NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging

To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56(dim) NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-gamma expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-gamma, TNF-alpha, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-gamma production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

Exploring protein-RNA interactions with site-directed mutagenesis and phage display

The importance of RNA as a mediator of genetic information is widely appreciated. RNA molecules also participate in the regulation of various post-transcriptional activities, such as mRNA splicing, editing, RNA stability and transport. Their regulatory roles for these activities are highly dependent on finely tuned associations with cognate proteins. The RNA recognition motif (RRM) is an ancient RNA binding module that participates in hundreds of essential activities where specific RNA recognition is required. We have applied phage display and site-directed mutagenesis to dissect principles of RRM-controlled RNA recognition. The model systems we are investigating are U1A and CUG-BP1. In this dissertation, the molecular basis of the binding affinity of U1A-RNA beyond individual contacts was investigated. We have identified and evaluated the contributions of the local cooperativity formed by three neighboring residues (Asn15, Asn16 and Glu19) to the stability of the U1A-RNA complex. The localized cooperative network was mapped by double-mutant cycles and explored using phage display. We also showed that a cluster of these residues forms a “hot spot” on the surface of U1A; a single substitution at position 19 with Gln or His can alter the binding properties of U1A to recognize a non-cognate G4U RNA. Finally, we applied a deletion analysis of CUG-BP1 to define the contributions of individual RRMs and RRM combinations to the stability of the complex formed between CUG-BP1 and the GRE sequence. The preliminary results showed RRM3 of CUG-BP1 is a key domain for RNA binding. It possibly binds to the GRE sequence cooperatively with RRM2 of CUG-BP1. RRM1 of CUG-BP1 is not required for GRE recognition, but may be important for maintaining the stability of the full-length CUG-BP1.

Investigations into the mechanism of orotidine 5'-monophosphate decarboxylase: Sources of substrate destabilization and transition state stabilization

Orotidine 5′-monophosphate decarboxylase (OMPDC) achieves a rarely paralleled rate acceleration, yet the catalytic basis prompting this enhancement have yet to be fully elucidated. To accomplish decarboxylation, OMPDC must overcome the high energy barrier due to the localized anionic charge of the intermediate. Mechanistic studies employing enzyme mutagenesis and product or intermediate analogues were used to investigate possible transition state stabilization by a carbene resonance structure. Viability of the carbene structure depends upon a key hydrogen bond between O4 of the substrate and the amide backbone of a conserved serine or threonine. Substitution of the conserved residue with Pro resulted in a kcat/KM of 1 M-1s-1; deletion of the FUMP O4 resulted in a product analogue that does not undergo H6 exchange or inhibit decarboxylation. Hence, indirect evidence reveals the O4-backbone interaction plays an important role for binding and catalysis. OMPDC likely has honed multiple mechanisms to attain its remarkable catalysis. The successful crystallizations of OMPDC a decade ago sparked hypotheses that structure and sequence conserved residues induced productive strain on the substrate-enzyme complex. Here, we demonstrate a new source of stress: a hydrophobic pocket adjacent to the OMP carboxylate that exhibits kinetic parameters characteristic of substrate destabilization. Substitution of these residues with hydrophilic side-chains, by providing hydrogen-bonding partners, decreased kcat by 10 to 10^4–fold. The same substitutions display very little change in the rate of product H6 exchange, supporting that this hydrophobic pocket affects the substrate-enzyme complex before the transition state. We also provide evidence that hydrophilic residues can insert water molecules into the pocket with detrimental effects to catalysis.

Novo roteiro para a Arte Nova em Aveiro e Ílhavo: proposta de uma aplicação mobile

