Tesi sul tema "X-Vectors"

X-Linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the common cytokine receptor gamma chain, yc, resulting in profound defects in both cellular and humoral immunity. Although allogeneic bone marrow transplantation has proved highly successful, HLA-mismatched procedures are associated with significant morbidity and mortality. This disease is therefore a good candidate for gene therapy and sustained correction of 19 SCID-X1 patients have been reported in two clinical trials. However, the occurrence of severe adverse advents in one trial has highlighted the potential side-effects of retroviral gene transfer and reinforced the need to develop safer gene therapy vectors. A series of self-inactivating (SIN) gammaretroviral and lentiviral vectors for the treatment of SCID-X1 have consequently been developed. To reduce the potential for insertional mutagenesis mediated by the duplicated viral LTR sequences, alternative internal regulatory elements have been incorporated into the vector backbone. These include both endogenous (human elongation factor la - EFS) and viral (spleen focus forming virus - SFFV) promoters. In vitro, the SIN retroviral vectors were able to regulate yc expression on the cell surface of SCID-X1 cell lines and restore the lymphoid differentiation potential of Il2rg" haematopoietic progenitor cells. Functional correction of the immunological defect in the SCID-X1 mouse model was also achieved at similar levels for the both the SIN retroviral vectors and the LTR-regulated clinical vector. To further improve upon safety, lentiviral vectors were developed incorporating the endogenous human IL2RG promoter to regulate physiological expression of yc. In vitro and in vivo analysis of the promoter indicated a degree of haematopoietic tissue specificity and restoration of functional yc-receptor complexes was achieved following transduction of a SCID-X1 T cell line with a lentiviral vector incorporating this promoter however phenotypic correction of the yc-deficient mouse was unsuccessful. These results demonstrate that SIN retroviral vectors for SCID-X1 are effective in restoration of the immune defect in the yc-deficient murine model. The SIN design together with an endogenous (EFS) promoter might provide a potentially less mutagenic but equally effective vector for gene therapy of SCID-X1.

Introduction. Chronic Granulomatous Disease (CGD) is caused by defects of the NADPH oxidase complex, responsible for the production of the oxidative burst in phagocytes. Patients present increased susceptibility to life-threatening fungal and bacterial infections and are treated with a lifelong prophylaxis. Currently, the only curative option is bone marrow transplantation. Gene therapy with hematopoietic stem cells (HSC) may represent a valid alternative to conventional transplant. Clinical trials for X-CGD employing gp91phox-expressing gammaretroviral vectors have been limited by insertional oncogenesis and lack of persistent engraftment. Methods. We developed a novel strategy based on regulated, self-inactivating lentiviral vectors (LVs) that target gp91phox expression to the differentiated myeloid cells while sparing HSC, to reduce the risk of genotoxicity and perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and a posttranscriptional, microRNA mediated regulation. We designed different therapeutic gp91phox-expressing LVs for CGD gene therapy: 1) PGK.gp91, in which gp91phox is driven by an ubiquitous cellular promoter; 2) MSP.gp91, to control the transgene expression at transcriptional level using a myeloid specific promoter; 3) PGK.gp91_126T(2), in which we exploited the miRNA system, incorporating miR-126 target sequences, to prevent the transgene off-target expression in HSC; 4) MSP.gp91_126T(2), combining the posttranscriptional de-targeting with the MSP. Vectors were tested in human cell line, human bone marrow HSC, their progeny differentiated in vitro and in the mouse model of X-CGD. Results. All vectors restored gp91phox expression and NADPH oxidase function in human X-CGD PLB-985 cell line and in myeloid cell lines and in macrophages from peripheral blood monocytes of 3 X-CGD patients (22-48%). While unregulated LVs ectopically expressed gp91phox in CD34+ cells, both transcriptionally and post-transcriptionally regulated LVs substantially reduced this off-target expression. By combining transcriptional and posttranscriptional targeting in the dual regulated vector, we achieved high levels of myeloid-specific transgene expression, entirely sparing the most primitive CD34+CD38-CD90+ HSC compartment (5-fold reduction). XCGD mice transplanted with all vectors engrafted and restored gp91phox expression, with 20-70% of granulocytes and monocytes expressing human gp91phox. MSP-driven vectors were superior in maintaining regulation during BM development as well as in peripheral blood B and T cells. Oxidase activity in corrected granulocytes was superior using MSP-driven vectors (38-59% of WT NADPH oxidase activity) as compared to PGK (17-23%). The MSP transcriptional control combined to miR-126 detargeting represent a promising approach for further clinical development of gp91phox therapeutic vectors.

Alzheimer's disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution. These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD.

