Rozprawy doktorskie na temat „Molecular diagnostic tool”

ABSTRACT The aim of this study is develop and test an inexpensive molecular tool kit to be used for diagnostic PCR for diseases such as Leber hereditary optic neuropathy (LHON) and Cystic fibrosis(CF). By developing and optimizing recombinant Taq polymerase and making a DNA size ladder from plasmids pPSU1 and pPSU2 the financial cost for the tool kit would be reduced significantly compared to the commercial components. With an inhouse method both the recombinant Taq polymerase and the pPSU1 and pPSU2 plasmids were purified from the E.coil strain DH5-α. Thereafter to analyse the components of the tool kit both conventional PCR and Real-time PCR to make sure that the tool kit would work for both types of PCRs.     The homemade Taq polymerase proved to be able to sustain in room temperature for at least 24 h and the polymerase also showed that it works with different primers such as LHON, CF and Beta-globin in both endpoint and probe base real-time PCR. The homemade size marker produced a reliable in agarose gel electrophoresis but requires optimization for continued usage for smaller PCR products.     In conclusion the homemade Taq polymerase will be used in future PCR analysis in the laboratory and the recombinant production process as well. Meanwhile the homemade size marker did not work sufficiency enough to be continuously used with gel electrophoresis in the laboratory without being further modified.

To further the understanding and knowledge about fusion plasmas and their behaviour during different conditions, it is important to be able to collect information about the plasma and the processes occurring within it. Visible spectroscopy, or the study of the visible wavelength light emitted by the plasma, is a useful tool in this search for knowledge.

This thesis is based on experiments where visible wavelength light has been measured and analysed in order to determine quantities about the emitting source. Doppler shift measurements of spectral lines have been utilised to determine the toroidal rotation velocities of plasma impurity ions and to study the correlation with mode rotation and the effect of active feedback control of the resistive wall modes. Information on the impurities present in the plasma has been determined and the calibrated intensities of spectral lines has yielded impurity concentrations, particle fluxes and electron temperature and densities. Ion temperatures have been determined from Doppler broadening measurements.

The measured vibrational and rotational band structure of deuterium molecular spectra has been analysed in order to calculate rotational and vibrational temperatures, relative populations and molecular particle fluxes. The effect of the molecular flux on simple calculations of atomic flux has also been studied. Specific molecular states and transitions of deuterium have also been probed with synchrotron radiation to study the level and transition energies.

The measurement and analysis of visible wavelength light has been demonstrated to be a sensitive diagnostic tool in the quest for increased knowledge about fusion plasmas and molecular structure.

In this thesis, the interactions between different proteins and small ligands were characterized by surface plasmon resonance spectroscopy (SPR) and fluorescence resonance energy transfer (FRET) based assays.    For the C-reactive protein (CRP), a new type of artificial binder was identified which allows designing diagnostic assays superior to commonly used standard assays. Furthermore, an interaction study with the endogenous ligand phosphocholine revealed the importance of the avidity of pentameric CRP for the distinction of different types of lipid membranes. The interaction study with calcium showed how SPR based assays can be used to study ion-protein interactions despite the low atomic weight of ions.    The transmembrane protease BACE1, an important drug target for Alzheimer’s disease, was immobilized to an SPR biosensor surface and embedded into a lipid membrane. An interaction study with a set of known BACE1 inhibitors showed that the transmembrane region has only minor effects on the interactions. Furthermore the pH-dependencies of the interactions were investigated and revealed new important conclusions for inhibitor design. Computer aided modelling showed that the protonation state of the aspartic dyad is dependent on the interacting inhibitor which offers new perspectives for in silico screenings. The SPR assay developed for BACE1 was adapted to a more complex membrane protein, the pentameric β3 GABAA receptor. The assay allowed the pharmacological characterisation for histaminergic and GABAergic ligands and gave further evidence for cross-talk between the two signal transduction pathways. This study shows that the immobilisation method used for BACE1 and the ß3 GABAA receptor has the potential to become a standard method for handling membrane proteins.   The identification of new drug leads from natural sources is a common strategy for drug discovery. A combination of SPR and FRET based activity assays were explored to increase the efficiency of this process. For HIV-1 protease, secreted aspartic protease (SAP) 1, 2 and 3 extracts from a marine vertebrate were identified containing potent inhibitors which interacted with the active site of the enzymes. The studies in this thesis show that the investigation of protein interactions is crucial for understanding protein functions and can help to develop novel drugs for the treatment of different diseases.

Le manioc (Manihot esculenta L. Crantz) est cultivé dans toute la zone intertropicale. Compte tenu de ses utilisations potentielles, le développement de la culture du manioc a considérablement augmenté au niveau mondial, mais il est encore considéré comme une culture de subsistance en particulier dans les régions pauvres où il est cultivé par les petits producteurs. La production du manioc est fortement contrainte par des facteurs biotiques et abiotiques, dont la bactériose vasculaire du manioc (CBB) et la nécrose bactérienne du manioc (CBN) qui sont deux maladies causées par les bactéries Xanthomonas axonopodis pv. les manihotis (Xam) et X. cassavae (Xc), respectivement. CBB est considérée comme la principale maladie bactérienne, notamment au Venezuela où la CBB a été signalée pour la première fois dans les années 70. Dans les années 90, des études ont été menées pour élucider la variabilité génétique de Xam dans différentes régions du pays, au moyen d’outils moléculaires, mettant en évidence un degré élevé de polymorphisme parmi les souches analysés.Le sujet de cette thèse porte sur la situation de CBB au Venezuela 20 ans après. Quelle est la diversité génétique actuelle de Xam au Venezuela? Quelle est la structure génétique des populations de Xam dans ce pays, et comment diffèrent-elles des souches de Xam recueillies dans les années 90? En outre, étant donné que Xam et Xc provoquent des symptômes semblables sur feuilles de manioc et présentent des caractéristiques physiologiques et morphologiques similaires, nous avons également visé à développer un nouvel outil de diagnostic moléculaire permettant une détection rapide et fiable de Xam et de Xc tout étant capable de les discriminer l’une vis à vis de l’autre.Pour atteindre nos objectifs, nous avons d'abord établi une PCR-duplex comme outil de détection moléculaire des xanthomonades infectant le manioc. Sur la base de l'analyse in silico des séquences du génome de 66 souches de Xam et une de Xc, nous avons pu sélectionner 6 paires d'amorces candidates spécifiques de Xam et six autres pour Xc. Nous avons pu développer un test de PCR-duplex qui a été validé en testant 53 souches de Xam et 25 souches de Xc issues de différents pays, 18 souches non cibles contrôles et cinq souches épiphytes associées au manioc. Cette technique représente un outil utile pour détecter et différencier Xam et Xc provenant de cultures in vitro mais aussi fonctionnelle à partir de tissues de plantes infectés.Deuxièmement, nous avons donc évalué la diversité des populations de Xam à l’aide de l’étude de microsatellites (ou MLVA pour « Multiple loci VNTR analysis »). Une enquête de terrain menée dans six États du Venezuela a permis d'évaluer la présence de la maladie, son statut et nous a permis d'établir une collection de souches de Xam dont l’analyse détaillée de la diversité a pu être réalisée. Au total nous avons isolé 202 souches de Xam issues de six localités situées dans quatre états. À l'aide d'un schéma 14-MVLA, nous avons analysé 12 populations et mis en évidence un indice élevé de la diversité génétique au sein de ces populations et entre elles, et principalement dans l'est du pays.Le développement de ce type de recherche est essentiel pour une gestion raisonnée des cultures dans le monde. Conjugué aux politiques agricoles existantes, il nous permettra de mieux comprendre les agents pathogènes d'importance agricole et les mécanismes impliqués dans leur établissement dans le temps et à travers l’espace. L'objectif à long terme est d'appliquer des mesures de contrôle efficaces et durables, ce qui conduira à des mesures de quarantaine plus efficaces pour prévenir la propagation de la maladie
Cassava (Manihot esculenta L. Crantz) belongs to the group of roots and tubers and is cultivated in the tropics worldwide. This species is a nutritional alternative in many populations where no optimal crop production, general conditions nor technological support exist. Considering its potential uses, the global development of cassava crop has increased significantly but it is still considered a subsistence crop in poor regions lead by smallholder producers. Cassava is affected by biotic and abiotic factors during its life cycle, which heavily limiting its optimal performance. A variety of pests and diseases are known to affect cassava production. Among them are those caused by Xanthomonas axonopodis pv. manihotis (Xam) and Xanthomonas cassavae (Xc), causal agents of Cassava Bacterial Blight (CBB) and Cassava Bacterial Necrosis (CBN) diseases, respectively. CBB is considered the major bacterial disease that affects cassava crop worldwide which is also the case in Venezuela where it was first reported in the 70s. Since the 90s, studies were conducted to elucidate Xam genetic variability in different regions in the country, by means of different molecular tools available at that time. A high degree of polymorphism among the isolates was reported, whether collected from the same or different fields. The Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin.Our questions deal with the situation of CBB in Venezuela 20 years later : what is the current genetic diversity of Xam populations in Venezuela? what is the genetic structure of Xam populations and how do they differ with respect to Xam strains collected in 90s. Moreover, because Xam and Xc cause similar symptoms on cassava leaves and display similar physiological and morphological characteristics, we also aimed at developing a new molecular diagnostic tool allowing for fast and reliable detection of Xam and able to discriminate with Xc.To achieve our goals, we first established a duplex-PCR as a molecular detection tool of cassava-infecting xanthomonads. Based on in silico analysis of the genome sequences of 66 Xam and 1 Xc strains, we were able to select 6 Xam and 6 Xc primers pairs candidates, of which one set of primers for each was selected for further studies. We were able to develop a duplex-PCR assay that was validated upon testing 53 Xam strains and 25 Xc strains from different countries, 18 non-target strains, and 5 epiphytic strains associated to cassava, proving this technique a useful tool to detect and differentiate Xam and Xc from in vitro cultures and in planta.Secondly we assessed the diversity of Xam populations through a variable number of tandem repeat analysis (MLVA). A field survey conducted in six states in Venezuela enabled to evaluate the occurence of the disease, its status and allowed us to establish a strain collection for detailed diversity analysis. We isolated 202 Xam strains from six localities, localized in four states. Using a MVLA14-scheme, we analyzed 12 populations highlighting a high index of genetic diversity among and within populations, mainly in the east of the country.The development of this type of research is essential in the management of crops in the world and coupled with the existing agricultural policies, it will allow us to have a deeper understanding of pathogens of agricultural importance and the mechanisms involved in their establishment over time and across regions. The long-term objective of this is to apply control measures that are effective in time, thus establishing more stringent quarantine measures to prevent the spread of the disease