A presente investigação tem como objeto de estudo a Arte Nova nas cidades de Aveiro e Ílhavo, traduzindo-se posteriormente na criação de uma aplicação mobile de um roteiro diferenciador referente ao património Arte Nova localizado nas duas cidades. Existe em Aveiro, uma tomada de consciência notória relativa ao património que se enquadra no espírito e nas características do estilo Arte Nova. Isso em muito se deve à originalidade e diversidade de interpretações que a caracterizam localmente. No entanto, apesar dos melhoramentos efetuados na comunicação deste património, especialmente depois da reabilitação do edifício Mário Belmonte Pessoa e da sua transformação em Museu Arte Nova, o roteiro atual não contempla todos os artefactos do estilo existentes na cidade e exclui aqueles que se encontram fora do centro urbano da cidade de Aveiro. Em Ílhavo a realidade é completamente díspar da encontrada na cidade vizinha, Aveiro, não existindo grande decoro pelo tratamento da informação referente ao estilo Arte Nova, apenas algumas referencias breves em guias culturais e um roteiro limitado no site da Câmara Municipal de Ílhavo. Paralelamente a isto, a região de Aveiro tornou-se numa das regiões com o espólio mais significativo do país. Dada à sua importância cultural e local, o roteiro diferenciador que propomos nesta investigação irá intervir de forma a melhorar o que já existe, passando pelo aprofundamento de conhecimentos sobre o tema, catalogação, cruzamento e agrupamento de toda a informação dos artefactos que se encontrem dispersos pelas duas cidades, de forma a tornar mais fácil a procura e o acesso à informação. Numa primeira fase, a investigação irá focar-se nos conteúdos afetos a cada um dos artefactos, sendo estes metodologicamente trabalhados através do método triangular de Francisco Providência, a interpretação autoral (autoria) que se traduz na evolução dos edifícios (tecnologia) e a relevância da sua história (programa) para o património nacional. Posteriormente, os conteúdos anteriormente referidos serão adaptados a uma aplicação mobile que facilitará o acesso à informação previamente selecionada referente a cada artefacto, apresentando uma breve história sobre as manifestações da Arte Nova nas cidades de Aveiro e Ílhavo. Esta aplicação mobile permitirá perceber a evolução dos edifícios desde a sua construção até à atualidade, ao nível de recuperação estrutural ou da falta de reabilitação e recuperação dos mesmos. Contribuirá para conhecer se os edifícios mantiveram (ou não) as suas características originais relativas ao desenho e tecnologia, para tal fará valerse de tecnologias como Realidade Aumentada, assim como os princípios de elaboração e leitura de QR codes, para facilitar o acesso, localização e compreensão dessa mesma informação, permitindo ainda que o seu utilizador embarque numa viagem no tempo e experiencie o roteiro de uma forma diferente. Paralelamente, pretende-se que este roteiro funcione como um roteiro único do património Arte Nova nas duas cidades, com o intuito de se expandir a outras cidades e se tornar num roteiro único do património Arte Nova na região de Aveiro. A diversidade do património Arte Nova nesta região assenta no cunho pessoal e social que os proprietários atribuíram aos seus artefactos, assim como na formação e a capacidade artística fortemente influenciada pela técnica pessoal, temperamento e sensibilidade dos seus autores, fazendo destes artefactos autênticas obras de arte, que merecem o seu estudo. Constatou-se, ao nível dos resultados que o protótipo da aplicação mobile, se adequaram ao que foi anunciado, a nível investigativo, e por isso, interessou a este estudo confirmar a demonstração do que foi enunciado. No entanto, concluiu-se que o respetivo protótipo necessita de ser ‘afinado’ em estudos futuros. Independentemente, das fragilidades encontradas, considera-se que este protótipo de aplicação mobile poderá servir como meio de excelência para a integração de conteúdos que vão mais além do que a visualização dos artefactos. Assim, contribui-se para o adensamento e acesso ao conhecimento sobre a história da Arte Nova em Portugal.

Rosarum monographia; or, A botanical history of roses to which is added, an appendix, for the use of cultivators, in which the most remarkable garden varieties are systematically arranged ...

Mode of access: Internet.

A atitude dos internautas face à Publicidade Display

Atualmente, com o aumento do consumo de internet e com a afirmação deste meio como veículo de comunicação e transmissão de publicidade, as marcas cada vez mais escolhem a internet para canalizar o seu investimento em publicidade, deste modo têm vindo a aumentar o seu investimento publicitário na internet. Neste canal existem vários tipos de publicidade, sendo que neste estudo académico será analisada a Publicidade Display, tendo como objetivo perceber a atitude e recordação face a este tipo de publicidade. Na metodologia foram utilizadas escalas que procuram analisar a atitude e a recordação dos internautas face à Publicidade Display, escortinando as dimensões Informação, Diversão, Irritação e Confiança bem como os fatores mais importantes para a recordação deste tipo de publicidade. A recolha de dados, teve como base um questionário online no qual foram inquiridos 190 participantes. Para analisar as hipóteses de estudo, foi feita uma regressão linear múltipla e correlações. Os principais resultados confirmam que as dimensões Informação, Entretenimento, Confiança e Irritação são preditores significativos das atitudes relacionadas com a publicidade display.