Le progrès rapide et la démocratisation de la technologie ont conduit à l’abondance des capteurs. Par conséquent, l’intégration de ces diverses modalités pourrait présenter un avantage considérable pour de nombreuses applications dans la vie réelle, telles que la reconnaissance biométrique ou la détection d’engagement des élèves. Dans le domaine de la multimodalité, les chercheurs ont établi des architectures variées de fusion, allant des approches de fusion précoce, hybride et tardive. Cependant, ces architectures peuvent avoir des limites en ce qui concerne des signaux temporels d’une durée courte, ce qui nécessite un changement de paradigme vers le développement de techniques d’apprentissage automatique multimodales qui promettent une précision et une efficacité pour l’analyse de ces données courtes. Dans cette thèse, nous nous appuyons sur l’intégration de la multimodalité pour relever les défis précédents, allant de l’identification biométrique supervisée à la détection non supervisée de l’engagement des étudiants. La première contribution de ce doctorat porte sur l’intégration de la Wavelet Scattering Transform à plusieurs couches avec une architecture profonde appelée x-vectors, grâce à laquelle nous avons amélioré la performance de l’identification du locuteur dans des scénarios impliquant des énoncés courts tout en réduisant le nombre de paramètres nécessaires à l’entraînement. En s’appuyant sur les avantages de la multimodalité, on a proposé une architecture de fusion tardive combinant des vidéos de la profondeur des lèvres et des signaux audios a permis d’améliorer la précision de l’identification dans le cas d’énoncés courts, en utilisant des méthodes efficaces et moins coûteuses pour extraire des caractéristiques spatio-temporelles. Dans le domaine des défis biométriques, il y a la menace de l’émergence des "deepfakes". Ainsi, nous nous sommes concentrés sur l’élaboration d’une méthode de détection des "deepfakes" basée sur des méthodes mathématiques compréhensibles et sur une version finement ajustée de notre précédente fusion tardive appliquée aux vidéos RVB des lèvres et aux audios. En utilisant des méthodes de détection d’anomalies conçues spécifiquement pour les modalités audio et visuelles, l’étude a démontré des capacités de détection robustes dans divers ensembles de données et conditions, soulignant l’importance des approches multimodales pour contrer l’évolution des techniques de deepfake. S’étendant aux contextes éducatifs, la thèse explore la détection multimodale de l’engagement des étudiants dans une classe. En utilisant des capteurs abordables pour acquérir les signaux du rythme cardiaque et les expressions faciales, l’étude a développé un ensemble de données reproductibles et un plan pour identifier des moments significatifs, tout en tenant compte des nuances culturelles. L’analyse des expressions faciales à l’aide de Vision Transformer (ViT) fusionnée avec le traitement des signaux de fréquence cardiaque, validée par des observations d’experts, a mis en évidence le potentiel du suivi des élèves afin d’améliorer la qualité d’enseignement
The rapid advancement and democratization of technology have led to an abundance of sensors. Consequently, the integration of these diverse modalities presents an advantage for numerous real-life applications, such as biometrics recognition and engage ment detection. In the field of multimodality, researchers have developed various fusion ar chitectures, ranging from early, hybrid, to late fusion approaches. However, these architec tures may have limitations involving short utterances and brief video segments, necessi tating a paradigm shift towards the development of multimodal machine learning techniques that promise precision and efficiency for short-duration data analysis. In this thesis, we lean on integration of multimodality to tackle these previous challenges ranging from supervised biometrics identification to unsupervised student engagement detection. This PhD began with the first contribution on the integration of multiscale Wavelet Scattering Transform with x-vectors architecture, through which we enhanced the accuracy of speaker identification in scenarios involving short utterances. Going through multimodality benefits, a late fusion architecture combining lips depth videos and audio signals further improved identification accuracy under short utterances, utilizing an effective and less computational methods to extract spatiotemporal features. In the realm of biometrics challenges, there is the threat emergence of deepfakes. There-fore, we focalized on elaborating a deepfake detection methods based on, shallow learning and a fine-tuned architecture of our previous late fusion architecture applied on RGB lips videos and audios. By employing hand-crafted anomaly detection methods for both audio and visual modalities, the study demonstrated robust detection capabilities across various datasets and conditions, emphasizing the importance of multimodal approaches in countering evolving deepfake techniques. Expanding to educational contexts, the dissertation explores multimodal student engagement detection in classrooms. Using low-cost sensors to capture Heart Rate signals and facial expressions, the study developed a reproducible dataset and pipeline for identifying significant moments, accounting for cultural nuances. The analysis of facial expressions using Vision Transformer (ViT) fused with heart rate signal processing, validated through expert observations, showcased the potential for real-time monitoring to enhance educational outcomes through timely interventions

X-linked lymphoproliferative disease (XLP) is a rare primary immunodeficiency characterised by severe immune dysregulation and is caused by mutations in the SH2D1A gene. Clinical manifestations vary and include haemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinaemia, often triggered by Epstein-Barr virus (EBV) infection. SLAM-associated protein (SAP) is a key regulator of immune function in T, NK, and NKT cells and defects in this protein lead to the cellular and humoral immune defects described in patients. Treatment options for XLP are limited and currently haematopoietic stem cell transplant (HSCT) is the only curative option. Results are variable and dependent on a good donor match and absence of active infection at transplant. Somatic gene therapy is now successfully used to correct certain severe immunodeficiencies and offers a potential cure in XLP. The use of self-inactivating (SIN) lentiviral vectors with transgene expression driven by non-viral promoters has improved the biosafety profile of haematopoietic stem cell gene therapy procedures. In this study we have successfully corrected both cellular and humoral defects in a SAP deficient murine model using a SIN lentiviral vector with a codon optimised SAP transgene under the transcriptional control of the elongation factor 1α short form (EFS) promoter. Initial attempts with a non-codon optimised version of SAP led to insufficient protein expression levels to restore immune function. We also assessed the CD2 locus control region (LCR) to evaluate any lymphoid specificity to permit more regulated SAP expression but were unable to demonstrate any benefit with this regulatory element. The results presented here provide proof of concept for the development of gene therapy for XLP and further work is warranted to improve the efficiency of gene transfer, secure engraftment of long term repopulating haematopoietic stem cell progenitors and additional characterisation of immune reconstitution after gene therapy.

