Dissertationen zum Thema „Diagnostic tool development“

In Automotive Industries, to be confident regarding the success of a planned operation, performing accurate methods in order to detect abnormal operating conditions, known as faults, is crucial. An effective method for diagnosis and fault recognition ensures the safety of the operation, reduces manufacturing cost and any other potential impacts. In addition, mobile solutions have been widely adopted among automotive manufactures during recent years and they have taken full advantage of mobile strategies. Accordingly, it is necessary for there to be a future-proof plan to control the diagnostic operations in advance. In this thesis, the immediate objective has been to offer a future-proof and user-friendly solution to assist engineers and service technicians in the monitoring, detecting, and diagnosing of faults on Toyota/BT/CESAB branded trucks. A mobile cross-platform framework is used to develop the diagnostic mobile solution which is not only able to be deployed on Android and iOS mobile platforms, but also provides wireless communication between truck machines and mobile devices through Bluetooth and Wi-Fi ad hoc technologies. The diagnostic mobile tool is capable of processing real-time controller area network messages and visualizing the condition of different sensors in a more user-friendly way through rich hybrid and client-side web user interfaces. The experience of evaluating a cross-platform diagnostic tool on different mobile operating systems proved that cross-platform mobile development methodology can be a reliable technique for developing projects that essentially require real-time data processing. In addition, it indicates that Apple iOS offers a better runtime performance than Google Android for the current tool.

En ny produkt, som utvecklats för att underlätta de många diagnoser och bedömningar avsjukdomen lymfödem, utvärderades och vidareutvecklades i denna studie. Tidiga mätningar avpatienter i riskzonen var viktigt för en tidig upptäckt av sjukdomen. Lymfödem yttrar sig somsvällande och förhårdnade kroppsdelar, ofta armar och ben, och uppkommer delvis till följd avcancerbehandling. De mätmetoder som användes inom vården när rapporten skrevs, bestod aven lång rad mätningar av kroppsdelars omkrets med ett vanlig måttband. Det fanns utrymme förförbättring av metoden som kunde göras mer konsekvent och effektiv. En risk fanns att klinikeranvände måttbandet på olika sätt när omkretsar på patienter mättes. Dessutom skulle denhårdhetsutvärdering av kroppsdelar, som genomfördes av klinikerna, kunna kvantifieras ochanalyseras om dessa utvärderingar sattes i siffror.Målen med detta projekt var att utvärdera den nya produktens noggrannhet och precision, samtutveckla och integrera en funktion för mätning av hudens hårdhet. Ett användargränssnittanpassad till klinikers krav och en konstruktion anpassad för testning och framtida tillverkning,var också projektmål. Med anledning av patientsäkerhetskrav gjordes en del av produktenutbytbar mellan mätningar av olika patienter.Under utvecklingen användes videoinspelningar av praktiska tester av prototypen ochundersökning som delades ut till kliniker som utförde lymfödembehandling. Mätningen avomkrets som utvecklats i denna studie var, enligt de tester som utförts, tillräckligt noggrannjämfört med den noggrannhet den vedertagna mätmetoden krävde. En automatisk beräkning aven bulkmodul K för vävnaden, utvecklades som en integrerad funktion för att kunna erhålla ettvärde på den hårdhet som kliniker kände med sina händer. K-värdet visade sig fungera som ettkoncept, genom mätningar av olika typer av skummaterial. Beträffande mätningar i vävnadvisade sig produktens bestämning av K-värden kunna detektera en ökning av muskelspänning,men resultaten var bara förutsägbara till en viss gräns i testet som presenteras i rapporten.Metoden kunde i denna studie, inte på detta sätt valideras för vävnad genom att jämförametoden med mätningar med en durometer. En av slutsatserna var dock att K-värdet fortfarandekunde vara en användbar indikator, som ett komplement till den bedömning som var gängse närrapporten skrevs. Utöver detta kunde produkten spara tid i de många mätningarna avlymfödempatienter. Denna sparade tid kunde användas för fler mätningar. Fler mätningar ökarchansen att upptäcka sjukdomen i ett tidigt stadium (Lawenda, Mondry, and Johnstone, 2009),vilket i sin tur är avgörande för en framgångsrik behandling.
To improve the frequently performed lymphedema measuring session – important for an earlydetection of the disease – a novel device was evaluated and further developed in this study.Lymphedema appears as limbs swelling, often arms and legs, partly because of cancertreatment. The current assessment technique includes several circumference measurements witha regular tape measure, and the method had room for improvement regarding consistency andeffectiveness. Different clinicians may pull the tape measure differently while measuring thecircumferences of a patient’s limb. An additional skin compliance evaluation, performed by theclinicians, could be quantified and analyzed if the assessments were put into numbers.The goals of this project were to evaluate and improve the accuracy and precision of this noveldevice, as well as develop and integrate a feature for measuring skin compliance. Furthermore,a user interface adapted to the clinicians’ demands and a design adapted to testing and futuremanufacturing was essential. Due to patient safety reasons, a part of the device was madereplaceable between patients.Developing the device, video recordings of hands-on testing were performed, as well as asurvey distributed to clinicians who provided lymphedema therapy. The circumferencemeasurement developed in this study was, according to the performed tests, accurate enoughcompared to the accuracy required the current measuring routine. An automatic calculation of askin bulk modulus K, was developed as a feature of the device to be able to obtain a value ofthe compliance clinicians felt for with their hands. The K-value was proven as a concept bymeasuring different foams. Measuring K-values of the tissue, the device was capable ofdetecting an increase in muscle tension, but results were only predictable to a certain limit inthe test presented in the report. The method could not yet be validated by comparing it to a skinhardness measurement with a durometer. A conclusion was however, that the K-value stillcould be a useful indicator in addition to the current assessment and that the device could savetime in the measuring session. This saved time could be used for more measurement sessions,beneficial in order to detect lymphedema early (Lawenda, Mondry, and Johnstone, 2009),which in turn was essential for a successful treatment.

The first step of debugging a deployed application is to reproduce the reported bug. But bugs that cause unpredictable behaviors without crashing the applica- tion can be difficult to reproduce, because the developer has to rely on bug-re- ports issued by users. The goal of this thesis has been to develop a diagnostic tool which makes user-reported bugs easier to reproduce. More specifically, the goal has been to develop such a tool for React Native applications implement- ing Redux, taking into consideration both state-data stored in Redux stores and React Native components. Requirements for the diagnostic tool were laid out and partitioned into proto- types that were implemented separately in iterations and then merged into the resulting tool. Requirements were also drawn for three sample applications to test and evaluate the tool, as well as a back-end to prove the tool's capability in uploading and downloading state-data from a server. The thesis resulted in a diagnostic tool that's imported into an already existing code-base as a third-party library, and which collects the complete state of the reported application instance and delivers it to the developer as a single JSON document. The tool can then inject the state-data into a fresh instance of the ap- plication to make it identical to the malfunctioning instance reported by the user; a process referred to as "reviving". Redux stores demonstrated state-over- write protection which complicates state-injection. A study was performed to compare the impact this difference has on performance. As expected, the study revealed that the diagnostic tool takes marginally longer to revive state-data in Redux stores for this reason.

Rodon is a diagnostic tool developed by Sörman. SAAB’s interest in Rodon regards the possibility to use the tool for development and operations of aircraft systems. The main goal of this thesis was to evaluate the capacity of Rodon and determine how SAAB can use the diagnostic tool during development and operations.

The tool uses model based diagnosis with artificial intelligence for fault isolation which is a powerful approach. If Rodon is introduced at SAAB, then detailed models of systems will be necessary to create, including the nominal behavior of the system and different faulty behaviors. In order to achieve high quality fault isolation, it is necessary to have complete and consistent models. To be able to use all applications that Rodon feature for a modeled system, preferable characteristics are that the model should be static, have discrete control signals, and have well defined system behavioral modes.

During development of a system Rodon can be used to improve and easy the work for failure analysis, guidance of sensor placements, evaluation of tests, generation of decision structures, and fault isolation. Since design of tests during development is a desirable application that Rodon does not have, two different methods are presented that utilizes Rodon to generate all possible limit checking tests.

In conclusion, Rodon can be very useful in several different aspects if introduced, but benefits gained by using Rodon will have to be compared to the labor cost of creating good models.

