Dendritic Cells in Normal and Inflamed Human Skin

Psoriasis is a skin disease originally thought to be a primary keratinocyte differentiation and maturation disease. Several T cell targeted theraputics were found to reverse disease, and thus subsequent research has focussed on the adaptive immune system, particularly effector CD8+ T cells infiltrating the epidermis. Recent studies, however, show that inhibitors of tumor necrosis factor (TNF) are also effective therapeutics. Activated dendritic cells (DCs) produce large ammounts of TNF which acts in an autocrine loop to increase DC maturation. Thus, TNF inhibitors may inhibit DC maturation and downstream T cell activation. This thesis elucidates DC subsets present in normal human skin and psoriasis lesional skin, and the mechanisms by which psoriatic inflammatory DCs activate Th17 T cells and downstream mediators to maintainan psoriatic cutanous inflammation. In normal blood, ther exists 3 non-overlapping subsets of myeloid dendritic all of which are Lin-CD11c+HLA-DR+ and either BDCA-1+ (24 ± 2%), CD16+ (70 ± 4%), or BDCA-3+ (5 ± 1%), in order of immunostimulatory capacity. Only two myloid dendritic cell populations exist in normal human dermis: Lin-CD11c+HLA-DR+CD16+ and either BDCA-1+ ( 90%) or BDCA-3+ ( 10%). In situ double-label immunofluorescence showed that approximately 10-15% of CD11c+ dermal cells cluster together in lymphoidlike structures and are BDCA-1+CD205+DC-LAMP+. Upon emigration from the dermis, 90-95% of BDCA-1+ cells expressed these mature antigens, stimulated allogeneic T cells, and increased immunostimulatory capacity after the addition of TNF, PGE2, IL-1, and IL-6. Functional studies were not performed on BDCA-3+ cells because of limited cell numbers. In normal dermis there also exists a large population of FXIIIA+CD163+ macrophages that are not immunostimulatory and phagocytose large particles. As in normal blood, psoriasis patient blood contained 3 non-overlapping subsets of myeloid DCs (BDCA-1+, CD16+, or BDCA-3+). In psoriatic skin the frequency and distribution of BDCA-1+ and BDCA-3+ cells is similar to normal, however, there was a 30-fold increase in “inflammatory“ CD11c+ cells that did not express either marker. Most BDCA-1+ cells expressed maturation markers CD205 and DC-LAMP, while most BDCA-1- inflammatory cells expressed CD209 immature DC/ macrophage marker. Some myeloid cells expressed TNF and inducible nitric oxide synthase (iNOS). Treatment of psoriasis patients with the TNF neutralizing antibody etanercept not only inhibited dendritic cells as expected, but also had inhibitory effects on a newly appreciated type of T cell – Th17 cells. Etanercept reduced inflammatory DC products that drive Th17 cell proliferation (IL-23) as well as Th17 products and downstream effector mollecules (IL-17, IL-22, CCL20, and DEFB4). In contrast, Th1 cellular products and effector molecules (IFN, LTA-1, and MX-1) were reduced late in disease resolution. Using affymetrix gene array we characterized a global set of 4 gene clusters modulated temporally over the course of etanercept treatment. TNF and IL-17 pathway genes were downmodulated with a similar velocity, while IFN pathway genes were downmodulated later

Engineering rotating apical-out airway organoid for assessing respiratory cilia motility

Motile cilia project from the airway apical surface and directly interface with inhaled external environment. Owing to cilia\u27s nanoscale dimension and high beating frequency, quantitative assessment of their motility remains a sophisticated task. Here we described a robust approach for reproducible engineering of apical-out airway organoid (AOAO) from a defined number of cells. Propelled by exterior-facing cilia beating, the mature AOAO exhibited stable rotational motion when surrounded by Matrigel. We developed a computational framework leveraging computer vision algorithms to quantify AOAO rotation and correlated it with the direct measurement of cilia motility. We further established the feasibility of using AOAO rotation to recapitulate and measure defective cilia motility caused by chemotherapy-induced toxicity and by CCDC39 mutations in cells from patients with primary ciliary dyskinesia. We expect our rotating AOAO model and the associated computational pipeline to offer a generalizable framework to expedite the modeling of and therapeutic development for genetic and environmental ciliopathies