Nous avons obtenu une preuve de concept pour la thérapie génique de 2 maladies génétiques perte-de-fonction.L'acrodysostose est une maladie osseuse et rénale causée par des mutations de la sous-unité régulatrice de la protéine kinase A (PRKAR1A). Nous avons testé les effets d'un rAAV9-CAG-humanPRKR1A dans un modèle murin KI. hPRKAR1A a été exprimée dans les chondrocytes de la plaque de croissance et les cellules tubulaires rénales. L'architecture des chondrocytes et la longueur du squelette ont été améliorées.L'AMN est une axonopathie tardive de la moelle épinière causée par des mutations du gène ABCD1.Nous avons construit et développé un vecteur, rAAV9- MAG-humanABCD1-HA (hABCD1), et testé ses effets dans un modèle murin KO de la maladie.L'expression de hABCD1-HA a été observée dans les oligodendrocytes (OL) et les astrocytes (A). Nous avons développé un vecteur ciblant les oligodendrocytes (OL), rAAV9-MAG-humanABCD1-HA. Les déficits neurologiques ont été prévenus avec un suivi de 2 ans.C26:0-lysoPC (VLCFA) était diminué dans la moelle épinière des souris traitées.Chez le primate, l'injection intrathécale du vecteur rAAV9-MAG a induit un niveau élevé d'expression de hABCD1-HA dans les OL et les A de la moelle épinière et du cervelet. Le ciblage OL n'avait pas été obtenu auparavant chez les primates avec d'autres vecteurs ou promoteurs, ce qui ouvre la porte à l'application humaine de notre vecteur dans diverses maladies du système nerveux central (SNC)
We have obtained proof-of concept for the gene therapy of two diseases.Acrodysostosis is a bone and kidney disease caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). We tested the effects of a rAAV9-CAG-humanPRKR1A in a knock-in mouse model. hPRKAR1A expression was found in growth plate chondrocytes, and kidney tubular cells. Chondrocyte architecture and skeleton length were improved.X-ALD AMN is a late-onset axonopathy of spinal cord caused by ABCD1 mutations. We made an original rAAV9-MAG-humanABCD1-HA (hABCD1) vector and tested its effects in a KO mouse model.hABCD1-HA expression was observed in numerous OL and astrocytes. Neurological deficits were prevented 24 months after injection. C26:0-lysoPC (VLCFA), was lower in spinal cord.In non-human primate, intrathecal injection of the rAAV9-MAG vector induced high hABCD1-HA expression in OL and astrocytes of spinal cord and cerebellum. OL targeting has not been obtained before in primates with other vectors or promoters. This opens the door to the human application of OL targeting in a number of CNS diseases

This thesis presents a joint experimental and theoretical study of the state-to-state resolved rotationally inelastic scattering of NO(X) with atoms and homonuclear diatoms. Emphasis is placed on measuring the correlations between the vectors which describe the system: k and k' the initial and final relative velocities, respectively, r the initial NO bond axis orientation, and j' the final rotational angular momentum of NO. Experimentally, the initial f Δ-doublet state of NO(X) is selected using a Hexapole state selector, and the scattered NO detected using (1+1') REMPI. In the first part of this thesis the k-k' correlation in the scattering of NO(X) with O2(X) is presented. A modified onion peeling algorithm is used to extracted the (partially) pair correlated angular distributions from the experimental ion images, as well as the relative populations of O₂ rotational states populated during scattering. Strong similarities are observed between the scattering of NO with Ar and O₂. The second part of this thesis focuses on steric effects (the r - k - k' correlation); the initial orientation of the NO bond axis is controlled using a static electric field, such that the collision partner approaches either the 'ends' or the 'sides' of the NO molecule. A new quantum mechanical theory is presented to describe the scattering of a symmetric top molecule in the presence of an arbitrarily directed electric field. The scattering of NO(X) with Ar at a collision energy of E_coll = 651cm^-1 is presented for 'end-on' and 'side-on' scattering on the spin-orbit changing and conserving manifolds, respectively. In both cases good agreement with full quantum mechanical calculations is observed. Scattering resulting in only weak deflection is shown to be very sensitive to the orientation of the NO bond axis prior to collision, and is maximised for collisions at the 'side' of the molecule. The integral steric asymmetry for scattering of NO with N₂(X) and O₂(X) is also presented, and information about the potential energy surfaces for such interactions inferred.

