Carbon nanotubes (CNT): Feasibility as nano-bio agents to target cancer.

With the advent of molecularly targeted agents in place of the traditionally employed chemotherapeutic cytotoxic compounds, cancer treatment has recently entered a new and exciting phase. Thus, the aim of this thesis was to explore the use of nanotechnology to deliver new-targeted cancer therapies using a carbon nanotube based interfering RNA (RNAi) gene delivery system. To consider carbon nanotubes (CNTs) as nano-bio agents for gene therapy, it is important first to understand how they behave at a cellular level. CNTs, in the form of double-walled CNTs, were oxidised and wrapped with biomolecules prior to incubation with cells. Evidence of CNT uptake and release was demonstrated through Raman spectroscopy, of single cells and cell lysates, from PCS and HeLa cancer cell lines. Results show a maximum uptake at 3 hours with 20% of CNTs being internalised by PC3 cells (5.85 mug/mL) and a consequent release within a 24-hour time frame (< 0.31 mug/mL). An increase of ID/IG ratio and loss of the outer diameter of the DWNT during incubation period suggests that CNTs are being degraded in the intracellular environment. However, the internalisation of complexes led to no significant changes in cell components, such as DNA/RNA, proteins and lipids. In addition, no significant stress, evaluated by activation of phosphorylated MAPK, was induced when cells were exposed to carbon nanotubes. Intracellular localisation and trafficking of carbon nanotubes was studied by means of antibody staining to specific cellular compartments and revealed that an endocytic pathway is involved in the internalisation of carbon nanotubes. In this endocytic pathway, carbon nanotubes were found to co-localise specifically with clathrin coated vesicles, early endosomes, lysosomes, and slow recycling endosomes. To study the feasibility of applying CNTs to deliver nucleic acids, green fluorescent protein (GFP) was employed as a reporter gene for both gene delivery and gene silencing in an in vitro model. Firstly, gene delivery protocols were optimised by transfecting GFP-encoding plasmid DNA (pDNA) to mammalian cells via CNTs. Secondly, CNTs were used to deliver siRNA to the target GFP gene integrated into the host cell genome (through Lipofectamine transfection of pDNA). Gene targeting was achieved by silencing the GFP gene through small interfering RNA-CNT complexes (siRNA-CNT) delivered to GFP expressing cells. However, results indicate that CNTs-nucleic acids (pDNA and sIRNA) complexes do not reach their target sites, resulting in poor GFP expression or silencing. Data analysed by fluorescent microscopy and flow cytometry revealed that less than 1 % of ceils were expressing the GFP gene (plasmid DNA delivery) or 2% of cells presented knockdown of GFP gene (siRNA delivery). The expression and silencing using CNTs was very low compared to delivery of constructs with transfection reagent (Lipofectamine), which led to 43% of cells expressing GFP and around 40 % of cells where the GFP gene was silenced. Nevertheless, the experience of other researchers has been that the GFP gene is very difficult to silence to a significant degree because of its high level of endogenous expression. We therefore used CNTs to deliver siRNA targeting survivin, an anti-apoptotic protein, which is known to be overexpressed in several cancer cells lines and is thought to contribute to their oncogenic character. Reduced survivin silencing effect by immunoblotting was observed when siRNA-CNTs were employed, in comparison with DharmaFECT transfection reagent. However, after extended incubation periods the silencing induced via survivin-siRNA delivered through CNT cause cells to undergo apoptosis