Scanning probe studies of thin films

The first part of this thesis deals with the phenomenon of thermoelectricity. It involves the improvement of the thermoelectric properties of silicon using innovative nanostructures. My contribution was to help fabricate these thermoelectric devices, and is the focus of this part of the thesis.

The second part and primary focus of this thesis is the analysis of thin films using scanning probe techniques. These surface techniques include atomic force microscopy, electric force microscopy, Kelvin probe force microscopy, and scanning tunneling microscopy. The thin films studied are graphene and molybdenum disulfide, two remarkable materials that display unique two-dimensional qualities. These materials are shown to be useful in studying the properties of adsorbates trapped between them and the substrate on which they rest. Moreover, these adsorbed species are seen to affect the structural and electronic properties of the thin films themselves. Scanning probe analyses are particularly useful in elucidating the properties of these materials, as surface effects play a significant role in determining their characteristics.

The final part of this thesis is concerned with the study of Akt in live cells using protein capture agents previously developed by my colleagues. The activation and degradation of Akt is investigated using various biological assays, including Western blots, in vitro kinase assays, and cell viability assays. Finally, the usefulness of synthetic capture agents in perturbing protein pathways and as delivery agents is assessed and analyzed.

GENETIC VARIATION, LOCAL ADAPTATION AND POPULATION STRUCTURE IN NORTH AMERICAN RED OAK SPECIES, QUERCUS RUBRA L. AND Q. ELLIPSOIDALIS E. J. HILL

Forest trees, like oaks, rely on high levels of genetic variation to adapt to varying environmental conditions. Thus, genetic variation and its distribution are important for the long-term survival and adaptability of oak populations. Climate change is projected to lead to increased drought and fire events as well as a northward migration of tree species, including oaks. Additionally, decline in oak regeneration has become increasingly concerning since it may lead to decreased gene flow and increased inbreeding levels. This will in turn lead to lowered levels of genetic diversity, negatively affecting the growth and survival of populations. At the same time, populations at the species’ distribution edge, like those in this study, could possess important stores of genetic diversity and adaptive potential, while also being vulnerable to climatic or anthropogenic changes. A survey of the level and distribution of genetic variation and identification of potentially adaptive genes is needed since adaptive genetic variation is essential for their long-term survival. Oaks possess a remarkable characteristic in that they maintain their species identity and specific environmental adaptations despite their propensity to hybridize. Thus, in the face of interspecific gene flow, some areas of the genome remain differentiated due to selection. This characteristic allows the study of local environmental adaptation through genetic variation analyses. Furthermore, using genic markers with known putative functions makes it possible to link those differentiated markers to potential adaptive traits (e.g., flowering time, drought stress tolerance). Demographic processes like gene flow and genetic drift also play an important role in how genes (including adaptive genes) are maintained or spread. These processes are influenced by disturbances, both natural and anthropogenic. An examination of how genetic variation is geographically distributed can display how these genetic processes and geographical disturbances influence genetic variation patterns. For example, the spatial clustering of closely related trees could promote inbreeding with associated negative effects (inbreeding depression), if gene flow is limited. In turn this can have negative consequences for a species’ ability to adapt to changing environmental conditions. In contrast, interspecific hybridization may also allow the transfer of genes between species that increase their adaptive potential in a changing environment. I have studied the ecologically divergent, interfertile red oaks, Quercus rubra and Q. ellipsoidalis, to identify genes with potential roles in adaptation to abiotic stress through traits such as drought tolerance and flowering time, and to assess the level and distribution of genetic variation. I found evidence for moderate gene flow between the two species and low interspecific genetic differences at most genetic markers (Lind and Gailing 2013). However, the screening of genic markers with potential roles in phenology and drought tolerance led to the identification of a CONSTANS-like (COL) gene, a candidate gene for flowering time and growth. This marker, located in the coding region of the gene, was highly differentiated between the two species in multiple geographical areas, despite interspecific gene flow, and may play a role in reproductive isolation and adaptive divergence between the two species (Lind-Riehl et al. 2014). Since climate change could result in a northward migration of trees species like oaks, this gene could be important in maintaining species identity despite increased contact zones between species (e.g., increased gene flow). Finally I examined differences in spatial genetic structure (SGS) and genetic variation between species and populations subjected to different management strategies and natural disturbances. Diverse management activities combined with various natural disturbances as well as species specific life history traits influenced SGS patterns and inbreeding levels (Lind-Riehl and Gailing submitted).

