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Introduction: The pathogenesis and treatment of allergic bronchopulmonary aspergillosis (ABPA), severe asthma-non fungal sensitised (SANFS) and severe asthma with fungal sensitization (SAFS) is poorly understood. IL-17A, IgE and microbiome may be associated with pathogenesis of asthma, but their role in fungal-associated asthma is uncertain. Further, the efficacy of voriconazole, posaconazole and nebulised amphotericin B (NAB) in ABPA and SAFS has not been fully studied. Aims and objectives: The aim of this PhD thesis was to evaluate the role of IL-17A, IgE and lung microbiome in patients with SANFS, SAFS and ABPA. We also studied the efficacy and safety of NAB, voriconazole and posaconazole. Methods: Airway lymphocytes and peripheral blood mononuclear cells (PBMC) from patients with ABPA (n=16), SAFS (n=15), SANFS (n=11), mild asthma (MA) (n=6) and NH (n=11) were characterized by flow cytometric analysis (FACS) to determine the % of CD (+) IL-17A expressing cells. We also evaluated microbiome population using culture and PCR plus sequencing from BAL of these patients. In chapter 3, we analysed total and specific IgE in blood from adult cohorts of SAFS (n=34) and ABPA (n=48) using ImmunoCAP 100. In chapter 5 we studied the efficacy of voriconazole and posaconazole and in chapter 6; we studied the efficacy of NAB.Results: %CD4+IL-17A expressing cells were significantly higher in patients with severe asthma and correlated positively with serum neutrophil and presence of fungi in the airways. ABPA, SAFS and SANFS were similar but all were significantly higher than MA and NH. There were no differences in IL-17A expression between blood and the lung. Fungi were more frequently associated with severe asthma and low FEV1. Steroid treatment significantly increased airway fungal load. IgE against staphylococcal aureus (SE-IgE) correlated positively with FEV1 and OCS dose. Voriconazole and posaconazole improved asthma severity and radiological abnormalities. NAB was associated bronchospasm, but was extrely effective in the few patients (n=3) that took treatment for >12 months. These responders had unique characteristics. Conclusions: IL-17A, SE-IgE, and lung microbiome are associated with asthma severity. Steroid use in these patients may increase airway fungal load. Whereas voriconazole and posaconazole are efficacious, the use of NAB is associated with significant bronchospasm. SE-IgE -high asthma patients may be a distinct asthma phenotype. Larger studies are needed.

En Viticulture de Précision, l’imagerie multi-spectrale est principalement utilisée pour des dispositifs de télédétection. Ce manuscrit s’intéresse à son utilisation en proxidétection, pour la caractérisation du feuillage. Il présente un dispositif expérimental terrestre mobile composé d’un GPS, d’une caméra multi-spectrale acquérant des images visible et proche infrarouge, et d’un Greenseeker RT-100 mesurant l’indice Normalized Difference Vegetation Index (NDVI). Ce système observe le feuillage de la vigne dans le plan de palissage, en lumière naturelle. La parcelle étudiée comporte trois cépages (Pinot Noir, Chardonnay et Meunier) plantés en carré latin. En 2013, six jeux de données ont été acquis à différents stades phénologiques.Pour accéder aux propriétés spectrales de la végétation, il est nécessaire de calibrer les images en réflectance. Cela requiert l’utilisation d’une mire de MacBeth comme référence radiométrique. Lorsque la mire est cachée par les feuilles, les paramètres de calibration sont estimés par une interpolation linéaire en fonction des images les plus proches sur lesquelles la mire est visible. La cohérence de la méthode d’estimation employée est vérifiée par une validation croisée (LOOCV).La comparaison du NDVI fournie par le Greenseeker avec celui déterminé via les images corrigées permet de valider les données générées par le dispositif. La polyvalence du système est évaluée via les images où plusieurs indices de végétation sont déterminés. Ils permettant des suivis de croissance de la végétation originaux offrant des potentialités de phénotypage ou une caractérisation de l’état sanitaire de la végétation illustrant la polyvalence et le gain en précision de cette technique. / Mutispectral imaging systems are widely used in remote sensing for Precision Viticulture. In