11,538 research outputs found

    Complement mediated synapse elimination in schizophrenia

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    Schizophrenia (SCZ) is a devastating psychiatric disorder with a typically age of onset in late adolescence. The heritability is estimated to be in between 60-80% and large-scale genome-wide studies have revealed a prominent polygenic component to SCZ risk and identified more than three-hundred common risk variants. Despite a better understanding of which genetic risk variants that increases SCZ risk, it has been challenging to map out the pathophysiology of the disorder. This has stalled the development of target drugs and current treatment options display moderate efficacy and are prone to produce side-effects. SCZ is generally considered a neurodevelopmental disorder and it was proposed more than forty years ago that physiological removal of less active synapses in adolescence, i.e., synaptic pruning, is increased in SCZ and hereby causes the core symptoms of the disorder. This theory has then been supported by post-mortem brain tissue and imaging studies displaying decreased synapse density in SCZ. More recently, it was then shown that the most strongly associated risk loci can largely be explained by copy numbers of a gene coding for the complement factor 4A (C4A). As microglia prune synapses with the help of complement signalling, we therefore decided to use a recently developed human 2D in vitro assay to assess microglial uptake of synaptic structures in models based on cells from individuals with SCZ and healthy controls (study I). We observed excessive uptake of synaptic structures in SCZ models and by mixing synapses from healthy controls with microglia from SCZ patients, and vice versa, we showed the contribution of microglial and neuronal factors contributing to this excessive uptake of synaptic structures. We then developed an in vitro assay to study neuronal complement deposition dependent on copy numbers of C4A in the neuronal lines. Complement 3 (C3) deposition increased by C4A copy numbers but was independent of C4B copy numbers (also unrelated to SCZ risk). Similar C4A copy numbers correlated with the extent of microglial uptake of synapses. Microglial uptake of synaptic structures could also be inhibited by the tetracycline minocycline that also decreased risk of developing SCZ in an electronic health record cohort. In study II, we cerebrospinal fluid (CSF) from first-episode psychosis patients to measure protein levels of C4A. In two independent cohorts, we observed elevated C4A levels (although not C4B levels) in first-episode patients that later were to develop SCZ and could show correlations with markers of synapse density. However, elevated C4A levels could not fully be explained by more copy numbers of C4A in individuals with SCZ and in vitro experiments revealed that SCZ-associated cytokines can induce C4A mRNA expression while also correlating with C4A in patient-derived CSF. In study III, we set-up a 3D brain organoid models to more fully comprehensively capture processes in the developing human brain and then also included innately developing microglia. We display synaptic pruning within these models and use single cell RNA sequencing to validate them. In conclusion, this thesis uses patient-derived cellular modelling to uncover a disease mechanism in SCZ that link genetic risk variants with bona fide protein changes in living patients

    Early Neanderthal social and behavioural complexity during the Purfleet Interglacial: handaxes in the latest Lower Palaeolithic.

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    Only a handful of ‘flagship’ sites from the Purfleet Interglacial (Marine Isotope Stage 9, c. 350-290,000 years ago) have been properly examined, but the archaeological succession at the proposed type-site at Purfleet suggests a period of complexity and transition, with three techno-cultural groups represented in Britain. The first was a simple toolkit lacking handaxes (the Clactonian), and the last a more sophisticated technology presaging the coming Middle Palaeolithic (simple prepared core or proto-Levallois technology). Sandwiched between were Acheulean groups, whose handaxes comprise the great majority of the extant archaeological record of the period – these are the focus of this study. It has previously been suggested that some features of the Acheulean in the Purfleet Interglacial were chronologically restricted, particularly the co-occurrence of ficrons and cleavers. These distinctive forms may have exceeded pure functionality and were perhaps imbued with a deeper social and cultural meaning. This study supports both the previously suggested preference for narrow, pointed morphologies, and the chronologically restricted pairing of ficrons and cleavers. By drawing on a wide spatial and temporal range of sites these patterns could be identified beyond the handful of ‘flagship’ sites previously studied. Hypertrophic ‘giants’ have now also been identified as a chronologically restricted form. Greater metrical variability was found than had been anticipated, leading to the creation of two new sub-groups (IA and IB) which are tentatively suggested to represent spatial and perhaps temporal patterning. The picture in the far west of Britain remains unclear, but the possibility of different Acheulean groups operating in the Solent area, and a late survival of the Acheulean, are both suggested. Handaxes with backing and macroscopic asymmetry may represent prehensile or ergonomic considerations not commonly found on handaxes from earlier interglacial periods. It is argued that these forms anticipate similar developments in the Late Middle Palaeolithic in an example of convergent evolution