A fronteira entre as deliberações nulas e anuláveis por vícios de conteúdo

O Código das Sociedades Comerciais, aprovado pelo Decreto-Lei nº 262/86, de 02 de setembro, prevê a possibilidade de impugnar uma deliberação dos sócios que seja desconforme com o sistema (lei ou contrato) e que possa, por essa razão, ser declarada nula ou anulada, com fundamento nos arts. 56º e 58º, respetivamente. Sendo o regime regra o da anulabilidade, só é possível apreender a abrangência deste desvalor da deliberação afastando os casos de nulidade, uma vez que o legislador remete para a anulabilidade sempre que não caiba a nulidade, tal como plasmado no art. 58º, n.º 1, al. a), do CSC. Assim, no que concerne à invalidade do conteúdo da deliberação, se a mesma não se enquadrar no âmbito da nulidade, por força das als. c) e d) do n.º 1 do art. 56º, será anulável nos termos da referida al. a) do nº 1 do art. 58º do CSC. Dado que a validade do conteúdo de uma deliberação se afere pelo respeito de determinados princípios, é por referência a estes que intentaremos distinguir ambas as situações, procurando detetar a quais o legislador pretendeu atender na consagração das normas. Tal indagação será de pendor tendencialmente casuístico, privilegiando a análise da forma como a doutrina e a jurisprudência caracterizam incidentalmente ambas as situações e uma proposta de clarificação, trazendo exemplos práticos que possibilitem delimitar a fronteira entre as deliberações nulas e anuláveis, por vícios de conteúdo, nessa referenciação, dando alguma materialidade à distinção entre as duas figuras.The Portuguese Companies Code, approved by Decree-Law nr. 262/86 of September 2nd, provides for the possibility to challenge a shareholders' resolution which is not in conformity with the system (law or contract) and that may, for that reason, be declared null or annulled, based on articles 56 and 58, respectively. As the rule is that of nullity, it is only possible to apprehend the scope of this devaluation of the resolution leaving aside the cases of nullity, since the legislator refers to nullity whenever nullity is not applicable, as set forth in article 58, no. 1, paragraph a) of the Portuguese Companies Code. Therefore, regarding the invalidity of the content of the resolution, if it does not fall within the scope of nullity, by virtue of paragraphs c) and d) of article 56 no. 1, it will be annulled in accordance with the referred paragraph a) of no. 1 of article 58 of the Portuguese Companies Code. Since the validity of the content of a resolution is assessed by the compliance with certain principles, it is by reference to these that we will attempt to distinguish both situations, trying to detect which ones the legislator intended to consider when establishing the rules. Such inquiry will tend to be casuistic in nature, privileging the analysis of how doctrine and jurisprudence incidentally characterize both situations and a clarification proposal, bringing practical examples that make it possible to delimit the boundary between null and annullable resolutions, due to content defects, in this referencing, giving some materiality to the distinction between the two figures

The use of antibody-antibiotic conjugates to fight bacterial infections

Copyright © 2022 Cavaco, Castanho and Neves. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.The emergence of antimicrobial resistance (AMR) is rapidly increasing and it is one of the significant twenty-first century’s healthcare challenges. Unfortunately, the development of effective antimicrobial agents is a much slower and complex process compared to the spread of AMR. Consequently, the current options in the treatment of AMR are limited. One of the main alternatives to conventional antibiotics is the use of antibodyantibiotic conjugates (AACs). These innovative bioengineered agents take advantage of the selectivity, favorable pharmacokinetic (PK), and safety of antibodies, allowing the administration of more potent antibiotics with less off-target effects. Although AACs’ development is challenging due to the complexity of the three components, namely, the antibody, the antibiotic, and the linker, some successful examples are currently under clinical studies.The project leading to these results has received funding from “la Caixa” Foundation (ID 100010434), under the agreement LCF/PR/HR17/52150011 and from Portuguese Funding Agency, Fundação para a Ciência e Tecnologia (FCT IP, grants PD/BD/128281/2017 and DL 7/2016/CP1451/CT0023).info:eu-repo/semantics/publishedVersio