Computational modelling to characterize dendrimer nanocarriers for siRNA delivery in chronic obstructive pulmonary disease

La Broncopneumopatia Cronica Ostruttiva (BPCO) è un’infiammazione polmonare progressiva caratterizzata da limitazione di flusso espiratorio. Ad oggi a livello mondiale si stima che 328 milioni di persone siano affette da BPCO e si prevede che diventi la quarta causa di morte entro il 2030 con più di 65 milioni di casi l’anno. Il successo dei trattamenti medici mediante acidi nucleici, come DNA e silencing interference RNA è fortemente legato al design di efficienti tecnologie di rilascio. I dendrimeri cationici rappresentano un’interessante possibilità come vettori grazie a noti vantaggi di efficienza, di basso costo, di produzione e di versatilità d’applicazione nel trattamento di molteplici malattie e disturbi. Il presente lavoro di tesi, svolto presso l’Istituto Dalle Molle di Studi sull’Intelligenza Artificiale di Lugano (CH), ha avuto lo scopo di investigare a livello computazionale su scala atomica il meccanismo di legame di diversi dendrimeri con un modello di siRNA, per l’ottenimento di efficienti sistemi di rilascio per il trattamento di BPCO. In primo luogo sono stati realizzati i modelli di tre dendrimeri con differente funzionalizzazione ed è stato eseguito uno studio preliminare di caratterizzazione in acqua. In seguito, mediante la dinamica molecolare è stato analizzato il complesso, esplorandone le caratteristiche di legame in termini energetici e strutturali. Si è evidenziato come i cambiamenti conformazionali e la presenza di cariche sulla superficie dei dendrimeri condizionassero il legame con il siRNA. La diversa funzionalizzazione dei dendrimeri infatti influisce fortemente sulla topologia e sulle proprietà fisico-chimiche che guidano l’interazione con gli agenti terapeutici e l’ambiente. I risultati di questo lavoro apportano un contributo significativo al futuro design di vettori di acidi nucleici selettivi, caratterizzati dal miglior compromesso tra stabilità del complesso e abilità di rilascio del sistema

Cellular and Circuit Level Responses to Neural Stem Cell Transplantation in the Rodent Cortex

Neural stem cell (NSC) transplantation is a promising strategy for the treatment of neurological disease and injury. NSC transplants have been documented to exert both neurotrophic and immunomodulatory effects in pathological contexts, but grafted cells frequently remain undifferentiated. The specific interactions between undifferentiated NSCs and the normal host microenvironment are not well understood. To investigate the functional impact of undifferentiated NSCs on host activity, a clonal NSC line (C17.2) was utilized. Network dynamics were monitored post-transplant in acute slices of somatosensory cortex using voltage sensitive dye imaging. Single and repetitive callosal stimuli elicited activity that originated in deep layers, propagated vertically along cortical columns, and spread horizontally across superficial layers. Very high levels of C17.2 engraftment (\u3e25%) interfered with parameters of cortical function, including the amplitude, spatial extent, velocity, and integration of evoked potentials. These levels also raised the current threshold required to activate cortical microcircuitry by ten-fold. Conversely, moderate levels of engraftment (\u3c15%) preserved network properties and induced only subtle changes in facilitation during repetitive stimulation. A binning analysis of cortical activity showed that deep cortical layers were more susceptible to the presence of ectopic NSCs than superficial layers. Pharmacological blockade of GABA-A signaling indicated that inhibition was not the predominant cause of circuit dampening in these layers. Instead, highly engrafted cortices showed a marked depletion in host neurons and associated neuronal metabolites. Microglial activation preceded neuronal loss in the transplanted brain and deactivation with doxycycline exerted a neuroprotective effect. Analysis of C17.2-conditioned supernatants showed they secrete a number of proinflammatory cytokines and chemokines. However, these factors did not induce direct toxicity, but rather enhanced microglial-mediated neuronal apoptosis in vitro via tumor necrosis factor alpha-dependent signaling. Primary NSCs from the postnatal subventricular zone showed similar effects on microglial-mediated cytotoxicity. Together, these results suggest that undifferentiated NSCs possess an inherent capacity to modulate microglial functions which can affect neuronal survivability and activity in the host brain