this work, this technique was applied in the proximal sensing context to characterize vine foliage. A mobile terrestrial experimental system is presented, composed of a GPS receiver, a multi-spectral camera acquiring visible and near infrared images, and a Greenseeker RT-100 which measures the Normalized Difference Vegetative Index (NDVI). This optical system observes vine foliage in the trellis plan, in natural sunlight. The experimental field is planted with Chardonnay, Pinot Noir and Meunier cultivars in a latin squared pattern. In 2013, six datasets were acquired at various phenological stages.Spectral properties of the vegetation are accessible on images when they are calibrated in reflectance. This step requires the use of a MacBeth colorchart as a radiometric reference. When the chart is hidden by leaves, the calibration parameters are estimated by simple linear interpolation using the results from resembling images, which have a visible chart. The performance of this method is verified with a cross-validation technique (LOOCV).To validate the data provided by the experimental system, the NDVI given by the Greenseeker was compared to those computed from the calibrated images. The assessment of the versatility of the system is done with the images where several indices were determined. It allows an innovative follow-up of the vegetative growth, and offering phenotyping applications. Moreover, the characterization of the sanitary state of the foliage prove that this technique is versatile and accurate.

Le système immunitaire adaptatif joue un rôle de premier plan dans la défense contre les infections. La réponse humorale, impliquant la production d'anticorps, est un élément important de la réponse immunitaire adaptative. Au cours d'une infection, des cellules B spécifiques du système immunitaire prolifèrent et libèrent de grandes quantités d'anticorps qui se lient sélectivement à la protéine cible (antigène) trouvée sur le pathogène invasif, induisant la destruction du pathogène.Cependant, le système immunitaire ne répond pas toujours suffisamment efficacement pour détruire les agents pathogènes, et les mécanismes de tolérance empêchent la génération d'anticorps contre les protéines humaines - comme les marqueurs de surface cellulaire sur les cellules cancéreuses ou les cytokines impliquées dans des maladies inflammatoires et auto-immunes - qui pourraient être des cibles thérapeutiques importantes. Par conséquent, il existe un grand intérêt pour la recherche et le développement d'anticorps spécifiques qui peuvent être utilisés pour le traitement des patients par immunothérapie. En raison de leur grande affinité et de leur liaison sélective aux antigènes, les anticorps monoclonaux (mAbs) sont apparus comme des agents thérapeutiques puissants. Les anticorps monoclonaux dérivés de cellules B individuelles ont une séquence unique et présentent une affinité de liaison pour un antigène spécifique. Cependant, jusqu'à maintenant, la découverte des mAbs a été limitée par l'absence de méthodes à haut débit pour le criblage direct et à grande échelle de cellules B primaires non immortalisées pour découvrir les rares cellules B qui produisent des anticorps spécifiques d'intérêt clinique. Ceci est maintenant possible avec l'émergence et l'amélioration des méthodes de compartimentation in vitro pour l'encapsulation et le criblage de cellules uniques dans des gouttelettes picolitriques. Dans mon projet de doctorat, je décris le développement d'immunodosages et de dispositifs microfluidiques pour le criblage phénotypique direct de cellules individuelles à partir de populations de cellules B enrichies. Ce développement a permis une analyse détaillée de la réponse immunitaire humorale, avec une résolution à l’échelle de la cellule unique. C’est aussi un élément essentiel d'un pipeline de détection d'anticorps couplant le criblage phénotypique de cellules individuelles au séquençage d'anticorps sur cellules uniques. Il est maintenant possible, pour la première fois, de cribler des millions de cellules B individuelles en fonction de l'activité de liaison des anticorps sécrétés et de récupérer les séquences d'anticorps / The adaptive immune system plays a leading role in defense against infection. The humoral response, involving the production of antibodies, is an important component of the adaptive