In this thesis, an experimental and theoretical study of two and three vector correlations in the inelastic scattering of NO(X) with various rare gas atoms is presented. Vector correlations for a selection of rare gas systems were determined experimentally, and the observations were interpreted using a variety of classical and quantum mechanical models. The experiment is able to provide state-to-state resolution of the dynamics by means of an electrostatic hexapole and 1+1' resonantly enhanced multi-photon ionisation (REMPI). The simplest vector correlation of interest is the differential cross section (DCS), given by the k-k' correlation. The DCSs were determined experimentally for the NO(X)--Kr and NO(X)--Xe collision systems, both characterised by the relatively deep (≈140cm^-1) attractive well and large extent of the attractive potential. The agreement between the experimental angular distributions and quantum mechanical DCS is very good for both systems. Classical calculations fail to correctly reproduce the form and magnitude of the DCS for either system, reflecting the inherently quantum mechanical nature of the collision. The classical calculations do however provide mechanistic insight into regions where the attractive part of the potential plays an important role in determining the dynamics. In order to investigate narrow angular features in the forward scattered direction, several experimental improvements to molecular beams and the detection ion-optic stack were made. Investigation into these structures revealed a strong contribution from molecular diffraction into the classical shadow of the NO(X), and the simple Fraunhofer model revealed a preference for scattering from an individual m→m' sub-state. Such measurements are in a region of the DCS where scattering is forbidden classically, and reveal the purely quantum nature of the collision interaction in the forward scattered direction. The low order k-k' correlation was then extended by using linearly or circularly polarised laser excitation. The interaction of the light with the molecular dipole allows the measurement of the k-k'-j' correlation. When linearly polarised light was used for the excitation laser, two of the rank two p^{2}_q(θ) renormalised polarisation dependent differential cross sections (PDDCSs), which describe rotational alignment, were obtained. With circularly polarised light, the rank one p^{1}_1-(θ) renormalised PDDCSs describing rotational orientation were determined. The collision induced alignment in NO(X)--Xe scattering was found to be well reproduced by classical and impulsive theories, highlighting the fact that the alignment is dominated by the propensity for the projection of j onto the kinematic apse to be conserved. The attractive part of the potential does augment the alignment renormalised PDDCSs, and this is most evident in states with strong features of the attractive part of the potential such as ℓ-type rainbows. The orientation is more strongly influenced by the attractive part of the potential and is also influenced by parity. In addition to the parity effect, there exist two limiting classical mechanisms which govern the orientation, one caused by attraction and the other repulsion. Finally, the bond axis of the NO(X) can be oriented by means of hexapole state selection combined with adiabatic orientation using a set of guiding rods. The integral steric effect, an r-k correlation, was measured for the NO(X)--Kr and NO(X)--Ar spin-orbit changing systems. There are large oscillations in the sign of the steric asymmetry which occur for scattering with the various rare gases. There are also large differences between the rare gases as the potentials become more attractive, and more isotropic. The steric asymmetry is well reproduced by quantum mechanics, however, a classical mechanism becomes dominant at high Δj.

The objective of this work is to study state-of-the-art deep neural networks based speaker verification systems called x-vectors on various conditions, such as wideband and narrowband data and to develop the system, which is robust to unseen language, specific noise or speech codec. This system takes variable length audio recording and maps it into fixed length embedding which is afterward used to represent the speaker. We compared our systems to BUT's submission to Speakers in the Wild Speaker Recognition Challenge (SITW) from 2016, which used previously popular statistical models - i-vectors. We observed, that when comparing single best systems, with recently published x-vectors we were able to obtain more than 4.38 times lower Equal Error Rate on SITW core-core condition compared to SITW submission from BUT. Moreover, we find that diarization substantially reduces error rate when there are multiple speakers for SITW core-multi condition but we could not see the same trend on NIST SRE 2018 VAST data.

Cílem konverze hlasu (voice conversion, VC) je převést hlas zdrojového řečníka na hlas cílového řečníka. Technika je populární je u vtipných internetových videí, ale má také řadu seriózních využití, jako je dabování audiovizuálního materiálu a anonymizace hlasu (například pro ochranu svědků). Vzhledem k tomu, že může sloužit pro spoofing systémů identifikace hlasu, je také důležitým nástrojem pro vývoj detektorů spoofingu a protiopatření.    Modely VC byly dříve trénovány převážně na paralelních (tj. dva řečníci čtou stejný text) a na vysoce kvalitních audio materiálech. Cílem této práce bylo prozkoumat vývoj VC na neparalelních datech a na signálech nízké kvality, zejména z veřejně dostupné databáze VoxCeleb. Práce vychází z moderní architektury AutoVC definované Qianem et al. Je založena na neurálních autoenkodérech, jejichž cílem je oddělit informace o obsahu a řečníkovi do samostatných nízkodimenzionýálních vektorových reprezentací (embeddingů). Cílová řeč se potom získá nahrazením embeddingu zdrojového řečníka embeddingem cílového řečníka. Qianova architektura byla vylepšena pro zpracování audio nízké kvality experimentováním s různými embeddingy řečníků (d-vektory vs. x-vektory), zavedením klasifikátoru řečníka z obsahových embeddingů v adversariálním schématu trénování neuronových sítí a laděním velikosti obsahového embeddingu tak, že jsme definovali informační bottle-neck v příslušné neuronové síti. Definovali jsme také další adversariální architekturu, která porovnává původní obsahové embeddingy s embeddingy získanými ze zkonvertované řeči. Výsledky experimentů prokazují, že neparalelní VC na nekvalitních datech je skutečně možná. Výsledná audia nebyla tak kvalitní případě hi fi vstupů, ale výsledky ověření řečníků po spoofingu výsledným systémem jasně ukázaly posun hlasových charakteristik směrem k cílovým řečníkům.

