Abstract Pteropid bats are species of zoonotic significance and are known to be reservoir hosts of many viral diseases recently emerged in Asia and Australia. The transfer of these organisms from bats into terrestrial hosts resulted in lethal illnesses in humans and animals. Implementing attentive strategies for the detection and monitoring of these organisms is essential to the protection of humans, animals and the balance of the global economy. Unfortunately the paucity of information on these mammals’ immune system and the absence of diagnostic tools for the detection and for the studying of the humoral response in these animals disengage us from responding promptly when such outbreaks occur and prevent us from describing the possible underling causes that may be responsible for the absence of symptoms in bats infected with such organisms. It was assumed that flying foxes have immunoglobulin like molecules that provide humoral response and are involved in mediating secondary effector functions such as complement fixation and activation of other components of the cellular response in a similar manner as it occurs in humans and other mammals. The aims of this study which include immunoglobulin isotype identification, purification and characterisation as well as generation of reagents that are immunoglobulin class specific, provide the primary platform that should enable us to begin examining these questions. IgG was purified from the black flying fox’s serum by affinity chromatography using Protein G and Protein A. Protein L was ineffective in purifying any antibody from the bat serum. The heavy chain of IgG was also purified by gel electroelution. IgG was digested with papain to yield Fab and Fc fragments. The identity of the bat IgG was confirmed by N-terminal sequencing of the heavy chain and light chain of immunoglobulins separated by SDS-PAGE and by N-terminal sequencing of Fc and Fc' fragments. IgM has also been purified using methods that have not been previously explored to our knowledge. These methods consisted of the purification of Fab specific antibodies from antisera generated against Fab, and using these antibodies to capture other immunoglobulin classes in samples that had been previously enriched by classical fractionation methods. Antisera against the whole IgG molecule, Fc, Fab, IgG heavy chain and IgM heavy chain have been produced in rabbits and tested by Western blot and ELISA. The antisera against the whole IgG molecule and against the Fc were also utilized to detect antibodies to Nipah virus in bats that were found positive to Serum Neutralisation Test. Failure to identify the bat IgA in the bat serum poses questions on the presence, abundance and functional significance of such molecule in bats. The tools that were generated in this study recognise immunoglobulin isotypes, which enabled us to detect and measure antibodies and will allow the study of the humoral response in infected bats to a large extent. Tedious approaches routinely adopted for the purification of antibodies involve a series of pre-fractionation steps or affinity chromatography which rely on the use of expensive immobilised novel or partially characterised ligands, with no guarantee of affinity for the immunoglobulin isotype of interest. The method adopted for immunoglubulin isotype purification described in this study proved to be an effective, reasonably quick and economic solution for immunoglubulin isotype purification from any mammalian species.

The superoxide radical is considered to play important roles in physiological processes as well as in the genesis of diverse cytotoxic conditions such as cancer, various cardiovascular disorders and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). The detection and quantification of superoxide within cells is of critical importance to understand biological roles of superoxide and to develop preventive strategies against free radical-mediated diseases. Cyclic nitrone spin traps such as DMPO, EMPO, DEPMPO, BMPO and their derivatives have been widely used in conjunction with ESR spectroscopy to detect cellular superoxide with some success. However, the formation of unstable superoxide adducts from the reaction of cyclic nitrones with superoxide is a stumbling block in detecting superoxide by using electron spin resonance (ESR). A chemiluminescent probe, lucigenin, and fluorogenic probes, hydroethidium and MitoSox, are the other frequently used methods in detecting superoxide. However, luceginen undergoes redox-cycling producing superoxide by itself, and hydroethidium and MitoSox react with other oxidants apart from superoxide forming red fluorescent products contributing to artefacts in these assays. Hence, both methods were deemed to be inappropriate for superoxide detection. In this study, an effective approach, a selective mechanism-based colorimetric detection of superoxide anion has been developed by using silylated azulenyl nitrones spin traps. Since a nitrone moiety and an adjacent silyl group react readily with radicals and oxygen anions respectively, such nitrones can trap superoxide efficiently because superoxide is both a radical and an oxygen anion. Moreover, the synthesized nitrone is designed to be triggered solely by superoxide and not by other commonly observed oxygen radicals such as hydroxyl radical, alkoxyl radicals and peroxyl radical. In vitro studies have shown that these synthesized silylated azylenyl nitrones and the mitochondrial-targeted guanylhydrazone analog can trap superoxide efficiently yielding UV-vis identifiable and even potentially fluorescence-detectable orange products. Therefore, the chromotropic detection of superoxide using these nitrones can be a promising method in contrast to other available methods.