immune response. During an infection, specific B cells of the immune system proliferate and release large amounts of antibodies which bind selectively to the target protein (antigen) found on the invading pathogen, inducing destruction of the pathogen. However, the immune system does not always respond efficiently enough to destroy pathogens, and tolerance mechanisms prevent the generation of antibodies against human protein - such as cell surface markers on cancer cells or cytokines involved in inflammatory and autoimmune disease - that could be important therapeutic targets. Hence, there is great interest in research and development of specific antibodies that can be used for immunotherapy of patients. Due to their high affinity and selective binding to antigens, monoclonal antibodies (mAbs) have emerged as powerful therapeutic agents. Monoclonal antibodies derived from single B cells have a unique sequence and display binding affinity for a specific antigen. However, until now, the discovery of mAbs has been limited by the lack of high-throughput methods for the direct and large-scale screening of non-immortalized primary B cells to uncover rare B cells which produce the specific antibodies of clinical interest. This is now becoming possible with the emergence and improvement of in vitro compartmentalization methods for single-cell encapsulation and screening in picoliter droplets. In my PhD project, I describe the development of binding immunoassays and microfluidic devices for the direct phenotypic screening of single-cells from enriched B cell populations. This development has enabled detailed analysis of the humoral immune response, with single-cell resolution and is an essential component of an antibody-discovery pipeline coupling single-cell phenotypic screening to single-cell antibody sequencing. It is now possible, for the first time, to screen millions of single B cells based on the binding activity of the secreted antibodies and to recover the antibody sequences

<div><div>In the forensic science community, there is a vast need for tools to help assist investigations when standard DNA profiling methods are uninformative. Methods such as Forensic DNA Phenotyping (FDP) and proteomics aims to help this problem and provide aid in investigations when other methods have been exhausted. FDP is useful by providing physical appearance information, while proteomics allows for the examination of difficult samples, such as hair, to infer human identity and ancestry. To create a “biological eye witness” or develop informative probability of identity match statistics through proteomically inferred genetic profiles, it is necessary to constantly strive to improve these methods. </div><div><br></div><div>Currently, two developmentally validated FDP prediction assays, ‘HIrisPlex’ and ‘HIrisplex-S’, are used on the capillary electrophoresis to develop a phenotypic prediction for eye, hair, and skin color based on 41 variants. Although highly useful, these assays are limited in their ability when used on the CE due to a 25 variant per assay cap. To overcome these limitations and expand the capacities of FDP, we successfully designed and validated a massive parallel sequencing (MPS) assay for use on both the ThermoFisher Scientific Ion Torrent and Illumina MiSeq systems that incorporates all HIrisPlex-S variants into one sensitive assay. With the migration of this assay to an MPS platform, we were able to create a semi-automated pipeline to extract SNP-specific sequencing data that can then be easily uploaded to the freely accessible online phenotypic prediction tool (found at https://hirisplex.erasmusmc.nl) and a mixture deconvolution tool with built-in read count thresholds. Based on sequencing reads counts, this tool can be used to assist in the separation of difficult two-person mixture samples and outline the confidence in each genotype call.<br></div><div><br></div><div>In addition to FDP, proteomic methods, specifically in hair protein analysis, opens doors and possibilities for forensic investigations when standard DNA profiling methods come up short. Here, we analyzed 233 genetically variant peptides (GVPs) within hair-associated proteins and genes for 66 individuals. We assessed the proteomic methods ability to accurately infer and detect genotypes at each of the 233 SNPs and generated statistics for the probability of identity (PID). Of these markers, 32 passed all quality control and population genetics criteria and displayed an average PID of 3.58 x 10-4. A population genetics assessment was also conducted to identify any SNP that could be used to infer ancestry and/or identity. Providing this information is valuable for the future use of this set of markers for human identification in forensic science settings. </div></div><div><br></div>