Personality Identification from Social Media Using Deep Learning: A Review

Social media helps in sharing of ideas and information among people scattered around the world and thus helps in creating communities, groups, and virtual networks. Identification of personality is significant in many types of applications such as in detecting the mental state or character of a person, predicting job satisfaction, professional and personal relationship success, in recommendation systems. Personality is also an important factor to determine individual variation in thoughts, feelings, and conduct systems. According to the survey of Global social media research in 2018, approximately 3.196 billion social media users are in worldwide. The numbers are estimated to grow rapidly further with the use of mobile smart devices and advancement in technology. Support vector machine (SVM), Naive Bayes (NB), Multilayer perceptron neural network, and convolutional neural network (CNN) are some of the machine learning techniques used for personality identification in the literature review. This paper presents various studies conducted in identifying the personality of social media users with the help of machine learning approaches and the recent studies that targeted to predict the personality of online social media (OSM) users are reviewed

A Cooperative Approach for Autonomous Landing of UAVs

This dissertation presents a cooperative approach for the autonomous landing of MRVTOL UAVs (Multi Rotor-Vertical Take-off and Landing Unmanned Aerial Vehicles). Most standard UAV autonomous landing systems take an approach, where the UAV detects a pre-set pattern on the landing zone, establishes relative positions and uses them to perform the landing. These methods present some drawbacks such as all of the processing being performed by the UAV itself, requiring high computational power from it. An additional problem arises from the fact most of these methods are only reliable when the UAV is already at relatively low altitudes since the pattern’s features have to be clearly visible from the UAV’s camera. The method presented throughout this dissertation relies on an RGB camera, placed in the landing zone pointing upwards towards the sky. Due to the fact, the sky is a fairly stagnant and uniform environment the unique motion patterns the UAV displays can be singled out and analysed using Background Subtraction and Optical Flow techniques. A terrestrial or surface robotic system can then analyse the images in real-time and relay commands to the UAV. The result is a model-free method, i.e independent of the UAV’s morphological aspect or pre-determined patterns, capable of aiding the UAV during the landing manoeuvre. The approach is reliable enough to be used as a stand-alone method, or be used along traditional methods achieving a more robust system. Experimental results obtained from a dataset encompassing 23 diverse videos showed the ability of the computer vision algorithm to perform the detection of the UAV in 93,44% of the 44557 evaluated frames with a tracking error of 6.6%. A high-level control system that employs the concept of an approach zone to the helipad was also developed. Within the zone every possible three-dimensional position corresponds to a velocity command for the UAV, with a given orientation and magnitude. The control system was tested in a simulated environment and it proved to be effective in performing the landing of the UAV within 13 cm from the goal

Genome-Wide Host Gene Expression Analysis Before and After the Initiation of Highly Active Antiretroviral Therapy And Natural Control of HIV in Therapy Naïve HIV+ Non-Progressors