Filarial infections (lymphatic filariasis and onchocerciasis) are amongst the major neglected tropical diseases, and together account for more than 120 million infections in tropical and subtropical regions. The gold-standard technique for the diagnosis of filariases relies on the detection of microfilariae (mf) either in blood smears (lymphatic filariasis) or in skin biopsies (onchocerciasis). The secretion of extracellular RNAs (exRNAs) by parasitic nematodes has opened new avenues for the development of novel biomarkers for helminthiases, including filariasis. However, rather little is known about the origin and regulation of these RNAs inside the nematodes. One outstanding question is whether the secretion of small RNAs is distinct across the developmental stages of parasitic nematodes. Similarly, it is not clear whether the secretion of miRNAs is affected by treatment with anthelminthic chemotherapy or their potential as biomarkers for infection. Litomosoides sigmodontis is a murine filarial nematode closely related to filarial nematodes of medical and veterinary importance, including Onchocerca spp. and Brugia spp. L. sigmodontis has been extensively used to decipher multiple aspects of filarial biology, including parasite development, vaccine, and host-pathogen interactions. Therefore, we decided to use this model to address fundamental questions regarding the secretion of small RNAs and their biomarker potential. Our in vitro studies demonstrate that some extracellular miRNAs are enriched in a sexand stage-specific manner in the Excretion/Secretion (ES) products from early larval and adult stages from the rodent filarial nematode Litomosoides sigmodontis. Moreover, our data demonstrates that the gravid adult female worms secrete a plethora of miRNAs enriched in the secretome of this developmental stage when compared to adult males or mf. Further characterization studies show that the miRNAs are likely to be secreted in association with extracellular vesicles (EVs), as previously reported for other parasitic nematodes, including the human pathogen Brugia malayi. Interestingly, Ivermectin, which is typically used to treat filarial infections, does not have consistent effects on the secretion of miRNAs by gravid adult female worms in vitro, requiring further in vivo experiments to determine the effect of IVM on detection of extracellular parasite-derived miRNAs. In vivo experiments, using murine models of infection with L. sigmodontis (gerbils and BALB/c mice), as well as human samples from patients infected with Onchocerca volvulus and cattle infected with Onchocerca ochengi, demonstrated the presence of filarial-derived miRNAs, including female-specific miRNA markers, in biofluids from infected hosts. Further statistical analysis showed that two parasite-derived miRNAs, miR-71 and miR-100d, can significantly discriminate infected animals from naïve controls with high sensitivity/specificity (> 80%/100%). The results presented in this PhD thesis provide an initial framework to understand the secretion of small RNAs throughout nematode development, the potential interactions between anthelminthic chemotherapy and small RNA trafficking and secretion, as well as the use of parasite-derived miRNAs for the development of a new generation of biomarkers for filarial infections.

There has been much interest in recent years about the potential of microRNA as a new source of biomarkers for the diagnosis of disease. The delivery of new diagnostic tools based on this potential has been limited by shortcomings in current microRNA detection techniques. This thesis explores the development of a new method of microRNA detection through the incorporation of conductive particles into oligonucleotide-functionalised polymers to form oligonucleotide cross-linked polymer composites. Such composites could provide a simple, rapid, and low-cost means of microRNA detection that could be easily multiplexed, providing a valuable tool for point-of-care medical diagnostics. This work presents oligonucleotide-functionalised carbon/polyacrylamide composites which demonstrate a selective swelling response in the presence of analyte oligonucleotide sequences and for which the electrical conductivity decreases with swelling. The composites were synthesised via UV-initiated free-radical polymerisation of carbon/- monomer mixtures upon custom electrode devices, consisting of interdigitated platinum electrodes fabricated upon a silicon substrate. The optimal cross-linker density and carbon loading concentration were determined as well as the best means of dispersing the carbon particles within the polymer. Various types of carbon particles, with differing sizes and aspect ratios, were compared and their performances as conductive additives for polymer swelling transduction evaluated. The swelling behaviour of these composites was evaluated by analysing images of composite microdroplets as they swell. The electrical characteristics of the composites were determined by measuring either the two-terminal resistance or the complex impedance of composite microdroplets on the electrode devices. Alternating and direct current measurement techniques were compared to determine the best approach for the transduction of composite swelling. The volumetric and electrical responses of oligonucleotide-functionalised carbon/polyacrylamide composites were analysed in solutions of analyte oligonucleotide and non-complementary controls. It has been demonstrated that, using carbon nanopowder composites and a direct current two-terminal resistance measurement, it is possible to differentiate between analyte and control solutions to concentrations as low as 10 nM, with single-base precision, in less than three minutes. However, the inability to detect at concentrations below this value, difficulties in differentiating between different analyte concentrations and thermal instability mean that, in their current form, oligonucleotide cross-linked polymer composites are unsuitable for the detection of circulating microRNA at clinically relevant concentrations. Potential avenues of work to address these challenges are discussed. Also presented are collaborative results for oligonucleotide-responsive polymers functionalised with morpholino nucleic acid analogues, in what is believed to be the first example of such a material. These morpholino-functionalised polymers offer significant advantages, in terms of stability and sensitivity, over their nucleic acid equivalents for bio-responsive polymer applications.