Thesis (M.Eng. and S.B.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2002.
Includes bibliographical references (leaves 76-79).
Barrett's esophagus (BE) is a condition of the lower esophagus caused by gastroesophageal reflux disease. Patients with BE have an increased probability of developing dysplasia, an abnormal growth or development of cells. This dysplasia in BE is a precursor to cancer of the esophagus, but is currently difficult to detect and diagnose. If the dysplasia is allowed to progress to cancer, it is very difficult to treat successfully. Treatment for dysplasia itself, however, is very effective if done at an early stage. The goal of this thesis project will be to develop a real-time tool that uses spectroscopy to improve upon the methods of detecting dysplasia in BE. This will involve analyzing spectra acquired from patients with BE using models and extracting quantitative information on different aspects of tissue morphology and biochemistry. Using this information, diagnostic algorithms will be developed, optimized and displayed to the physician through a useful interface.
by Austin H. Kim.
M.Eng.and S.B.

The development of a clinical diagnostic scheme for ciguatera fish poisoning has been accomplished through the utilization of three assays, specifically a competitive enzyme-linked immunosorbent assay (ELISA) for quantification, and two procedures for separation of toxic potentials, i.e., a liquid-liquid partition method and an immunoaffinity column system. For the ELISA assay, quality control parameters were evaluated including the coating binding efficiency and the binding capability of the conjugate (okadaic acid-bovine serum albumin). A 100% recovery was obtained and reproducibility values were under 30% coefficient of variation (CV) for concentrations from 0.0001 to 100 ng okadaic acid/mL serum or g fish flesh. Additionally, the usefulness of the method was evaluated by determining sensitivity and specificity parameters (66.7 and 95%, respectively) from where the positive and negative predictive values of the test as a diagnostic instrument were assessed. A rapid extraction procedure utilizing a mixture of methanol:chloroform:water was adapted for extraction and isolation of toxins associated with ciguatera fish poisoning. The method has been optimized for the analytical evaluation of potentially ciguatoxic fish samples and human serum. Applications of the extraction procedure include the clinical diagnosis of ciguatera illness and, if leftover fish portions responsible for the intoxication event are available, analysis of the seafood. It can also be used as a tool for monitoring seafood products for food safety. Recovery values of the extraction method with and without an intermediary defatting step for spiked fish samples were of 105 and 86%, respectively. By attaching anti-okadaic acid monoclonal antibody onto an inert matrix (AH-Sepharose®), an immunoaffinity column specific to ciguatera-related toxins has been developed, also for serum analysis, but with the potential application in other body fluids. The binding efficiency was 82% and reproducibility values for spiked test portions at concentrations as low as 250 pg were below 10%.

Parkinson’s disease and Essential tremor are the two most prevalent movement disorders in the world, but due to overlapping clinical symptoms, accurate differential diagnosis is difficult. As a result, approximately 60% of patients with movement disorders symptoms will have their diagnosis changed at least once before death. By their subjective nature, clinical exams are inherently imprecise, leading to the desire to create an objective, quantifiable test for movement disorders; a test that currently is elusive. Eye movements have been studied for a century, and are widely appreciated to be quantifiably affected in those with neurological disease. Through a collaborative effort between the VA hospital and VCU, over 1,000 movement disorder subjects had their eye movements recorded, utilizing an SR Research Eyelink 2. Patients with Parkinson’s disease exhibited an ocular gaze tremor during fixation, normal reflexive saccades, and reduced blink rate. Subjects with Essential tremor exhibited slowed saccadic dynamics, with increased latencies, in addition to a larger number of square wave jerk interruptions of otherwise stable fixation. After diagnostic features of each disorder were identified, prospective data collection could occur in a blinded fashion, and oculomotor features used to predict clinical diagnoses. It was determined that measures of fixation stability were capable of almost perfectly differentiating subjects with PD, and a novel, combined parameter was capable of similar results in ET. As a group, it appears as if these symptoms do not progress as the disease does, but subanalyses show that individual patients on constant pharmaceutical doses tracked over time do slightly change and progress. The near perfect separation of disease states suggest the ability of oculomotor recording to be a powerful biomarker to be used for the differential diagnosis of movement disorders. This tool could potentially impact and improve the lives of millions of people the world over.

For decades the problems of access to and sustained use of water and sanitation (WATSAN) technologies in developing countries has dominated the political agendas of international organisations and governments. Despite the significant investments made and the apparent appropriateness of technologies transferred, the effective implementation and sustained use of WATSAN technologies remains a chimera. More importantly, improving access to water and sanitation does not necessarily guarantee the longevity of those systems transferred. Lessons from past interventions suggest that the success of WATSAN interventions depends on the ability of ensuring users‟ broad acceptance of the technologies and sustained used after donor assistance ends. Yet, in the academic literature users‟ feedback and experiences in the post-implementation stage of technologies has received scarce attention. Against this background, this thesis aims to contribute to understanding the dynamics involved in the process of WATSAN technology adoption and sustained use in developing countries by reporting the design and evaluation of a diagnostic post- implementation tool, called RECAP, to address and investigate the problem. This research employs a multiple case study approach to evaluate users‟ post-implementation experience of WATSAN technologies in South Africa and Indonesia. Semi-structured interviews with technology users as well as in depth interviews with local governments and health clinics were conducted in three case studies. By comparing and contrasting technology intended performance and users‟ experiences in the post-implementation stage this study aims to identify potential challenges to technology sustained used. Conclusions relate to the existence of discrepancies between performance and experience, manifested in the post-implementation stage, which suggest the necessity to develop evolving mechanisms to routinely assess users‟ feedbacks of the technologies and assist them with appropriate interventions. Further conclusions relate to the validity, reliability and flexibility of a post-implementation diagnostic tool in investigating user experiences, diagnosing emerging challenges and suggesting remedial intervention to contribute to sustained technology use.

The Oil Pollution Diagnostic System (OPDS) is a proof of concept prototype pattern recognition system designed to assist chemists in assessing the type and source of refined oil spills that occur in freshwater. The pollution of inland waters by refined oils can cause damage to the local environment that takes many months to repair since oils are toxic to many of the flora and fauna often found in these habitats. The issue of assessing the exact type and source of the spill based on chemical analysis are separate but closely linked problems. The lesser of the two problems relates to the reliable identification of the oil type based upon distinctive patterns contained in the analysis data. The problem of identifying the likely source is more challenging however and the work describes an approach where the patterns contained in small sections of the analysis data are used as the basis of a match. Manual methods of analysing chemical data to determine the type and source of a spill have existed for some time. However in a courtroom situation where the analysis is presented as evidence in the attempt to secure a prosecution, manual methods of matching could be seen as subjective and unreliaole and possibly be exploited by the defence. The OPDS was developed as an alternative to the manual methods already in use. The system illustrates the advantages of transformable oil type templates, the advantages of automatic calibration to reduce the impact of retention time drift and the use of the lesser peaks between the major peaks for oil source fingerprinting. The major contribution in this section of the development project was to incorporate all the features mentioned in a system dedicated to the matching of gas cl1romatograms (GC), and furthermore to incorporate the facility to allow the system to automatically match GCs with minimal input from the user. The template based approach for type matching required the construction of a dynamic type template for each oil type to be identified. The template modelled a collection of distinctive features identified from the weathered information provided by the Environment Agency (of England and Wales). The templates were dynamic since they model the way the distinctive features are altered by the effects of weathering. This ability to negate the effects of weathering from the pattern together with some calibration allowing for instrument variation allowed the comparison of a sample to a set of templates via a metric based on the Euclidean distance between matched peaks. A similar approach was adopted with the problem of source identification where the emphasis was more on matching small collections of GC peaks that are present between the major features. This approach was based on a suggestion contained in the feasibility study. The EA provided a number of GCs relating to actual pollution incidents and these formed the basis of a set of case studies which were then used to develop and test the source matching system. Tests carried out on the type matching component of the system using independent test data produced correct classification figures of greater than 90% and kappa values of greater than 0.9. The source matching testing was less extensive than that done on the type matching component however a correct classification figure of 88% was observed. This thesis contains an explanation of the problems caused by spills of refined m inland waters, an explanation of the teclmiques used to analyse oil spills, a review of pattern recognition and chemometric tecrwiques, at1 overview of the software design and development the results achieved and the conclusions that can be drawn from these.

Serial peak expiratory flow measurements (PEF) are recommended as an initial investigation in the confirmation of occupational asthma. Plotting measurements in Oasys gives reproducible results and can be used by non-experts. I report a new analysis, the area between curves (ABC) score, which gives 72% sensitivity and 100% specificity using a cut off of 15 L/min/h. Two-hourly measurements of PEF require 8 work days and 3 rest days for sensitive and specific analysis. Serial PEF records with long periods off work (≥ 4 consecutive days) show improved sensitivity from 73% to 80%, implying that 7 more workers in every 100 would be diagnosed. In a comparison of forced expiratory volume in one second (FEV1) to PEF, PEF was more sensitive to diurnal changes than FEV1, although FEV1 was more reproducible. Exhaled breath nitric oxide (FENO) showed similar ABC scores between those with normal and raised FENO. FENO was significantly correlated to methacholine reactivity. In shift workers, mean ABC scores were increased on morning shifts compared to nights, but the cut off of 15 L/min/h would be applicable across all shift types. The ABC score is a new robust method of confirming occupational asthma requiring shorter records than the Oasys score.