Tuberculosis (TB) remains one of the world’s leading causes of mortality due to a single infectious agent, with approximately 1.5 million deaths and 9.2 million new cases per year as estimated in 2006. It is assumed that about 5-10% of individuals infected with M. tuberculosis develop TB and the remaining 90-95% contain M. tuberculosis through their immune systems, but have a latent tuberculosis infection (LTBI). To effectively control TB, it is essential to detect individuals with LTBI and to reliably diagnose active TB. Conventional TB diagnosis continues to rely on smear microscopy and culture that have several known limitations in terms of both speed and sensitivity that delay the diagnosis and, consequently, hold-up TB treatment and increase the spread of infection in the community. M. tuberculosis infection remains widespread, but the disease is generally limited to the primary infection stage. Patients with an immune defect or impaired immunity are more prone to develop the disease. In LTBI, the host immune response is capable of controlling the infection by the release of chemokines and cytokines produced by T helper (Th) cells, critical for the outcome of the infection. Several cells of the immune system are involved in the control of TB, from the macrophages and dendritic cells, called antigen presenting cells (APC) to the T cells, CD4, CD8, gamma delta T cells. Activation of these cells with excessive pro inflammatory responses can lead to tissue damage, with the need of mechanisms to counteract this, such as Th2 and T regulatory cells (Treg)-mediated responses. The optimal scenario would therefore seem to have balanced Th1, Th2 and Treg response, suited to the immune challenge. The balance between these types of response is reflected in the resultant host resistance against infection. Therefore the aims of the thesis were to find new approaches for diagnosis of active TB (First Part) and LTBI (Second Part). In this work we wanted to explore the immune mechanisms of TB pathogenesis with particular focus on the impact of Treg on suppressing M. tuberculosis-specific response (Third Part). For the diagnosis of active TB, we describe an alternative PCR methodology based on the amplification of small DNA fragments, originated from cells dying throughout the body (transrenal DNA; Tr-DNA) and detected in urine. It was found that small M. tuberculosis DNA fragments were specifically detected in the cell-free fraction of urine specimens from pulmonary TB patients. To detect LTBI, we compared the performances of two short-incubation interferon (IFN)-g release assays (IGRAs), the commercial QuantiFERON TB-Gold and the in-house whole blood stimulation with region of difference (RD)-1 proteins, with those of a 7-day whole blood stimulation and tuberculin skin test (TST). In an effort to find new markers for LTBI diagnosis, we also evaluated the production of pro-inflammatory cytokines [interleukin (IL)-1, IL-2, IL-6 and Tumor Necrosis Factor (TNF)-alfa], anti-inflammatory cytokines (IL-4, IL-10, IL-13) and chemokines [inducible protein (IP)-10, Macrophage Inflammatory Protein (MIP)-alfa, MIP-1beta, IL-8] after specific stimulation. The results raise the hypothesis that short-incubation IGRAs mainly detect recent or ongoing infection with M. tuberculosis, while prolonged-incubation IGRAs seem to be more sensitive for the diagnosis of past latent infection. Moreover we found that IL-2 and IP-10 may be additional markers for TB infection after RD1 specific stimulation. Finally we wanted to evaluate the impact of Treg on suppressing M. tuberculosis-specific response. Using classical markers for Treg recognition, discordant results were found in terms of Treg expansion during active TB disease. Recently CD39 has been shown to be an accurate marker for Treg detection. Objectives of this part of the thesis were: 1) to identify Treg expressing CD39 in patients with TB and to compare the results with those obtained by the standard phenotypic markers; 2) to evaluate if Treg are expanded in vitro by exogenous IL-2 or by antigen-specific stimulation; 3) to characterize Treg function on the modulation of antigen-specific responses. In this study we demonstrated that CD39 is a useful marker to detect Treg because within CD4+CD25high cells, it identifies a cell subset characterized by high production of transforming growth factor (TGF)-beta1 and the absence of IFN-gamma expression. Moreover, we showed that CD39+ Treg are expanded by M. tuberculosis-specific stimulation in patients with active TB disease.

Improved diagnosis, prognosis and disease follow-up is a fundamental procedure and a constant challenge in medicine.  Among the different molecular biomarkers, proteins are the essential regulatory component in blood; hence, by developing enhanced specific and sensitive molecular tools will gives great insight into the different processes in disease treatment.  In this thesis, we build on the proximity ligation assay to develop and apply new adaptable methods to facilitate protein detection. In paper I, I present a variant of the proximity ligation assay (we call PLARCA) using micro titer plate for detection and quantification of protein using optical density as readout in the fluorometer. PLARCA detected femtomolar levels of these proteins in patient samples, which was considerably below the detection threshold for ELISA. In paper II, we developed and adapted a new method into the in situ PLA methods for detection and identification of extracellular vesicles (EVs) using flow cytometry as readout (a method we call ExoPLA).  We identified five target proteins on the surface of the Evs and using three colors, we identified the EV using flow cytometer. In paper III, we aim to improve the efficiency of in situ PLA by creating and developing new designs and versions of the assay we called Unfold probes Through comparison of detection of protein using in situ PLA versus Unfold probes, we observed considerable decrease in non-specific signals, and also a lower detection threshold. In paper IV, we describe the development of a solid phase proximity extension (sp-PEA) assay for protein detection and quantification. We compared detection of IL-8, TNF-alpha, IL-10 and IL-6 using spPEA and PEA; spPEA demonstrations over 2 orders of magnitudes in the lower detection concentrations by decreased in background noise.

La brucellose est une zoonose causée par le genre bactérien Brucella (B.) dont l’incidence mondiale est estimée à 500 000 cas humains par an. Le réservoir est animal, touchant principalement les espèces de rente. Les espèces les plus importantes pour l’Homme sont B. melitensis, B. abortus et B. suis qui partagent plus de 90% d’identité de séquence. Bien qu’elles soient très apparentées sur le plan génétique, elles présentent une diversité de caractéristiques phénotypiques, de préférence d’hôte et de pathogénicité. L’homogénéité génétique de ces espèces peut apparaître comme un atout pour le développement d’outils de diagnostic universels robustes. En revanche, il s’agit d’un challenge pour les distinguer, rendant difficile la caractérisation précise des isolats issus d’un même foyer. Dans le cadre de cette thèse, un outil de diagnostic moléculaire de PCR en temps réel ciblant le genre Brucella a été développé et optimisé. L’outil a été évalué sur des prélèvements de lait de ruminants, ces prélèvements peuvent être une source importante de Brucella et peuvent être utiles au dépistage de la maladie à l’échelle du troupeau. Basée sur la détection de l’élément d’insertion IS711, une séquence présente en plusieurs exemplaires dans le génome, cette méthode affiche des valeurs de sensibilité et de spécificité qui la rendent intéressante pour un schéma global de lutte contre la brucellose. D’autre part, en vue d’améliorer la compréhension de la stabilité génétique de B. melitensis, un panel original de souches isolées dans le cadre d’un foyer et impliquant 4 espèces d’hôtes différentes a été comparé. Ainsi à l’aide de différentes approches complémentaires, leurs séquences génomiques, les caractères phénotypiques ainsi que leurs comportements dans un modèle in vitro ont été comparés. Nos résultats n’ont pas mis en évidence marqueurs qui laisserait à penser que des mutations dans le génome soient indispensables pour s’adapter à un nouvel hôte
Brucellosis is a zoonotic disease caused by the bacterial genus Brucella (B.), whose global incidence is estimated at 500,000 human cases per year. The reservoir is animal, affecting mainly livestock. The most important species for humans are B. melitensis, B. abortus and B. suis, which share more than 90% sequence identity. Although highly genetically related, Brucella spp. exhibit a variety of phenotypic characteristics, host preference and pathogenicity. The genetic homogeneity of these species may appear as an asset for the development of robust universal diagnostic tools. On the other hand, it is a challenge to distinguish them, making it difficult to precisely characterize isolates from the same outbreak. As part of this thesis, a real-time PCR molecular diagnostic tool targeting the genus Brucella was developed and optimized. The method has been evaluated on ruminant milk samples; these samples may be an important source of Brucella and may be useful for herd-scale disease screening. Based on the detection of the IS711 insertion element, a sequence present in several copies within the genome, this method displays sensitivity and specificity values that make it interesting for a global scheme to fight against brucellosis. On the other hand, in order to improve the understanding of the genetic stability of B. melitensis, an original panel of strains isolated in an outbreak and involving four different host species was compared. Thus, using different complementary approaches, their genomic sequences, phenotypic characteristics and their behavior in an in vitro model were compared. Our results did not highlight markers that would suggest that mutations in the genome are essential to adapt to a new host