Motivating the use of public transit through a mobile glanceable display

With increasing concerns about the impact of global warming on human life, policy makers around the world and researchers have sought for technological solutions that have the potential to attenuate this process. This thesis describes the design and evaluation of an information appliance that aims to increase the use of public transportation. We developed a mobile glanceable display that, being aware of the user’s transportation routines, provides awareness cues about bus arrival time, grounded upon the vision of Ambient Intelligence. We present the design process we followed, from ideation to building a prototype and conducting a field study, and conclude with a set of guidelines for the design of relevant personal information systems. More specifically we seek to test the following hypotheses: 1) That the tangible prototype that provides ambient cues will be used more frequently than a similar purpose mobile app, 2) That the tangible prototype will reduce the waiting time at the bus stop, 3) That the tangible prototype will result to reduced anxiety on passengers, 4) That the tangible prototype will result to an increase in the perceived reliability of the transit service, 5) That the tangible prototype will enhance users’ efficiency in reading the bus schedules and 6) That the tangible prototype will make individuals more likely to use public transit. In a field study, we compare the tangible prototype against the mobile app and a control condition where participants were given no external support in obtaining bus arrival information, other than their existing routines. Using qualitative and quantitative data, we test the aforementioned hypotheses and explore users’ reactions to the prototype we developed.

Prototype and proof of concept for a display system in retail stores

Selling devices on retail stores comes with the big challenge of grabbing the customer’s attention. Nowadays people have a lot of offers at their disposal and new marketing techniques must emerge to differentiate the products. When it comes to smartphones and tablets, those devices can make the difference by themselves, if we use their computing power and capabilities to create something unique and interactive. With that in mind, three prototypes were developed during an internship: a face recognition based Customer Detection, a face tracking solution with an Avatar and interactive cross-app Guides. All three revealed to have potential to be differentiating solutions in a retail store, not only raising the chance of a customer taking notice of the device but also of interacting with them to learn more about their features. The results were meant to be only proof of concepts and therefore were not tested in the real world.

Motivating the use of public transit through a mobile glanceable display

With increasing concerns about the impact of global warming on human life, policy makers around the world and researchers have sought for technological solutions that have the potential to attenuate this process. This thesis describes the design and evaluation of an information appliance that aims to increase the use of public transportation. We developed a mobile glanceable display that, being aware of the user’s transportation routines, provides awareness cues about bus arrival time, grounded upon the vision of Ambient Intelligence. We present the design process we followed, from ideation to building a prototype and conducting a field study, and conclude with a set of guidelines for the design of relevant personal information systems. More specifically we seek to test the following hypotheses: 1) That the tangible prototype that provides ambient cues will be used more frequently than a similar purpose mobile app, 2) That the tangible prototype will reduce the waiting time at the bus stop, 3) That the tangible prototype will result to reduced anxiety on passengers, 4) That the tangible prototype will result to an increase in the perceived reliability of the transit service, 5) That the tangible prototype will enhance users’ efficiency in reading the bus schedules and 6) That the tangible prototype will make individuals more likely to use public transit. In a field study, we compare the tangible prototype against the mobile app and a control condition where participants were given no external support in obtaining bus arrival information, other than their existing routines. Using qualitative and quantitative data, we test the aforementioned hypotheses and explore users’ reactions to the prototype we developed.

ABC transporters of the A subfamily: potential prognostic markers and candidates for antibody selection from phage-display libraries for therapeutic use in Ewing sarcoma