This thesis investigates the implications for muon decay of a gauge theory that incorporates modifications to the standard electro-weak theory, the Weinberg-Salam model. The Higgs sector of the original model is broadened to include an iso-vector or triplet Higgs field. Such an addition naturally provides for massive Majorana-type neutrinos and permits the decay modesμ → e and μ → 3e which would not otherwise be allowed. The particles most responsible for these decay modes are new, charged, physical, scalar Higgs particles not present in the Weinberg-Salam model. The decay rates found, although considerably more favorable to muon decay than the simple addition of neutrino mass to the standard theory, are found to be much below the present limits of experimental detectability. The lengthy calculations required, including the derivation and utilization of the relevant Feynman diagrams, are displayed in some detail.
Science, Faculty of
Physics and Astronomy, Department of
Graduate

Le retrait des nématicides rend urgent la mise au point de méthodes alternatives de lutte contre les nématodes parasites des cultures et la création de variétés résistantes est une voie prometteuse. En vignoble, le nématode Xiphinema index a un impact économique élevé en transmettant le Grapevine fanleaf virus (GFLV), principal virus du court-noué de la vigne et première virose de la vigne à l’échelle mondiale. Des porte-greffe résistants vis-à-vis du vecteur X. index basés sur la source de résistance muscadine (Muscadinia rotundifolia) sont en cours de sélection chez la vigne afin de stopper ou retarder l’infection. Sur cette culture, une étude antérieure avait montré que ce nématode parthénogénétique méiotique est aussi capable de se reproduire (rarement) de façon sexuée. Un travail préliminaire de phylogéographie avait permis de révéler les groupes prédominants de diversité et de sélectionner des populations représentatives pour la création de lignées monofemelles. La durabilité de la résistance doit prendre en compte la diversité du nématode. Dans ce contexte, la thèse a d’abord complété et approfondi l’approche phylogéographique en utilisant une très large gamme d’échantillons originaires de l’aire mondiale de répartition de la vigne. Nos résultats permettent de proposer des hypothèses fortes afin de localiser l’aire native du nématode X. index au Moyen-Orient et de retracer ses itinéraires de dissémination à partir de l’Antiquité. IIs illustrent également le lien étroit depuis cette époque entre la dissémination du nématode et celle de la vigne domestiquée par l’homme. La deuxième partie de la thèse a évalué la durabilité de la résistance de matériel porte-greffe issu de la muscadine en serre (nématodes non virulifères sur plants entre 3 et 6 ans) et en vignoble (nématodes virulifères sur plants âgés de 16 ans). En serre, des accessions résistantes F1 ou BC1, préalablement obtenues à partir d’in vitro ou de boutures ligneuses, ont été inoculées avec un mélange de 4 lignées représentatives, chaque lignée étant traçable avec des marqueurs microsatellites. Nous avons montré que les nématodes issus de plants obtenus par multiplication in vitro surmontent progressivement la résistance tandis que le matériel issu de boutures exprime une résistance durable. La multiplication progressive des nématodes sur le matériel résistant uniquement dans le cas où il est issu d’in vitro écarte a priori l’hypothèse d’une adaptation génétique du nématode. Elle apparaît liée à une architecture différente du système racinaire chez les plants issus de ce type de multiplication, multiplication qui pourrait induire des changements physiologiques discrets mais durables dans les tissus racinaires apicaux à partir desquels les nématodes se nourrissent. Le génotypage des nématodes par microsatellites a permis de détecter un taux bas mais croissant d’individus hybrides entre lignées sur les plants âgés de 4 à 6 ans, ce qui confirme l’aptitude de multiplication sexuée précédemment observée en vignoble. Du fait que l’observation d’individus hybrides apparaît indépendante du type de propagation et du statut de résistance de la plante, nos résultats écartent l’hybridation comme mode d’adaptation du nématode qui serait à même d’expliquer le contournement de la résistance chez les plants issus d’in vitro. En vignoble, après 16 années, les nématodes ont été quasi-impossibles à détecter sur l’accession résistante BC1 qui est également peu affectée par les attaques virales, tandis que des effectifs de nématodes plus élevés ont été retrouvés sur une accession témoin sensible dont les plants sont par contre très majoritairement morts ou en dépérissement. Considérés globalement, nos résultats montrent que la stratégie de résistance basée sur la muscadine apparaît durable. Cette stratégie ciblée sur le nématode vecteur contribuera à réduire significativement l’impact du GFLV transmis par X. index
The ban of most nematicides renders urgent control alternatives against plant-parasitic nematodes and breeding for resistant plant varieties is promising. In vineyards, the nematode Xiphinema index has a high economical impact by transmitting Grapevine fanleaf virus (GFLV), the main virus of ‘Court-noué’ disease and the first grapevine viral disease worldwide. Resistant rootstocks are being selected in grapevine, using Muscadinia rotundifolia (muscadine) as a resistance source to the vector, in order to arrest or delay GFLV transmission. In this crop, a previous study had shown that this meiotic parthenogenetic nematode is able to reproduce sexually (rarely) in the field. A preliminary phylogenetic work had allowed to reveal the predominant diversity groups and to select representative populations for the creation of single-female lines. Resistance durability is a real challenge that must consider the key information of the nematode diversity. In this context, the PhD project first completed and deepened our phylogeographical approach using an extended geographic coverage of the worldwide nematode distribution. Our results allow proposing strong hypotheses to locate the native area of X. index in the Middle-East and trace its dissemination routes from the Antiquity. They also highlight the close link since this epoch between dissemination of the nematode and domesticated grapevine by man. The second part of the PhD project has then evaluated the durability of muscadine-derived rootstock material in greenhouse (non viruliferous nematodes on plants aged 3 to 6 years) and field (viruliferous nematodes on plants aged 16 years) conditions. In the greenhouse, F1 and BC1 resistant accessions, previously obtained from both in vitro and hardwood-cutting propagation, were inoculated with 4 mixed representative X. index lines, traceable each with microsatellite markers. We showed that nematodes from plants obtained from in vitro progressively overcame the resistance while the material obtained from cuttings displayed a durable resistance. Nematode progressive multiplication in resistant accessions obtained only from in vitro removes a priori the hypothesis of a nematode genetic adaptation and appears linked to a different architecture of the root system in this propagation type. This type may have induced discrete but durable physiological changes in apical root tissues from where nematodes feed. Nematode microsatellite genotyping allowed detecting a low but increasing rate of hybrid individuals from 4 to 6 years, which confirms data from the vineyard. As the hybrid occurrence appears independent from the propagation type and the resistance status of the plant, our data discard hybridization as the mode of adaptation of the nematode underlying resistance breakdown from in vitro plants. In field conditions, after 16 years, nematodes were almost undetectable on the resistant BC1 accession, also almost unaffected by the viral attacks, while higher numbers were detected on a susceptible control accession, whose plants were by contrast in high majority dead or poorly vigorous. Taken all together, our results show that the muscadine-derived resistance strategy appears durable. This strategy focused on vector control will significantly contribute to reduce the impact of GFLV transmitted by X. index