Mestrado em Biomedicina Molecular
Nowadays it is still difficult to perform an early and accurate diagnosis of dementia, therefore many research focus on the finding of new dementia biomarkers that can aid in that purpose. So scientists try to find a noninvasive, rapid, and relatively inexpensive procedures for early diagnosis purpose. Several studies demonstrated that the utilization of spectroscopic techniques, such as Fourier Transform Infrared Spectroscopy (FTIR) and Raman spectroscopy could be an useful and accurate procedure to diagnose dementia. As several biochemical mechanisms related to neurodegeneration and dementia can lead to changes in plasma components and others peripheral body fluids, blood-based samples and spectroscopic analyses can be used as a more simple and less invasive technique. This work is intended to confirm some of the hypotheses of previous studies in which FTIR was used in the study of plasma samples of possible patient with AD and respective controls and verify the reproducibility of this spectroscopic technique in the analysis of such samples. Through the spectroscopic analysis combined with multivariate analysis it is possible to discriminate controls and demented samples and identify key spectroscopic differences between these two groups of samples which allows the identification of metabolites altered in this disease. It can be concluded that there are three spectral regions, 3500-2700 cm -1, 1800-1400 cm-1 and 1200-900 cm-1 where it can be extracted relevant spectroscopic information. In the first region, the main conclusion that is possible to take is that there is an unbalance between the content of saturated and unsaturated lipids. In the 1800-1400 cm-1 region it is possible to see the presence of protein aggregates and the change in protein conformation for highly stable parallel β-sheet. The last region showed the presence of products of lipid peroxidation related to impairment of membranes, and nucleic acids oxidative damage. FTIR technique and the information gathered in this work can be used in the construction of classification models that may be used for the diagnosis of cognitive dysfunction.
Atualmente, não é possível fazer um diagnóstico precoce e diferencial da doença de Alzheimer, deste modo, é necessário encontrar biomarcadores que o permitam. Para isso, os cientistas tentam encontrar um procedimento nãoinvasivo, rápido, e relativamente barato. Os resultados de vários estudos demonstraram que a utilização de técnicas espectroscópicas, tais como a Espectroscopia de Infravermelho Transformada de Fourier (FTIR) e / ou espectroscopia de Raman, podem ser ferramentas úteis para diagnosticar a DA. Uma vez que, na DA, alguns mecanismos bioquímicos podem levar a mudanças em componentes do plasma, podem então ser utilizadas amostras de sangue nas análises espectroscópicas o que torna a técnica simples e menos invasiva. Com este trabalho pretende-se confirmar algumas das hipóteses de estudos anteriores em que o FTIR foi usado no estudo de amostras de plasma de possíveis doentes com DA e respetivos controlos e verificar a reprodutibilidade desta técnica espectroscópica na análise deste tipo de amostras. Através da análise espectroscopia combinada com análise multivariada é possível discriminar as amostras controlos e dementes e identificar as principais diferenças espectroscópicas entre estes dois grupos de amostras que permitem identificar os metabolitos alterados nesta patologia. Pode-se concluir que existem três regiões espectrais, 3500-2700 cm-1, 18001400 cm-1 e 1200-900 cm-1 onde se pode extrair informação espectroscópica relevante. Na primeira região, a principal conclusão que é possível tirar é que há um desequilíbrio entre o teor de lípidos saturados e insaturados. Na região entre 1800-1400 cm-1, é possível observar a presença de agregados de proteínas e a alteração na conformação das proteínas para folha β paralela altamente estável. A última região revelou a presença de produtos de peroxidação lipídica relacionados com a insuficiência de membranas, e danos oxidativos nos ácidos nucleicos. A técnica de FTIR e a informação reunida neste trabalho pode ser utilizada na construção de modelos de classificação que possam vir a ser utilizados para o diagnóstico de disfunções cognitivas.

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.

La tuberculose est une maladie ancienne et ré-émergente qui constitue un véritable problème de santé publique dans le monde. L’émergence de la tuberculose à souches de M. tuberculosis multirésistantes et ultrarésistantes aux antituberculeux en plus de la pandémie du VIH/Sida, représentent un défi majeur dans la lutte contre la tuberculose pour son contrôle et son élimination. Ce contrôle de la tuberculose nécessite des mesures en santé publique et au niveau de l’individu. Ces mesures concernent la disponibilité et l’accessibilité à des tests de diagnostic rapides, des traitements efficaces et des outils de surveillance et de contrôle.Nos travaux concernent la recherche, le développement et la validation de méthodes moléculaires multiplexées, souvent basées sur le polymorphisme des loci CRISPR (Clustered Regularly Interspersed Palindromic Repeats). Elles sont rapides, à haut débit, moins onéreuses et applicables pour la santé publique (transmission de la tuberculose sensible et multirésistante, évaluation des programmes nationaux de tuberculose) mais aussi pour un meilleur diagnostic dans l’intérêt du patient (antibiogramme moléculaire, identification infra-spécifique). C’est ainsi que nous avons développé et validé le spoligoriftyping (méthode de génotypage combiné à la détection moléculaire de la résistance de M. tuberculosis à la rifampicine), le “TB-SPRINT” (Spoligoriftyping plus la détection moléculaire de la résistance à l’isoniazide) et le sous-typage de M. africanum. Ces différentes méthodes, aux performances (sensibilité/spécificité) satisfaisantes (99/100% pour le spoligoriftyping, 95/100% en moyenne pour le “TB-SPRINT”) ont servi à des études d’épidémiologie moléculaire dans des pays comme le Pakistan, le Nigéria et le Brésil. D’autres travaux en cours portent sur le génotypage basé sur les CRISPR d’autres espèces (Salmonella enterica, Legionella pneumophila) et sur des études de génomique comparative. Nos tests, utilisés en routine, replacent le laboratoire au cœur de la lutte anti-tuberculeuse et permettront d’importantes avancées en Santé Publique et Microbiologie médicale et environnementale
Tuberculosis (TB) remains a major public health concern worldwide despite all the efforts to fight this disease. The emergence of multi drug and extensively drug resistant TB and the pandemic of HIV/AIDS constitute major threats and challenge for the TB control and eradication. TB control requires measures in public health and in individual level as accessibility to tests for early diagnostic, effective treatment and tools for tuberculosis surveillance and control.The goals of this work were research, development and validation of new molecular multiplexed methods based on polymorphism of the CRISPR (Clustered Regularly Interspersed Palindromic Repeats) loci and single nucleotides polymorphisms. These methods are rapid, high throughput, cheap and can be applied both for public health purposes (transmission of susceptible and multi-drug resistant tuberculosis, evaluation of national TB programs) as for interest of TB patient (drug resistance testing, infra-specific identification). Thus we developed spoligoriftyping and “TB-SPRINT” tests that allow genotyping and rifampicin or rifampicin and isoniazide resistance detection. Another test was developed for subtyping of M. africanum. All these methods had high performances (sensitivity/specificity), 99/100% for the spoligoriftyping and about 95/100% for the “TB-SPRINT” and were applied for molecular epidemiology studies of countries as Nigeria, Brazil and Pakistan. Other ongoing work and developments of genotyping methods are the spoligotyping of L. pneumophila and S. enterica and comparative genomics projects.Used in routine, our methods may play key roles in TB control and would allow important advances in Public Health, in medical and environmental Microbiology

Nucleic acids are fundamental molecules of living organisms functioning essentially as the molecular information carriers of life. From how an organism is built to how it responds to external conditions, all of it, can be found in the form of nucleic acid sequences inside every single cell of every life form on earth. Therefore, accessing these sequences provides key information regarding the molecular identity and functional state of any living organism, this is very useful for areas like biomedicine, where accessing and understanding these molecular signatures is the key to develop strategies to understand, treat and diagnose diseases. Decades of research and technological advancements have led to the development of a number of molecular tools and engineering technologies that allow accessing the information contained in the nucleic acids. This thesis provides a general overview of the tools and technologies available for nucleic acid analysis, and proposes an illustrative concept on how molecular tools and emergent technologies can be combined in a modular fashion to design methods for addressing different biomedical questions. The studies included in this thesis, are focused on the particular use of the molecular tools named: padlock and selector probes, rolling circle amplification, and fluorescence detection of single molecules in combination with microfluidics and portable microscopy. By using this combination, it became possible to design and demonstrate novel approaches for integrated nucleic acid analysis, inexpensive digital quantification, mobile-phone based diagnostics and the description of viral infections. These studies represent a step forward towards the adoption of the selected group of tools and technologies, for the design and building of methods that can be used as powerful alternatives to conventional tools used in molecular diagnostics and virology.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 1: Manuscript.

Les vaques de llet sofreixen pèrdues de calci (Ca) quan comença la lactació i poden veure’s afectades per la hipocalcèmia clínica (sèrum Ca < 6mg/dL) o subclínica (Ca < 8.5mg/dL). Donat que la incidència clínica és del 5%, el problema més rellevant es troba en els casos subclínics (SCHC) perquè tenen una prevalença més elevada. Aquests casos no poden ser tractats degut a la falta d’eines de diagnosi. Per tal d’estudiar l’impacte, les conseqüències i la regulació de SCHC, i per desenvolupar estratègies de diagnòstic i de prevenció, es van dur a terme quatre estudis. Al primer estudi, es va avaluar l’associació de SCHC amb vàries malalties del peripart. El 75% de les vaques tenien SCHC. El desplaçament d’abomàs, la ketosi, la retenció de placenta i la metritis van ser més propenses a trobar-se en vaques amb SCHC. Les vaques afectades tenien una producció de llet més elevada. El zel apareix abans en vaques normocalcèmiques. A més, la gravetat de les diferents malalties està relacionada amb la intensitat de la SCHC. Per entendre els mecanismes involucrats, un segon estudi es va dur a terme per clarificar els papers de la calcitonina a l’inici de la malaltia i a la prevenció de la hipocalcèmia sota acidosi metabòlica. Una pujada de calcitonina en vaques amb SCHC greu després de parir perjudica la recuperació del Ca sanguini perquè la resposta del receptor de la PTH (PTHR) no és suficient com per activar la vitamina D i compensar l’efecte de la calcitonina. L’acidosi metabòlica prevé la hipocalcèmia perquè l’expressió de PTHR s’incrementa al ronyó. A més, l’activitat de la calcitonina es veu perjudicada a pHs baixos i això incrementa el rol hipercalcèmic de la PTH. Basat en aquest fet, al tercer estudi es va intentar una estratègia per prevenir la hipocalcèmia basada en immunització passiva contra calcitonina. Anticossos policlonals van neutralitzar la calcitonina in vitro i a un model de rata, augmentant la concentració de Ca en sang. Un mètode assequible per produir en massa va ser derivat d’una naïve phage library de ScFv. El ScFv B10 va reconèixer i neutralitzar la calcitonina in vitro, però no va afectar els nivells de Ca en sang quan es va administrar en vedells o vaques. Més estudis seran necessaris per entendre les dificultats de l’estratègia proposada. Un sistema de diagnòstic seria molt útil per identificar i tractar vaques hipocalcèmiques. En el quart i últim estudi, es va desenvolupar un sistema analític semiautomàtic i portable basat en transistors de camp ió-selectius amb membranes fotocurables selectives per ions de Ca2+. Aquest sensor determina concentracions de Ca en sang de boví de forma ràpida i fiable i pot ser aplicat en mesures en vaques al camp.
Dairy cows suffer blood calcium (Ca) losses as lactation begins and might be affected by hypocalcemia in its clinical (serum Ca < 6mg/dL) or subclinical state (serum Ca < 8.5mg/dL). Since clinical incidence is only about 5%, the most relevant problem concerns subclinical cases (SCHC) because of a higher prevalence. These cases cannot be treated due to the lack of diagnostic tools. In order to study the impact, consequences and regulation of SCHC and to develop preventive and diagnostic strategies, four studies were conducted. In the first study, the association of SCHC with several periparturient diseases was evaluated. Seventy five percent of cows incurred SCHC. Displaced abomasum, ketosis, retained placenta, and metritis were more likely to happen in cows with SCHC. Affected cows had a greater milk production. Normocalcemic cows showed their first heat sooner. Also, the severity of SCHC is related to the severity of the different periparturient diseases. In order to understand the exact mechanisms involved, a second study was performed to clarify the potential roles of calcitonin in the onset of SCHC and in the prevention of hypocalcemia under metabolic acidosis. A calcitonin rise in severe SHCH cows after calving impairs the recovery of blood Ca because PTH receptor (PTHR) response is not sufficient to activate vitamin D and compensate the calcitonin effect. Metabolic acidosis prevents hypocalcemia because the expression of PTHR is up-regulated in kidney. Moreover, an impairment of calcitonin activity at low pH enhances the hypercalcemic role of PTH. Based on this calcitonin role, in the third study an approach to prevent hypocalcemia through passive immunization against calcitonin was tested. Polyclonal antibodies neutralized calcitonin in vitro and in a rat model, raising the blood Ca concentration. An affordable method of mass-production was designed from a naïve ScFv phage library. The ScFv B10 recognized and neutralized calcitonin in vitro, but it did not affect blood Ca levels when administered to cattle requiring further research to understand the main difficulties of the proposed strategy. A diagnostic system would be very useful to identify and treat hypocalcemic cows. In the fourth and last study we developed a portable semiautomatic analytical system based on ion-selective field effect transistors with Ca2+ ion selective photocurable membranes. This sensor determines bovine serum calcium concentration in a reliable and fast way and can be applied in the field in cow-side measurements.