DNA topoisomerases are essential enzymes in the control and in the maintenance of the topological state of the DNA. One of the best studied topoisomerases is the human Topoisomerase IB (hTop1). This enzyme has gained great interest because it is the only target of the anti-cancer drugs of the camptothecin (CPT) family. HTop1 accomplishes its function by breaking and rejoining the DNA backbone to solve torsional stresses occurring during essential DNA metabolic processes, such as transcription and replication. The efficiency of CPT is dependent on the intracellular hTop1 activity. For this reason, the detection of hTop1 activity possesses a great clinical potential in the cancer diagnostics and therapy. The standard assays for the detection of the activity of hTop1 do not permit to measure the activity of low concentration of enzyme, such as in clinical samples. In this thesis, two different novel techniques to detect the activity of hTop1 at very low concentration are presented. First, we improved the Rolling Circle Amplification (RCA)-based assay, using magnetic sepharose beads in order to concentrate the rolling circle products, produced by the amplification of DNA substrates circularized by hTop1 and detected by annealing with fluorescent probes. The sensitivity of the assay was considerably increased, with a detection limit of 0.3 pM hTop1. Second, a graphene-based biosensor was developed. The DNA substrates were designed to decouple the cleavage and the religation steps. The activity of 300 pM hTop1 can be detected by measuring electrically the sensor response. This method is label-free, fast and permit to measure the activity of hTop1 in real-time.

This case study explored the use of site-visitation as a diagnostic tool for school improvement. Nine charter schools in New Orleans were selected for the study. Based on qualitative research and systems theory, a within- and cross-case analysis of nine semi-structured interviews with school leaders were conducted. The school leaders’ experiences with the state-run site-visitation model and their use of the findings for school improvement was explored. The findings led to the development of a hybrid accountability model that encompasses the components school leaders believe will lead to school improvement. This study aims to assist educators, policy makers, and researchers to better understand site-visitation and its role in school improvement.

Att sjuksköterskor ställer giltiga omvårdnadsdiagnoser är betydelsefullt för att patienter ska få den omvårdnad som de behöver. Trots att denna förmåga är något som sjuksköterskeutbildningen är ålagd att utrusta blivande sjuksköterskor med är omvårdnadsdiagnostisk förmåga inte utrett hos sjuksköterskestudenter. Inget verktyg för att mäta denna förmåga finns tillgänglig i Sverige idag. Syftet med denna metodologiska studie var att utveckla ett verktyg som avsåg mäta omvårdnadsdiagnostisk förmåga. Genom kognitiva intervjuer, i dialog med experter samt genom att studenter och sjuksköterskor testade att genomföra en omvårdnadsdiagnostisk uppgift utifrån två patientfall utvecklades verktyget stegvis. Verktyget validerades mot omvårdnadslitteratur och mätinstrument samt mot en expertgrupp. Förutom den omvårdnadsdiagnostiska uppgiften utgjordes verktyget även av ett poängsättningsformulär, manual samt en rättningsmall. Studien frambringade ett verktyg som var känsligt för kunskapsvariationer och föreföll ha utrymme för förbättrade resultat. Poängsättningsformuläret med tillhörande manual och rättningsmall visade en hög reliabilitet. Det framtagna verktyget mäter förmågan att urskilja relevanta kännetecken, göra en analys av kännetecken och etiologi samt att rubricera diagnosen; d.v.s. analytisk förmåga, logiskt resonemang och till viss del yrkeskunnande i omvårdnad. Omvårdnadsdiagnostisk förmåga utgörs av fler delförmågor och alla dessa går inte att mäta med det framtagna verktyget.
The fact that nurses identify valid nursing diagnoses is important in order for patients to recieve the nursing care they need. Even though it is imposed on the nurse education to equip the future nurses with this ability, diagnostic competency in nursing isn’t tested among nurse students. There is in Sweden today no tool available for measuring diagnostic competency. The aim of this methodological study was to develop a tool that indended to measure diagnostic competency in nursing. The tool was developed gradually through cognitive interviews, in dialogue with experts and by having students and nurses complete a test in nursing diagnostics based on two case studies. The tool was validated in relation to nursing literature and measuring instruments along with an expert group. In addition to the test in diagnostic competency, the tool was also constituted of a point form, a manual for awarding points and a correcting model. The study created a tool that was sensitive for variations in knowledge within the test group and that seemed to have room for improved results. The point form, the manual for awarding points and the correcting model showed a high reliability. The abilities that the tool measure is the capability to distinguish relevant characteristics, make an analysis of the characteristics and the etiology together with labeling the diagnosis; i.e. analytic ability, logical reasoning and to some extent professional skills in nursing. Diagnostic competency embodies several different abilities and all of these can’t be measured by the developed tool.

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

Every year approximately 2800 Swedes are diagnosed with malignant melanoma, the form of cancer that is most rapidly increasing in incidence in the Western world. The earlier we can identify and diagnose a malignant melanoma, the better is the prognosis. In Sweden, 155 000 benign naevi, harmless skin tumours or moles, are surgically excised each year, many of them because melanoma cannot be dismissed by non-invasive methods. The excisions result in substantial medical costs and cause unrest and suffering of the individual patient. For untrained physicians, it is often difficult to make an accurate diagnosis of melanoma, thus a tool that could help to strengthen the diagnosis of suspected melanomas would be highly valuable. This thesis describes the development and assessment of a non-invasive method for early skin cancer detection. Using near infrared (NIR) and skin impedance spectroscopy, healthy and diseased skin of various subjects was examined to develop a new instrument for detecting malignant melanoma. Due to the complex nature of skin and the numerous variables involved, the spectroscopic data were analysed multivariately using Principal Component Analysis (PCA) and partial leas square discriminant analysis (PLS-DA). The reproducibility of the measurements was determined by calculating Scatter Values (SVs), and the significance of separations between overlapping groups in score plots was determined by calculating intra-model distances. The studies indicate that combining skin impedance and NIR spectroscopy measurements adds value, therefore a new probe-head for simultaneous NIR and skin impedance measurements was introduced. Using both spectroscopic techniques it was possible to separate healthy skin at one body location from healthy skin at another location due to the differences in skin characteristics at various body locations. In addition, statistically significant differences between overlapping groups of both age and gender in score plots were detected. However, the differences in skin characteristics at different body locations had stronger effects on the measurements than both age and gender. Intake of coffee and alcohol prior to measurement did not significantly influence the outcome data. Measurements on dysplastic naevi were significantly separated in a score plot and the influence of diseased skin was stronger than that of body location. This was confirmed in a study where measurements were performed on 12 malignant melanomas, 19 dysplastic naevi and 19 benign naevi. The malignant melanomas were significantly separated from both dysplastic naevi and benign naevi. Overall, the presented findings show that the instrument we have developed provides fast, reproducible measurements, capable of distinguishing malignant melanoma from dysplastic naevi and benign naevi non-invasively with 83% sensitivity and 95% specificity. Thus, the results are highly promising and the instrument appears to have high potential diagnostic utility.