Le dé cit cognitif est une cause majeure de handicap de la personne âgée. Du fait de l’augmentation de la durée de vie, le nombre de personnes âgées qui pourraient béné cier d’une prise en charge spécialisée dans le but de ralentir leur perte fonctionnelle va croitre. En dépit de traitement validé qui stoppe le processus neurodégénératif, des interventions spéci ques peuvent en ralentir les manifestations. De nombreuses études expérimentales, neuroscienti ques et comportementales ont démontré la relation étroite entre cognition en motricité.Ce travail doctoral propose d’explorer si certaines caractéristiques de la marche sont des biomarqueurs non-invasifs d’un trouble cognitif et des chutes, et de mieux comprendre les relations entre la cognition et le contrôle de la marche. Le cadre théorique était l’hypothèse de la « Perte de Complexité ». Des analyses multivariées ont été appliquées aux critères de jugement dynamiques de marche en relation avec le statut cognitif-et-chute (Le Chapitre 1).Le Chapitre 2 est une revue systématique de la littérature. Nous avons recensé 20 études longitudinales de prédiction de trouble de la marche et de dé cit cognitif. La vitesse de marche était associée à la cognition, son ralentissement était un prédicteur de la perte cognitive. Cette revue a aussi mis en évidence la nécessité d’améliorer la mesure des phénomènes concernésL’objectif du Chapitre 3 était de déterminer ce qu’apportait l’évaluation cognitive (MMSE, mémoire et fonctions exécutives) et des paramètres dynamiques précis de la marche, au bilan habituel du risque de chute. La précision de la classi cation entre chuteurs et non- chuteurs a augmenté avec l’ajout de la cognition et des paramètres de marche de AUC=0.86 à AUC=0.93. La spéci cité du modèle de classi cation a, quant à elle, augmenté de 60% à 72% avec l’ajout des mesures cognitives, et de 72% à 80% avec l’ajout des paramètres dynamiques de la marche. Dans leur ensemble, ces résultats montrent l’intérêt d’une approche multidimensionnelle incluant l’évaluation des fonctions cognitives et de la marche, dans la prédiction du risque de chute chez la personne âgée.Le Chapitre 4 détermine les paramètres de marche les plus associés au déclin cognitif. La vitesse de marche, la régularité, la prévisibilité et la stabilité ont montré que les simples et doubles-tâches étaient les plus discriminantes (score VIP moyen de 1.12). La marche des patients avec troubles cognitifs est plus lente, moins régulière et moins stable que celle de personnes âgées saines. Cependant, les résultats montrent également que la discrimination des patients âgés avec et sans dé cit cognitif, fondée sur l’exploitation des paramètres de marche uniquement, est faible, avec 57% (simple tâche) et 64% (double-tâche) des patients classés de manière erronée.Le Chapitre 5 présente une étude pilote prospective dans laquelle les paramètres de marche les plus discriminants du Chapitre 4 ont été étudiés. Les résultats ont montré qu’un déclin cognitif signi catif observé après 14.4 mois en moyenne était corrélé avec une marche plus régulière (ρ=0.579*) et plus prévisible (ρ=0.486*) mesurée pendent les mesures de ligne de base, mais pas avec la vitesse de marche de la ligne de base (ρ=0.073). Une augmentationde la régularité et de la prévisibilité de la marche est le re et d’une perte de la complexité de la marche, témoin d’une détérioration future des fonctions cognitives chez les patients âgés.Les résultats de ce travail doctoral sont résumés et discutés dans le Chapitre 6. / The rising life expectancy will result in an increased number of ‘older old adults’ who will need specialized geriatric care to slow functional decline. Cognitive impairment is a major cause of disability in geriatric patients. Even though there is no cure yet to reverse neurodegeneration, tailored interventions can slow disease progression and reduce symptoms. Because of the abundant evidence from experimental, neuroscienti c, and behavioral studies that underscored the close link between motor- and cognitive function, the present thesis proposed to use gait characteristics as non-invasive indicators of cognitive impairment and falls in geriatric patients. The main objective therefore was to increase our understanding of the relationship between gait and cognition in this vulnerable population, in which gait outcomes were calculated from 3D-acceleration signals of the lower trunk that were collected with an iPod Touch 4G. The ‘Loss of Complexity’ hypothesis provided a theoretical framework. Multivariate analyses were applied to dynamic gait outcomes in relation to cognitive- and fall-status (Chapter 1).Chapter 2 presents a systematic literature review including 20 longitudinal studies that examined associations between baseline gait function and future cognitive decline. A slow gait speed was associated with future decline in global cognition and in speci c cognitive functions, and with an increased risk for Mild Cognitive Impairment (MCI) and dementia (maximal odds and hazard ratios of 10.4 and 11.1, respectively) in 4.5 years on average. The review projected that future research could increase the speci city of the gait-cognition link by indexing gait and cognition in more detail.From this perspective, Chapter 3 examined whether an extensive cognitive evaluation (global cognition, memory, and executive functioning) and ne-grained, dynamic gait outcomes could add to a usual fall-risk screening. The overall classi cation accuracy of fallers and non-fallers increased from Area Under the Curve (AUC) =0.86 to AUC=0.93. The speci city of the fall-classi cation model increased from 60% to 72% when cognitive outcomes were added, and from 72% up to 80% when gait dynamics were added to the model. The results underscored the need for a multifactorial approach in fall risk assessment in geriatric patients, including a detailed evaluation of cognitive- and gait function.Chapter 4 explored what gait outcomes are most susceptible to change with cognitive decline, and examined multiple gait outcomes in relation to cognitive impairment. Outcomes related to gait speed, regularity, predictability, and stability revealed with the highest discriminative power, indicated by the Variable