Accurate smoltification and disease management in Atlantic salmon (Salmo salar) are key issues for the aquaculture industry. Due to their anadromous lifecycle the transfer of salmon from fresh water (FW) to seawater (SW) is crucial to their survival; too early can cause mortality, too late can cause desmoltification and long-term health problems. Both scenarios can increase susceptibility to four viral diseases: Salmon alphavirus (SAV), Infectious salmon anemia virus (ISAV), orthoreovirus (PRV), and Piscine myocarditis virus (PMCV). They all show similar clinical and histopathological symptoms and can easily spread throughout farms. Understanding the initial innate immune response to these viruses may provide biomarkers that could help identify and monitor infections. An in house and onsite Na+/K+ ATPase (NKA) qRT-PCR assay was developed for the salmon biomarker ATPase to test smoltification readiness in salmon smolts. Tested against NKA enzymatic assays it showed a similar success rate over 3 years: NKA qRT-PCR (57%), NKA activity assay (60%). Onsite tests confirmed that the ATPase mRNA transcript is a useful biomarker for smoltification detection. An in-lab and mobile multiplex qRT-PCR assay was developed for detection of SAV, PRV and PMCV. The analytical sensitivity of the SAV (86.5% SE 0.11), PRV (90.94%, SE 0.09) and PMCV (100.46%, SE 0.19) assays was 102 copies for PMCV and 103 for SAV and PRV. Initial results suggest individual assays could be run on site at farms. Addition of an internal control, probit analysis and viral positive tests are still required for multiplex assay integration. Salmon erythrocytes were infected with ISAV, SAV and Poly I:C to investigate whether they induce and up-regulate innate immune response genes. All genes were expressed at low levels in all parameters investigated including non-infected control erythrocytes. These findings suggest erythrocytes act as an initial buffer to viral infections and may help stimulate the innate immune response.

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo. Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase. This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Thesis submitted in fulfilment of the requirements for the degree Master of Technology: Environmental Health in the Faculty of Applied Sciences at the Cape Peninsula University of Technology, 2013
Water is an important daily requirement and in a clean, pure form, it promotes health and well-being. In addition to South Africa being one of the driest countries in the world, water availability is also being compromised by massive pollution of remaining water sources. The Berg- and Plankenburg Rivers are two of the surface water sources in the Western Cape, South Africa, which are highly polluted by sewage, industrial and agricultural run-off. The current investigation was aimed at comparing diagnostic tools, which are employed by municipalities and food industries, and molecular based techniques to routinely monitor water for indicator organisms in time- and cost-effective manner. These rivers were sampled twice a month (July 2010 to January 2011) at the sites closest to the informal settlements of Kayamandi in Stellenbosch (Plankenburg River) and Mbekweni in Paarl (Berg River). The contamination levels of the two river systems were evaluated by the enumeration of Escherichia coli and coliforms using the Colilert 18® system, Membrane Filtration (MF) and Multiple Tube Fermentation (MTF) techniques. The highest faecal coliform count of 9.2 × 106 microorganisms/100 ml was obtained in weeks 21 and 28 from the Plankenburg River system by the MTF technique, while the lowest count of 1.1 × 103 microorganisms/100 ml was obtained in week one for both river systems by the MTF technique. The highest E. coli count of 1.7 × 106 microorganisms/100 ml was obtained from the Berg River system (week 9) using the MTF technique, while the lowest count of 3.6 × 102 microorganisms/100 ml was obtained by the MF technique from the Plankenburg River system. The coliform and E. coli counts obtained by the enumeration techniques thus significantly (p > 0.05) exceeded the guidelines of 2000 microorganisms/100 ml stipulated by the Department of Water Affairs and Forestry (DWAF, 1996) for water used in recreational purposes. Overall the results obtained in this study showed that the water in the Berg- and Plankenburg River systems is highly polluted, especially where these water sources are used for irrigational and recreational purposes. For the coliform and E. coli counts obtained using the three enumeration techniques, it was noted that the MTF technique was more sensitive and obtained higher counts for most of the sampling weeks. However, the media (Membrane lactose glucuronide agar) used in the MF technique also effectively recovered environmentally stressed microbial cells and it was also better for the routine selection and growth of coliforms and E. coli. While E. coli and total coliforms were detected utilising the Colilert 18® system, accurate enumeration values for these two indicator groups was not obtained for the entire sampling period for both river systems. It has previously been shown that dilutions (up to 10-3) of highly polluted waters increase the accuracy of the Colilert 18® system to enumerate colifoms and E. coli in marine waters. As the results obtained utilising the Colilert 18® system were also not comparable to the MF and MTF techniques it is recommended that highly polluted water samples be diluted to increase the accuracy of this system as a routine enumeration technique. Water samples were directly inoculated onto MacConkey, Vile Red Bile (VRB) agar and the Chromocult Coliform agar (CCA) and single colonies were inoculated onto nutrient agar. Chromocult coliform agar proved to be more sensitive than MacConkey and VRB agar for the culturing of E. coli and coliforms. Preliminary identification of these colonies was done using the RapID ONE and API 20 E systems. The most isolated Enterobacteriaceae species by both systems, included Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli and Enterobacter cloacae in both river systems. The API 20 E system was more sensitive in the preliminary identification of the various isolates, as greater species diversity was obtained in comparison to the RapID ONE system. The Polymerase Chain Reaction (PCR) was firstly optimised using positive Enterobacteriaceae species. The optimised method was then applied to the analysis of river water samples, which were centrifuged to harvest the bacterial cells, with DNA extracted using the boiling method. The extracted DNA was amplified using conventional PCR with the aid of species specific primers. The Enterobacteriaceae species that were detected throughout the study period in both river systems include Serratia marcescens, Escherichia coli, Klebsiella pneumoniae and Bacillus cereus. Conventional PCR was the most reliable and sensitive technique to detect Enterobacteriaceae to species level in a short period of time when compared to RapID ONE and the API 20 E systems. Multiplex PCR was optimised using the positive pathogenic E. coli strains namely, Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (EIEC), Enterohaemorrhagic E. coli (EHEC) and Enteroaggregative E. coli (EAEC). It was then employed in river water sample analysis and enabled the detection of EAEC, EHEC, and EIEC strains in Berg River system, with only the EAEC detected in the Plankenburg River system. Real-time PCR was used to optimise the multiplex PCR in the amplification of E. coli strains and successfully reduced the time to obtain final results when using control organisms. Real-time PCR was found to be more sensitive and time-effective in the identification of E. coli strains, and also more pronounced DNA bands were observed in real-time PCR products compared to conventional-multiplex PCR amplicons. To sustain the services provided by the Berg- and Plankenburg Rivers in the Western Cape (South Africa), these water sources should frequently be monitored, results assessed and reported according to the practices acknowledged by responsible bodies. It is therefore recommended that the enumeration techniques be used in conjunction with the very sensitive PCR technique for the accurate detection of coliforms and E. coli in river water samples.

Barrett's oesophagus is a complication of gastro-oesophageal reflux disease and is the single most important predisposing factor for the development of adenocarcinoma of the oesophagus. New molecular markers are needed for early diagnosis and to monitor disease progression. Telomerase is a ribonuclear protein with reverse transcriptase activity, which uses its own RNA component as a template for the addition of telomeric repeats to the end of chromosomes. Telomerase activity has been studied during the neoplastic progression of Barrett's oesophagus using a TRAP based ELISA technique, which found telomerase to be reactivated early during . disease progression. A non-isotopic method of in situ hybridisation for the detection of the RNA component of telomerase has also been successfully developed. Plasminogen activation is an inducible extracellular proteolytic system involved in the regulation of cellular interactions and invasion. The components of the urokinase-type Plasminogen Activator system have been fully investigated during the progression of Barrett's oesophagus to adenocarcinoma utilising immunohistochemistry and ELISA techniques. Changes in the expression of this invasive phenotype were found to occur late during disease progression in malignant tissues. Two-oesophageal cell-lines have been characterised using molecular biological techniques to detect a range of molecular markers to produce ex vivo models of oesophageal adenocarcinoma and oesophageal squamous cell carcinoma. In order to assess the effects of bile salts and acidity on oesophageal tissues these celllines were then utilised as ex vivo models. Exposure to acidic conditions both alone and with bile salts altered the morphological appearance of the cells and disrupted adhesion molecules in the cellular membrane. Investigations into both telomerase reactivation and the plasminogen activator system have provided new information concerning the nature and timing of molecular changes during the Barrett's metaplasia/dysplasia/adenocarcinoma sequence.

