9,083 research outputs found
Complement mediated synapse elimination in schizophrenia
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
Quantum dots based superluminescent diodes and photonic crystal surface emitting lasers
This thesis reports the design, fabrication, and electrical and optical characterisations of GaAs-based quantum dot (QD) photonic devices, specifically focusing on superluminescent diodes (SLDs) and photonic crystal surface-emitting lasers (PCSELs). The integration of QD active regions in these devices is advantageous due to their characteristics such as temperature insensitivity, feedback insensitivity, and ability to utilise the ground state (GS) and excited state (ES) of the dots.
In an initial study concerning the fabrication of QD-SLDs, the influence of ridge waveguide etch depth on the electrical and optical properties of the devices are investigated. It is shown that the output power and modal gain from shallow etched ridge waveguide is higher than those of deep etched waveguides. Subsequently, the thermal performance of the devices is analysed. With increased temperature over 170 ºC, the spectral bandwidth is dramatically increased by thermally excited carrier transition in excited states of the dots.
Following this, an investigation of a high dot density hybrid quantum well/ quantum dot (QW/QD) active structure for broadband, high-modal gain SLDs is presented. The influence of the number of QD layers on the modal gain of hybrid QW/QD structures is analysed. It is shown that higher number of dot layer provides higher modal gain value, however, there is lack of emission from QW due to the requirement of large number of carriers to saturate the QD. Additionally, a comparison is made between “unchirped QD” and “ chirped QD” of hybrid QW/QD structure in terms of modal gain and spectral bandwidth. It is showed that “chirped” of the QD can improve the “flatness” of the spectral bandwidth.
Lastly, the use of self-assembled InAs QD as the active material in epitaxially regrown GaAs-based PCSELs is explored for the first time. Initially, it is shown that both GS and ES lasing can be achieved for QD-PCSELs by changing the grating period of the photonic crystal (PC). The careful design of these grating periods allows lasing from neighbouring devices at GS ( ~1230 nm) and ES (~1140 nm), 90 nm apart in wavelength. Following this, the effect of device area, PC etch depth, PC atom shape (circle or triangle or orientation) on lasing performance is presented. It is shown that lower threshold current density and higher slope efficiencies is achieved with increasing the device size. The deeper PC height device has higher output power due to more suitable height and minimal distance to active region. The triangular atom shape has slightly higher slope efficiency compared to triangular atom shape which is attributed to breaking in-plane symmetry and increase out-of-plane emission
Anuário científico da Escola Superior de Tecnologia da Saúde de Lisboa - 2021
É com grande prazer que apresentamos a mais recente edição (a 11.ª) do Anuário Científico da Escola Superior de Tecnologia da Saúde de Lisboa. Como instituição de ensino superior, temos o compromisso de promover e incentivar a pesquisa científica em todas as áreas do conhecimento que contemplam a nossa missão. Esta publicação tem como objetivo divulgar toda a produção científica desenvolvida pelos Professores, Investigadores, Estudantes e Pessoal não Docente da ESTeSL durante 2021. Este Anuário é, assim, o reflexo do trabalho árduo e dedicado da nossa comunidade, que se empenhou na produção de conteúdo científico de elevada qualidade e partilhada com a Sociedade na forma de livros, capítulos de livros, artigos publicados em revistas nacionais e internacionais, resumos de comunicações orais e pósteres, bem como resultado dos trabalhos de 1º e 2º ciclo. Com isto, o conteúdo desta publicação abrange uma ampla variedade de tópicos, desde temas mais fundamentais até estudos de aplicação prática em contextos específicos de Saúde, refletindo desta forma a pluralidade e diversidade de áreas que definem, e tornam única, a ESTeSL. Acreditamos que a investigação e pesquisa científica é um eixo fundamental para o desenvolvimento da sociedade e é por isso que incentivamos os nossos estudantes a envolverem-se em atividades de pesquisa e prática baseada na evidência desde o início dos seus estudos na ESTeSL. Esta publicação é um exemplo do sucesso desses esforços, sendo a maior de sempre, o que faz com que estejamos muito orgulhosos em partilhar os resultados e descobertas dos nossos investigadores com a comunidade científica e o público em geral. Esperamos que este Anuário inspire e motive outros estudantes, profissionais de saúde, professores e outros colaboradores a continuarem a explorar novas ideias e contribuir para o avanço da ciência e da tecnologia no corpo de conhecimento próprio das áreas que compõe a ESTeSL. Agradecemos a todos os envolvidos na produção deste anuário e desejamos uma leitura inspiradora e agradável.info:eu-repo/semantics/publishedVersio
Targeting Fusion Proteins of HIV-1 and SARS-CoV-2