Importance in Projection (VIP)-values for single- and dual-tasking (average VIP-score of 1.12, with a VIP-score>1 indicating a high discriminative power). Geriatric patients walked slower, less regular, and less stable than healthy old controls. However, the discrimination of geriatric patients with- and without cognitive impairment based on gait outcomes alone was poor, with 57% (single-task) and 64% (dual-task) of the patients being misclassi ed.In Chapter 5, the gait outcomes with the highest discriminative power in chapter 4 were studied in a prospective pilot study. Signi cant cognitive decline (in global cognition, memory, and executive functioning) over 14.4 months on average correlated with a moreregular (ρ=0.579*) and more predictable (ρ=0.486*) gait at baseline, but not with baseline gait speed (ρ=0.073). The increased gait regularity and predictability re ected a loss of gait complexity and this loss of gait complexity may thus predict future cognitive decline in geriatric patients.The results are summarized and discussed in Chapter 6 of this thesis.

Ventilator associated pneumonia (VAP) and acute respiratory distress syndrome (ARDS) are two respiratory conditions unique to mechanically ventilated patients. The diagnosis of these conditions, and therefore any subsequent treatment, are befuddled by uncertainty. VAP rates vary considerably according to the diagnostic or surveillance criteria used. The pathogenesis of ARDS is well understood but when the internationally agreed consensus criteria are employed, the histological hallmarks are absent about half the time, indicating a disconnection between the clinical diagnosis and what is known about the biology of this condition. It is argued that tests of biological function should be considered in addition to clinical characteristics in order to improve the utility of diagnosis. Given that the pathological sequelae of both VAP and ARDS are driven by an over exuberant host neutrophil response, the activated neutrophil was selected as a potential biological imaging target. Optical molecular imaging uses visible and near visible wavelengths from the electromagnetic spectrum to derive or visualize information based on the optical properties of the target tissue. Optical wavelengths are safe and cheap to work with, producing much higher resolution images than those relying on x-rays or gamma radiation. The imaging modality can be coupled with exogenously applied chemistry to identify specific biological targets or processes. The hypothesis that optical molecular imaging could be used to detect activated neutrophils in real time in the alveolar region of patients was tested. A bespoke optical molecular imaging agent called Neutrophil Activation Probe (NAP), designed in-house, was used to test the hypothesis. NAP is a dendrimeric compound delivered to the alveolar region of a patient in microdoses (≤100 micrograms), becoming fluorescent only on contact with activated neutrophils, and can be detected by optical endomicroscopy. Both the imaging agent and the endomicroscope are delivered to the distal lung via routine bronchoscopy. The agent was tested extensively in the laboratory to demonstrate function, specificity, and safety. Ex vivo testing took place using human and ovine lungs. A regulated dose escalation Phase I clinical trial of investigational medicinal product (CTIMP) in healthy volunteers, patients with bronchiectasis, and mechanically ventilated patients with a pulmonary infiltrate on chest radiography (NCT01532024) was designed and conducted. The aim of the Phase I study was to demonstrate the safety of the technique and to confirm proof of concept. In order to support the requirement for a technique that interrogates alveolar neutrophils two supplementary clinical studies were performed. Firstly, two VAP surveillance techniques (CDC surveillance and HELICS European VAP surveillance) were compared with clinically diagnosed VAP across consecutive admissions in two large tertiary centres for one year. Secondly, the utility of circulating neutrophils to permit discrimination between acute respiratory illnesses was examined. Blood samples from mechanically ventilated patients with and without ARDS underwent flow cytometric assessment using eight clusters of differentiation and internal markers of activation to determine neutrophil phenotype. All clinical studies received the appropriate regulatory, ethical, and/or Caldicott guardian approval prior to commencement. NAP became fluorescent only in the presence of three processes specific to neutrophil activation: active pinocytosis, progressive alkalinization of the phagolysosome, and the activity of human neutrophil elastase. High optical signal was detected following the application of NAP in the alveolar regions of explanted lungs from patients with cystic fibrosis, known to be rich in activated neutrophils. Using an ex vivo ovine lung ventilation and perfusion model optical signal was demonstrated following segmental lung injury. The safety and specificity of the technique in a small cohort of healthy volunteers and mechanically ventilated patients was demonstrated. The technique was tested on a small cohort of patients with bronchiectasis, which provided the first opportunity to obtain broncho-alveolar lavage samples for laboratory correlation. Fluorescent signal was shown in the lavaged neutrophils, labeling that could only have taken place in the alveolar region. The supportive clinical studies found the concordance between actual VAP events was virtually zero even though the reported VAP rates were similar. Furthermore, the rate at which clinicians initiate antibiotics for VAP was approximately five times higher than either surveillance VAP rate. The study of circulating neutrophils from the blood of healthy volunteers and mechanically ventilated patients with and without ARDS indicated circulating neutrophil activation phenotype was not capable of discriminating between clinically diagnosed ARDS and other acute respiratory illnesses. In summary, an ambitious programme of work was completed to develop and support an optical molecular imaging technique that meets the rigorous requirements for human application and can be applied at the bedside to yield immediate visual results. The spatiotemporal relationship of neutrophil activation in real time both in the laboratory and in volunteers and patients was visualized. The visualization of neutrophil activation at such a resolution has never been achieved before in humans, healthy or unhealthy. The Phase I study was not powered to determine utility but recruitment has begun to a Phase II CTIMP (NCT02804854) to investigate the utility, accuracy, and precision of the imaging technique in a large cohort of mechanically ventilated patients. Ultimately, it is proposed that the technique will facilitate diagnosis, stratify patients for treatment and monitor treatment response using this technique.