Diploma project is aimed to load force vector decomposition to x, y and z direction for complicated Skoda car parts. The methodology of decomposition is based on measuring of car parts deformation by strain gauges and mathematical identifation of force compoments by neural network (ANN). ANN will be trained by results of FE model simulations or by calibration on real car parts.

Le retrait des nématicides rend urgent la mise au point de méthodes alternatives de lutte contre les nématodes parasites des cultures et la création de variétés résistantes est une voie prometteuse. En vignoble, le nématode Xiphinema index a un impact économique élevé en transmettant le Grapevine fanleaf virus (GFLV), principal virus du court-noué de la vigne et première virose de la vigne à l’échelle mondiale. Des porte-greffe résistants vis-à-vis du vecteur X. index basés sur la source de résistance muscadine (Muscadinia rotundifolia) sont en cours de sélection chez la vigne afin de stopper ou retarder l’infection. Sur cette culture, une étude antérieure avait montré que ce nématode parthénogénétique méiotique est aussi capable de se reproduire (rarement) de façon sexuée. Un travail préliminaire de phylogéographie avait permis de révéler les groupes prédominants de diversité et de sélectionner des populations représentatives pour la création de lignées monofemelles. La durabilité de la résistance doit prendre en compte la diversité du nématode. Dans ce contexte, la thèse a d’abord complété et approfondi l’approche phylogéographique en utilisant une très large gamme d’échantillons originaires de l’aire mondiale de répartition de la vigne. Nos résultats permettent de proposer des hypothèses fortes afin de localiser l’aire native du nématode X. index au Moyen-Orient et de retracer ses itinéraires de dissémination à partir de l’Antiquité. IIs illustrent également le lien étroit depuis cette époque entre la dissémination du nématode et celle de la vigne domestiquée par l’homme. La deuxième partie de la thèse a évalué la durabilité de la résistance de matériel porte-greffe issu de la muscadine en serre (nématodes non virulifères sur plants entre 3 et 6 ans) et en vignoble (nématodes virulifères sur plants âgés de 16 ans). En serre, des accessions résistantes F1 ou BC1, préalablement obtenues à partir d’in vitro ou de boutures ligneuses, ont été inoculées avec un mélange de 4 lignées représentatives, chaque lignée étant traçable avec des marqueurs microsatellites. Nous avons montré que les nématodes issus de plants obtenus par multiplication in vitro surmontent progressivement la résistance tandis que le matériel issu de boutures exprime une résistance durable. La multiplication progressive des nématodes sur le matériel résistant uniquement dans le cas où il est issu d’in vitro écarte a priori l’hypothèse d’une adaptation génétique du nématode. Elle apparaît liée à une architecture différente du système racinaire chez les plants issus de ce type de multiplication, multiplication qui pourrait induire des changements physiologiques discrets mais durables dans les tissus racinaires apicaux à partir desquels les nématodes se nourrissent. Le génotypage des nématodes par microsatellites a permis de détecter un taux bas mais croissant d’individus hybrides entre lignées sur les plants âgés de 4 à 6 ans, ce qui confirme l’aptitude de multiplication sexuée précédemment observée en vignoble. Du fait que l’observation d’individus hybrides apparaît indépendante du type de propagation et du statut de résistance de la plante, nos résultats écartent l’hybridation comme mode d’adaptation du nématode qui serait à même d’expliquer le contournement de la résistance chez les plants issus d’in vitro. En vignoble, après 16 années, les nématodes ont été quasi-impossibles à détecter sur l’accession résistante BC1 qui est également peu affectée par les attaques virales, tandis que des effectifs de nématodes plus élevés ont été retrouvés sur une accession témoin sensible dont les plants sont par contre très majoritairement morts ou en dépérissement. Considérés globalement, nos résultats montrent que la stratégie de résistance basée sur la muscadine apparaît durable. Cette stratégie ciblée sur le nématode vecteur contribuera à réduire significativement l’impact du GFLV transmis par X. index
The ban of most nematicides renders urgent control alternatives against plant-parasitic nematodes and breeding for resistant plant varieties is promising. In vineyards, the nematode Xiphinema index has a high economical impact by transmitting Grapevine fanleaf virus (GFLV), the main virus of ‘Court-noué’ disease and the first grapevine viral disease worldwide. Resistant rootstocks are being selected in grapevine, using Muscadinia rotundifolia (muscadine) as a resistance source to the vector, in order to arrest or delay GFLV transmission. In this crop, a previous study had shown that this meiotic parthenogenetic nematode is able to reproduce sexually (rarely) in the field. A preliminary phylogenetic work had allowed to reveal the predominant diversity groups and to select representative populations for the creation of single-female lines. Resistance durability is a real challenge that must consider the key information of the nematode diversity. In this context, the PhD project first completed and deepened our phylogeographical approach using an extended geographic coverage of the worldwide nematode distribution. Our results allow proposing strong hypotheses to locate the native area of X. index in the Middle-East and trace its dissemination routes from the Antiquity. They also highlight the close link since this epoch between dissemination of the nematode and domesticated grapevine by man. The second part of the PhD project has then evaluated the durability of muscadine-derived rootstock material in greenhouse (non viruliferous nematodes on plants aged 3 to 6 years) and field (viruliferous nematodes on plants aged 16 years) conditions. In the greenhouse, F1 and BC1 resistant accessions, previously obtained from both in vitro and hardwood-cutting propagation, were inoculated with 4 mixed representative X. index lines, traceable each with microsatellite markers. We showed that nematodes from plants obtained from in vitro progressively overcame the resistance while the material obtained from cuttings displayed a durable resistance. Nematode progressive multiplication in resistant accessions obtained only from in vitro removes a priori the hypothesis of a nematode genetic adaptation and appears linked to a different architecture of the root system in this propagation type. This type may have induced discrete but durable physiological changes in apical root tissues from where nematodes feed. Nematode microsatellite genotyping allowed detecting a low but increasing rate of hybrid individuals from 4 to 6 years, which confirms data from the vineyard. As the hybrid occurrence appears independent from the propagation type and the resistance status of the plant, our data discard hybridization as the mode of adaptation of the nematode underlying resistance breakdown from in vitro plants. In field conditions, after 16 years, nematodes were almost undetectable on the resistant BC1 accession, also almost unaffected by the viral attacks, while higher numbers were detected on a susceptible control accession, whose plants were by contrast in high majority dead or poorly vigorous. Taken all together, our results show that the muscadine-derived resistance strategy appears durable. This strategy focused on vector control will significantly contribute to reduce the impact of GFLV transmitted by X. index