Variação na concordância verbal - um estudo sobre o verbo haver existencial

A estrutura com o verbo haver existencial encontra-se em variação no sistema. Possui uma forma normativa consagrada em gramáticas e dicionários que é ensinada nas escolas, mas também uma forma não normativa que é utilizada pelos falantes, tanto que, apesar de ser normativamente um verbo impessoal transitivo direto, é possível encontrar dados empíricos em textos veiculadores da norma-padrão em que manifesta concordância com o seu argumento interno. Segundo Raposo (2013b), a construção sintática com concordância em número dá-se principalmente quando o verbo haver está no pretérito imperfeito do indicativo ou quando ocorre como verbo pleno em perífrases verbais. O verbo haver existencial não atribui papel temático externo e ainda assim atribui Caso acusativo, contrariando a Generalização de Burzio (Burzio, 1986) e tornando-se um verbo atípico no sistema. As estruturas com concordância vão no sentido de o tornar mais regular e de o fazer funcionar como os outros verbos inacusativos em que o argumento interno passa a funcionar como sujeito gramatical e recebe caso nominativo. Esta reanálise do verbo haver existencial como sendo um verbo inacusativo é um fenómeno que pode ser enquadrado com outros fenómenos na língua portuguesa, nos quais também se verifica variação. Pode enquadrar-se numa família de construções que envolvem expletivos na posição de sujeito (Cardoso, Carrilho & Pereira, 2011; Carrilho, 2003), à semelhança do que se verifica, por exemplo, no inglês com o contraste entre os expletivos it e there. Também no caso do verbo haver existencial poderão ocorrer dois expletivos nulos: um com traços de pessoa e número e que verifica a concordância com o verbo (construção sem concordância, 3ª pessoa do singular); e outro que não tem traços de pessoa - número e faz uma relação de associação com o SN pós-verbal e é esse SN que acaba por verificar os traços (construção com concordância, 3ª pessoa do plural). O objetivo deste trabalho foi estudar o grau de aceitabilidade das estruturas com e sem concordância do verbo haver existencial e verificar até que ponto as estruturas com concordância estão integradas na gramática implícita dos falantes e quais os fatores condicionadores dos padrões de concordância. Neste estudo, 28 falantes monolingues de português europeu, a frequentar o ensino superior, realizaram duas tarefas de juízo de aceitabilidade: uma com pressão de tempo e outra sem pressão de tempo. Os participantes tinham de avaliar a aceitabilidade de frases com o verbo haver numa escala de Likert. Foram manipuladas diferentes variáveis linguísticas, incluindo a concordância (com e sem concordância), o tipo de forma verbal (simples ou perifrástica), o tempo verbal (pretérito perfeito ou imperfeito) e o tipo de verbo auxiliar (semiauxiliar modal ou temporal). A análise mostrou que, na tarefa sem pressão de tempo, as construções com concordância são mais aceites com formas verbais perifrásticas do que com verbos simples, com os quais tendem a ser rejeitadas; e as construções sem concordância são mais aceites com verbos simples do que com formas perifrásticas. Na tarefa com pressão de tempo, as construções com concordância são aceites tanto com formas verbais simples como perifrásticas; e as construções sem concordância são mais aceites com as formas verbais simples do que com as perifrásticas. Estes resultados confirmam que existe sensibilidade a diferentes fatores linguísticos na manifestação de concordância com o verbo haver e parecem indicar que a construção com concordância, em alguns contextos, está já integrada na gramática implícita dos falantes.The structure with the existential verb haver is in variation in the system. It has a normative form enshrined in grammars and dictionaries that is taught in schools, but also a non-normative form that is used by speakers, so much so that, although it is normatively a direct transitive impersonal verb, it is possible to find empirical data in standard texts in which it occurs in agreement with its internal argument. According to Raposo (2013b), the syntactic construction with number agreement occurs mainly when the verb haver is in the imperfect tense of the indicative or when it occurs as a main verb in verbal periphrases. The existential verb haver does not assign an external thematic role and yet it assigns accusative case, contrary to Burzio's generalization (Burzio 1986) and becoming an atypical verb in the system. The structures with agreement make it more regular and make it function like the other unaccusative verbs, where the internal argument functions as the grammatical subject and receives nominative case. This reanalysis of the existential verb haver as an unaccusative verb is a phenomenon that can be framed with other phenomena in the Portuguese language, in which variation also occurs. It may fit into a family of constructions involving expletives in the subject position (Cardoso, Carrilho & Pereira, 2011; Carrilho, 2003), similarly to what happens, for example, in English with the contrast between the expletives it and there. Also, in the case of the existential verb haver there may be two null expletives: one with person-number features that check the agreement with the verb (construction without agreement, 3rd person singular); and another that has no person-number features and establishes an association relation with the post-verbal NP and it is this NP that ends up checking the features (construction with agreement, 3rd person plural). The objectives of this dissertation are to study the degree of acceptability of the structures with and without agreement of the existential verb haver and to examine to what extent the structures with agreement are part of the implicit grammar of the speakers and what factors condition the agreement patterns. In this study, 28 monolingual speakers of European Portuguese, attending higher education, performed two acceptability judgement tasks: a speeded task and an untimed task. Participants had to rate the acceptability of sentences with the verb haver on a Likert scale. Different linguistic variables were manipulated, including agreement (with and without agreement), verb form (simple vs. periphrastic), verb tense (preterite vs. imperfect) and auxiliary verb (modal or temporal semiauxiliary). The analysis showed that, in the untimed task, constructions with agreement are more accepted with periphrastic verb forms than with simple verbs, with which they tend to be rejected; and constructions without agreement are more accepted with simple verbs than with periphrastic forms. In the timed task, constructions with agreement are accepted with both simple and periphrastic verb forms; and constructions without agreement are accepted more with simple than with periphrastic verb forms. These results confirm that different linguistic factors condition the use of agreement with the verb haver and seem to indicate that the construction with agreement, in some contexts, is already integrated into the speakers’ implicit grammar