Fasciolosis caused by infection with Fasciola hepatica and Fasciola gigantica is a zoonotic disease of worldwide importance, with an estimated 91 million people considered at risk of infection and livestock losses expected to exceed US $3 billion/year. Despite the significant human and animal health impacts, no test is capable of ante-mortem Fasciola spp. differentiation in areas of parasite sympatry. The aim of this thesis was to design, validate and deploy a suite of highly sensitive molecular diagnostic tools for Fasciola spp. differentiation from faecal samples to enable ante-mortem screening of livestock in Northern Laos. In combination with a traditional sedimentation method and with the use of a high-speed benchtop homogeniser, the detection and quantification of Fasciola spp. infection in 100% of cattle with low faecal egg loads (<25 EPG) was possible. The point of first detection of F. hepatica infection in experimentally-infected sheep was then examined and compared to the sedimentation and a commercially-available coproantigen ELISA. Faecal samples were first considered positive at 6, 7 and 8 weeks post infection (WPI) by coproELISA, real-time PCR and sedimentation, respectively. A simplified method using un-concentrated faecal samples was developed to increase sample throughput. The limit of detection using this method was 10 and 20 EPG for sheep and cattle, respectively. Finally, several single nucleotide polymorphism assays were developed to differentiate Fasciola spp. in faecal samples alongside a Next Generation Sequencing method to determine the contribution of nucleotides from each species. These assays were applied to 153 faecal samples collected from local cattle across 27 villages in Northern Laos to detect F. hepatica translocation in an area of SE Asia with frequent international livestock trade. Of the 91 positive samples, 11 were identified as containing F. hepatica DNA, indicating establishment of the F. hepatica lifecycle in Northern Laos.

Ixodídeos (carraças de corpo-duro), são importantes vetores de agentes patogénicos, responsáveis por doenças emergentes como a doença de Lyme (DL). Esta zoonose é causada por espiroquetas do complexo Borrelia burgdorferi sensu lato (s.l.) transmitidas por carraças da espécie Ixodes ricinus, o principal vetor na Europa. A DL é uma doença multisistémica com diversas apresentações clínicas e diagnóstico complexo. Em Portugal é ainda pouco diagnosticada e a notificação, apesar de obrigatória, é escassa. A presente investigação teve como principal objetivo desenvolver ferramentas moleculares, nomeadamente um algoritmo de PCR em tempo real e uma amplificação isotérmica, para identificar as espécies de B. burgdorferi s.l. mais prevalentes em Portugal. O desenvolvimento deste objetivo permitiu também avaliar as características bioecológicas da ixodofauna presente em nove distritos do país (norte, centro e sul) nos quais o vetor I. ricinus havia sido anteriormente reportado, e ainda determinar a taxa de infeção por B. burgdorferi s.l. no vetor e hospedeiros. Os resultados obtidos são apresentados sob a forma de artigos científicos (dez), dos quais sete estão publicados, dois em submissão e um em preparação. Do conjunto dos resultados alcançados, importa realçar as variações observadas na distribuição das carraças para possivelmente para novas regiões, provavelmente relacionadas com alterações ao nível da paisagem, clima e vegetação, às quais as carraças são muito sensíveis. Acresce o facto de várias espécies de B. burgdorferi s.l. terem sido detetadas em carraças que não aquelas até agora reconhecidas como vetores. A espécie B. lusitaniae foi a mais prevalente no vetor, estando este presente em seis dos nove distritos selecionados. Surpreendentemente no decurso deste estudo, identificaram-se três espécies do complexo Borrelia recorrente em carraças da vegetação, nomeadamente, B. miyamotoi numa ninfa I. ricinus, e duas possíveis ‘novas’ espécies do complexo Borrelia recorrente em carraças da espécie Haemaphysalis punctata e Rhipicephalus sanguineus. Paralelamente, foi identificado DNA de B. burgdorferi s.l. em amostras biológicas de animais de estimação (cães e gatos) e de animais silváticos (javalis), confirmando a importância destes animais, principalmente os de estimação, enquanto sentinela para uma deteção precoce da DL, ajudando na determinação do risco de transmissão das espiroquetas de B. burgdorferi s.l. a humanos e outros animais com importância económica (ex. bovinos), em áreas geográficas restritas. Foi também possível otimizar dois protocolos moleculares para o diagnóstico laboratorial da DL, um dos quais, um algoritmo de PCR em tempo real que permite a identificação de quatro das espécies de B. burgdorferi s.l. mais prevalentes em Portugal, apresentando elevada sensibilidade e especificidade e contribuindo para um diagnóstico mais preciso da DL. Alguns dos aspetos introduzidos e explorados nesta tese necessitam ainda de uma investigação mais detalhada. No entanto, este trabalho alerta para a introdução de possíveis ‘novas’ espécies do complexo de Borrelia recorrente em carraças de corpo-duro da vegetação, cuja patogenicidade é ainda desconhecida, mas que poderão tornar-se um risco para a saúde pública; contribui para a atualização espacial de áreas importantes na ecoepidemiologia da DL em Portugal; e inova no diagnóstico molecular desta zoonose, constituindo um valioso suporte para os clínicos, permitindo uma terapêutica mais direcionada dos doentes.
Ixodids (hard-body ticks), are the most important vectors of pathogenic agents, responsible for emerging diseases like Lyme disease (LD). This zoonosis is caused by spirochetes of Borrelia burgdorferi sensu lato (s.l.) complex, transmitted by Ixodes ricinus tick, the main vector in Europe. LD is a multisystem disorder with different clinical presentations, and a complex diagnosis. In Portugal is still underdiagnosed and the notification, although mandatory, is scarce. The main goal of this research was to develop molecular protocols, namely a TaqMan realtime PCR algorithm and an isothermal amplification, for the identification of the most prevalent genospecies of B. burgdorferi s.l. in Portugal. The development of this goal, also allowed to evaluate the bio-ecological characteristics of the ixodofauna in nine districts of the country (north, center and south), where the presence of I. ricinus tick was previously reported, and also to determinate B. burgdorferi s.l. infection rate in the vector and hosts. The obtained results are presented in 10 scientific papers, from which seven are published, two in submission and one in preparation. Of all the achieved results, is important to highlight the variations in the distribution of ticks possible into new regions, probably related to changes in the landscape, climate and vegetation, to which ticks are very sensitive. Moreover, several B. burgdorferi s.l. species were detected in ticks, apart from those commonly recognized as vectors. B. lusitaniae species was the most prevalent species in the vector, which was collected in six of the nine selected districts. Unexpectedly, during this study, three species from Relapsing Fever Borrelia complex were identified in questing ticks, namely B. miyamotoi in an I. ricinus nymph, and two possible new Relapsing Fever like-Borrelia in Haemaphysalis punctata and Rhipicephalus sanguineus tick species. Alongside, B. burgdorferi s.l. DNA was identified in biological samples from pets (dogs and cats) and sylvatic animals (wild boars), confirming the importance of these animals, mostly pets, as sentinel for early detection of emerging LD, helping to access the risk of B. burgdorferi s.l. spirochetes transmission to humans and other animals with economic importance (e.g. cattle), in restricted geographical areas. It was also possible to optimized two molecular protocols for LD laboratory diagnosis, one of which, a TaqMan real-time PCR algorithm allowing the identification of four of the most prevalent species of B. burgdorferi s.l. in Portugal, presenting high sensitivity and specificity, and contributing for a more accurate diagnosis of LD. Some of the subjects introduced and developed in this thesis deserve more detailed investigation. However, this work alerts for the introduction of possible ‘new’ Relapsing Fever Borrelia species in questing hard-body ticks, whose pathogenicity is still unknown, but it may become a risk to public health; contributes to the spatial update of important areas for LD eco-epidemiology in Portugal; and innovates in the molecular diagnosis of this zoonosis, being a valuable tool to clinicians, allowing a more accurate therapy of the patients.

The requirements for prompt diagnosis of foot-and-mouth disease (FMD) during outbreaks, and the need to establish robust laboratory testing capacity within FMD-endemic countries, have motivated the development of point-of-care tests (POCTs) to support current diagnostic strategies. Despite numerous publications detailing the design of platforms and assays for this purpose, the majority have only been evaluated in laboratory settings, using protocols incompatible for use in challenging environments. To address this gap, this thesis describes the development of an end-to-end molecular toolbox for the detection and characterisation of FMD virus (FMDV) RNA in decentralised settings. A critical review and multiway comparison of seven assay formats and 11 sample preparation methods revealed that reverse transcription loop-mediated isothermal amplification (RT-LAMP) and real-time reverse transcription PCR (rRT-PCR) POCT-formats exhibited comparable analytical and diagnostic sensitivity to their laboratory-based equivalents. Additionally, reagent lyophilisation provided a solution for cold chain and storage considerations, whilst not compromising assay performance. Both assays were compatible with simple sample preparation methods, removing the requirement for nucleic acid extraction. For example, dilution of samples in nuclease-free water enabled FMDV RNA to be detected in multiple sample types (epithelial tissue suspensions, serum, oesophageal-pharyngeal fluid and lesion swabs), from as early as one day post infection. Notably, when the robust field-ready protocols were deployed into challenging low-resource laboratory and field-settings within East Africa, POCT results (rRT-PCR = 144; RT-LAMP = 145) were consistent with clinical observations and a reference rRT-PCR, with FMDV detected from acutely infected as well as convalescent cattle. Furthermore, transitioning of East Africa-specific FMDV-typing rRT-PCR assays (for serotypes O, A, Southern African Territories [SAT] 1 and SAT 2) into a multiplex POCT-format enabled rapid identification of FMDV serotype in situ, confirming active outbreaks of both O and A. This thesis also describes the development of GoPrime, a novel real-time PCR (rPCR) primer/probe validation tool. By parameterising GoPrime with experimental data, collected to investigate the effects of primer/probe-template mismatches on cycle threshold and limit of detection, it was possible to quantitatively predict the performance of rPCR assays in silico. The work of this thesis supports the deployment of molecular POCTs into non-specialised, resource-limited and challenging settings for simple, highly sensitive and rapid detection and/or characterisation of FMDV.

Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: South Africa is regarded as one of the top wine producing countries in the world. One of the threats to the sustainability of the wine industry is viral diseases of which Grapevine leafroll-associated virus 3 (GLRaV-3) and Grapevine virus A (GVA) are considered to be the most important and wide spread. Scion material is regularly tested for viruses; however scion material is often grafted onto rootstocks that have questionable phytosanitary status. Virus detection in rootstocks is challenging due to low and varying titres, but is imperative as a viral control mechanism. An additional viral control mechanism is the use of transgenic grapevine material which offers resistance to grapevine infection. The objective of this project was to establish a detection system using real time PCR (qPCR) techniques, to accurately and routinely detect GLRaV-3 and GVA in rootstock propagation material. qPCR would furthermore be used to perform molecular characterisation of transgenic plants containing a GLRaV-3 antiviral ΔHSP-Mut construct. A severely infected vineyard (Nietvoorbij farm) in the Stellenbosch area was screened throughout the grapevine growing season to investigate virus prevalence throughout the season and to determine the optimal time for sensitive virus detection. A large scale screening of nursery propagation material for GLRaV-3 infection was also conducted. The qRT-PCR results were compared to DAS-ELISA results to compare the efficacy and sensitivity of the two techniques. For the severely infected vineyard, the ability to detect GLRaV-3 increased as the season progressed towards winter. qRT-PCR was more sensitive and accurate in detecting GLRaV-3 than DASELISA, as the latter technique delivered numerous false positive results later in the season. The best time to screen for GLRaV-3 in the Western Cape region was from the end of July to September. For the nursery screenings, our qRT-PCR results were compared to the results of the DAS-ELISA performed by the specific nurseries. No GLRaV-3 infection was detected in the specific samples received from the two different nurseries. The results for all the samples correlated between the two techniques. This confirms that the propagation material of these nurseries has a healthy phytosanitary status with regards to GLRaV-3. However, the detection of GVA in the severely infected vineyard yielded inconsistent results. Detection ability fluctuated throughout the season and no specific trend in seasonal variation and virus titre fluctuation could be established. The highest percentage of GVA infected samples were detected during September, April and the end of July. Previously published universal primers were used for the detection of GVA, but further investigation indicated that they might not be suitable for sensitive detection of specific GVA variants present in South Africa. Vitis vinifera was transformed with a GLRaV-3 antiviral construct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) and qRT-PCR were utilised as alternative methods for molecular characterisation of transgenic plants. The qPCR and Southern blot results correlated for 76.5% of the samples. This illustrated the ability of qPCR to accurately estimate transgene copy numbers. Various samples were identified during qRT-PCR amplification that exhibited high mRNA expression levels of the transgene. These samples are ideal for further viral resistance studies. This study illustrated that the versatility of real time PCR renders it a valuable tool for accurate virus detection as well as copy number determination.
AFRIKAANSE OPSOMMING: Suid Afrika word geag as een van die top wyn produserende lande ter wereld. Die volhoubaarheid van die wynbedryf word onder andere bedreig deur virus-infeksies. Grapevine leafroll associated virus 3 (GLRaV-3) en Grapevine virus A (GVA) is van die mees belangrike virusse wat siektes veroorsaak in Suid-Afrikaanse wingerde. Wingerd bo-stok materiaal word gereeld getoets vir hierdie virusse, maar hierdie materiaal word meestal geënt op onderstokmateriaal waarvan die virus status onbekend is. Virus opsporing in onderstokke word egter gekompliseer deur baie lae en variërende virus konsentrasies, maar opsporing in voortplantingsmateriaal is ‘n noodsaaklike beheermeganisme vir virus-infeksie. Die doel van die projek was om ‘n opsporingsisteem te ontwikkel via kwantitatiewe PCR (qPCR) tegnieke vir akkurate en gereelde toetsing van GLRaV-3 en GVA in onderstokmateriaal. qPCR sal ook verder gebruik word vir molekulêre karakterisering van transgeniese plante wat ‘n GLRaV-3 antivirale ΔHSP-Mut konstruk bevat. ‘n Hoogs geïnfekteerde wingerd was regdeur die seisoen getoets om seisoenale fluktuasies in viruskonsentrasie te ondersoek en om die optimale tydstip vir sensitiewe virus opsporing te bepaal. ‘n Grootskaalse toetsing van kwekery voortplantingsmateriaal vir GLRaV-3 infeksie was ook uitgevoer. Die qRT-PCR resultate is met die DAS-ELISA resultate vergelyk om die effektiwiteit en sensitiwiteit van die twee tegnieke te vergelyk. Vir die hoogs geïnfekteerde wingerd het die GLRaV-3 opsporing toegeneem met die verloop van die seisoen tot en met winter. qRT-PCR was meer sensitief en akkuraat as DAS-ELISA in die opsporing van GLRaV-3, weens verskeie vals positiewe resultate wat later in die seisoen deur die laasgenoemde tegniek verkry is. Die beste tyd om vir GLRaV-3 te toets is vanaf einde Julie tot September. Tydens die kwekery toetsings was qRT-PCR resultate met die DAS-ELISA resultate van die spesifieke kwekerye vergelyk. Geen GLRaV-3 infeksie was waargeneem in die spesifieke monsters wat vanaf die kwekerye ontvang is nie. Die resultate van die twee tegnieke het ooreengestem vir al die monsters wat v getoets is. Dit het bevestig dat die voortplantingsmateriaal van hierdie kwekerye gesonde fitosanitêre status met betrekking tot GLRaV-3 gehad het. Die opsporing van GVA in die geïnfekteerde wingerd het egter wisselvallige resultate gelewer. Opsporing van die virus het ook regdeur die seisoen gefluktueer en geen spesifieke neiging in seisoenale opsporingsvermoë kon gemaak word nie. Die hoogste persentasie GVA geïnfekteerde monsters was waargeneem tydens September, April en die einde van Julie. Voorheen gepubliseerde universele inleiers was gebruik vir die opsporing van GVA, maar verdere ondersoeke het getoon dat hierdie inleiers nie noodwendig geskik is vir sensitiewe opsporing van GVA variante wat teenwoordig is in Suid-Afrika nie. Vitis vinifera was getransformeer met ‘n GLRaV-3 antivirale konstruct, ΔHSP-Mut. SYBR Green Real time PCR (qPCR) en qRT-PCR was ingespan as alternatiewe metodes vir molekulêre karaterisering van transgeniese plante. Die qPCR en Southern-klad resultate het ooreengestem vir 76.5% van die monsters. Dit illustreer die vermoë van qPCR om akkurate kopie-getalle van transgene te bepaal. Verskeie plante is geïdentifiseer tydens qRT-PCR amplifisering wat hoë vlakke van transgeen mRNA uitdrukking getoon het. Hierdie monsters is ideaal vir verdere virus weerstandbiedendheids studies. Hierdie studie het die veelsydigheid van real time PCR bewys en getoon dat dit ‘n kosbare tegniek is vir akkurate virus opsporing sowel as kopie-getal bepaling.

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

Allogeneic haematopoietic-stem-cell transplantation (HSCT) is the treatment of choice for many malignant and non-malignant disorders. The development of novel strategies such as donor leukocyte infusion, nonmyeloablative HSCT, and cord blood transplantation allowed expanding the indications for allogeneic HSCT over the last several years, especially among older patients. However, the major toxicity of allogeneic HSCT, Graft-versus-Host Disease (GvHD), remains a complication that limits its wider application. Despite advances in post-transplantation immunosuppressive therapy, GvHD remains a major life-threatening post-HSCT complication, developing in a substantial number of patients and resulting in poor outcome. Although in the last three decades the risk of GvHD has been reduced by modifying the transplant program and the stem cell source, yet significant challenges remain. The best hope for continued progress lies in the development of innovative treatments, thanks to a better understanding of GvHD pathogenesis, and in the identification of new easily measurable disease markers able to predict GvHD onset and therapy response. Along these hypotheses, the project comprises two lines of research. 1)The first one is focused on the potential role of Chemerin, an immunoregulatory molecule, in the pathogenesis of GvHD, with the aim to define new diagnostic tools and therapeutic targets for improving the management of post-transplant GvHD. 2)The second line of research is focused on the immunosuppressive factors produced by mesenchymal stromal cells (MSCs), a novel very promising therapy for steroid-resistant GvHD.

博士
國立臺灣大學
高分子科學與工程學研究所
106
In a single-cell study, isolating and identifying single cells are essential, but these processes often require a large investment of time or money. The aim of this study was to isolate and analyse single cells using a novel platform, the PanelChip™ Analysis System, which includes 2500 microwells chip and a digital real-time polymerase chain reaction (dqPCR) assay, in comparison with a standard RT-PCR (qPCR) assay. Through the serial dilution of a known concentration standard, namely pUC19, the accuracy and sensitivity levels of two methodologies were compared. The two systems were also tested on the basis of expression levels of the genetic markers vimentin, E-cadherin, N-cadherin and GAPDH in A549 lung carcinoma cells at two known concentrations. Furthermore, the influence of a known PCR inhibitor commonly found in blood samples, heparin, was evaluated in both methodologies. Finally, mathematical models were proposed and separation method of single cells was verified; moreover, gene expression levels during epithelial–mesenchymal transition in single cells under TGFβ1 treatment were measured. The drawn conclusion is that dqPCR performed using PanelChip™ has good linearity (R^2=0.9974) at 3.4 to 3.4 x 10^8 copies/μL, and it is superior to the standard qPCR in terms of, reproducibility (at 2pg/μL, dqPCR CV:1.97 < qPCR CV:3.94 ; at 200pg/μL , dqPCR CV:1.30 < qPCR CV:8.11) and heparin tolerance (dqPCR IC50: 0.02IU/mL > qPCR IC50: 0.002IU/mL). The dqPCR assay is a potential tool for clinical diagnosis and single-cell applications.