Since its discovery 25 years ago, the HIV virus has infected more than 34 million people in the world; the infection caused by this virus has led the world to its most terrible pandemic, and the greatest global health crises of our times. The host-virus interaction and natural history of the disease are influenced by the distinctive interface the virus has with each infected individual. The infection with HIV-1 is characterized by the destruction of CD4+ T-cells during the typical course of the infection, but HIV has the arsenal to infect practically all the major blood leukocytes. Taken from these observations, it is apparent that HIV has the innate ability to subvert and manipulate the host gene machinery at the transcriptomic level. The highly active anti-retroviral therapy (HAART) consists of a combination of powerful drugs, which serve as potent defence mechanism against the ways in which the HIV virus attacks the human bod y. Although these drugs are not able to rid the body of HIV virus, they can significantly delay the onset of AIDS and reduce the incidence of opportunistic infections, morbidity and mortality related to HIV infection. After the introduction of HAART treatment, it is observed that most patients with good adherence respond to HAART, which is defined by a decrease of plasma viral load to undetectable levels and an immune reconstitution with a significant increase of CD4+ T cell levels. Around 30% of the patients fail to achieve this response and continue to express high plasma viral load and low CD4+ T cell numbers. In contrast, some rare HIV+ patients maintain below detectable levels of plasma viremia without the treatment. These are termed long-term nonprogressors(LTNPs), less commonly called elite controllers. These rare individuals are infected with HIV, but have the natural ability to control the infection with the strength of their immune system. Many of th ese patients have been HIV positive for 30 years or more and! off the rapy for the entire duration of their infection, showing high CD4+T cells counts and no progression to the disease. In this context, it is important to mention that the genomic basis of this natural effective immunological control of viremia in LTNPs, as opposed to drug-mediated control of HIV, remains unknown. The development of high throughput microarray platforms and bioinformatic platforms to visualize and analyse the complex dataset has enabled considerable progress in the field of viral genomics, and also the visualization of host-virus interactions at the molecular level. In chapter III, we carried out a comparative genome-wide (encompassing all 25,000 human genes) pharmacogenomic study using whole primary peripheral blood mononuclear cells (PBMC) derived from 14 HIV+ patients at two time points: pre-HAART (TP-1 with detectable viremia) and post (TP2: below detectable level (BDL) of plasma HIV <40 copies of HIV RNA/mL plasma), to ascertain how genomically distinct viremic phase is from the phase in which virus is fully controlled with HAART. Another goal was to define the underlying pharmacogenomic basis of HIV control during HAART. In the second study shown in Chapter IV, we compared the two time points against the 9 LTNPs to unravel the genomic basis of natural control of viremia in therapy naïve LTNPs showing below detectable levels of viremia (<20 copies of HIV RNA/mL plasma) and high and stable CD4+T cell counts. Genomic RNA extracted from the PBMCs was used in genome-wide microarray analysis, using HT-12v3 Illumina chips. Quantile normalization was performed to normalize the data and inter-patient variability. Illumina®BeadStudio Data Analysis Software wa s used to obtain differentially expressed (DE) genes. Only the significant genes with p value <0.01 and FDR of <5% (for the comparison between TP1 and TP2) and FDR <1% (for the comparison between LTNP vers us TP1 and LTNP versus TP2) were considered appropriate for ! analysis . Pathway analysis was performed in MetaCoreTM from GeneGo, Inc to derive functional annotations. Functionally significant genes were validated by quantitative real time PCR. Between TP1 and TP2, 234 genes were differentially expressed. Within these genes, 212 were down-regulated and 22 upregulated. Between the comparison between LTNP vs TP1, 965 genes were differently expressed (706 genes were up-regulated and 259 genes were down-regulated), and when LTNP was compared to the TP2 group, we found 1181 DE genes (with 727 genes up-regulated and 454 genes downregulated). In the first part of this study, comparing the TP1 and TP2 only, we found that of the top 10 pathways, 8 belonged to the immune response system. This was the most significant pathway up-regulated in TP1 when compared to TP2. This comprised of genes that were involved in antiviral action of interferon (IFN) and their signalling function, antiviral response, dendritic cell maturation a nd migration, and cell metabolism. Map folder and enrichment analysis corroborated with our findings, thereby confirming an intrinsic role of the immune, inflammatory and interferon response family-related genes during HIV viremia in the absence of treatment. But a closer examination of this contrast also showed a mirror down-regulation of genes involved in innate and adaptive immunity, inflammation, apoptosis and antiviral functions. This directly implies a functional relevance of these pathways, through their modulation in TP1 and TP2 stages. Although these data are intuitive and expected, such analysis has never been performed before. The second part of this study, on comparisons between LTNP and TP1 and LTNP against TP2, we show the first evidence demonstrating that the natural control of HIV in LTNP is guided by the genes enriched in the immune response, cytoskeleton remodelling, apoptosis and T cell signalling pathways. Another striking ob servation was that, even though the LTNP and TP2 groups maintai ned BDL of plasma viremia (<40 copies), the LTNP group was genomically distinct from the TP2, which controlled viremia with HAART. This highlights the qualitative distinction and critical role of enriched pathways in natural control of viremia in the LTNPs. Seventeen genes encompassing all these pathways were validated by q-PCR, which showed consistent trends between microarray and q-RT PCR. One gene in particular, the thrombospondin (THBS1) (R2= 0.942) was identified as a biomarker in our study, discriminating between viremic patients and LTNPs at the genomic (R2= 0.942, p<2.654e.08) and proteomic levels (p<0.003761). The levels of expression of THBS1 showed excellent correlation with plasma viremia (R2=0.81557;p<.0.0003761), the first description of such an important protein. This is the most unique finding of this work, which has significance in HIV disease prognostics and diagnostics, in addition to predicting the strength of the ho st immune system, as evident from its down-regulation and low expression in the natural controllers. Overall, through these analyses, we have shown that, although there are common set of key genes associated with HIV at all stages, each stage also showed unique molecular signatures. This was demonstrated with the identification of molecular signatures for the control of virus with HAART therapy, as well as for the natural control of HIV in LTNPs. Especially for the LTNPs, the downregulation of the apoptosis was the most significant feature, which may have important implications in therapy, especially in the context of using apoptosis as a target for future therapies