    A suite of quantum algorithms for the shortestvector problem

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    Crytography has come to be an essential part of the cybersecurity infrastructure that provides a safe environment for communications in an increasingly connected world. The advent of quantum computing poses a threat to the foundations of the current widely-used cryptographic model, due to the breaking of most of the cryptographic algorithms used to provide confidentiality, authenticity, and more. Consequently a new set of cryptographic protocols have been designed to be secure against quantum computers, and are collectively known as post-quantum cryptography (PQC). A forerunner among PQC is lattice-based cryptography, whose security relies upon the hardness of a number of closely related mathematical problems, one of which is known as the shortest vector problem (SVP). In this thesis I describe a suite of quantum algorithms that utilize the energy minimization principle to attack the shortest vector problem. The algorithms outlined span the gate-model and continuous time quantum computing, and explore methods of parameter optimization via variational methods, which are thought to be effective on near-term quantum computers. The performance of the algorithms are analyzed numerically, analytically, and on quantum hardware where possible. I explain how the results obtained in the pursuit of solving SVP apply more broadly to quantum algorithms seeking to solve general real-world problems; minimize the effect of noise on imperfect hardware; and improve efficiency of parameter optimization.Open Acces

    Cis-Regulation of Gremlin1 Expression during Mouse Limb Bud Development and its Diversification during Vertebrate Evolution

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    Embryonic development and organogenesis rely on tightly controlled gene expression, which is achieved by cis-regulatory modules (CRMs) interacting with distinct transcription factors (TFs) that control spatio-temporal and tissue-specific gene expression. During organogenesis, gene regulatory networks (GRNs) with selfregulatory feedback properties coordinately control growth and patterning and provide systemic robustness against genetic and/or environmental perturbations. During limb bud development, various interlinked GRNs control outgrowth and patterning along all three limb axes. A paradigm network is the epithelial-mesenchymal (e-m) SHH/GREM1/AER-FGF feedback signaling system which controls limb bud outgrowth and digit patterning. The BMP antagonist GREMLIN1 (GREM1) is central to this e-m interactions as its antagonism of BMP activity is essential to maintain both AER-Fgf and Shh expression. In turn, SHH signaling upregulates Grem1 expression, which results in establishment of a self-regulatory signaling network. One previous study provided evidence that several CRMs could regulate Grem1 expression during limb bud development. However, the cis-regulatory logics underlying the spatio-temporal regulation of the Grem1 expression dynamics remained obscure. From an evolutionary point of view, diversification of CRMs can result in diversification of gene regulation which can drive the establishment of morphological novelties and adaptions. This was evidenced by the observed differences in Grem1 expression in different species that correlates with the evolutionary plasticity of tetrapod digit patterning. Hence, a better understanding of spatio-temporal regulation of the Grem1 expression dynamics and underlying cis-regulatory logic is of interest from both adevelopmental and an evolutionary perspective. Recently, multiple candidate CRMs have been identified that might be functionally relevant for Grem1 expression during mouse limb bud development. For my PhD project, I genetically analyzed which of these CRMs are involved in the regulation of the spatial-temporal Grem1 expression dynamics in limb buds. Therefore, we generated various single and compound CRM mutant alleles using CRISPR/Cas9. Our CRMs allelic series revealed a complex Grem1 cis-regulation among a minimum of six CRMs, where a subset of CRMs regulates Grem1 transcript levels in an additive manner. Surprisingly, phenotypic robustness depends not on threshold transcript levels but the spatial integrity of the Grem1 expression domain. In particular, interactions among five CRMs control the characteristic asymmetrical and posteriorly biased Grem1 expression in mouse limb buds. Our results provide an example of how multiple seemingly redundant limb-specific CRMs provide phenotypical robustness by cooperative/synergistic regulation of the spatial Grem1 expression dynamics. Three CRMs are conserved along the phylogeny of extant vertebrates with paired appendages. Of those, the activities of two CRMs recapitulate the major spatiotemporal aspects of Grem1 expression in mouse limb buds. In order to study their functions in species-specific regulation of Grem1 expression and their functional diversification in tetrapods, I tested the orthologous of both CRMs from representative species using LacZ reporter assays in transgenic mice, in comparison to the endogenous Grem1 expression in limb buds of the species of origin. Surprisingly, the activities of CRM orthologues display high evolutionary plasticity, which correlates better with the Grem1 expression pattern in limb buds of the species of origin than its mouse orthologue. This differential responsiveness to the GRNs in mouse suggests that TF binding site alterations in CRMs could underlie the spatial diversification of Grem1 in limb buds during tetrapod evolution. While the fish fin and tetrapod limb share some homologies of proximal bones, the autopod is a neomorphic feature of tetrapods. The Grem1 requirement for digit patterning and conserved expression in fin buds prompted us to assess the enhancer activity of fish CRM orthologues in transgenic mice. Surprisingly, all tested fish CRMs are active in the mouse autopod primordia providing strong evidence that Grem1 CRMs are active in fin buds and that they predate the fin-to-limb transition. Our results corroborate increasing evidence that CRMs governing autopodial gene expression have been co-opted during the emergence of tetrapod autopod. Furthermore, as part of a collaboration with Dr. S. Jhanwar, I contributed to the study of shared and species-specific epigenomic and genomic variations during mouse and chicken limb bud development. In this analysis, Dr. S. Jhanwar identified putative enhancers that show higher chicken-specific sequence turnover rates in comparison to their mouse orthologues, which defines them as so-called chicken accelerated regions (CARs). Here, I analyzed the CAR activities in comparison to their mouse orthologues by transgenic LacZ reporter assays, which was complemented by analysis of the endogenous gene expression in limb buds of both species. This analysis indicates that diversified activity of CARs and their mouse orthologues could be linked to the differential gene expression patterns in limb buds of both species