Molte tecnologie sviluppate in campo diagnostico si basano sull’individuazione di specifici marcatori presenti nei campioni biologici. In questo ambito è ampiamente sfruttata la capacità intrinseca degli anticorpi di riconoscere il loro antigene. Pertanto, è fondamentale implementare i vari processi che permettono il rapido isolamento degli anticorpi e che ne garantiscono un alto livello di produzione. Grazie al continuo avanzamento delle tecnologie del DNA ricombinante, sono state sviluppate tecnologie in vitro che permettono l'isolamento su larga scala di anticorpi monoclonali. Il phage display è una tecnologia basata sullo screening di una libreria di anticorpi contro un dato antigene. La libreria di anticorpi è di solito composta da domini variabili delle catene leggere e pesanti di una immunoglobulina combinati casualmente, questi domini sono fusi con una proteina del capside del batteriofago M13. Ogni fago espone sulla sua superficie un singolo peptide, quindi si può facilmente risalire alla sequenza codificante dei frammenti anticorpali. L'obiettivo principale di questo lavoro è quello di ottimizzare l’intera pipeline che consente lo sviluppo di anticorpi monoclonali ricombinanti interessanti in ambito diagnostico. Questo processo inizia con l'isolamento di cloni in grado di riconoscere l'antigene partendo da una libreria fagica naïve. Pertanto, questo lavoro è iniziato con la creazione e la caratterizzazione di una libreria fagica naïve. Una volta costruita la libreria, questa è stata validata mediante NGS, in questo modo è stato possibile avere un'istantanea della sua diversità. La libreria è stata ulteriormente validata mediante il classico sequenziamento di Sanger in modo da poter anche valutare la qualità delle sequenze codificanti dei frammenti anticorpali che la compongono. Per ottimizzare e validare l'intera pipeline, come antigene “pilota” è stato scelto Interferone γ, una proteina rilevante da un punto di vista diagnostico perché è utilizzata nella diagnosi della tubercolosi. Diverse procedure di biopanning hanno permesso di isolare, complessivamente, 25 scFv diversi. Questi cloni sono stati testati anche su una piattaforma automatizzata progettata per i test immunodiagnostici e questo ne ha permesso una caratterizzazione più approfondita. In questo modo è stato possibile isolare i cloni che mostravano le prestazioni migliori. Tra questi, sono stati scelti due cloni per essere ulteriormente maturati in modo da migliorare la loro affinità nei confronti dell'antigene. Considerato che il ruolo chiave svolto dalla VH nel riconoscimento dell'antigene è ben noto, è stato deciso di creare due nuove librerie di display fagico composte da scFv tutti recanti la VH dei due cloni parentali e un pannello di VL. La procedura di selezione ha permesso di isolare due cloni che sembrano funzionare meglio rispetto il clone parentale quando testati sulla piattaforma automatizzata. In campo diagnostico, generalmente, sono utilizzate immunoglobuline nel loro formato nativo. Quindi, una volta selezionati i frammenti anticorpali, questi devono essere clonati in vettori che ne consentano l'espressione in linee cellulari di mammifero. In questo lavoro è stata costruita una serie di vettori che consentono l'espressione delle IgG, IgA, IgM umane e IgG di topo. Tutti i vettori sono stati trasfettati e la produzione di anticorpi è stata buona anche in termini di corretto folding dell'immunoglobulina. La fase finale di convalida dell'intera pipeline consiste nell'espressione dei tre cloni gli anti-interferone γ come immunoglobuline in formato nativo e nel test della loro funzionalità sulla piattaforma automatizzata come reagenti in un kit diagnostico della tubercolosi commerciale.
In diagnostics, many tools rely on the detection of specific markers in biological specimens and widely exploit the ability of antibodies to recognize their antigen. Thus, the implementation of the various processes ranging from antibody isolation to their high rate production is fundamental. Thanks to the advantages of recombinant DNA technologies, in vitro display methods have been developed for large scale isolation of monoclonal antibodies. Phage display is a technology based on the screening of an antibody library against a given antigen. The antibody library is usually composed of randomly combined immunoglobulin’s light and heavy variable domains fused with a coat protein of the bacteriophage M13. Each phage exposes on its surface a single peptide, so the coding sequence of the antibody fragments can be easily retrieved. The main objective of this work is to optimize a pipeline for recombinant monoclonal antibody development against antigens of diagnostic interest. This process begins with the isolation from a naïve phage library of clones able to recognize the antigen. Therefore, this work started with the creation and the characterization of a naïve phage display library. Once constructed the library, it has been validated with NGS to have an actual snapshot about library diversity and with Sanger sequencing to assess the quality of the coding sequences. To optimize and validate the whole pipeline, as a “pilot” antigen it has been chosen Interferon γ, a diagnostically relevant protein given its employment in the detection of tuberculosis. Different biopanning procedures allowed to isolate, overall, 25 different scFv. Thanks to a deeper characterization, performed on an automated platform designed for immunodiagnostic testing, it was possible to point out the best performing clones. Among them, two were chosen to be maturated to further enhance their affinity towards the antigen. Since it is well established the key role played by the VH in antigen recognition, it has been decided to create two new phage display libraries composed of scFv all bearing the VH of the two parental clones and a panel of VLs. The tailored selection procedure allowed to isolate two clones that seemed to perform better than the parental one on the automated platform. In a diagnostic setting, usually, full-size immunoglobulins are required, so, once selected, the antibody fragments are cloned in vectors allowing the expression in mammalian host cell lines. In this work a set of vectors allowing the expression of the human IgG, IgA, IgM and mouse IgG has been built. All the vectors have been transfected and antibody production was good also in terms of correct folding of the full-size immunoglobulin. The final step of validation of this whole pipeline consists in the expression of all the three anti-Interferon γ scFv as full-size immunoglobulin and the assay of their functionality on an automated platform as reagents in a commercial tuberculosis diagnostic kit.

Cette recherche est basée sur l’idée que le tourisme stimule l’essor économique d’une région en créant de la valeur ajoutée, améliorant ainsi le niveau de vie de ses habitants (Bensahel, Donsimoni, 1999: 27). Son but est de démontrer que le tourisme peut être un moteur de développement pour la région étudiée. Notre hypothèse principale est que dans une région riche en ressources exploitables touristiquement mais dont le niveau de développement est faible (PIB en dessous de la moyenne européenne) la multiplication des projets touristiques de mise en valeur des ressources permettrait de stimuler et de soutenir le développement de la région tout en respectant les principes du développement durable. La thèse est divisée en deux parties. La première est conceptuelle et méthodologique et se focalise sur l’aménagement basé sur le projet territorial de développement touristique. La deuxième partie est consacrée aux étapes principales d’un projet territorial de développement touristique : diagnostic territorial et propositions de développement futur. Pour le diagnostic territorial nous avons conçu un outil permettant de calculer la valeur touristique totale des divisions territoriales et d’analyser les données collectées concernant les pratiques touristiques déjà en œuvre dans la région. Cet outil nous a permis d’identifier 4 systèmes touristiques localisés qui peuvent être considérés comme modèle d’organisation de la région des Souscarpates situées entre les Vallées de l’Olt et du Motru
The current research is based on the assumption that tourism stimulates the economic growth of an area through creating the added value, thus, enhancing the standard of living in the area in question (Bensahel, Donsimoni, 1999: 27). We aim to demonstrate that in the targeted area tourism can (re)produce development. Noticing the weak development (DGP below the European average) in a region rich in resources that may be exploited through tourism, we think that the multiplication of such projects may boost and support the development of the region, in compliance with the principles of sustainability. The thesis is divided in two main parts. The former, conceptual and methodological in nature, focuses on territorial planning, based on the territorial project for tourism development. The latter is concerned with the main stages of a territorial project for tourism development : territorial diagnosis and future development proposal. In the case of territorial diagnosis, we designed a tool to calculate the total touristic value of the territorial divisions in question and to collect data using a survey of tourism practices in the area. The findings allowed us to identify 4 local tourism systems that we suggest as touristic organisation types in the Sub-Carpathians, situated between the Olt Valley and the Motru Valley

Usability evaluation results in several usability problems and the non-UE developer is often not a part of the evaluation as it might deter the participant from reporting all the errors and also, conducting usability evaluation is a usability engineerâ s responsibility. Thus the evaluator needs to create unambiguous usability problem reports, which will help the developer fix the usability problems. This research involves the development and evaluation of the Usability Problem Diagnosis tool, which supports problem diagnosis through analysis and storage in a common database shared between the evaluation and the development team. This tool uses the User Action Framework as an underlying knowledge base to support problem diagnosis.
Master of Science

Bridge fault extractors are tools that analyze chip layouts and produce a realistic list of bridging faults within that chip. FedEx, previously developed at Texas A&M University, extracts all two-node intralayer bridges of any given chip layout and optionally extracts all two-node interlayer bridges. The goal of this thesis was to further develop this tool. The primary goal was to speed it up so that it can handle large industrial designs in a reasonable amount of time. A second goal was to develop a graphical user interface (GUI) for this tool which aids in more effectively visualizing the bridge faults across the chip. The final aim of this thesis was to perform FedEx output analysis to understand the nature of the defects, such as variation of critical area (the area where the presence of a defect can cause a fault) as a function of layer as well as defect size.