Back pain is responsible for British employees taking 5 million sick days per year. Low back pain (LBP) has a controversial aetiology, with 95% of cases caused by mechanical, non-pathological causes. Current medical treatment for mechanical LBP is an exercise regime designed to restore lumbar stability. Unfortunately this is often a painful process, and therefore difficult to complete. Electromyography (EMG) variables have been shown to be able to discriminate between subjects with and without mechanical LBP. If these variables could be shown to have discriminatory abilities before the actual onset of LBP they could be used to predict future episodes of LBP in currently otherwise asymptomatic individuals and allow the rehabilitation process to begin before the onset of symptoms. However a number of problems persist with EMG measurement. The test must be administered under closely controlled conditions in order to record clean signals, and interpretation of this data requires special tools and training. This thesis aims to make contributions in three main areas; Automated Analysis: Manual analysis of a large store of EMG raw data files is a time consuming process. If outcome variables that require manual interpretation are included this effect is magnified, with necessary questions being raised as to the accuracy and consistency levels that can be maintained. A successfully implemented automated system would reduce analysis time and improve confidence in the outcome variables recorded. Investigations will also be carried out into the addition of error detection and correction algorithms that could be performed during the analysis procedure. ECG Contamination Removal: Previous studies have identified ECG as a potential source of contamination of lumbar EMG signals. Compensation for this effect is non-trivial as the ECG frequencies overlap an area of interest in the EMG spectrum, and the ECG signal characteristics would change over a fatiguing EMG test. The Independent Component Analysis method will be used to attempt to extract and remove the ECG component of a recorded signal whilst preserving the underlying EMG data. If this is successful an analysis of the effect that removing ECG contamination has on EMG outcome variables will be presented. Colour Map Diagnostic Method: Colour maps are an excellent method of presenting a large amount of signal data to a researcher, and have been used to discriminate between LBP and non-LBP subjects. The usefulness of this diagnostic display too has been somewhat limited however by the difficulty in producing such maps. Investigations will be carried out into methods that will be able to quickly and accurately produce these colour maps to the same specification as earlier studies. Colour maps of subjects that did not report LBP at the time of testing, but who then did report LBP at their next presentation, will be examined to assess whether or not EMG colour maps can be used as a predictor for low back pain.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Robert L. Larson / Bradley J. White / There are multiple modalities available to evaluate health or stress status of animals. The objective of my research was to evaluate different modalities including behavior, rectal and nasal temperature, and blood samples to determine the relationship with these outcomes of interest in bovine respiratory disease (BRD) events, environmental conditions, transportation, and Mannheimia haemolytica challenge model. The objective for the final project was to determine whether diagnostic sensitivity or specificity resulted in greater economic value for the industry using simulation models for identification of BRD. There was a positive association with rectal temperature and probability of not finishing the production cycle normally, but this relationship was not linear. Rectal temperature of feedlot calves at first treatment for BRD had limited value as a prognostic indicator of whether those calves would finish the production cycle normally. A positive association between rectal temperature and ambient temperature and temperature-humidity index was determined. Environmental conditions must be considered when rectal temperature is used as a diagnostic tool. At 48 hours after initiation of transportation there were no differences in body weight, rectal temperature, and time spent at various locations in the pen detected between transported and non-transported control heifers. Transportation of heifers during periods of high ambient temperatures caused transient changes in physiologic and behavioral indices of heifers. Calves challenged with Mannheimia haemolytica had more changes in behavior, body weight, and blood biomarkers during high ambient temperatures compared to control calves. Results of this study may guide research in development of objective assessment tools for identification and management of cattle affected with BRD during extreme summer conditions. For both low and high apparent prevalence cohorts, increasing diagnostic specificity resulted in more rapid, positive change in net returns compared to change in increasing sensitivity. Improvement of diagnostic specificity, perhaps through a confirmatory test or pen-level diagnostics, can increase diagnostic value. Mortality risk was the primary driver for net returns. Results from this study are important for determining future research priorities to analyze diagnostic techniques for BRD and provide a novel way for modeling diagnostic tests.