    Investigating Glioblastoma Multiforme Sub-Proteomes: A Computational Study of CUSA Fluid Proteomic Data

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    Based on our previous proteomic study on Cavitating Ultrasound Aspirator (CUSA) fluid pools of Newly Diagnosed (ND) and Recurrent (R) glioblastomas (GBMs) of tumor core and periphery, as defined by 5-aminolevulinc acid (5-ALA) metabolite fluorescence, this work aims to apply a bioinformatic approach to investigate specifically into three sub-proteomes, i.e., Not Detected in Brain (NB), Cancer Related (CR) and Extracellular Vesicles (EVs) proteins following selected database classification. The study of these yet unexplored specific datasets aims to understand the high infiltration capability and relapse rate that characterizes this aggressive brain cancer. Out of the 587 proteins highly confidently identified in GBM CUSA pools, 53 proteins were classified as NB. Their gene ontology (GO) analysis showed the over-representation of blood coagulation and plasminogen activating cascade pathways, possibly compatible with Blood Brain Barrier damage in tumor disease and surgery bleeding. However, the NB group also included non-blood proteins and, specifically, histones correlated with oncogenesis. Concerning CR proteins, 159 proteins were found in the characterized GBM proteome. Their GO analysis highlighted the over-representation of many pathways, primarily glycolysis. Interestingly, while CR proteins were identified in ND-GBM exclusively in the tumor zones (fluorescence positive core and periphery zones) as predictable, conversely, in R-GBM they were unexpectedly characterized prevalently in the healthy zone (fluorescence negative tumor periphery). Relative to EVs protein classification, 60 proteins were found. EVs are over-released in tumor disease and are important in the transport of biological macromolecules. Furthermore, the presence of EVs in numerous body fluids makes them a possible low-invasive source of brain tumor biomarkers to be investigated. These results give new hints on the molecular features of GBM in trying to understand its aggressive behavior and open to more in-depth investigations to disclose potential disease biomarkers

    Investigating PAX6 and SOX2 dynamic interactions at the single molecule level in live cells