L'obésité sarcopénique (OS) est une condition caractérisée par la coexistence de l'obésité et de la sarcopénie, c'est-à-dire une réduction de la masse et de la fonction musculaires. Diagnostiquer l'OS est très complexe en raison de l'absence de critères diagnostiques universellement acceptés, ce qui entraîne des diagnostics imprécis et une estimation hautement variable de sa prévalence. Face à ces limitations, l'objectif de cette thèse était de développer un outil de diagnostic empirique pour l'OS aidé d'intelligence artificielle, basé sur l'analyse de la composition corporelle. Nous avons élaboré l'AIM-SO score dans une population de sujets en surpoids/obésité puis nous l'avons testé dans deux autres populations, l'une de patients avec obésité sévère subissant une chirurgie bariatrique, l'autre dans la population générale de l'UK Biobank. Une étude longitudinale a également été menée, avec un suivi à un an chez les sujets ayant subi une chirurgie bariatrique. Nous avons étudié les corrélations cliniques, en particulier cardiométaboliques et hépatiques, notamment histologiques per-opératoires, dans la cohorte bariatrique. La prévalence d'OS a été très proche entre ces trois cohortes. L'OS diagnostiquée par l'AIM-SO score était associée à de multiples comorbidités cardiométaboliques, ainsi qu'à une forme plus sévère d'atteinte inflammatoire et fibrosante du foie. Malgré la perte de poids, le bénéfice métabolique (rémission des comorbidités) après chirurgie bariatrique était moindre chez les patients ayant une OS. Des analyses préliminaires de la cohorte UK Biobank ont montré une association significative entre l'OS diagnostiquée par l'AIM-SO score et des paramètres de fonctionnalité musculaire, en particulier la force musculaire. Nous proposons ce nouvel outil diagnostique pour standardiser le diagnostic d'OS et identifier des patients en situation d'obésité avec sarcopénie dont le phénotype cardiométabolique et hépatique est plus sévère. Le diagnostic d'OS pourrait également renseigner le bénéfice attendu des différentes interventions à visée de réduction pondérale participant ainsi à une prise en charge médicale personnalisée
Sarcopenic obesity (SO) is a condition characterized by the coexistence of obesity and sarcopenia, the latter defined as a reduction in muscle mass and function. Diagnosing SO is highly complex due to the lack of universally accepted diagnostic criteria, leading to imprecise diagnoses and highly variable prevalence estimates. Given this scenario, this thesis aimed to develop an empirical diagnostic tool for SO using artificial intelligence, based on the analysis of body composition. We developed the AIM-SO score in a population of patients with overweight/obesity and tested it in two other populations: patients with severe obesity undergoing bariatric surgery (BS) and the general population of the UK Biobank. A longitudinal study with a one-year follow-up was conducted in subjects who underwent BS. We examined clinical correlations, particularly cardiometabolic and hepatic, including perioperative histological findings in the bariatric cohort. The prevalence of SO was similar across these three cohorts. SO diagnosed by the AIM-SO score was associated with multiple cardiometabolic comorbidities and more severe inflammatory and fibrosing liver damage. Despite weight loss, the metabolic benefit (remission of comorbidities) after BS was lower in patients with SO. Preliminary analyses of the UK Biobank cohort showed a significant association between SO diagnosed by the AIM-SO score and parameters of muscular functionality, particularly muscle strength. We propose this new diagnostic tool to standardize the SO diagnosis and identify patients with obesity and sarcopenia who exhibit more severe cardiometabolic and hepatic phenotype. Diagnosing SO could also inform the expected benefit of various weight loss interventions, thus contributing to personalized medical management

The Australian potato industry is threatened by inadequate measures to control the virus health of seed potatoes. Potatoes are vegetatively propagated; therefore infection can result in disease spreading through generations. This has the potential to cause significant economic losses. Virus testing on tuber sprouts is currently conducted by ELISA, however a significant time delay of several weeks can occur while tubers sprout. There is a considerable need for a rapid, quantitative and cost effective virus test directly on bulked samples of dormant tubers to identify infected lots during seed multiplication. The potato viruses of economic importance in Western Australia are Potato virus S, (PVS), Potato virus X, (PVX), Potato leafroll virus, (PLRV) and Tomato spotted wilt virus (TSWV). The main aim of this project was to develop reliable PCR-based methods for multiplex real-time quantitative detection of these viruses in bulked potato tuber samples for seed certification for domestic and export markets. Knowledge of the distribution of the viruses within tuber tissue was needed to develop more effective methods of RNA extraction. The distribution of the viruses in histological sections of potato tubers was investigated using immunohistochemistry and in situ hybridization. All four viruses were found to be distributed at the stolon end of freshly harvested infected potato tubers. Extraction of RNA from tuber tissue is problematic because it contains starches and phenolics which inhibit RT-PCR. Extracting RNA from the tuber peelings would overcome this problem; however one of the viruses, PLRV, is restricted to the phloem in potato tubers. The distribution of the phloem in the superficial tissue of potato tubers was therefore investigated using histological staining and transmission electron microscopy. The vascular tissue was found to be within 2 mm below the epidermis of the tuber. With this knowledge, total RNA was extracted rapidly and efficiently from bulked potato peelings equivalent to 300 dormant tubers to detect single infections of PLRV, PVX, PVS and TSWV. For the quantitative detection of these viruses in potato leaves and tuber tissue, specific primers and fluorescent-labeled TaqMan® probes were designed. A realtime multiplex, single tube RT-PCR assay was developed. The main tasks involved primer design, optimization of reagents, standardization of RNA samples from which standard curves for analysis were generated, and identification of a baseline on which to interpret results. Limits of detection sensitivity were established using a range of virus transcript copy numbers (8 x 101 to 8 x 109 copies of PVX and PVS, 1 x 102 to 1 x 1010 copies of PLRV and 1 x 103 to 1 x 1010 copies of TSWV). The multiplexed assay was validated in blind studies. This high-throughput test is accurate and sensitive, and as a result this test is in the process of being commercialized and used by the seed potato industry of Western Australia as a cost-effective diagnostic tool to detect viruses reliably in bulked samples of dormant potato tubers.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references.
The immune system distinguishes self from non-self to combat pathogenic incursions. Evasion tactics deployed by viruses, microbes, or malignant cells may impede an adequate response. In such cases, therapeutic interventions aid in the elimination of pathogens and the restoration of physiological homeostasis. A major road block in the development of such therapies is the reliance on imperfect detection methods to identify site(s) of infection, or to monitor immune cell recruitment to sites of infection or inflammation in vivo. The goal of this thesis is overcome at least some of these limitations by utilizing novel tools that have been developed and refined in the laboratory to facilitate in vitro and in vivo characterization of specific immune subsets. We then track their recruitment to sites of active immune responses, such as infection or tumor progression sites. These tools consist of two components: one that confers specificity for immune cells and the other offers a site for labeling in a controlled manner. Single-domain antibodies (VHHs) from camelids are amongst the smallest (15 KDa) proteins that can recognize a diverse set of targets with excellent specificity. Chemoenzymatic labeling of molecules using sortase allows site-specific attachment of a single label of interest to the target protein containing the sortase recognition sequence LPXTG. VHHs specific for immune cell determinants labeled with sortase technology facilitate non-invasive and efficient monitoring of cells that infiltrate immunological niches in vivo in a manner not possible until now. This thesis presents the development of novel methods to allow in vitro and in vivo detection and imaging of specific immune subsets and their recruitment to sites of an active immune response. This thesis aims to (1) use DNA oligomers as a scaffold to push the limits of fluorescence labelling yield (2) create small and efficient biosensors for the rapid capture of specific lymphocyte subsets from peripheral blood samples using VHHs and graphene oxide nanosheets (3) develop radioisotope-labeled VHHs to track immune cell subsets to elucidate the roles of innate and adaptive immune components in the course of infection. Chapter 1 describes a new method for protein labeling via site-specific modification of proteins using a DNA scaffold. To avoid self-quenching of multiple fluorophores localized in close proximity, Holliday junctions were used to label proteins site-specifically with fluorophores. Holliday junctions enable the introduction of multiple fluorophores with reasonably precise spacing to improve fluorescence yield for both single domain and full-sized antibodies, without deleterious effects on antigen binding. Chapter 2 presents a biosensor generated for characterization of leukocytes from whole blood using a graphene oxide surface coated with single domain antibody fragments. This format allows quick and efficient capture of distinct white blood cell subpopulations from small samples of whole blood in a format that does not require any specialized equipment such as cell sorters or microfluidic devices. Chapter 3 documents a non-invasive immune-PET imaging method for tracing CD8+ T cells in the course of influenza A infection to better elucidate their protective mechanism(s) and immunopathological effects.
by Zeyang Li.
Ph. D.