碩士
國立高雄應用科技大學
機械與精密工程研究所
101
In this paper, we investigated the characteristics of YBa2Cu3O7-x (YBCO) superconducting quantum interference device (SQUID) integrated magnetometers for vector magnetic field measurement. The devices were made from YBCO thin films grown on bicrystal SrTiO3 (STO) substrates by using a radio frequency magnetron sputtering system. Before device fabrication, the thin films were grown on STO single crystal substrates for several times to find out the optimal sputtering conditions for achieving the highest superconducting transition temperature, which was determined by measuring the resistance-temperature curve of the film with the four point method. The magnetometer elements were patterned from YBCO films grown on bi-crystal STO substrates via photolithography and wet etching. The patterned device was mounted inside a homemade cryogenic electrical-property measurement probe to characterize its voltage-current and voltage-field curves. The magnetometer has a field sensor and two field gradient sensors, of which can be operated with flux-locked loops by using the modulation coil comprising a field exciter and two orthogonal field gradient exciters. Prior to mounting the modulation coil, its field distributions are checked with a two-dimensional magnetic scanning system to confirm the simulated result of the magnetic field map. By employing a flux deflector, the proposed magnetometer can be turned into a three-axis magnetic field sensor, which can be applied to a variety of weak vector field measurement, such as nondestructive evaluation and active biomagnetic field scanning.

碩士
國立中興大學
生物科技學研究所
102
Foot-and-mouth disease virus (FMDV) is a major plague in animal farming. The present vaccine used is produced by chemical inactivation which may has the risk of live virus contamination. Therefore, the subunit vaccine can be considering as a safer choice for future vaccine production. Accordingly, a Bamboo mosaic virus (BaMV) based viral vector expressing FMDV epitope - BVP1 recombinant virus had been established in previous study, expressing FMDV VP1 epitope by fusing it to the N-terminal of BaMV coat protein (CP). But since the BaMV host range is relatively narrow, that plants can be applied in vaccine production are still restrained. Potato virus X (PVX) has been widely used on foreign protein expressing in plants, it has a wide host range that includes several edible plants, such as potato, tomato, cucumber, spinach, cowpea and celery. Thus, we combined BaMV with PVX viral vector system to improve the applicability of the BVP1 CP viral expressing system. Expressing BVP1 CP via PVX viral vector in edible plants would allow the vaccine product put into use directly or simply with some extraction process, omitting the costly purification process. Furthermore, due to the PVX CP ORF replacement with our gene of interest (GOI), the systemic transport ability and infectious virion forming are eliminated. In fact, to minimize the modification on the PVX genome, we only constructed XΔCP-BVP1 originally. But after the test, we found that the protein yield from XΔCP-BVP1 was far from enough. In that case, we introduced the HcPro silencing suppressor from Tobacco etch virus (TEV) and compared the expression with another newly constructed XΔTC-BVP1. Though after the Coomassie blue staining and Western blot analysis, it is confirmed that BVP1 CP can express with both two strategy but BVP1 CP were still undetectable in some hosts, possibly due to the low expression. That is, before we can put this system into application, there’s still a lot more research and study to do in the future.