Der ischämische Schlaganfall ist global eine der bedeutendsten Volkskrankheiten. Die derzeit verfügbaren kurativen Therapieoptionen werden vorrangig durch ein enges therapeutisches Zeitfenster limitiert. Ziel der aktuellen Schlaganfallforschung ist die Entwicklung von über dieses Zeitfenster hinaus wirksamen Therapien. Ein vielversprechender neuer Ansatz ist die experimentelle Behandlung mit humanen Nabelschnurblutzellen. Diese Arbeit erforscht das therapeutische Zeitfenster für die systemische Therapie des ischämischen Schlaganfalls mittels mononukleärer Nabelschnurblutzellen (hUCB MNC) in spontanhypertensiven Ratten nach permanentem Verschluss der Arteria cerebri media (pMCAO). Hierzu wurden die Therapiezeitpunkte 4, 24, 72, 120 Stunden und 14 Tage nach experimentellem Schlaganfall in einem komplexen Studiendesign inklusive neurofunktioneller Tests, magnetresonanztomographischer und immunhistochemischer Verfahren untersucht. In vitro wurde der Einfluss kokultivierter hUCB MNC auf Nekrose und Apoptose in neuralem Gewebe unter Sauerstoff-Glukose-Deprivation betrachtet. Die Studie ergab eine verbesserte funktionelle Rekonvaleszenz und eine geringere Ausprägung von Atrophie und Astroglianarbe bei Therapie innerhalb eines 72- Stunden-Zeitfensters. In vitro wurde eine signifikante Reduktion von Nekrose und Apoptose durch kokultivierte hUCB MNC beobachtet. Eine histologische Relokalisierung der intravenös applizierten Zellen war in keiner Therapiegruppe möglich. Die Integration der hUCB MNC ins Hirnparenchym stellt somit keine conditio sine qua non für die funktionelle Erholung nach Schlaganfall dar. Trotz des beobachteten erweiterten Zeitfensters ist die Translation dieses Therapieansatzes in die klinische Realität kritisch zu diskutieren, da weiterführende Studien unserer Arbeitsgruppe eine limitierte Wirksamkeit unter sehr praxisnahen Bedingungen (z.B. Einsatz kryokonservierter hUCB MNC) gezeigt haben. / Experimental treatment strategies using human umbilical cord blood mononuclear cells (hUCB MNCs) represent a promising option for alternative stroke therapies. An important point for clinical translation of such treatment approaches is knowledge on the therapeutic time window. Although expected to be wider than for thrombolysis, the exact time window for hUCB MNC therapy is not known. Our study aimed to determine the time window of intravenous hUCB MNC administration after middle cerebral artery occlusion (MCAO). Male spontaneously hypertensive rats underwent MCAO and were randomly assigned to hUCB MNC administration at 4h, 24h, 72h, 120h or 14d. Influence of cell treatment was observed by magnetic resonance imaging on days 1, 8 and 29 following MCAO and by assessment of functional neurological recovery. On day 30, brains were screened for glial scar development and presence of hUCB MNCs. Further, influence of hUCB MNCs on necrosis and apoptosis in post-ischemic neural tissue was investigated in hippocampal slices cultures. Transplantation within a 72h time window resulted in an early improvement of functional recovery, paralleled by a reduction of brain atrophy and diminished glial scarring. Cell transplantation 120h post MCAO only induced minor functional recovery without changes in the brain atrophy rate and glial reactivity. Later transplantation (14d) did not show any benefit. No evidence for intracerebrally localized hUCB MNCs was found in any treatment group. In vitro hUCB MNCs were able to significantly reduce post-ischemic neural necrosis and apoptosis. Our results for the first time indicate a time window of therapeutic hUCB MNC application of at least 72 hours. The time window is limited, but wider than compared to conventional pharmacological approaches. The data furthermore confirms that differentiation and integration of administered cells is not a prerequisite for poststroke functional improvement and lesion size reduction.

