Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Stabilising suppressor of cytokine signalling 3 (SOCS3) protein levels to limit neointimal hyperplasia

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Characterization of TRIB2-mediated resistance to anti-cancer drugs

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2016

Prevalence of SOS-mediated control of integron integrase expression as an adaptive trait of chromosomal and mobile integrons

BACKGROUND Integrons are found in hundreds of environmental bacterial species, but are mainly known as the agents responsible for the capture and spread of antibiotic-resistance determinants between Gram-negative pathogens. The SOS response is a regulatory network under control of the repressor protein LexA targeted at addressing DNA damage, thus promoting genetic variation in times of stress. We recently reported a direct link between the SOS response and the expression of integron integrases in Vibrio cholerae and a plasmid-borne class 1 mobile integron. SOS regulation enhances cassette swapping and capture in stressful conditions, while freezing the integron in steady environments. We conducted a systematic study of available integron integrase promoter sequences to analyze the extent of this relationship across the Bacteria domain. RESULTS Our results showed that LexA controls the expression of a large fraction of integron integrases by binding to Escherichia coli-like LexA binding sites. In addition, the results provide experimental validation of LexA control of the integrase gene for another Vibrio chromosomal integron and for a multiresistance plasmid harboring two integrons. There was a significant correlation between lack of LexA control and predicted inactivation of integrase genes, even though experimental evidence also indicates that LexA regulation may be lost to enhance expression of integron cassettes. CONCLUSIONS Ancestral-state reconstruction on an integron integrase phylogeny led us to conclude that the ancestral integron was already regulated by LexA. The data also indicated that SOS regulation has been actively preserved in mobile integrons and large chromosomal integrons, suggesting that unregulated integrase activity is selected against. Nonetheless, additional adaptations have probably arisen to cope with unregulated integrase activity. Identifying them may be fundamental in deciphering the uneven distribution of integrons in the Bacteria domain.

Role of the Tryptophan Residues in the Specific interaction of the Sea Anemone Stichodacty la helianthus’s Actinoporin Sticholysin II with Biological Membranes

Actinoporins are pore-forming toxins from sea anemones. Upon interaction with sphingomyelin-containing bilayers, they become integral oligomeric membrane structures that form a pore. Sticholysin II from Stichodactyla helianthus contains five tryptophans located at strategic positions; its role has now been studied using different mutants. Results show that W43 and W115 play a eterminant role in maintaining the high thermostability of the protein, while W146 provides specific interactions for protomer−protomer assembly. W110 and W114 sustain the hydrophobic effect, which is one of the major driving forces for membrane binding in the presence of Chol. However, in its absence, additional interactions with sphingomyelin are required. These conclusions were confirmed with two sphingomyelin analogues, one of which had impaired hydrogen bonding properties. The results obtained support actinoporins’ Trp residues playing a major role in membrane recognition and binding, but their residues have an only minor influence on the diffusion and oligomerization steps needed to assemble a functional pore.

Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNA

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Estudo sobre os mecanismos de combate à utilização indevida do contrato de prestação de serviço em relações de trabalho subordinado

A investigação que doravante se apresenta tem como objetivo a análise dos mecanismos de combate à utilização indevida do contrato de prestação de serviço em relações de trabalho subordinado, e está estruturada em duas partes distintas, a que correspondem dois capítulos. A primeira parte é dedicada aos problemas inerentes ao contrato de trabalho e ao contrato de prestação de serviço, mormente, a delimitação entre as duas figuras, detendo-nos em particular na questão da determinação da subordinação jurídica. A semelhança entre os aludidos contratos aumenta, na prática, a dificuldade de delimitação entre ambos. A presunção de laboralidade assume um papel fundamental na ajuda necessária no seu campo de atuação. De facto, a presunção de laboralidade tende a facilitar a prova, conforme veremos. O método indiciário e o método tipológico podem ser utilizados na delimitação dos elementos do contrato de trabalho. O método tipológico ajuda a procurar elementos que apontem que aquele contrato é ou não um contrato de trabalho, este método usado pelos tribunais, apesar de adequado, não resolve o problema daquelas relações jurídicas que se encontram na fronteira, também conhecida como “zona cinzenta”. No que toca ao método indiciário este tem como objetivo procurar indícios da existência de subordinação jurídica para, assim, se poder afirmar a existência de um contrato de trabalho. Os indícios não são elementos determinantes da existência de um contrato de trabalho, pelo que, por não existir um indício, não significa automaticamente que não exista um contrato de trabalho. Os indícios devem ser analisados todos em conjunto, para se aferir se existe ou não subordinação jurídica. Na segunda parte, estabelecemos como comando investigativo o estudo dos mecanismos de combate à utilização indevida do contrato de prestação de serviço em sentido amplo, no que concerne a relações de trabalho subordinado. Para tal analisaremos o estado da arte antes e depois da Lei nº 63/2013, de 27 de agosto. A ação de reconhecimento da existência de contrato de trabalho é inovadora no sentido em que já não caminha em direção ao interesse egoístico do trabalhador, mas sim tendo em conta o interesse público, conforme melhor veremos. É pela novidade do tema, que optamos por entrevistar pessoalmente profissionais, que estejam de alguma forma ligados a esta realidade, e que com a sua experiência contribuíram, e muito, para alicerçarmos as nossas conclusões. Para tal, iniciamos os nossos trabalhos, entrevistando Tiago Gillot, no dia 22-01-2015 e 14-10-2015 em representação da Associação “Precários Inflexíveis,” que encabeçou o movimento de cidadãos nesta iniciativa legislativa que deu corpo à Lei nº 63/2013, de 27 de agosto, o Dr. Jorge Pinhal, em representação da ACT, que nos recebeu no dia 02-02-2015, o Ministério Público representado pela Exma. Procuradora Dra. Cristina Faleiro, no dia 08-04-2015. Entrevistamos por escrito o Exmo. Sr. Dr. António Ramos, Juiz do TRP, no dia 14-10-2015. Por fim, tentaremos de acordo com o estudo desenvolvido apresentar algumas medidas contributivas no combate à utilização indevida do contrato de prestação de serviço, assente sobretudo no plano de emergência social, apresentado pela Associação do Combate à precariedade, que consideramos apaziguar o flagelo que se instalou nas relações laborais e ao que parece dificilmente sairá da nossa linha de preocupação.