[ES] El síndrome de Usher (USH) es un trastorno raro autosómico recesivo definido principalmente por sordera neurosensorial (SNHL), y una distrofia retiniana conocida como retinosis pigmentaria (RP). La patología muestra heterogeneidad genética, puesto que se conocen al menos 10 genes responsables. No obstante, las mutaciones en USH2A son la causa más frecuente de la enfermedad, en gran medida por la recurrencia de la variante patogénica c.2299delG. En esta tesis se ha desarrollado un ensayo de edición génica para revertir dicha anomalía genética por medio del sistema CRISPR/Cas9. Se diseñaron y probaron varios complejos CRISPR específicos de locus, y el más eficiente fue usado para la corrección de la mutación c.2299delG en células derivadas de pacientes. La tasa de corrección de la mutación obtenida fue del 2.5%. Otro objetivo de esta tesis ha sido la caracterización genética de pacientes USH aún sin diagnóstico molecular. Una primera fase implicó la secuenciación masiva dirigida de las regiones codificantes de todos los genes asociados a la enfermedad. Este estudio, cuya cohorte incluyó 58 pacientes no escrutados previamente, permitió la identificación de 42 nuevas mutaciones presuntamente patológicas, y una tasa general de detección de alelos responsables de la enfermedad de prácticamente el 83%. Sorprendentemente, uno de los sujetos presentaba mutaciones en CEP250, uno de los últimos genes correlacionados con la enfermedad. Una exhaustiva revisión clínica reveló que la degeneración retiniana se trataba en realidad de una distrofia de conos y bastones en lugar de RP clásica, lo cual permitió consolidación del gen CEP250 como responsable de un fenotipo similar al USH. El resto de casos sin resolver induce a sospechar de la existencia de otros genes vinculados con USH. Así pues, se analizó el exoma íntegro de dichos casos negativos del panel por medio de secuenciación de exoma completo, lo cual proporcionó resultados relevantes en seis de las muestras estudiadas. Uno de tales sujetos resultó ser un claro caso de fenocopia de USH, al albergar mutaciones patogénicas en dos genes independientes, TECTA y REEP6, siendo el primero responsable de la SNHL y el segundo de la RP. De forma parecida, en otro paciente se detectaron variantes patológicas para RP en el gen EYS, pero no se identificó paralelamente ningún cambio genético que explicara la SNHL. Tres individuos adicionales resultaron haber sido erróneamente diagnosticados como USH, dada la conclusiva inexistencia o ambigüedad de la sordera. Uno de ellos fue definido como homocigoto de una mutación en CNGB1, ya reconocido como responsable de RP. En el segundo de dichos sujetos se identificó una mutación en homocigosis en el gen GRN, cuyos defectos en estado heterocigoto están asociados a demencia frontotemporal y más raramente combinada con RP si ambos alelos se encuentran alterados. Por otro lado, el tercer paciente fue resuelto como heterocigoto compuesto de variantes en WDR19, un gen asociado en mayor medida a una distrofia retiniana acompañada de trastornos renales y, más raramente, a la forma aislada del síntoma. En el último de los seis casos resaltados de este objetivo se detectó una mutación homocigota sin sentido en el gen ASIC5, cuyo papel en el organismo todavía se desconoce. Sin embargo, se han correlacionado funciones visuales y auditivas para miembros de la misma familia proteica. En conjunto, los hallazgos obtenidos en este trabajo avalan la importancia del uso de las más novedosas tecnologías en la búsqueda de soluciones para enfermedades raras, las cuales presentan por ahora un pronóstico terapéutico bastante desamparado. Asimismo, otras consecuencias positivas en cuanto a la caracterización genética de los pacientes son la corroboración (o rectificación) del diagnóstico inicial, así como la contribución a la estimación demográfica y correlaciones de genotipo-fenotipo, que en definit
[CAT] La síndrome d'Usher (USH) és una malaltia rara autosòmic recessiu definit principalment per sordera neurosensorial (SNHL) i una distròfia retiniana coneguda com a retinosi pigmentària (RP). La patologia mostra heterogeneïtat genètica, ja que es coneixen almenys 10 gens responsables. No obstant això, les mutacions en USH2A són la causa més freqüent de la malaltia, a causa de la recurrència de la variant patogènica c.2299delG. En aquesta tesi s'ha desenvolupat un assaig d'edició gènica per a revertir la dita anomalia genètica per mitjà del sistema CRISPR/Cas9. Es van dissenyar i van probar diversos complexos CRISPR específics de locus, i el més eficient va ser usat per a la correcció de la mutació en cèl·lules derivades de pacients. La taxa de correcció de la mutació obtinguda va ser del 2.5%. Un altre objectiu d'aquesta tesi ha sigut la caracterització genètica de pacients USH encara sense diagnòstic molecular. Una primera fase va implicar la seqüenciació massiva dirigida de les regions codificants de tots els gens associats a la malaltia. Aquest estudi, la cohort de la qual va incloure 58 pacients no escrutats prèviament, va permetre la identificació de 42 noves mutacions presumptament patològiques, i una taxa general de detecció d'al·lels responsables de la malaltia de pràcticament el 83%. Sorprenentment, un dels subjectes presentava mutacions en CEP250, un dels últims gens correlacionats amb la malaltia. Una exhaustiva revisió clínica va revelar que la degeneració retiniana es tractava en realitat d'una distròfia de cons i bastons en lloc de RP clàssica. Aquestes troballes han permés la consolidació del gen CEP250 com a responsable d'un fenotip similar al USH. La resta de casos sense resoldre induïx a sospitar de l'existència d'altres gens vinculats amb USH. Així, doncs, es va analitzar l'exoma íntegre dels casos negatius del panell a través de seqüenciació d'exoma complet, cosa que va proporcionar resultats rellevants en sis de les mostres estudiades. Un de tals subjectes va resultar ser un clar cas de fenocopia d'USH, a l'albergar mutacions patogèniques en dos gens independents, TECTA i REEP6, sent el primer responsable de la SNHL i el segon de la RP. De forma semblant, en un altre pacient es van detectar variants patològiques per a RP al gen EYS, però no es va identificar paral·lelament cap canvi genètic que explicara la SNHL. Tres individus addicionals van resultar haver sigut erròniament diagnosticats com USH, donada la final inexistència o ambigüitat de la sordera. Un d'ells va ser definit com a homozigot d'una mutació en CNGB1, ja reconegut com a responsable de RP. En el segon d'aquestes subjectes es va identificar una mutació en homozigosi en el gen GRN, els defectes del qual estan associats a demència frontotemporal en estat heterozigot, i més rarament en combinació amb RP si ambdós al·lels es troben alterats. D'altra banda, el tercer pacient va ser resolt com a heterozigot compost de variants en WDR19, un gen associat en major grau a una distròfia retiniana acompanyada de trastorns renals i, més rarament, a la forma aïllada del símptoma. En l'últim dels sis casos ressaltats d'aquest objectiu es va detectar una mutació homozigota sense sentit en el gen ASIC5, el paper en l'organisme del qual encara es desconeix. Amb tot, s'han correlacionat funcions visuals i auditives per a membres de la mateixa família proteica. En conjunt, les troballes obtingudes en aquest treball avalen la importància de l'ús de les més noves tecnologies en la recerca de solucions per a malalties rares, les quals presenten per ara un pronòstic terapèutic prou desemparat. Així mateix, altres conseqüències positives quant a la caracterització genètica dels pacients són la corroboració (o rectificació) del diagnòstic inicial, així com la contribució a l'estimació demogràfica i correlacions de genotip-fenotip, que en definitiva ajuden en la compressió d'US
[EN] Usher syndrome (USH) is a rare autosomal recessive disorder defined essentially by sensorineural hearing loss (SNHL) and a retinal dystrophy known as retinitis pigmentosa (RP). The condition shows a genetic heterogeneity, since there are at least 10 genes known to be causative of the syndrome. However, mutations in USH2A are the most frequent cause of the disease, due in a large measure to the recurrence of the c.2299delG pathogenic variant. A gene editing assay to reverse this specific genetic anomaly was developed in this thesis by means of the groundbreaking CRISPR/Cas9 system. Several locus-specific CRISPR complexes were designed and tested, and the most efficient was used to proceed with the c.2299delG mutation correction on patient-derived cells. The trial resulted in a mutation correction rate of 2.5%. Another goal of this thesis was the genetic characterization of molecularly undiagnosed USH patients. Given the genetic diversity of the disease, the procedure required the implementation of high-throughput sequencing, a technology that enables in bulk sequencing of any number of selected loci (or the indiscriminate totality) of the genome. The first phase implied the targeted sequencing of the coding-relevant regions of all known causative or disease-associated genes at the moment. The study, comprising a cohort of 58 previously unscreened patients, enabled the identification of 42 novel putative pathogenic mutations, and an etiologic-allele detection ratio shy of 83%. Remarkably, one of the subjects harbored nonsense mutations in CEP250, which is one of the latest USH-associated genes. However, an exhaustive review of the clinical features unmasked the retinal degeneration as a cone-rod dystrophy rather than RP, which reinforced the linkage of the gene to an USH-like phenotype. The remaining portion of unresolved cases lead to suspicion of the existence of other genes accountable for USH. Hence, the complete exome of such panel-negative cases was screened through whole exome sequencing. This venture provided relevant findings in six of the surveyed samples. One subject was plainly exposed as an USH phenocopy by harboring pathogenic splice-site mutations in two independent genes, TECTA and REEP6, the former responsible for the SNHL and the latter for the RP. Similarly, RP-causative variants in EYS were detected in another patient, yet no pathogenic changes explaining the HL were discovered. Three additional individuals were ultimately unveiled as USH misdiagnosed cases, being the HL actually absent or ambiguous. One of the patients in this set was homozygous for a mutation in CNGB1, already known to be accountable for RP. The other two cases showed a more peculiar outcome being compound heterozygous for putatively pathogenic variants in genes generally associated to other disorders. One presented a homozygous mutation in GRN, a gene associated to frontotemporal dementia under heterozygous condition and less commonly to combined RP for homozygous alterations. The third subject was found to be a carrier of mutations in WDR19, a gene best associated with retinal disorders accompanied by renal signs and rarely with the isolated visual symptom. The last case presented a homozygous nonsense variant in the ASIC5 gene, whose role has yet to be learned. However, some correlations to visual and hearing functions have been reported for members of the same protein family. Altogether, the results obtained from this work attest to the importance of applying the most up-to-date technologies in the search of solutions for rare diseases that realistically pose a despairing therapeutic prognosis. In addition, the positive consequences of the genetic characterization of the patients are the corroboration (or else correction) of the initial diagnosis, and the contribution to the appraisal of demographic and genotype-phenotype correlations, which ultimately aid in the understanding USH and other related diseases.
This work was financially supported by the Institute of Health Carlos III and FEDER funds (ISCIII; grants PI13/00638, PI16/00425, PI16/00539, and PIE13/00046), Fundación ONCE (grant 2015/0398), XVIII Fundaluce-FARPE, and “Telemaratón: Todos Somos Raros, Todos Somos Únicos” (grant IP58). C.F.-G. is a recipient of a fellowship (grant IFI14/00021) from the ISCIII. R.P.V.-M. is a Miguel Servet researcher (grant CP11/00090 funded by ISCIII, Madrid, Spain). The funds from the ISCIII are partially supported by the European Regional Development Fund. R.-P.V.M. is also a Marie Curie fellow (grant CIG322034 from the European Commission).
Fuster García, C. (2020). Therapeutic approaches and development of genomic diagnostic tools for Usher syndrome [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137034
TESIS