This thesis presents the design, layout, and measurements of a bi-directional amplifier with variable vector (in-phase / quadrature) gain control that will be part of an electronically steered phased array system. The electronically steered phased array has many advantages over the conventional mechanically steered antennas including rapid scanning of the beam and adaptively creating nulls in desired locations. The 10-bit bi-directional Vector Variable Gain Amplifier (VVGA) is part of the transmit and receive module of each antenna element where transmit and receive functionality is determined through a simple switch. The VVGA performs amplification of the IF IQ pair by an adjustable complex coefficient. At receive, the VVGA functions as a Vector Variable Gain Current Amplifier (VVGCA) and at transmit, the VVGA functions as a Vector Variable Gain Transadmittance Amplifier (VVGTA). Design procedure, layout entry, schematic and parasitic extracted simulation results, and measurements are presented in this thesis.

碩士
國立東華大學
化學系
98
The nuclear receptor pregnane X receptor (PXR) is a ligand-regulated transcription factor that responds to a wide range of endogenous and exogenous molecules. Upon activation with ligands, PXR can increase induction levels of metabolism enzymes. Therefore, PXR plays a critical role in phase I and phase II metabolism and excretion. Identifying the chemicals that activate this promiscuous protein can be of great help to predict adverse drug interaction. An in silico model was developed to predict the action of PXR using the newly invented pharmacophore ensemble/support vector machine (PhE/SVM) scheme based on the data compiled from the literature. The predictions by the PhE/SVM model are in good agreement with the experimental observations for those molecules in the training set (n = 32, r2 = 0.86, q2 = 0.80, RMSE = 0.37, s = 0.21), the test set (n = 120, r2 = 0.80, RMSE = 0.25, s = 0.19) and the outlier set (n = 8, r2 = 0.91, RMSE = 0.15, s = 0.12). In addition, this generated model also completely met with those validation criteria generally adopted to gauge the predictivity of a theoretical model. When compared with crystal structures, the calculated results are consistent with the published hPXR-ligand co-complex structure and the plasticity nature of hPXR is also revealed. Thus, this PhE/SVM model is an accurate, fast and robust model and can be utilized for predicting the activation of hPXR to facilitate drug discovery and drug development.

博士
國立中央大學
物理學系
102
High quality polycrystalline samples of Mn3-xCoxTeO6 (x = 0, 1, 2) perovskite were synthesized using the solid-state reaction technique, that a portion of the Mn2+ ions are substituted by the Co2+ ions for samples x = 1, 2. The temperature dependence of X-ray and high resolution neutron diffraction were performed to check the nuclear phase transition and to detect the long-range antiferromagnetic ordering of the samples x = 1, 2. From the results of General Structure Analysis System (GSAS) refinement, the substitution of Co2+ for Mn2+ in Mn3TeO6 will not alters the symmetry of crystal structure (hexagonal, ), even the temperature was cooled down to 3 K. Order parameters analysis of neutron diffraction and χ-T measurement show that the transition temperature of antiferromagnetic order are changed from 35 to 40 K for x = 1 to 2, and relocations of Mn/Co ions are observed below 35 and 40 K for Mn2CoTeO6 and MnCo2TeO6, respectively. The thermal variation of lattice constants of Mn3TeO6 can be described by T4 phonon term, but that of Mn2CoTeO6 and of MnCo2TeO6 can be described by T2 conduction electron term. From magnetization and susceptibility measurements, another magnetic transition is revealed for Mn2CoTeO6 and for MnCo2TeO6 at the temperature ~ 185 K due to the order of moments of Co2+, and the magnetic domains in the system are changed with the increasing of applied magnetic fields. The incommensurate propagation vectors are shifted from k = [0, 0, 0.481] to [0, 0, 0.515] as the concentration of Co2+ increased from x = 1 to 2, and it changes from k = [0, 0, 0.4302] in Mn3TeO6 to k = [0, 0, 0.515] in MnCo2TeO6, that a reduction of k with increased concentration of Co is toward the commensurate propagation vector k = [0, 0, 0.5]. The unique Mn/Co spin is split into two magnetically different orbits through the refinement of magnetic structure.

"Bone marrow tyrosine kinase in chromosome X (BMX) is a major member of the TEC family kinases and has been implicated in tumorigenecity, motility, proliferation and differentiation. BMX is highly overexpressed in prostate cancer and it is involved in the adaptive compensatory mechanism of castrate-resistance prostate cancer to androgen deprivation therapy. Besides, it suppresses a core component of the intrinsic apoptotic pathway, granting tumor cells the ability to escape apoptosis induced by chemotherapeutic drugs. BMX knockout mouse have a normal lifespan, without an obvious altered phenotype, suggesting that therapies based on BMX inhibition, may have limited side effects. We developed a series of BMX-IN-1 analogues that showed an increased inhibitory capacity when compared to BMX-IN-1. Since crystallographic information is essential to understand the molecular basis of BMX inhibition by covalent inhibitors we establish a baculovirus-insect expression system protocol for the production of the recombinant human BMX. Here we report the full biochemical and biophysical characterization of human BMX alone and in complex with different covalent ligands.(...)"