Design of double-walled carbon nanotubes for biomedical applications

Double-walled carbon nanotubes (DWNTs) prepared by catalytic chemical vapour deposition were functionalized in such a way that they were optimally designed as a nano-vector for the delivery of small interfering RNA (siRNA), which is of great interest for biomedical research and drug development. DWNTs were initially oxidized and coated with a polypeptide (Poly(Lys:Phe)), which was then conjugated to thiol-modified siRNA using a heterobifunctional cross-linker. The obtained oxDWNT–siRNA was characterized by Raman spectroscopy inside and outside a biological environment (mammalian cells). Uptake of the custom designed nanotubes was not associated with detectable biochemical perturbations in cultured cells, but transfection of cells with DWNTs loaded with siRNA targeting the green fluorescent protein (GFP) gene, serving as a model system, as well as with therapeutic siRNA targeting the survivin gene, led to a significant gene silencing effect, and in the latter case a resulting apoptotic effect in cancer cells

Conservação das argamassas de Cabo Verde

Mestrado em Engenharia CivilO presente trabalho tem como base o estudo das argamassas antigas de Cabo Verde aplicadas em diversos tipos de edifícios, de vários locais, construídos nos finais do século XIX e início do século XX. O estudo das argamassas antigas englobou pesquisa bibliográfica, assim como a recolha, análise e ensaio destas. A partir dos resultados obtidos procedeu-se à caracterização das argamassas antigas de Cabo Verde com determinação de propriedades físicas, identificação dos materiais que entram na sua composição e as proporções utilizadas. Paralelamente foram também analisadas as propriedades de materiais locais que integram a constituição das argamassas antigas de Cabo Verde.This project is based on a research about the old Capeverdean mortars applied in a variety of building’ types. These buildings were constructed between the end of the XIX Century and the beginning of the XX century in many locations in Cape Verde. Besides bibliographic research this project about the Capeverdean mortars also included collection, analysis and laboratorial testing of samples. Relying on the results, the Capeverdean old mortars were characterized taking into account their physical properties, their composition materials and the rates of these materials’ utilization. At the same time, the local materials found in the Capeverdean old mortars were also analyzed

Pitoresco constante - um percurso na arquitectura inglesa entre os séculos XVIII e XXI