Viruses are disease-causing pathogenic agents that require host cells to replicate. Fusion of host and viral membranes is critical for the lifecycle of enveloped viruses. Studying viral fusion proteins can allow us to better understand how they shape immune responses and inform the design of therapeutics such as drugs, monoclonal antibodies, and vaccines. This thesis discusses two approaches to targeting two fusion proteins: Env from HIV-1 and S from SARS-CoV-2. The first chapter of this thesis is an introduction to viruses with a specific focus on HIV-1 CD4 mimetic drugs and antibodies against SARS-CoV-2. It discusses the architecture of these viruses and fusion proteins and how small molecules, peptides, and antibodies can target these proteins successfully to treat and prevent disease. In addition, a brief overview is included of the techniques involved in structural biology and how it has informed the study of viruses. For the interested reader, chapter 2 contains a review article that serves as a more in-depth introduction for both viruses as well as how the use of structural biology has informed the study of viral surface proteins and neutralizing antibody responses to them. The subsequent chapters provide a body of work divided into two parts. The first part in chapter 3 involves a study on conformational changes induced in the HIV-1 Env protein by CD4-mimemtic drugs using single particle cryo-EM. The second part encompassing chapters 4 and 5 includes two studies on antibodies isolated from convalescent COVID-19 donors. The former involves classification of antibody responses to the SARS-CoV-2 S receptor-binding domain (RBD). The latter discusses an anti-RBD antibody class that binds to a conserved epitope on the RBD and shows cross-binding and cross-neutralization to other coronaviruses in the sarbecovirus subgenus.</p
Application of advanced fluorescence microscopy and spectroscopy in live-cell imaging
Since its inception, fluorescence microscopy has been a key source of discoveries in cell biology. Advancements in fluorophores, labeling techniques and instrumentation have made fluorescence microscopy a versatile quantitative tool for studying dynamic processes and interactions both in vitro and in live-cells. In this thesis, I apply quantitative fluorescence microscopy techniques in live-cell environments to investigate several biological processes. To study Gag processing in HIV-1 particles, fluorescence lifetime imaging microscopy and single particle tracking are combined to follow nascent HIV-1 virus particles during assembly and release on the plasma membrane of living cells. Proteolytic release of eCFP embedded in the Gag lattice of immature HIV-1 virus particles results in a characteristic increase in its fluorescence lifetime. Gag processing and rearrangement can be detected in individual virus particles using this approach. In another project, a robust method for quantifying Förster resonance energy transfer in live-cells is developed to allow direct comparison of live-cell FRET experiments between laboratories. Finally, I apply image fluctuation spectroscopy to study protein behavior in a variety of cellular environments. Image cross-correlation spectroscopy is used to study the oligomerization of CXCR4, a G-protein coupled receptor on the plasma membrane. With raster image correlation spectroscopy, I measure the diffusion of histones in the nucleoplasm and heterochromatin domains of the nuclei of early mouse embryos. The lower diffusion coefficient of histones in the heterochromatin domain supports the conclusion that heterochromatin forms a liquid phase-separated domain. The wide range of topics covered in this thesis demonstrate that fluorescence microscopy is more than just an imaging tool but also a powerful instrument for the quantification and elucidation of dynamic cellular processes
High intensity proton beam impact at 440 GeV/c on Mo and Cu coated CfC/graphite and SiC/SiC absorbers for beam intercepting devices
The authors would like to acknowledge the support of CERN's Sources, Targets and Interactions (STI) Group, the Accelerator Consolidation Project at CERN, as well as M. Wendt and F. Caspers. This program is partially supported by JSPS KAKENHI Grant Number JP16H03994. This support is gratefully acknowledged. The research leading to these results has received funding from the transnational access activity ARIES which is co-funded by the European Union's Horizon 2020 Research and innovation programme under Grant Agreement no. 730871.Beam Intercepting Devices (BIDs) are essential protection elements for the operation
of the Large Hadron Collider (LHC) complex. The LHC internal beam dump (LHC Target Dump
Injection or LHC TDI) is the main protection BID of the LHC injection system; its main function
is to protect LHC equipment in the event of a malfunction of the injection kicker magnets during
beam transfer from the SPS to the LHC. Several issues with the TDI were encountered during LHC
operation, most of them due to outgassing from its core components induced by electron cloud
effects, which led to limitations of the injector intensity and hence had an impact onLHCavailability.
The absorbing cores of the TDIs, and of beam intercepting devices in general, need to deal with high
thermo-mechanical loads induced by the high intensity particle beams. In addition, devices such as
the TDI—where the absorbing materials are installed close to the beam, are important contributors
to the accelerator impedance budget. To reduce impedance, the absorbing materials that make up
the core must be typically coated with high electrical conductivity metals. Beam impact testing of
the coated absorbers is a crucial element of development work to ensure their correct operation.