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.

Glioblastomas multiforme (GBM) are the most common malignant primary brain tumors in adults. They are highly aggressive and have an overall survival of <15 months despite maximal surgical resection and chemoradiation (Ostrom, 2019). GBMs are typically heterogeneous with a wide range of genetic and epigenetic variations among tumor cells. Extracellular vesicles (EVs) represent one of the plausible ways through which can be obtained a better understanding of the heterogeneous subpopulations of GBM / molecular signatures. EVs hold promise for the discovery of potential tumours biomarkers useful in clinical managment for GBM patient diagnosis and follow-up. Isolating EVs from body fluids and screening their protein content may serve as a complementary approach to assess the heterogeneous molecular landscape of GBM as tumors evolve. Many reports support the idea that Hsps are implicates in the pathogenesis and in the progression of different human neoplasms, by uncertain metabolic mechanism. Athough Hsps perform their canonical “chaperoning” functions in both prokariotic and eukaryotic cells, they have also acquired, probly during evolution, “extra-chaperoning” roles. Among these roles, there are some involved in the mechanism of cancerogenesis. In this study, we evaluate the exspression of some Hsps (in particular Hsp10, Hsp27, Hsp60, Hsp70, and Hsp90) through experiments of immnohistochemistry in samples of GBM and healthy controls, and also by immunofluorescence analysis on priamry and secondary cell lines of GBM. We also focused to research these proteins in EVs isolated from plasma obatained from patients with GBM, before and after surgery. The isolation was followed by a morphological and biochemical characterization of the EVs, in order to better study the characteristics of these potential natural carriers for the tool development for diagnostic and, possibly, also follow-up biopsy.

Thesis (S.M. in Real Estate Development)--Massachusetts Institute of Technology, Program in Real Estate Development in Conjunction with the Center for Real Estate , 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 141-142).
Real estate investment firms have the ever increasing need for understanding their firm's strengths and weaknesses, their performance relative to their peers and competitors, and for developing assessment tools for facilitating more informed investment and management decisions. One potentially very useful tool to further these objectives, a tool that is so far underutilized and underappreciated, is investment performance attribution analysis. Such performance attribution may be broadly characterized as the partitioning of the total investment return of a particular manager or portfolio in order to quantify and help to understand and assess the components and determinants of the overall investment performance. Traditional investment attribution analysis, adopted from the securities investment industry, has focused primarily on the portfolio level, where property selection and allocation factors are the two primary attributes of total return that can be parsed and benchmarked. In the case of real estate investments, property-level investment functions such as operational management and asset transaction execution, which are not captured by a traditional attribution analysis, also play a major role in the overall investment returns. During the past two decades a system to drill the investment performance attribution down to a deeper level, separating the asset "selection" component into further breakouts, including income return and components of the capital return (cash flow change and yield change), have been propounded by influential firms such as the Investment Property Databank (IPD) based in the UK. In a 2003 article David Geltner proposed a system for property-level performance attribution (PPA) based on the since-inception IRR of each individual property investment. This thesis furthered Geltner's work on PPA by an in depth exploration of the application of the IRR-Based Property-Level Performance Attribution analysis based on a large-scale, real-world-based case study of a complete set of actual core-asset round-trip transactions completed by several internally managed funds in the institutional investment industry. Furthermore, this thesis explored the use of PPA for organizational management diagnostics, and thereby demonstrated the potential of using the PPA analysis as an investigative tool for developing plausible hypotheses about a firm's investment management strengths and weaknesses.
by Tony Feng.
S.M.in Real Estate Development

Research has demonstrated that academic engagement is an important resource for students, promoting their learning and achievement. Less well documented is the possibility that students' classroom engagement may also be a valuable resource for their teachers, capable of influencing how teachers treat their students over time. The current study sought to examine the relationship between student motivation and teacher behavior to better understand how teachers perceive and respond to their students' classroom motivation and whether these motivational states contain diagnostic information about the types of supports students may need in order to be engaged, enthusiastic learners. The observable manifestations of motivation, engagement and disaffection, may contain valuable information about students' inner experiences that educators can use to optimize their teaching. Thus, the goal of the current study was to examine the reciprocal effects of student motivation on teachers' provision of support by using a longitudinal design, a more comprehensive assessment of behavioral and emotional engagement and disaffection, and a person-centered approach to investigate whether potential factors influencing the quality of students' classroom engagement can help inform more targeted intervention efforts. Data from 1018 3rd through 6th grade students and their teachers were used to create two sets of teacher-reported student motivation profiles, namely, a theory-driven and an empirically-derived set of profiles. Using both sets of profiles, the current study failed to provide evidence that student engagement and disaffection profiles influenced changes in the quality of support students' received from their teachers over the school year. The current study also examined whether knowledge of the motivation profile into which a student falls can tell us something meaningful about their unobservable, inner experiences or self-system processes (SSP's) such that we can use their profile to "diagnose" motivational issues stemming from these student inner experiences. Results indicated that, with one exception, students in different profiles did not report differential levels of the three SSP's; rather, if students in a given profile had low levels of one self-system process, they had low levels of all three. Finally, for two of the ten student motivation profiles, (At Risk and Checked-out) students in the high teacher support subgroup and the low teacher support subgroup experienced differential changes in their self-reported engagement from fall to spring such that the students who received the "treatment" (high levels of teacher support) started and ended higher than those who received low levels of teacher support, but also showed steeper declines over the year, because students with low teacher support started low and remained low (but did not lose any more ground) across the year. Discussion focuses on the utility and potential drawbacks of using person-centered approaches to examining student motivation and potential causes for the lack of supported hypotheses. Implications discuss the need for further research and how we can help teachers gain a more nuanced and differential view of their students' motivated actions and emotions in the classroom.