A presente dissertação tem como objecto a evolução do movimento pitoresco na cultura arquitectónica inglesa.Estuda o contexto do seu aparecimento no séc. XVIII e as transformações que provocou na arquitectura e no paisagismo até aos dias de hoje. A análise realizada tem como objectivo compreender a importância do movimento nas suas principais manifestações como a relação com a paisagem exterior, a criação de uma paisagem interior, a importância do movimento para a apreensão do espaço e, finalmente, as condições da criação da noção de "Townscape", que consiste na importação dos princípios que o pitoresco desenvolveu nos jardins para o contexto da cidade.Foca-se a análise destes temas em obras dos diferentes períodos que o estudo atravessa, procurando aferir a sua presença e relevância no período contemporâneo e compreender o percurso de revisão e de re-interpretação a que estiveram sujeitos. Constata-se que a presença deste movimento, desde o seu aparecimento, é uma das constantes da arquitectura inglesa, alternando entre períodos de maior evidência e outros em que é algo subliminar.The current dissertation focuses on the Picturesque movementʼs evolution in English architectural culture. It studies the context out of which it emerged in the eighteenth-century and the transformations it has originated both in architecture and landscape design until nowadays. This study aims to understand the movementʼs relevance in its most important manifestations such as the relation with exterior landscape, the conception of an interior landscape, the relevance of movement to spacial apprehension and, fi nally, the conditions for the criation of the notion "Townscape", which is the transposition to the urban context of the principles the Picturesque had developed in gardens.The themesʼ analysis is focused on works from the different times throughout the study, searching to confi rm its presence in the contemporary period and to understand the course of revision and re-interpretation it has gone through. It realizes that this movementʼs presence and importance, from its emergence, is persistent in English architecture, shifting from periods of greater obviousness and others in which it is an underlying presence

Beliefs on Marital Violence and Self-Reported Dating Violence: A Comparative Study of Cape Verdean and Portuguese Adolescents

In this study, we compare Portuguese and Cape Verdean youths’ upholding of traditional beliefs about intimate partner violence (IPV) and the frequency of selfreported violent behavior in dating relationships. The sample (n =404) consisted of 183 Cape Verdean and 221 Portuguese secondary school students of both sexes (56 % female; mean age = 16). We used two questionnaires that had previously been validated in the Portuguese population. The results revealed that young Cape Verdean adolescents uphold stronger traditional beliefs than Portuguese adolescents do, but there were no differences in overall prevalence of abuse between the two samples. The relationship between traditional beliefs and self-reported violence was significant only in the Cape Verdean sample, suggesting that campaigns against IPV have not, so far, been as effective in Cape Verde as in Portugal.info:eu-repo/semantics/publishedVersio

Antibodies for the treatment of brain metastases, a dream or a reality?

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).The incidence of brain metastases (BM) in cancer patients is increasing. After diagnosis, overall survival (OS) is poor, elicited by the lack of an effective treatment. Monoclonal antibody (mAb)-based therapy has achieved remarkable success in treating both hematologic and non-central-nervous system (CNS) tumors due to their inherent targeting specificity. However, the use of mAbs in the treatment of CNS tumors is restricted by the blood-brain barrier (BBB) that hinders the delivery of either small-molecules drugs (sMDs) or therapeutic proteins (TPs). To overcome this limitation, active research is focused on the development of strategies to deliver TPs and increase their concentration in the brain. Yet, their molecular weight and hydrophilic nature turn this task into a challenge. The use of BBB peptide shuttles is an elegant strategy. They explore either receptor-mediated transcytosis (RMT) or adsorptive-mediated transcytosis (AMT) to cross the BBB. The latter is preferable since it avoids enzymatic degradation, receptor saturation, and competition with natural receptor substrates, which reduces adverse events. Therefore, the combination of mAbs properties (e.g., selectivity and long half-life) with BBB peptide shuttles (e.g., BBB translocation and delivery into the brain) turns the therapeutic conjugate in a valid approach to safely overcome the BBB and efficiently eliminate metastatic brain cells.This research was funded by the Portuguese Funding Agency, Fundação para a Ciência e Tecnologia, FCT IP, grants PD/BD/128281/2017, PTDC/BBB-BQB/1693/2014 and PTDC/BBB-NAN/1578/2014.info:eu-repo/semantics/publishedVersio