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    The abundance of transcription factor (TF) molecules in the nuclei of eukaryotic cells are in the range of thousands. However, the functional binding sites of most TFs lie in the range of hundreds. This suggests that there is a surplus of the number of molecules for many TFs, relative to their binding sites at any given time. Nevertheless, precise TF levels are instrumental for normal development and maintenance, with haploinsufficiency (namely lowering the dosage of a TF by half) being a hallmark of many TF-related human developmental disorders. Qualitative methods assessing TF binding such as chromatin immunoprecipitation, provide static information, from fixed cell populations and so fail to provide insight into TF dynamic behaviour. Live-cell imaging methodologies such as Fluorescence Correlation Spectroscopy (FCS) offer the ability to measure kinetics of binding to chromatin, protein-protein interactions, absolute concentrations of molecules and the underlying cell-to-cell variability. SOX2 and PAX6 TFs exhibit haploinsufficiency in humans. Heterozygous point mutations, deletions or insertions in these genes can lead to a plethora of abnormal ocular developmental disorders (e.g. coloboma, aniridia, microphthalmia, anopthalmia). SOX2 encodes a high-mobility group (HMG) domain-containing TF, essential for maintaining self-renewal of embryonic stem cells and is expressed in proliferating central nervous system (CNS) progenitors. PAX6 contains two DNA binding domains; a PAIRED domain (PD) and a homeodomain (HD). Both DNA binding domains present in PAX6 (PD and HD) can function either jointly, or separately, to regulate a plethora of genes implicated in the development and maintenance of the CNS, the eye and the pancreas. Despite existing genetic and phenotypic evidence, it remains unclear how PAX6 and SOX2 influence each other at the molecular level and how sensitive their stoichiometry is during ocular development. In this thesis I investigated the dynamic interplay between PAX6/SOX2 and chromatin in live cells, at the molecular level. I compared wild-type protein function with pathogenic missense variants using advanced fluorescence microscopy techniques and assessed how these mutations quantitatively and qualitatively affected molecular behaviour. My results showed that both SOX2 and PAX6 pathogenic missense mutants display differential subnuclear localisation, as well as altered protein-protein and protein-chromatin interactions, linking molecular diffusion to pathogenic phenotype in humans. More importantly, I identified a novel role of SOX2 in stabilising PAX6- chromatin complexes in live cells, providing further insight into the complex and dynamic relation of PAX6 and SOX2 in ocular tissue specification, maintenance and development