Membrane protein nanoclustering as a functional unit of immune cells : from nanoscopy to single molecule dynamics

State-of-the-art biophysical techniques featuring high temporal and spatial resolution have allowed for the first time the direct visualization of individual transmembrane proteins on the cell membrane. These techniques have revealed that a large amount of molecular components of the cell membrane do not organize in a random manner but they rather grouped together forming so-called clusters at the nanoscale. Moreover, the lateral behavior of these clusters shows a great dependence on the compartmentalization of the cell membrane by, e.g., the actin cytoskeleton at multiple temporal and spatial scales. Since these lateral and temporal organizations have been shown to be crucial for the regulation of the biological activity by these transmembrane proteins, the understanding of the spatiotemporal behavior of membrane receptors, and of proteins in general, is a necessary step towards understanding the biology of the cell. Protein nanoclustering and membrane compartmentalization have been shown to play a crucial role on leukocytes, particularly on the surface of antigen presenting cells. Hence, the direct visualization of membrane proteins on the cell membrane of antigen presenting proteins represents a crucial step in understanding how an immune response can be controlled by leukocytes at the molecular level. In Chapter 1, the immune system, the membrane receptor DC-SIGN and the antigen presenting protein CD1d are briefly introduced. Moreover, recent advances in superresolution microscopy and single particle tracking techniques which allow the study of membrane proteins at the nanoscale are discussed. Finally, an updated review of protein nanoclustering on the cell membrane shows examples of the importance of protein nanoclustering in regulating biological function in the immune system. Chapter 2 presents the quantitative methodology for analyzing STED nanoscopy images and multi-color single particle tracking data used throughout this thesis. Chapter 2 also describes the single-molecule fluorescence sensitive microscopes implemented in this thesis for multi-color single particle tracking experiments and the corresponding data analysis. At the end of Chapter 2, cartography maps combining high temporal with micron-scale spatial information on the basis of single-molecule detection are presented. The following chapters in this thesis describe the major results obtained on two important receptors of the immune system. In Chapter 3, we address the role of the neck region of DC-SIGN in fine-tuning the nanoclustering degree of DC-SIGN on the cell membrane. Moreover, Chapter 3 also links the nanoclustering capability of DC-SIGN with its virus binding capability. The meso-scale organization of DC-SIGN and its dependence on a glycan-based connectivity is addressed on Chapter 4. This glycosylation network enhances the interaction between DC-SIGN and clathrin beyond stochastic random encountering. In Chapter 5, we showed that DC-SIGN shows subdiffusive behavior and weak ergodicity breaking (wEB) that cannot be described using the continuous time random walk (CTRW) model. Instead, our data are more consistent with a model in which the plasma membrane is composed of "patches" that change in space in time. In Chapter 6, we demonstrate that the antigen presenting protein CD1d organizes in nanoclusters on the cell membrane of antigen presenting cells whose size and density are tightly controlled by the actin cytoskeleton. Moreover, we also showed that this cytoskeletal control of the CD1d nanoclustering predominantly occurs on the pool of CD1d that has undergone lysosomal recycling, including under inflammatory conditions. Finally, in Chapter 7 we summarize the main results of this thesis and highlight future experiments that will expand the knowledge obtained so far regarding the role of plasma