanisms behind these steps by teasing apart specific their biochemical reactions. By measuring the individual microscopic rate constants of the reactions we have shed light on both the proper sequence of events in the regulation of CRLs as well as how they are in fact controlled.

Prior to this work, it was believed that CSN deactivated CRLs by binding them and enzymatically removing the activating post-translation modification Nedd8. It was believed that CSN could not bind to CRLs while they were active due to the steric hindrance by the CRL substrates, and that they would remain bound to deneddylated CRLs as a sequestering agent until a new substrate could displace it. We now have some insight that substrates themselves cannot inhibit CSN very well, but that the active ubiquitination by an E2 enzyme precludes CSN binding and activity. When the substrate for a CRL becomes depleted, CSN then binds to the CRL in a low affinity, low activity conformation. This triggers a conformational change that pulls the autoinhibitory Ins-1 loop away from the active site in the catalytic subunit Csn5, resulting in a large increase in affinity and cleavage of the isopeptide bond between CRLs and Nedd8. Upon dissociation of Nedd8, CSN rapidly returns to the low affinity state and dissociates from the CRL, allowing it reenter its activation cycle.

Three essays on corporate taxation : financial reporting, value, and firm creation

Doutoramento em Gestão

A implementação de preços de transferência interna : o caso Xerox

Mestrado em Contabilidade, Fiscalidade e Finanças Empresariais

Aplicações biotecnológicas do supressor de silenciamento de cucumber mosaic virus

Tese de Doutoramento, Ciências Agrárias, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Conocimientos, actitudes y sintomatología sobre el climaterio en mujeres de 45 a 65 años que acuden a consulta externa de ginecología del Hospital Vicente Corral Moscoso (HVCM). Cuenca. 2015

Antecedentes: La función hormonal ovárica comienza a declinar luego de los 45 años, determinando así el final de la vida reproductiva en la mujer, estos cambios que se experimentan antes, durante y después de la menopausia se define como climaterio. La pérdida de la función ovárica provoca la deficiencia de estrógenos por lo cual las mujeres cursan con un gran número de síntomas que alteran su conducta, además de las complicaciones cardiacas y óseas que se presentan tardíamente y comprometen la calidad de vida aumentando la morbimortalidad en esta etapa. Objetivo general: Determinar el grado de conocimiento que tienen las mujeres sobre el climaterio y cuál es su actitud de respuesta frente a los cambios que desencadena esta nueva etapa de vida, valorando los principales síntomas percibidos por las mujeres. Metodología: Material y método: Estudio descriptivo y transversal, universo de estudio es 9336 mujeres entre 45 y 65 años que acuden a consulta externa de Ginecología en el HVCM, la muestra en estudio es de 370 mujeres. La información fue obtenida por entrevista, posterior a consentimiento informado. Los datos obtenidos se analizaron en el paquete estadístico SPSS15. Resultados: El 15,7% tienen conocimiento sobre el climaterio, un 47% de mujeres encuestadas, tienen una actitud positiva frente al climaterio y el 17,3% manifiestan una actitud negativa refiriendo temor a envejecer. El principal síntoma presentando es aumento de peso con un 49.5%, seguido de disminución de la libido con el 40.5% (150) de encuestadas.

The transition from health to sickness via the chloroplast

Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. This talk presents results showing the chloroplast as a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

Flow around a hemisphere-cylinder at high angle of attack and low Reynolds number. Part II: POD and DMD applied to reduced domains

Three-dimensional direct numerical simulations (DNS) have been performed on a finite-size hemispherecylinder model at angle of attack AoA = 20◦ and Reynolds numbers Re = 350 and 1000. Under these conditions, massive separation exists on the nose and lee-side of the cylinder, and at both Reynolds numbers the flow is found to be unsteady. Proper orthogonal decomposition (POD) and dynamic mode decomposition (DMD) are employed in order to study the primary instability that triggers unsteadiness at Re = 350. The dominant coherent flow structures identified at the lower Reynolds number are also found to exist at Re = 1000; the question is then posed whether the flow oscillations and structures found at the two Reynolds numbers are related. POD and DMD computations are performed using different subdomains of the DNS computational domain. Besides reducing the computational cost of the analyses, this also permits to isolate spatially localized oscillatory structures from other, more energetic structures present in the flow. It is found that POD and DMD are in general sensitive to domain truncation and noneducated choices of the subdomain may lead to inconsistent results. Analyses at Re = 350 show that the primary instability is related to the counter rotating vortex pair conforming the three-dimensional afterbody wake, and characterized by the frequency St ≈ 0.11, in line with results in the literature. At Re = 1000, vortex-shedding is present in the wake with an associated broadband spectrum centered around the same frequency. The horn/leeward vortices at the cylinder lee-side, upstream of the cylinder base, also present finite amplitude oscillations at the higher Reynolds number. The spatial structure of these oscillations, described by the POD modes, is easily differentiated from that of the wake oscillations. Additionally, the frequency spectra associated with the lee-side vortices presents well defined peaks, corresponding to St ≈ 0.11 and its few harmonics, as opposed to the broadband spectrum found at the wake.