    Estudo do IPFS como protocolo de distribuição de conteúdos em redes veiculares

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    Over the last few years, vehicular ad-hoc networks (VANETs) have been the focus of great progress due to the interest in autonomous vehicles and in distributing content not only between vehicles, but also to the Cloud. Performing a download/upload to/from a vehicle typically requires the existence of a cellular connection, but the costs associated with mobile data transfers in hundreds or thousands of vehicles quickly become prohibitive. A VANET allows the costs to be several orders of magnitude lower - while keeping the same large volumes of data - because it is strongly based in the communication between vehicles (nodes of the network) and the infrastructure. The InterPlanetary File System (IPFS) is a protocol for storing and distributing content, where information is addressed by its content, instead of its location. It was created in 2014 and it seeks to connect all computing devices with the same system of files, comparable to a BitTorrent swarm exchanging Git objects. It has been tested and deployed in wired networks, but never in an environment where nodes have intermittent connectivity, such as a VANET. This work focuses on understanding IPFS, how/if it can be applied to the vehicular network context, and comparing it with other content distribution protocols. In this dissertation, IPFS has been tested in a small and controlled network to understand its working applicability to VANETs. Issues such as neighbor discoverability times and poor hashing performance have been addressed. To compare IPFS with other protocols (such as Veniam’s proprietary solution or BitTorrent) in a relevant way and in a large scale, an emulation platform was created. The tests in this emulator were performed in different times of the day, with a variable number of files and file sizes. Emulated results show that IPFS is on par with Veniam’s custom V2V protocol built specifically for V2V, and greatly outperforms BitTorrent regarding neighbor discoverability and data transfers. An analysis of IPFS’ performance in a real scenario was also conducted, using a subset of STCP’s vehicular network in Oporto, with the support of Veniam. Results from these tests show that IPFS can be used as a content dissemination protocol, showing it is up to the challenge provided by a constantly changing network topology, and achieving throughputs up to 2.8 MB/s, values similar or in some cases even better than Veniam’s proprietary solution.Nos últimos anos, as redes veiculares (VANETs) têm sido o foco de grandes avanços devido ao interesse em veículos autónomos e em distribuir conteúdos, não só entre veículos mas também para a "nuvem" (Cloud). Tipicamente, fazer um download/upload de/para um veículo exige a utilização de uma ligação celular (SIM), mas os custos associados a fazer transferências com dados móveis em centenas ou milhares de veículos rapidamente se tornam proibitivos. Uma VANET permite que estes custos sejam consideravelmente inferiores - mantendo o mesmo volume de dados - pois é fortemente baseada na comunicação entre veículos (nós da rede) e a infraestrutura. O InterPlanetary File System (IPFS - "sistema de ficheiros interplanetário") é um protocolo de armazenamento e distribuição de conteúdos, onde a informação é endereçada pelo conteúdo, em vez da sua localização. Foi criado em 2014 e tem como objetivo ligar todos os dispositivos de computação num só sistema de ficheiros, comparável a um swarm BitTorrent a trocar objetos Git. Já foi testado e usado em redes com fios, mas nunca num ambiente onde os nós têm conetividade intermitente, tal como numa VANET. Este trabalho tem como foco perceber o IPFS, como/se pode ser aplicado ao contexto de rede veicular e compará-lo a outros protocolos de distribuição de conteúdos. Numa primeira fase o IPFS foi testado numa pequena rede controlada, de forma a perceber a sua aplicabilidade às VANETs, e resolver os seus primeiros problemas como os tempos elevados de descoberta de vizinhos e o fraco desempenho de hashing. De modo a poder comparar o IPFS com outros protocolos (tais como a solução proprietária da Veniam ou o BitTorrent) de forma relevante e em grande escala, foi criada uma plataforma de emulação. Os testes neste emulador foram efetuados usando registos de mobilidade e conetividade veicular de alturas diferentes de um dia, com um número variável de ficheiros e tamanhos de ficheiros. Os resultados destes testes mostram que o IPFS está a par do protocolo V2V da Veniam (desenvolvido especificamente para V2V e VANETs), e que o IPFS é significativamente melhor que o BitTorrent no que toca ao tempo de descoberta de vizinhos e transferência de informação. Uma análise do desempenho do IPFS em cenário real também foi efetuada, usando um pequeno conjunto de nós da rede veicular da STCP no Porto, com o apoio da Veniam. Os resultados destes testes demonstram que o IPFS pode ser usado como protocolo de disseminação de conteúdos numa VANET, mostrando-se adequado a uma topologia constantemente sob alteração, e alcançando débitos até 2.8 MB/s, valores parecidos ou nalguns casos superiores aos do protocolo proprietário da Veniam.Mestrado em Engenharia de Computadores e Telemátic

    Characterisation of the unfolded protein response in prostate cancer, and investigation of the ATF6 interactome using a modified mammalian expression system.

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    Prostate cancer (PCa) cells grow in an environment which is known to cause endoplasmic reticulum (ER) stress. This activates a process called the unfolded protein response (UPR) which PCa utilises in order to survive and adapt to the adverse environmental conditions. Using interdisciplinary approaches and tissue culture models representing different stages of PCa, this project investigated the link between the UPR and the key oncogenic driver of PCa, the androgen receptor. It has been observed that hormone responsive PCa utilises all three UPR arms in order to promote ER homeostasis and cell survival. The importance of the UPR during the progression of PCa to the castrate resistant stage was also assessed. Interestingly, UPR signalling was inactivated in castrate-resistant models of PCa, and the cells were instead dependent on the ER-associated degradation (ERAD) pathway in order to resolve the stress and survive. These findings have identified potential UPR vulnerabilities that can be targeted to prevent disease progression. Little is known about the structure and interaction partners of the UPR sensor ATF6, as protein expression has been shown to be problematic. To resolve this issue a stable tetracycline-inducible HEK293S GnTI(-) cell line for the expression of ATF6 was generated and mass spectrometry performed to characterise the ATF6 interactome. Sixty novel interaction partners of ATF6 were identified, most of which are associated with the cytoskeleton, such as Spectrin β-II and p195, which were validated by immunoblotting. It is hoped that the use of this modified expression system will provide an advantage in the process of expression, solubilisation and structure determination of ATF6 and of other membrane proteins
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