membrane organization and biological regulation.Gracias a su alta resolución temporal y espacial, las técnicas biofísicas de última generación han permitido la observación directa de proteínas de transmembrana de forma individual en la membrana celular. Estas técnicas han mostrado que la organización de una gran parte de las proteínas de transmembrana no es aleatoria sino que éstas están agrupadas en la membrana celular formando nano-agregados, o "clusters". En el caso concreto del sistema inmune, se ha demostrado que el agrupamiento de proteínas y los compartimentos de la membrana celular juegan un papel determinante en las células presentadoras de antígenos a la hora de controlar la iniciación de una respuesta inmune. Por tanto, la visualización directa de proteínas de membrana en células presentadoras de antígenos a la escala nanométrica representa un paso crucial en el entendimiento del sistema inmune y en un futuro desarrollo de terapias basadas en el sistema inmune humano. En el primer capítulo de esta tesis, se presentará al lector una breve introducción del sistema inmune y una descripción general de las dos proteínas que se han estudiado extensivamente en esta tesis: el receptor reconocedor de patógenos DC-SIGN y la proteína presentadora de antígenos glicolipídicos CD1d. Se discutirán además los últimos avances en técnicas de microscopía de fluorescencia con alta resolución temporal y espacial que permiten el estudio de proteínas a la escala nanométrica. Finalmente, el primer capítulo concluye con una revisión de los últimos avances en la caracterización de la organización lateral de proteínas de membrana mostrando cómo dicha organización determina la función biológica de estas proteínas. En el capítulo 2, se presentan los distintos tipos de metodología utilizados en esta tesis para cuantificar imágenes de microscopía de super-resolución STED así como para analizar datos provenientes del seguimiento de partículas individuales usando varios colores. Al final del capítulo 2 se presenta una nueva metodología desarrollada en esta tesis que permite el estudio lateral de proteínas de membrana con una alta resolución temporal y una escala espacial de orden de micras y a la que hemos denominado mapas cartográficos. Los siguientes capítulos de esta tesis se enfocan en el estudio de dos importantes proteínas involucradas en el sistema inmune. En el capítulo 3 se describe como la parte central de la estructura del receptor captador de patógenos DC-SIGN determina su grado de nano-agrupamiento sobre la membrana celular. A su vez, este agrupamiento tiene una incidencia clave en la capacidad de DC-SIGN en unirse a partículas virales. La organización de DC-SIGN a la escala mesoscópica y la dependencia de dicha organización de una conectividad en la membrana celular basada en la glicosilación de proteínas es descrita en el capítulo 4. En el capítulo 5 descubrimos que DC-SIGN tiene un comportamiento que no solo es sub-difusivo en la membrana celular sino que también conlleva a la ruptura de ergodicidad por parte de este receptor. Esta rotura de ergodicidad no puede ser descrita por el modelo "continous time random walk" (CTRW) sino por un modelo nuevo donde la difusión de la partícula cambia constantemente en el espacio y en el tiempo. En el capítulo 6 de esta tesis describimos como la molécula CD1d forma nano-agrupamientos en la membrana celular cuyo tamaño y densidad son controlados por el citoesqueleto de actina. Además, observamos que dicho control mayoritariamente sucede cuando CD1d ha sido reciclado a través de compartimentos lisosomales, incluyendo procesos inflamatorios. Finalmente, en el capítulo 7 se discuten las conclusiones generales de esta tesis y se sugieren experimentos a futuro de manera de incrementar, en base a los resultados obtenidos en esta tesis, nuestro conocimiento de la membrana celular y el papel que la organización espacial y temporal juega en el control del sistema inmune