IntroduktionDe humana blodgrupperna består av A, B, AB samt O. Antigen kan detekteras av alloantikroppar, att en reaktion sinsemellan kan ske är ett krav för att en molekyl ska kunna kallas antigen. Antigen och antikroppar är vitala för analys inom transfusionsmedicin och möjliggör transfusioner av blod samtidigt som man förhindrar allvarliga transfusionsreaktioner. För att effektivisera manuella serologiska analyser används saltlösning med låg jonstyrka. Syftet med arbetet var att verifiera lösningen MLB 2 (Bio-Rad Medical Diagnostics GmbH, Dreieich, Tyskland). Denna lösning ska förstärka reaktioner i antikroppsidentifiering, blodgruppskontroll med antikropps-screening samt förenlighetsprövning, men även för fenotypning av erytrocyter gällande antigenen s, Fya, Fyb, Kpa, Kpb samt Lua. MetodTotalt fenotypades 61 prover varav 31 var positiva och 30 var negativa för sökt fenotyp. Av de 31 positiva var 17 fenotyper av känt heterozygot anlag. Vid varje analystillfälle utfördes kontroller som bestod av heterozygot positiva samt negativa celler från Örebropanelen för vardera fenotyp. För antikroppsidentifiering ämnade två antikroppar, anti-E samt anti-Lea, att analyseras från två prover där de tidigare identifierats. Beträffande blodgruppskontroll med antikroppsscreening samt förenlighetsprövning analyserades tre prover från patienter som varit i behov av transfusioner och som tidigare påvisat antikroppar. Resultat och slutsatsSamtliga provers resultat överensstämde fullständigt vid fenotypning. För antikroppsidentifiering bekräftas tidigare påvisad anti-E för ett av proven, men för det andra provet erhölls inte resultat fullständigt överensstämmande med patientens anti- Lea. För blodgruppskontroller med antikropps-screen samt förenlighetsprövning överensstämde samtliga resultat. Effekten av förstärkningslösningen MLB 2 bedöms som god. Tydligt blir dock att gelteknik, som idag används som golden standard, är överlägsen de äldre rörteknikerna. / BackgroundHuman blood groups are defined as A, B, AB and O. Antigens are detectable by alloantibodies and a reaction between them is necessary in order to denominate as such. Antigens and antibodies respectively are essential within immunohematology to enable transfusion therapy while evading severe transfusion reactions. To improve the efficiency of manual serological test, low ionic strength saline is used. The purpose of this study was to verify MLB 2 (Bio-Rad Medical Diagnostics GmbH, Dreieich, Germany) for use as low ionic saline solution (LISS) in indirect agglutination technique (IAT) tube-tests for antibody identification, blood group verification with antibody-screen and compatibility test, also for phenotyping antigens s, Fya, Fyb, Kpa, Kpb and Lua. MethodsA total of 61 samples, of which 31 were previously known positive and 31 negative were tested. Of the 31 positive, 17 were heterozygous. At every assay, positive heterozygous and negative cells for each of the phenotype was applied as control, from the Örebro panel. For antibody identification, two previously positive samples were used. For blood group verification with antibody-screen and compatibility test, three samples with previous need for transfusion therapy and positive antibody screen were used. Results and conclusionAll samples phenotyped present fully consistent results compared to the previous ones. For antibody identification the results for one of the samples are completely confirmed. The second sample showed inconsistencies to the previous result. For blood group verification with antibody-screen and compatibility test all samples had equivalent results. The effect from MLB 2 in IAT-LISS tube tests are adequate. A distinct observation can be made; gel column technology is superior to the tube tests.