In the work covered by this paper, the behaviour of several metal-coated absorber materials
was investigated when exposed to high intensity and high energy proton beams in the HiRadMat
facility at CERN. Different coating configurations based on copper and molybdenum, and absorbing
materials such as isostatic graphite, Carbon Fibre Composite (CfC) and Silicon Carbide reinforced
with Silicon Carbide fibres (SiC-SiC), were tested in the facility to assess the TDI’s performance
and to extract information for other BIDs using these materials. In addition to beam impact tests and
an extensive Post Irradiation Examination (PIE) campaign to assess the performance of the coatings
and the structural integrity of the substrates, extensive numerical simulations were carried out.CERN's Sources, Targets and Interactions (STI) GroupAccelerator Consolidation Project at CERNMinistry of Education, Culture, Sports, Science and Technology, Japan (MEXT)Japan Society for the Promotion of ScienceGrants-in-Aid for Scientific Research (KAKENHI) JP16H03994transnational access activity ARIES - European Union 73087
A comprehensive review on laser powder bed fusion of steels : processing, microstructure, defects and control methods, mechanical properties, current challenges and future trends
Laser Powder Bed Fusion process is regarded as the most versatile metal additive manufacturing process, which has been proven to manufacture near net shape up to 99.9% relative density, with geometrically complex and high-performance metallic parts at reduced time. Steels and iron-based alloys are the most predominant engi-neering materials used for structural and sub-structural applications. Availability of steels in more than 3500 grades with their wide range of properties including high strength, corrosion resistance, good ductility, low cost, recyclability etc., have put them in forefront of other metallic materials. However, LPBF process of steels and iron-based alloys have not been completely established in industrial applications due to: (i) limited insight available in regards to the processing conditions, (ii) lack of specific materials standards, and (iii) inadequate knowledge to correlate the process parameters and other technical obstacles such as dimensional accuracy from a design model to actual component, part variability, limited feedstock materials, manual post-processing and etc. Continued efforts have been made to address these issues. This review aims to provide an overview of steels and iron-based alloys used in LPBF process by summarizing their key process parameters, describing thermophysical phenomena that is strongly linked to the phase transformation and microstructure evolution during solidifica-tion, highlighting metallurgical defects and their potential control methods, along with the impact of various post-process treatments; all of this have a direct impact on the mechanical performance. Finally, a summary of LPBF processed steels and iron-based alloys with functional properties and their application perspectives are presented. This review can provide a foundation of knowledge on LPBF process of steels by identifying missing information from the existing literature
Investigating the role of R2TP-like co-chaperone complexes during axonemal dynein assembly
Motile cilia are specialised cell-types which in humans have important roles in
the linings of the airways, the reproductive system and the brain. The
movement, required for this type of cilia to function, is facilitated by structures
called axonemal dynein motor complexes. These are large, multi-subunit
structures, and so it is crucial that they are assembled correctly. In humans, if
the motility of these is defective, it can lead to a disorder called Primary Ciliary
Dyskinesia, or PCD. This is a heterogeneous, autosomal recessive disorder –
symptoms of which include abnormally positioned organs, chronic respiratory
infections and infertility. Therefore, the development and structure of the motile
cilia is tightly regulated by multiple proteins including chaperones, dynein
axonemal assembly factors (DNAAFs), microtubule inner proteins (MIPs), the
outer arm docking complex (ODA-DC) and the nexin-dynein regulatory
complex (N-DRC). Chaperones work with co-chaperones to regulate their
many functions within the cell. One of these co-chaperones is the R2TP
complex, which was originally discovered in yeast but is conserved in higher
organisms. This multi-protein co-chaperone is involved in the assembly of
multi-subunit complexes such as the axonemal dynein motors. Two of the
R2TP subunits, Pontin and Reptin, are involved in many cellular functions both
in this co-chaperone complex and independently. It is thought that as some
DNAAFs share similar protein domains to the components of the R2TP
complex, they may form R2TP-like complexes. However, the specific details
surrounding the roles of these complexes during the assembly process
remains unclear. The structure of motile cilia is highly conserved throughout
evolution and Drosophila melanogaster has been shown previously to be an
excellent model for furthering understanding into the development and function
of these structures as only two cell types in the fly contain axonemal dynein
motor complexes. These are the chordotonal neuron, which has a motile
ciliated dendrite essential for its mechanosensory function, and the sperm
flagellum. In this thesis, I use the Drosophila model to further characterise
putative ciliary genes (Wdr16 and Dpcd) identified by a transcriptome analysis
previously carried out in the lab. RNAi knockdown experiments as well as
expression analysis supported motile cilia functions. The diversity which has
been identified regarding the roles of these two putative ciliary genes highlights
how proteins can be involved in motile cilia in different ways. I also use this
genetically tractable model to further understand the roles of the individual
proteins of a previously identified R2TP-like complex (R2DP3). Electron
microscopy, proteomics and investigation into how the localisation of dynein
subsets was affected in null mutants (generated using CRISPR/Cas9) allowed
for the role of this R2TP-like complex in the dynein assembly process to be
further specified. Using co-immunoprecipitation and affinity purification, we
identified an additional protein complex featuring Pontin and Reptin of the
R2TP complex, alongside the DNAAF Heatr2 and the putative DNAAF Dpcd.
As well as a role in dynein assembly, both DNAAFs are additionally expressed
in the neuroblasts of the CNS, and disruption to their function results in a late
larval lethality. Therefore, we have found these genes to not be specific to the
dynein assembly process and hypothesise that Dpcd may have an additional
function (working with Pontin, Reptin and potentially Heatr2) in the regulation
of AKT signalling and therefore impact cell proliferation
Investigating PAX6 and SOX2 dynamic interactions at the single molecule level in live cells
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
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