Le Bti est un bioinsecticide mondialement utilisé dans la lutte contre les moustiques. La toxicité du Bti est due à un cristal, constitué de quatre toxines principales, produit par la bactérie Bacillus thuringiensis subsp. israelensis. Le Bti représente une bonne alternative aux insecticides chimiques car il est peu persistant dans l’environnement et spécifique des insectes ciblés. La sélection d’une souche de moustique (Aedes aegypti) de laboratoire au Bti a entrainé une résistance modérée au Bti (3.5 fois) mais plus élevée aux toxines testées séparément (jusqu’à 60 fois). Ce résultat suggère que l’adaptation des moustiques au Bti en populations naturelles peut être corrélée à une résistance accrue à chacune des toxines. Pour pouvoir détecter une adaptation au Bti, il est donc nécessaire de mieux caractériser la résistance à chaque toxine qui compose le Bti.Cette thèse se structure en trois axes. 1. L’analyse du transcriptome par RNA-seq des souches résistantes à chaque toxine et au Bti a permis de mettre en évidence plusieurs gènes candidats potentiellement impliqués dans la résistance. La résistance développée au laboratoire est complexe, combinant des mécanismes de résistances spécifiques de chaque toxine et généralistes. 2. L’analyse du transcriptome d’une souche sensible exposée au Bti a permis d’identifier des gènes impliqués dans la réponse à l’intoxication, notamment les phosphatases alcalines. De plus, l’exposition à chaque toxine et au Bti induit une modification de l’expression de ces gènes bien plus importante chez les souches résistantes que chez la souche sensible. 3. L’implication de la réponse immunitaire dans la résistance et dans la réponse à une exposition au Bti est discutée. L’exposition au Bti accroit la compétence vectorielle d’A. aegypti pour deux maladies tropicales (Dengue et Chikungunya), différemment selon si la souche est sensible ou résistante au Bti. Cette thèse permet de mieux comprendre les mécanismes de résistances au Bti et à ses toxines ; ces connaissances sont nécessaires pour mieux anticiper la résistance sur le terrain. / Bti is a bioinsecticide used worldwide for mosquito control. Bti toxicity is due to a crystal, composed of four main toxins, produced by the bacteria Bacillus thuringiensis subsp. israelensis. Bti represents a good alternative to chemical insecticides because it is known to have a low persistence in the environment and to be specific to the targeted insects. The selection of a laboratory mosquito (Aedes aegypti) strain with a persistent form of Bti led to moderate resistance to Bti (3.5-fold) but to higher resistance (up to 60-fold) to Bti toxins tested separately. This result suggests that the adaptation of mosquitoes to Bti in the field may be correlated to a higher resistance to each toxin. In order to detect an adaptation to Bti, it is thus necessary to characterize better the resistance against each Bti toxin.This thesis is organized in three parts. 1. The transcritpome analysis of strains resistant to each toxin and to Bti using RNA-seq technique enabled to highlight genes and mechanisms potentially involved in the resistance. The resistance developed in the laboratory is complex, combining generalist and specific mechanisms to resist to each toxin. 2. The transcriptome analysis of a susceptible strain exposed to Bti enabled identifying genes involved in the response to the intoxication, especially alkaline phosphatases. Furthermore, exposure to Bti and to each toxin resulted in gene expression modification being far higher in resistant strains than in susceptible strain. 3. The role of immunity in the resistance and in the response to Bti exposure is discussed. The Bti exposure increases the vector competence of A. aegypti for two tropical diseases (Dengue and Chikungunya) differently, depending on the strain being susceptible or resistant to Bti. This thesis enables better understanding of resistance mechanisms to Bti and its toxins; this knowledge is necessary for anticipating field resistance development.