<div>Leaf Area Index (LAI) is an important variable for both for characterizing plant canopy and as an input to many crop models. It is a dimensionless quantity broadly defined as the total one-sided leaf area per unit ground area, and is estimated over agriculture row crops by both direct and indirect methods. Direct methods, which involve destructive sampling, are laborious and time-consuming, while indirect methods such as remote sensing-based approaches have multiple sources of uncertainty. LiDAR (Light Detection and Ranging) remotely sensed data acquired from manned aircraft and UAVs’ have been investigated to estimate LAI based on physical/geometric features such as canopy gap fraction. High-resolution point cloud data acquired with a laser scanner from any platform, including terrestrial laser scanning and mobile mapping systems, contain random noise and outliers. Therefore, outlier detection in LiDAR data is often useful prior to analysis. Applications in agriculture are particularly challenging, as there is typically no prior knowledge of the statistical distribution of points, description of plant complexity, and local point densities, which are crop dependent. This dissertation first explores the effectiveness of using LiDAR data to estimate LAI for row crop plants at multiple times during the growing season from both a wheeled vehicle and an Unmanned Aerial Vehicle (UAV). Linear and nonlinear regression models are investigated for prediction utilizing statistical and plant structure-based features extracted from the LiDAR point cloud data and ground reference obtained from an in-field plant canopy analyzer and leaf area derived from destructive sampling. LAI estimates obtained from support vector regression (SVR) models with a radial basis function (RBF) kernel developed using the wheel-based LiDAR system and UAVs are promising, based on the value of the coefficient of determination (R2) and root mean squared error (RMSE) of the residuals. </div><div>This dissertation also investigates approaches to minimize the impact of outliers on discrete return LiDAR acquired over crops, and specifically for sorghum and maize breeding experiments, by an unmanned aerial vehicle (UAV) and a wheel-based ground platform. Two methods are explored to detect and remove the outliers from the plant datasets. The first is based on surface fitting to noisy point cloud data based on normal and curvature estimation in a local neighborhood. The second utilizes the deep learning framework PointCleanNet. Both methods are applied to individual plants and field-based datasets. To evaluate the method, an F-score and LAI are calculated both before and after outlier removal for both scenarios. Results indicate that the deep learning method for outlier detection is more robust to changes in point densities, level of noise, and shapes. Also, the predicted LAI was improved for the wheel-based vehicle data based on the R2 value and RMSE of residuals. </div><div>The quality of the extracted features depends on the point density and laser penetration of the canopy. Extracting appropriate features is a critical step to have accurate prediction models. Deep learning frameworks are increasingly being used in remote sensing applications. In the last objective of this study, a feature extraction approach is investigated for encoding LiDAR data acquired by UAV platforms multiple times during the growing season over sorghum and maize plant breeding experiments. LAI estimates obtained with these inputs are used to develop support vector regression (SVR) models using plant canopy analyzer data as the ground reference. Results are compared to models based on estimates from physically-based features and evaluated in terms of the coefficient determination (R2). The effects of experimental conditions, including flying height, sensor characteristics, and crop type, are also investigated relative to the estimates of LAI.</div><div><br></div>

<p> Despite the continuous breeding efforts towards improving yield, seed quality, and yield-related traits, there is still little understanding of several aspects of soybean breeding; however, crop breeding is ever-evolving, and plant breeding technologies offer immense potential for accelerating genetic improvement in soybeans. This thesis explores different frameworks to further characterize tradeoffs among seed quality traits, soybean maturity's genetic architecture, and selections for yield. We explored the interactions of carbohydrate traits with other seed traits, flowering, and maturity using data from a large panel of <em>G. max </em>accessions from the USDA soybean germplasm collection. We found a negative correlation between sucrose and protein and a negative correlation between protein and oil, representing a significant challenge for improving seed quality. In contrast to other well-documented correlations, such as protein and oil, correlations between raffinose and oil content seem more specific to populations and environments and are unlikely to generalize to the whole specie; however, the correlations of sucrose with protein and seed size appears to be more stable. In addition, we performed a genome-wide association analysis (GWA) to detect novel QTLs for flowering (R1) time, maturity (R8) time, and reproductive length (RL) using a soybean panel with the same genotype for major <em>E </em>genes (<em>e1-as/E2/E3). </em>While major maturity <em>E</em> genes are known to have pleiotropic effects on R1 and R8, we found two QTLs associated with R8 and RL that do not control R1, suggesting minor-effect, trait-specific loci are also involved in controlling R1 and R8. In addition, w<em>e identified six genes that may play essential roles in regulating R1, R8, and RL; however, further validation of the QTLs and f</em>ine mapping and map-based cloning studies of the candidate genes are necessary before they can be used in breeding programs. Lastly, we conducted a selection experiment in progeny row (PR) populations of four breeding programs to compare the agronomic performance of lines selected by breeders using their usual selection methods to lines selected through prediction of yield performance using new sources of data and information. Our results suggest that aerial average canopy coverage (ACC) used as a secondary trait in combination with field spatial variation adjustment is an efficient high throughput methodology to effectively select high-yielding lines from non-replicated experiments at the PR stage. </p>

No description available.