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Renal transplantation has become the treatment of choice for patients with end-stage renal disease. Recognizable improvements in early graft survival and long-term graft function have made kidney transplantation a more cost-effective alternative to dialysis. Moreover, the discovery of new immunosuppressive agents has made a significant impact on short-term graft survival. Despite these improvements a substantial portion of grafts develop progressive dysfunction and fail within a decade by a process known as chronic rejection. Ongoing monitoring of kidney transplants is crucial to avoid the development of this condition. The most common approaches to monitor renal allograft function are the measurement of the serum creatinine levels, whose variations are not specific for rejection and sometimes there is a need to perform renal biopsies, which is a risky process and only diagnoses rejection once it is installed. The main objective of this study was to create an immunological and cellular profile associated with chronic dysfunction establishment that could serve as a diagnostic tool. For this purpose, a series of different analyses were carried out on 71 patients with stable/normal renal function after the transplant and the results compared to the same tests performed on 27 patients who have been diagnosed with chronic graft rejection. The development of an antibody screening method in the urine of renal transplant patients was one of the most relevant points in the study with more than half the patients diagnosed with chronic rejection presenting anti-HLA Class I antibodies in urine. The normalized gene expression values found in the urinary sediment of patients diagnosed with chronic rejection confirm the involvement of inflammation in the development of chronic rejection. Furthermore, the increased normalized gene expression levels for B cells markers (CD19 and CD79B) in chronic rejection group suggest the existence of B cells clusters that can function as a tertiary lymphoid tissue which can harbour B cell maturation into memory B cells and antibody producing plasma cells. According to the receiving operating characteristic curves, CD19, CXCL10 and TNF3 were the genes with the highest diagnostic values for chronic rejection. This study demonstrates that analysis of the urine of renal transplant patients could give valuable information that may allow the monitoring of the transplant without resort to invasive methods. Nevertheless, only the combination of results obtained in the urine and blood samples can provide a complete and accurate assessment of the allograft condition.
A transplantação renal tornou-se a terapia de eleição para doentes com insuficiência renal crónica terminal. As melhorias, em termos de sobrevivência do enxerto e da função renal a longo prazo, fizeram com que a transplantação renal tenha uma melhor relação custo/benefício do que a diálise. A acrescentar a isto, a introdução de novos agentes imunossupressores teve um impacto significativo na sobrevivência do enxerto a curto prazo. Apesar destas melhorias, uma quantidade substancial dos enxertos desenvolvem uma disfunção progressiva com perda de função total no prazo de uma década, através de um processo denominado rejeição crónica. A monitorização funcional dos rins transplantados torna-se crucial para tentar evitar o desenvolvimento desta condição. Os métodos mais comuns para realizar a monitorização da função do enxerto são as medições dos níveis de creatinina no soro, sendo que estas variações não são específicas para a rejeição e ainda, por vezes, é necessário realizar biopsias renais, que é um processo arriscado e que só diagnostica esta rejeição depois de esta estar instalada. O principal objetivo deste estudo foi desenvolver um perfil imunológico e celular, associado ao estabelecimento da disfunção crónica, que possa servir como ferramenta de diagnóstico. Tendo isto em vista, um conjunto de análises foram efetuadas em 71 transplantados renais com uma função renal estável e os resultados, comparados com os mesmos testes efetuados em transplantados renais que tinham sido diagnosticados com rejeição crónica. O desenvolvimento de um novo método de seleção de anticorpos na urina de transplantados renais foi um dos pontos mais importantes neste estudo, sendo que mais de metade dos transplantados com rejeição crónica apresentou anticorpos anti-HLA Classe I na urina. Os valores da expressão génica normalizada encontrados para as células do sedimento urinário dos transplantados renais, confirmaram o envolvimento da inflamação no desenvolvimento da rejeição crónica. Além disso, os valores mais elevados de expressão génica normalizada para os marcadores da célula B (CD19 e CD79B), no grupo da rejeição crónica, sugerem a existência de aglomerados de células B que funcionam como um órgão linfoide terciário, capaz de albergar a maturação da célula B em células de memória e em células do plasma produtoras de anticorpos. De acordo com a análise feita das curvas características de operação do recetor, os genes CD19, CXCL10 e TNF3 foram os que obtiveram maior valor de diagnóstico para a rejeição crónica. xv Este estudo comprova que a análise da urina de transplantados renais, no futuro breve, pode fornecer informações valiosas, permitindo assim, a monitorização funcional destes doentes sem recurso a procedimentos invasivos. No entanto, só a combinação dos resultados obtidos na urina e no sangue pode oferecer uma avaliação completa e precisa da condição do enxerto

Thesis (Ph. D.)--University of Georgia, 2003.
Directed by Mark W. Jackwood. Includes articles submitted to Avian diseases, Virus genes, and The journal of clinical microbiology. Includes bibliographical references.

Immunotherapy has had a revolutionary impact on cancer treatment, providing valuable tools to be used in combination or as an alternative to chemotherapy (Weiner G.J., 2015). Such wide success has been demonstrated by the fact that, since 1997, twelve monoclonal antibodies have been approved by the FDA for the treatment of a variety of solid tumors and haematological malignancies, along with an increasing number of new antibodies that are now being tested at different stages of clinical trials (ClinicalTrials.gov). Nevertheless, there are limitations to the use of monoclonal antibodies mainly due to their big size, which is detrimental especially for their applications in clinics and diagnostics. For these reasons, the arise of recombinant antibodies, which conjugate small size with antigen specificity has represented a big achievement in the oncological setting. Another crucial point is the identification of cell antigens, which can be exploited to target cancer cells. To this purpose, we have focused on inotropic Glutamate Receptor 4 (iGluR4), which is involved in many CNS pathological conditions and, moreover, has recently emerged to be implicated in many aspects of cancer progression (Stepulak A. et al., 2011; Stepulak A. et al., 2014; Ribeiro MP et al., 2016). We have also focused on hERG1 voltage-gated ion channel, that it is known to be a role player in cancer progression (Bianchi L. et al., 1998; Lastraioli E. et al., 2015). More recently, it has emerged as a novel target for cancer therapy and as a marker for patients’ stratification (Arcangeli A. et al., 2009; Pointer KB et al., 2016). Moreover, it has been demonstrated that hERG1 forms complexes in particular with β1 subunit of integrin receptors, thus mediating its effects in cancer cell behavior (Crociani et al. 2013). The present work has focused on developing new antibodies, that have demonstrated their potential from a diagnostic or a therapeutic point of view. We have produced two clones B5 and C4 of a monoclonal antibody directed against ionotropic Glutamate Receptor 4 (iGluR4), providing evidences that this molecule is able to recognize the antigen, also in a pathological scenario such as that of tuberous sclerosis complex (TSC) disease. Moreover, C4 anti-iGluR4 clone has emerged as a Abstract possible positive allosteric modulator of the receptor; such behavior will be better characterized in the future, to exploit its potential application as a channel regulator (Stepulak A. et al., 2014). We are also assembling a construct for the production of a scFv-iGluR4 directed against the same antigen as the monoclonal antibody, to overcome the problems related to the passage of the blood brain barrier (BBB). In fact, it is known that recombinant protein therapeutics are too large to cross the BBB, however, recombinant proteins can be reengineered as BBB-penetrating IgG fusion proteins, where the IgG part is a genetically engineered monoclonal antibody (MAb) (Pardridge WM and Boado RJ, 2012). The recombinant antibody we have produced will be conjugated with NPs (already capable to cross an in vitro model of BBB), to set up a drug-delivery system. In parallel, we have developed a single-chain variable fragment antibody, scFv-hERG1- G3 (scFv-hERG1 construct), that has already been validated, after labelling with Alexa 488 fluorophore, as a tool for optical in vivo imaging (Lastraioli E. et al., 2016). Moreover, we have produced a new antibody scFv-hERG1-D8-Cys (scFv-hERG1-Cys construct) (via mutagenesis of the scFv-hERG1 construct), in which we have reintroduced one of the cysteine that is in a fundamental position for the formation of the disulfide bonds, within the VH chain of the scFv antibody. In fact, scFv-hERG1-G3 antibody, in this position, showed a phenylalanine amino acid. We have given evidence that the restoration of the Cys deeply affects the protein yield, without affecting the antibody binding capacities. scFv-hERG1-D8-Cys has also demonstrated to have effects on cell growth and invasiveness, as demonstrated by the viability experiments performed on 2D tumour cell lines and on 3D spheroid tumour cell cultures (patent in preparation). In the third part of this work, we have focused on the expression and characterization of a scDb bispecific antibody directed against hERG1-β1 oncogenic unit. The antibody has been also characterized with IF experiments performed on different substrates, showing the capacity to recognize hERG1-β1complex. These evidences allow us to conclude that both scFv-hERG1-D8-Cys and anti-scDbhERG1- β1, after a proper validation, will represent valuable tools for cancer therapy.

Mycobacterium avium Complex (MAC) comprises microorganisms that affect a wide range of animals including humans. The most relevant are Mycobacterium avium subspecies hominissuis (Mah) with a high impact on public health affecting mainly immunocompromised individuals and Mycobacterium avium subspecies paratuberculosis (Map) causing paratuberculosis in animals with a high economic impact worldwide. In this work, we characterized 28 human and 67 porcine Mah isolates and evaluated the relationship among them by Multiple-Locus Variable number tandem repeat Analysis (MLVA). We concluded that Mah population presented a high genetic diversity and no correlations were inferred based on geographical origin, host or biological sample. For the first time in Portugal Map strains, from asymptomatic bovine faecal samples were isolated highlighting the need of more reliable and rapid diagnostic methods for Map direct detection. Therefore, we developed an IS900 nested real time PCR with high sensitivity and specificity associated with optimized DNA extraction methodologies for faecal and milk samples. We detected 83% of 155 faecal samples from goats, cattle and sheep, and 26% of 98 milk samples from cattle, positive for Map IS900 nested real time PCR. A novel SNPs (single nucleotide polymorphisms) assay to Map characterization based on a Whole Genome Sequencing analysis was developed to elucidate the genetic relationship between strains. Based on sequential detection of 14 SNPs and on a decision tree we were able to differentiate 14 phylogenetic groups with a higher discriminatory power compared to other typing methods. A pigmented Map strain was isolated and characterized evidencing for the first time to our knowledge the existence of pigmented Type C strains. With this work, we intended to improve the ante mortem direct molecular detection of Map, to conscientiously aware for the existence of Map animal infections widespread in Portugal and to contribute to the improvement of Map and Mah epidemiological studies.