Onchocerciasis is a severely debilitating yet neglected tropical disease (NTD) currently affecting approximately 15.5 million people, including 12.2 million people with skin disease and 1.025 million with vision loss. The disease causes social stigma, generates and perpetuates poverty, and leads ultimately to irreversible unilateral or bilateral blindness if untreated. Consequently, onchocerciasis is a major impediment to socioeconomic development in addition to being a major public health concern in some African countries. Many control programs have been launched against the disease with moderate successes achieved in Africa. These limited outcomes are partially due to the unavailability of reliable, non-invasive and easily applicable diagnostic tools for mapping endemic regions, monitoring control program successes, determining treatment endpoints and post-elimination surveillance. The current WHO recommendations for certification of elimination include the use of the Ov16 antibody detection ELISA which is flawed by an intrinsic systematic error as 15-25% of infected populations may not produce antibodies against this antigen due to genetic restrictions. With the recent shift in the global health goal of onchocerciasis from control to elimination, there is a need for the development of novel appropriate tools. These tools include amongst others, drugs, diagnostics, and vaccines. In this work, bioinformatics analyses combined with immunological assays were applied in a bid to develop potential tools for the current elimination programs. With regards to chemotherapy, Ivermectin which has been the sole drug for onchocerciasis treatment for over 30 years kills only the microfilariae (mf) leaving the adult worms intact which continue to produce the mf. Moreover, there is a recent problem of development of parasite resistance to this drug. In addition, moxidectin which was recently approved for treatment is contra-indicated in pregnant women and children under 12 who could continue to serve as reservoirs for infection. There is therefore a need to develop new treatment strategies, preferably for macrofilaricidal drugs. For a total eradication of onchocerciasis, diagnosis and treatment must be complemented with vaccine development. The aim of this work was therefore (i) to characterise an O. volvulus antigen, Ov58GPCR and (ii) to design an epitope-based chimeric antigen, which we designated, Ov-DKR-1, within the framework of the development of onchocerciasis control tools. In order to achieve the first goal, towards diagnosis, synthetic peptides representing linear B-epitopes and the recombinant extracellular domain of a G-protein coupled receptor (GPCR) with diagnostic potential were tested for their immune responses using serum from onchocerciasis-infected individuals and various controls. The results obtained indicate that (i) the O. volvulus antigen, Ov58GPCR is a G-protein coupled receptor (GPCR) conserved in related nematodes, (ii) synthetic peptides predicted to be in the extracellular domain (ECD) of Ov58GPCR are indeed immunogenic epitopes in onchocerciasis-infected individuals, (iii) synthetic peptide cocktails discriminate between actively-infected individuals, treated non-infected individuals and healthy African controls, (iv) polyclonal antibodies against one of the peptides or against the bacterially-expressed ECD reacted specifically with the native antigen in O. volvulus total and surface extracts, (v) Ov58GPCR is transcribed in both larvae and adult parasite stages, (vi) IgG and IgE responses to the recombinant ECD decline with Ivermectin treatment. All these findings suggest that the recombinant extracellular domain and synthetic peptides of Ov58GPCR, as well as the specific immune responses generated, could be harnessed in the context of disease diagnosis and surveillance. To assess the potential role of Ov58GPCR in drug or vaccine target development, preliminary examination on the essentiality of the Ov58GPCR for parasite survival was evaluated through RNA interference. A short-interfering RNA (siRNA) sequence targeting the gene designed and tested by soaking with O. volvulus male worms resulted in a reduction in motility. Results indicated that the gene may be involved primarily in motility. Further investigations are recommended in this light. With regards to the second goal, towards the development of a potential immune-protective tool, many indicators reveal the possibility of the development of protective tools against onchocerciasis. Consequently, an immuno-informatics approach was applied to design a filarial-conserved multi-epitope subunit vaccine candidate consisting of B-and T-cell epitopes of proteins reported to be potential novel vaccine candidates. Conservation of the selected proteins in other nematode parasitic species and predicted epitopes suggests that the generated chimera protein (Ov-DKR-1) could be vital in cross-protection. The 3D structure was predicted, refined and validated bioinformatically. Protein-protein docking of the chimeric vaccine candidate with the TLR4 receptor predicted efficient favourable binding. Immune simulation predicted significantly high levels of IgG1, T-helper, T-cytotoxic cells, INF-γ, and IL-2 responses. Overall, the designed chimeric peptide demonstrated antigenicity superior to the current vaccine candidates. / L’onchocercose est une maladie tropicale sévèrement débilitante mais négligée qui touche actuellement environ 15,5 millions de personnes, dont 12,2 millions de souffrant de maladies de la peau et 1,025 millions de souffrant de perte de vision. La maladie provoque une stigmatisation sociale, génère et perpétue la pauvreté et finit par conduire à une cécité unilatérale