The prognostic value of ABC transporters in Ewing sarcoma is still poorly explored and controversial. We described for the first time the impact of various ABCs on Ewing sarcoma prognosis by assessment of their gene expression in two independent cohorts of patients. Unexpected associations with favourable outcomes were observed for two ABCs of the A-subfamily, ABCA6 and ABCA7, whereas no associations with the canonical multidrug ABC transporters were identified. The ABCs of the A-subfamily are involved in cholesterol/phospholipids transportation and efflux from cells. Our clinical data support the drug-efflux independent contribution to cancer progression of the ABCAs, which has been confirmed in PDX-derived cell lines. The impact of these ABCA transporters on tumor progression seems to be mediated by lowering intracellular cholesterol, supporting the role of these proteins in lipid transport. In addition, the gene expression of ABCA6 and ABCA7 is regulated by transcription factors which control lipid metabolism: ABCA6 was induced by the binding of FoxO1/FoxO3a to its promoter and repressed by IGF1R/Akt signaling, whereas the expression of ABCA7 was regulated by p53. The data point to ABCA6 and ABCA7 as potential prognostic markers in Ewing sarcoma and suggest the IGF1/ABCA/lipid axis as an intriguing therapeutic target. Agonist monoclonal antibodies towards ABCA6/7 or inhibitors of cholesterol biosynthesis, such as statins or aminobiphoshonates, may be investigated as therapeutic options in combination with chemotherapy. Considering that no monoclonal antibodies selectively targeting extracellular domains of ABCA6/7 are available, the second part of the project has been dedicated to the generation of human antibody phage-display libraries as tools for selecting monoclonal antibodies. A novel synthetic human antibody phage-display library has been designed, cloned and characterized. The library takes advantages of the high variability of a designed naïve repertoire to be a useful tool for isolating antibodies towards all potential antigens, including the ABCAs.

Les machines du pouvoir. Technique et politique entre l'Iran sassanide et le califat abbasside

Dans l’Antiquité, la recherche sur la technique permet les premières réalisations de dispositifs ingénieux, tels que des appareils qui accomplissent une série d’actions par le biais de stimulus externes et de mécanismes cachés. Les organismes politiques et religieux saisissent rapidement la puissance communicative de ces machines, en devenant les promoteurs et patrons privilégiés de leur production. L’Empire sassanide (224-650) ne constitue pas une exception. En effet, les souverains perses consacrent, au moins à l’époque tardive, une grande attention à la conception et au déploiement de dispositifs savants. De même, un siècle plus tard, dans le milieu du califat islamique, les Abbassides (750-1258) semblent s’entourer de tels dispositifs. La continuité entre les deux empires dans plusieurs domaines, de la théorie politique à l’administration, est bien connue. Cependant, la question de la réutilisation du patrimoine technique et scientifique ancien, et notamment sassanide, par la cour abbasside, demeure encore largement inexplorée. L’étude d’un corpus de sources, aussi vaste qu’hétérogène, rassemblant des ouvrages historiographiques, géographiques, poétiques et d’adab, ainsi que des traités scientifiques et techniques en plusieurs langues, permet d’analyser différents aspects de la production et de l’usage politique des machines. Au sein de la cour sassanide, comme de la cour abbasside, la machine s’avère constituer un véhicule préférentiel de représentation et de diffusion de l’idéologie politique. À travers sa mise en scène publique, elle contribue de manière substantielle à la définition de l’espace du pouvoir, en participant à la création d’une image de la cour comme un microcosme au cœur duquel le Roi des rois, et plus tard le calife, occupaient le rôle cardinal de maître incontesté du monde. La continuité entre les empires sassanide et abbasside dans le domaine technique ne se limite donc pas à une récupération de savoirs, mais s’opère aussi sous la forme d’une véritable réactivation d’un patrimoine symbolique

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Primary Relapse Site Pattern In Women With Triple-negative Breast Cancer.

Despite the remarkable improvements in breast cancer (BC) characterization, accurate prediction of BC clinical behavior is often still difficult to achieve. Some studies have investigated the association between the molecular subtype, namely the basal-like BC and the pattern of relapse, however only few investigated the association between relapse pattern and immunohistochemical defined triple-negative breast cancers (TNBCs). The aim of this study was to evaluate the pattern of relapse in patients with TNBC, namely the primary distant relapse site. One-hundred twenty nine (129) invasive breast carcinomas with follow-up information were classified according to the molecular subtype using immunohistochemistry for ER, PgR and Her2. The association between TNBC and distant relapse primary site was analyzed by logistic regression. Using multivariate logistic regression analysis patients with TNBC displayed only 0.09 (95% CI: 0.00-0.74; p=0.02) the odds of the non-TNBC patients of developing bone primary relapse. Regarding visceral and lymph-node relapse, no differences between in this cohort were found. Though classically regarded as aggressive tumors, TNBCs rarely development primary relapse in bone when compared to non-TNBC, a clinical relevant fact when investigating a metastasis of an occult or non-sampled primary BC.