Memoria en publicidad y mercadeo

La psicología y la publicidad son dos disciplinas que desde hace algunos años se han venido complementando, la publicidad es la disciplina que se ha visto beneficiada con esta alianza, ya que el principal aporte que le hace la psicología a la publicidad es poder determinar aquellos factores o estrategias que generan en el consumidor; atención, asociación entre marca y producto, para luego almacenarse en la memoria y así generar una posible conducta de compra en el individuo. Los medios de comunicación cumplen un papel muy importante, ya que son los instrumentos utilizados para la transmisión de la información publicitaria, dependiendo del medio utilizado ya sea prensa, televisión o radio, las estrategias implementadas para dar a conocer la información deben ir dirigidas al campo perceptivo, que se utiliza para recibir la información y lograr una adecuada asociación y posterior almacenamiento en la memoria. La publicidad y el mercadeo hacen uso de una prueba de evaluación; top of mind, que muestra la jerarquía de recuerdo en la memoria de los individuos ante la mención de una categoría de servicio de un producto.

Memoria de los consumidores expuestos a la publicidad

La psicología y la publicidad son dos disciplinas que se han complementado, la publicidad se ha visto beneficiada con esta alianza, ya que el principal aporte que le hace la psicología a la publicidad es poder determinar aquellos factores o estrategias que generan en el consumidor: atención, asociación entre marca y producto, para luego almacenarse en la memoria y así generar una posible conducta de compra en el individuo. Los medios de comunicación cumplen un papel importante en la transmisión de la información publicitaria, dependiendo del medio utilizado se enfoca la información teniendo en cuenta el campo perceptivo que recibirá la información, para lograr asociación y posterior almacenamiento en la memoria. Para poder medir la jerarquía del recuerdo en la memoria de los individuos ante la mención de una categoría de servicio de un producto, la publicidad y el mercadeo hacen uso de la prueba Top of Mind.

Vehicle routing and location routing problems with minimum latency: algorithms and models

Latency can be defined as the sum of the arrival times at the customers. Minimum latency problems are specially relevant in applications related to humanitarian logistics. This thesis presents algorithms for solving a family of vehicle routing problems with minimum latency. First the latency location routing problem (LLRP) is considered. It consists of determining the subset of depots to be opened, and the routes that a set of homogeneous capacitated vehicles must perform in order to visit a set of customers such that the sum of the demands of the customers assigned to each vehicle does not exceed the capacity of the vehicle. For solving this problem three metaheuristic algorithms combining simulated annealing and variable neighborhood descent, and an iterated local search (ILS) algorithm, are proposed. Furthermore, the multi-depot cumulative capacitated vehicle routing problem (MDCCVRP) and the multi-depot k-traveling repairman problem (MDk-TRP) are solved with the proposed ILS algorithm. The MDCCVRP is a special case of the LLRP in which all the depots can be opened, and the MDk-TRP is a special case of the MDCCVRP in which the capacity constraints are relaxed. Finally, a LLRP with stochastic travel times is studied. A two-stage stochastic programming model and a variable neighborhood search algorithm are proposed for solving the problem. Furthermore a sampling method is developed for tackling instances with an infinite number of scenarios. Extensive computational experiments show that the proposed methods are effective for solving the problems under study.