L’efficacia di PCR quantitativa basata su una sonda TaqMan specifica per il genere è stata confermata come preciso strumento di rilevamento di DNA di Phytophthora su suolo artificialmente infettato, in suolo di invasatura contaminato proveniente da vivaio ed in campioni di trappole aeree. Nessuna quantificazione di DNA è avvenuta dopo due settimane dalla morte indotta del patogeno e, in confronto con i metodi di isolamento tradizionali, è stata dimostrata una significativa maggiore efficienza come strumento diagnostico. La fluttuazione stagionale di Phytophthora in aria è stata quantificata e descritta per il periodo di campionamento. La tecnica di sequenziamento 454 è stata utilizzata per identificare la tassonomia delle specie di Phytophthora in un hotspot biologico in Western Australia ed al fine di descrivere la patogenicità delle due specie sequenziate con maggior frequenza è stato realizzato un esperimento in serra. Le tecniche di laboratorio utilizzate in questo studio hanno fornito nuove nozioni sull’ecologia di Phytophthora. La PCR quantitativa basata su sonda TaqMan è testata e proposta come efficace strumento di prevenzione verso l’arrivo di specie invasive. Importanza dell’impatto di questo studio L’efficienza della gestione di Phytophthora dipende dalla conoscenza delle caratteristiche patogene di gruppi ristretti di specie o di una singola specie . Ricerche come queste forniscono le basi scientifiche per comprendere l’epidemiologia di una malattia ed applicare un controllo risolutivo. A quantitative PCR technique based on a genus specific TaqMan probe was confirmed as a precise method for detecting Phytophthora DNA in artificially infested soil under laboratory condition, in naturally infested soil and tissues of potted nursery plants and in the filters of air traps. No positive DNA quantification occurred in soil after two weeks from pathogen induced death and a significant higher efficiency as diagnostic tool was demonstrated compared to traditional isolation methods both in soil and plant tissues. Seasonal fluctuation of aerial spread of Phytophthora was also quantified and described. A 454 sequencing approach was used to identify the Phytophthora species present in a biological hotspot area in Western Australia, and a glass house experiment was performed in order to describe the pathogenicity traits of the two most frequently detected species. The lab procedures used in this study provided a more precise knowledge of Phytophthora ecology. The quantitative PCR assay based on designed TaqMan probe was demonstrated to be very efficient and is proposed as a reliable early detection instrument of prevention against the income of invasive species. Efficient management of Phytophthora depends to the knowledge of pathogenicity traits in restrict groups or single species. Investigations like those presented in this thesis contribute the scientific bases to understand the epidemiology of disease and to apply a successful control.

I capsidi virali sono strutture stabili e robuste composte da multiple copie di una o più tipi di subunità proteiche organizzate con simmetrie ordinate (icosaedriche o filmentose). I capsidi virali possono essere prodotti in sistemi vegetali in consistenti quantità sia tramite l’infezione delle piante sia tramite l’espressione delle subunità capsidiche. Data la semplicità e l’elevata stabilità, le Chimeric Virus Particles (CVPs) e le empty Virus Like Particles (eVLPs) hanno attirato l’attenzione della comunità scientifica per lo sviluppo di reagenti che possono trovare impiego nell’ambito delle nano-biotecnologie. In questo lavoro di tesi, le CVPs e le eVLPs sono state sfruttate per l’espressione di peptidi funzionali, al fine di stabilizzarli e di produrli a basso costo. In particolare, le piattaforme di espressione virali scelte si basano sui seguenti virus vegetali: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) e il Turnip Mosaic Virus (TuMV). La prima applicazione descritta riguarda il trattamento di due malattie autoimmuni che hanno un forte impatto sociale: il Diabete Melito di tipo 1 (T1D) e l’Artrite Reumatoide (AR). Attualmente, esistono trattamenti che sono solo in grado di arginare e gestire gli effetti di queste patologie senza però bloccarne definitivamente l’azione. In questo lavoro, virus vegetali che espongono peptidi associati al T1D e all’AR sono stati utilizzati per lo sviluppo rispettivamente di un trattamento preventivo e terapeutico. Le particelle virali sono state geneticamente modificate in modo da esporre peptidi autoantigenici associati al T1D e all’AR, espresse in pianta, purificate dal tessuto vegetale ed utilizzate per studi pre-clinici in animali modello. I risultati ottenuti mostrano che la struttura delle particelle virali è in grado di agire come adiuvante aumentando la capacità immunomodulante dei peptidi autoantigenici selezionati ed esposti sui capsidi virali. La seconda parte di questo lavoro di tesi, si concentra sull’utilizzo delle CVPs esprimenti peptidi associati alla Sindrome di Sjögren (SjS) e all’AR per lo sviluppo di innovativi kit diagnostici. Queste due malattie autoimmuni sono attualmente difficili da diagnosticare principalmente per la presenza di un sottogruppo di pazienti che risulta essere negativo alla presenza dei più comuni marcatori sierologici utilizzati per la diagnosi. Questo progetto si è focalizzato sull’espressione in pianta di CVPs di struttura filamentosa che, tramite l’espressione di specifici peptidi, hanno permesso di migliorare notevolmente le performance diagnostiche di un test ELISA sviluppato con tali particelle in confrontato con lo stesso sviluppato utilizzo i peptidi sintetici. Inoltre, sono state espresse in pianta ulteriori CVPs che espongono due peptidi associati all’AR. Tali CVPs saranno in futuro utilizzate per la messa a punto di un kit per la diagnosi dell’AR. L’ultima parte di questo lavoro, riguarda altre possibili applicazioni dei virus vegetali come strumenti nano-biotecnologici. Nello specifico, eVLPs sono state espresse in pianta al fine di esprimere un peptide antimicrobico (AMP) e un cell penetrating peptide (CPP) per ottenere rispettivamente un innovativo eco-pesticida e uno strumento biotecnologico per l’internalizzazione di peptidi funzionali o proteine di interesse nelle cellule.
The capsids of most plant viruses are simple and robust structures consisting of multiple copies of one or few types of protein subunits arranged with either icosahedral or helical ordered symmetry. In many cases, capsids can be produced in large quantities either by the infection of plants or by the expression of the subunits. In view of their relative simplicity, stability and easy production, plant chimeric virus particles (CVPs) or empty virus-like particles (eVLPs) have attracted attention as potential reagents for applications in bionanotechnology. In this work CVPs and eVLPs have been exploited for the expression of functional peptides, in order to stabilize them and avoid peptide low intrinsic stability and susceptibility to degradation. In particular, the viral expression platforms chosen for the expression of target peptides are based on four plant viruses widely used as scaffold for peptide display: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) and Turnip Mosaic Virus (TuMV). The first application explored in this work regards the therapy of Type 1 diabetes (T1D) and Rheumatoid arthritis (RA), two autoimmune diseases that share a strong social impact. Currently, there are treatments able to manage and/or stem the effects of these disorders. In particular, plant viruses displaying peptides associated to T1D and RA have been used respectively for the development of a preventive and therapeutic drug. Virus particles displaying autoantigenic peptide specific for these diseases have been expressed and used for pre-clinical studies in T1D and RA animal models. The results observed suggest that the use of viral structure for peptide display works as an adjuvant by increasing peptide modulation capability. The second part of this work regards the use of plant viruses displaying peptide as reagents for the development of innovative kit for the Sjögren’s Syndrome (SjS) and RA diagnosis. These two autoimmune diseases are difficult to be diagnosed and either for SjS of RA there are subgroups of patient seronegative to the main diagnostic serological markers. In this work, the use of filamentous particles for the display of specific SjS peptide allowed to increase the diagnostic performances of an ELISA kit in comparison to the use of the peptide alone. Moreover, autoantigenic peptides associated to RA were successfully expressed in plants on the surface of viral particles that will be exploited in the future for the development of a kit for seronegative RA diagnosis. A third part of this PhD thesis regards another possible application of plant viruses as tools for peptide display. In particular, viral particles have been used for the expression of a antimicrobial peptide (AMP) that could be exploited as eco-friendly pesticide and for “nanoagriculture” application. Finally, the possibility of developing a biotechnological tool for peptide internalisation into the cells has been exploited by fusing on the surface of an icosahedral plant virus a cell-penetrating peptide (CPP) derive from HIV. Regarding this third part, CVPs and eVLPs displaying the selected peptides have been successfully expressed in plants; however, several drawbacks have been encountered in the purification process.

Skin homeostasis is dependent on a tightly coordinated network of signaling pathways, resulting in a spatial and temporal balance of proliferation, growth arrest, differentiation, senescence and apoptosis. The knowledge of mechanisms that regulate keratinocyte growth and proliferation is attractive for several biotechnological fields, such as cosmetics, tissue engineering and skin regeneration. DNp63alfa, as critical pro-proliferative factor and marker of epidermal stemness, is essential for morphogenesis of organs/tissues developing by epithelial-mesenchimal interactions such as the epidermis, teeth, hair and glands (Mills, 1999). Using a proteomic approach my research group had identified YB-1 as a DNp63alfa molecular partner (Amoresano, 2010). Y Box Binding protein 1 (YB-1) is a transcription/translation factor involved in a wide variety of cellular functions, including cell proliferation and migration, DNA repair, multidrug resistance and stress response to extracellular signals (Lyabin, 2014). My PhD project was aimed to advance in the characterization of the functional interplay between DNp63alfa and Y-box binding 1 proteins using in cell approaches to elucidate their roles in skin proliferation. First, I have validated DNp63alfa-YB-1 interaction in distinct cell contexts and demonstrated that this interaction causes accumulation of YB-1 into the nuclear compartment and influences its localization-dependent functions (Di Costanzo, 2012). Then, I extended our knowledge on the YB-1 and DNp63alfa functional interplay demonstrating that, being able to sustain DNp63alfa gene expression, YB-1 is part of a complex molecular network linking DNp63alfa to the PI3K/AKT/PTEN pathway and that establishment of a positive feedback loop, coupling induction of DNp63alfa expression with PI3K/AKT activation, may be a relevant step in the progression of squamous carcinogenesis (Troiano, 2015). During my PhD program, I have spent six months at the Blizard Institute - Queen Mary University of London collaborating with Professor Bergamaschi's team. Based on my experience on squamous carcinoma I explored if p63 and YB-1 are also involved in melanoma pathogenesis and/or progression. Although my data are still preliminary, they show that DNp63alfa controls YB-1 protein integrity and nuclear localization also in melanoma cells and their functional cross-talk plays a role in melanoma cell survival. Finally, thanks to the collaboration with Prof. Piera Quesada's team, I had the opportunity to perform experiments aimed to investigate on the role of p53 family members in the response of cancer cells to Topoisomerase I and poly(ADP-ribose)polymerase (PARP1) inhibitors. My results point to a role for p63 in the cell response to treatment with TOP I and PARP-1 inhibitors. Highlighting the different outcome (i.e cell cycle arrest vs apoptosis) of PARP-1 activation/inhibition, my studies give right of the use of PARP inhibitor as chemotherapic adjuvant and/or in monotherapy also in TAp63/DNp63alfa proficient cancer cells (Montariello, 2013; Montariello, 2015).