ou bilatérale irréversible si elle n'est pas traitée. En conséquence, l’onchocercose est un obstacle majeur au développement socioéconomique en plus d’être une préoccupation majeure pour la santé publique. De nombreux programmes de lutte ont été lancés contre la maladie, avec quelques succès en Afrique. Ces résultats sous-optimaux (limités) sont en partie dus à l’absence d’outils fiables, non invasifs et facilement applicables pour la cartographie des régions endémiques, le suivi des succès des programmes de contrôle, la détermination des paramètres de traitement et la surveillance post-élimination. Les recommandations actuelles de l’OMS pour la certification de l’élimination incluent l’utilisation du test ELISA de détection d’anticorps Ov16, entaché d’une erreur systématique intrinsèque puisque 15 à 25% des populations infectées peuvent ne pas produire d’anticorps contre cet antigène en raison de restrictions génétiques. Avec l’évolution récente de l’objectif de santé mondial de l’onchocercose de passé de la lutte à l’élimination, il est donc nécessaire de mettre au point de nouveaux outils appropriés. Ces outils nécessaires incluent, entre autres, des médicaments, des diagnostics et des vaccins. Dans ce travail, des analyses bio-informatiques combinées à des tests immunologiques ont été appliquées dans le but de développer des outils potentiels pour les programmes d'élimination actuels. En ce qui concerne la chimiothérapie, l’ivermectine, qui est le seul médicament utilisé depuis plus de 30 ans pour le traitement de l’onchocercose, ne tue que les microfilaires (mf) laissant intacts les vers adultes qui continuent à produire le mf. A ceci joint, il y a le problème récent du développement de la résistance des parasites à ce médicament. En outre, un traitement récemment approuvé, la moxidectine, est contre-indiquée chez les femmes enceintes et les enfants de moins de 12 ans qui pourraient continuer à servir de réservoirs d’infection. Il est donc absolument nécessaire d’élaborer de nouvelles stratégies de traitement, de préférence pour les médicaments macrofilaricides. Pour une éradication totale de l’onchocercose, le développement du vaccin doit être complété par le diagnostic et le traitement. Le but de ce travail est donc de caractériser un antigène d'O. volvulus, Ov58GPCR et de concevoir un antigène chimérique à base d'épitope, que nous avons appelé Ov-DKR-1, dans le cadre du développement d'outils de contrôle de l'onchocercose.Concernant le premier objectif, des peptides synthétiques représentant des épitopes B linéaires et le domaine extracellulaire (DEC) recombinant d'un récepteur couplé à la protéine G (GPCR) présentant un potentiel diagnostique ont été testés pour déterminer leur réponse immunitaire en utilisant du sérum d'individus infectés par l'onchocercose et divers témoins. Les résultats obtenus indiquent que (i) l'antigène d'O. volvulus, Ov58GPCR est un récepteur couplé à la protéine-G (GPCR) conservé dans les nématodes apparentés (ii) les peptides synthétiques prédits comme localisés dans le domaine extracellulaire de Ov58GPCR sont bien des epitopes immunogéniques chez les individus infectés par l’onchocercose, (iii) les cocktails de peptides synthétiques établissent une distinction entre les individus activement infectés avec l’onchocercose, les individus non-infectés traités et les témoins africains en bonne santé, (iv) les anticorps polyclonaux contre un des peptides ou le domaine extracellulaire exprimé au bactéries réagisent spécifiquement avec l'antigène natif dans les extraits total et de surface d'O. volvulus, (v) Ov58GPCR est transcrit aux stades larvaire et adulte, (vi) les niveaux détectés d’IgG et IgE grâce à le DEC recombinant diminuent au cours du traitement par l'ivermectine. Toutes ces découvertes suggèrent que le domaine extracellulaire recombinant et les peptides synthétiques de Ov58GPCR, ainsi que les réponses immunitaires spécifiques générées, pourraient être exploités dans le contexte du diagnostic et de la surveillance de la maladie. Pour évaluer le rôle potentiel d’Ov58GPCR dans le développement de médicaments ou de vaccins cible, un examen préliminaire de l’indispensabilité du gène Ov58GPCR pour la survie du parasite a été évalué par interférence d’ARN. Une séquence d'ARN interférant court (ARNic) ciblant le gène conçu et testé par trempage avec des vers mâles d'O. volvulus a entraîné une réduction de la motilité. Les résultats ont indiqué que le gène pourrait être impliqué principalement dans la motilité. Des investigations complémentaires sont recommandées dans cette optique.Concernant le deuxième objectif, de nombreux indicateurs révèlent la possibilité de développer des outils de protection contre l’onchocercose. En conséquence, une approche immuno-informatique a été appliquée pour concevoir un candidat-vaccin des sous-unités de multi-épitopes conservées-filarienne consistant des épitopes de cellules B et T de protéines qui seraient de nouveaux candidats vaccins. La conservation des protéines sélectionnées chez d'autres espèces parasitaires de nématodes et d'épitopes prédits suggère que la protéine chimère générée (Ov-DKR-1) pourrait être vitale pour la protection croisée. La structure 3D a été prédite, raffinée et validée bioinformatiquement. La fixation protéine-protéine du candidat vaccin chimère au récepteur TLR4 prédit une liaison favorable efficace. La simulation immunitaire prédit des niveaux significativement élevés d'IgG1, de réponses T-helper, de cellules T-cytotoxiques, de INF-γ et d'IL-2. Globalement, le peptide chimère conçu a démontré une antigénicité supérieure aux candidats vaccins actuels. / Option Biologie moléculaire du Doctorat en Sciences / info:eu-repo/semantics/nonPublished