The aim of this study was to evaluate new methods to improve detection and investigation of the effects of chronic or subclinical infection with Trypanosoma evansi in various mammalian species. Some of the more resistant host species, including pigs and buffaloes, are present in large feral populations in the northern parts of Australia, the area where T. evansi is most likely to gain entry to the country. Existing tests are not sufficiently reliable to detect all cases of disease and they cannot distinguish acute from chronic infections. Furthermore, the tests have different sensitivities in different host species. Surveillance for trypanosomiasis in Australia is problematic because of the need to work in remote parts of northern Australia where provision of a cold-chain for traditional blood and serum storage is difficult. An existing dried blood storage system was modified by treating cotton lint filter paper (Whatman #903) with a commercial post coating buffer (TropBio, Queensland). This treatment increased the longevity of antibodies to T. evansi in serum and blood stored on the paper (detected using an antibody-detection ELISA) compared to samples stored on plain paper, especially when the papers were stored under humid conditions and at high ambient temperatures. Attempts were made to improve the diagnostic utility and repeatability of antibody-ELISAs through the use of 2 recombinant T. brucei antigens (PFRA and GM6) and to optimize a competitive ELISA using RoTat 1.2 variable surface antigen and its monoclonal antibody. Antibody-detection using the two recombinant proteins was not sufficiently specific to enable their use for the detection of T. evansi. The RoTat 1.2 cELISA had good sensitivity and specificity (75% and 98% respectively) when used to test serum from cattle and buffaloes experimentally infected with T. evansi and uninfected animals. However, the test was not able to detect anti-T. evansi antibodies in serum from wallabies, pigs, a dog or a horse that were experimentally infected with T. evansi. The inability of the cELISA to detect anti-T. evansi antibodies may be due to the small number of samples tested or the lack of RoTat 1.2 specific antibodies in the animals tested. The feasibility of using an enzymatic test to detect trypanosome aminotransferase or antibodies to this enzyme was evaluated. Prior publications suggested that the detection of TAT was an appropriate diagnostic tool for the detection of T. evansi infection in camels. However, the results from this study did not support the use of this test for the detection of T. evansi infection in cattle or buffaloes with low to moderate parasitaemia. Trypanosomiasis is an immunological disease that affects most of the body’s organs, with more severe disease developing over time. Attempts were made to determine key cytokine and biochemical patterns that would distinguish infected from uninfected animals and acute from chronic infections. The results from this study showed that there was no specific pattern in serum cytokines or serum biochemistry that could be used to distinguish infected from uninfected animals, or different stages of disease. Immunohistochemistry was used on tissues from buffaloes and mice experimentally infected with T. evansi and T. brucei gambiense respectively to characterise the cellular immune response that was present. The immune response was predominantly cell mediated, with CD3+ T lymphocyte and macrophage infiltration occurring in most tissues. In end stage disease there was often suppression of the immune system with disruption of the architecture of the spleen and a decrease in B lymphocytes in the circulation. Trypanosomes were rarely visible in the tissues and were only seen in those animals with high parasitaemia. Lesions generally became more severe over time, but there was a large variation between animals, which suggests that immunohistochemistry is unsuitable as a diagnostic tool.

Bitter pit is an important physiological disorder of many apple cultivars where the low uptake and poor distribution of calcium within the cortex of apples pervades. Controlled atmosphere storage and application of 1-MCP (SmartFreshSM) can delay the onset of bitter pit symptoms by delaying maturity and senescence; however, significant losses may occur in long-term stored apples. It is hard to detect internal bitter pit using external examination alone. Previous studies have focused on improving pre-harvest prediction and curative treatments before harvest. Present prediction models are based on history of orchards, mineral analysis 2-3 weeks before harvest and quality assessments and monitoring over storage time. This study aimed to identify a greater understanding of the storage potential of fruit based on destructive standard quality assessments, biochemical and molecular analysis, also a non-destructive monitoring method by chlorophyll fluorescence at the point of harvest and monitoring during storage for developing more reliable prediction models to improve storage management. The role of free and conjugated calcium in maintaining cellular integrity and the relationship between biochemical and fluorescence changes and development of bitter pit were investigated. A diagnostic model based on comparison of changes of ascorbic acid during storage was developed. Another diagnostic model based on changes in the proportion of calcium oxalate content during storage in comparison with harvest was developed to identify samples with higher propensity to bitter pit. Also chlorophyll fluorescence was investigated as a non-destructive method for monitoring fruit during storage and prediction models for detecting changes in the maturity of fruit and developing bitter pit and reduction of fluorescence during storage as an alert to identify incidence of bitter pit were developed. Furthermore, changes in gene expression profiles of a limited number of genes like calmodulin showed the differences in patterns of transcripts between apples suffering from bitter pit and healthy apples. All the suggested methods have potential of being commercialised and applied practically to improve apple fruit store management. It would be possible to build a multi variate model for predicting the onset of bitter pit development in apple by combination of two or more suggested diagnostic tools.

Doctor of Philosophy
Department of Chemistry
Stefan Bossmann
Numerous Proteases are implicated in cancer initiation, survival, and progression. Therefore, it is important to diagnose the levels of protease expression by tumors and surrounding tissues, which are reflected in blood and tissue samples. Nanoplatforms for Cathepsin(CTS) B and L, matrix metalloproteinases(MMP) 1, 2, 3, 7, 9, 13 and urokinase plasminogen activator(uPA) detection have been synthesized. Nanoplatforms feature a central dopamine-coated core/shell Fe/Fe₃O₄ nanoparticle. Cyanine 5.5 is permanently tethered to the dopamine ligands via amide bonds. Tetrakis(4-carboxy-phenyl)porphyrin (TCPP) is co-tethered to Fe/Fe₃O₄/dopamine by means of protease consensus sequences. In the presence of a relevant protease sequence, it is cleaved, releasing TCPP from the nanoplatform. In contrast, Cy 5.5 will remain permanently tethered to the nanoparticle. Therefore, an extensive increase of emission intensity of the fluorescence signal from TCPP is observed. This permits the detection of the activity of proteases at femtomolar levels in biospecimens by fluorescence spectroscopy. 46 breast cancer and 20 healthy human blood serum samples were analyzed. Based on the expression pattern of analyzed enzymes, human breast cancer can be detected at stage I. By monitoring CTS B and L stage 0 detection may be achieved. This study demonstrates the feasibility of minimally invasive successful early cancer diagnosis. Immunosuppression is one of the hallmarks of aggressive cancers. Arginase is overexpressed in cancer patients, resulting in systemic immunosuppression. Two nanoplatforms for arginase detection have been synthesized. Both feature a central dopamine-coated core/shell Fe/Fe₃O₄ nanoparticle to which cyanine 7.0 or cyanine 7.5 is tethered via amide bonds. In both nanoplatforms, cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. According to our results obtained from fluorescence spectroscopy, the oligopeptides GRRRRRRRG and GOOOOOOOG differ in their chain dynamics. In the presence of arginase, and dependent on arginase activity, fluorescence increase of both nanoplatforms is observed, which is an indication that proton-transfer quenching decreases when arginine gets converted to ornithine. The novel assays permit the detection of active arginase within an hour. Additionally, Förster Resonance Energy Transfer (FRET) is observed in nanoplatforms featuring cy 5.5/7.0 pairs, resulting in picomolar detection limits. This is the first example of a “post-translational” enzyme sensor, in which the tether is subjected to chemical transformations of the aminoacid side chains and not cleaved by an enzyme, resulting in the modified mobility of the tether. The nanoplatforms do not show a fluorescence increase when incubated with NO-reductase, an enzyme indicative of immunoactivation, which also uses arginase as substrate. Copper dependent inhibitory activity of 10000 compound library has been studied against of Staphylococcus aureus. 53 copper- dependent hit molecules were recognized featuring extended thiourea core structure with NNSN motif. NMR titrations, UV/Vis studies have been performed for characterization of metal complexation and structure modeling. Chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, in spite of other compound groups in chemical screening libraries. This will lead to the development of novel class of antibacterial agents against Staphylococcus aureus.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Aeronautics and Astronautics, 2013.
This thesis was scanned as part of an electronic thesis pilot project.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 107-109).
A low power, Hall-effect type plasma thruster known as the MIT-Cylindrical Cusped- Field Thruster (MIT-CCFT) has been developed and simulated using a fully-kinetic plasma model, the Plasma Thruster particle-in-cell (PTpic) model. Similar to the Diverging Cusped-Field Thruster (DCFT) previously developed in the Massachusetts Institute of Technology Space Propulsion Laboratory, this thruster uses cusped magnetic fields aligned in alternating polarity in order to confine electrons, thus slowing their flow to the anode and readily ionizing neutral gas, which is then electrostatically accelerated by the anode. The design methodology for the CCFT will be discussed, with significant emphasis on the effects of magnetic topology on thruster performance. In particular, while the topology is similar to that of the DCFT in that it also confines the discharge plasma away from the channel walls to limit wall erosion, the CCFT was also designed to minimize plume divergence. To predict the CCFTs performance and plasma dynamics, the design has been modeled and simulated with PTpic. From multiple simulations of the CCFT under different operating conditions, the thruster performance and plume characteristics were found and compared to past simulations of the DCFT. Specifically, the predicted nominal total efficiency ranged from 25 to 35 percent, providing 4-9 mN of thrust at a fixed xenon mass flow rate of 4.0 sccm, whilst consuming 90-400 W of power and with a corresponding nominal specific impulse of 1050 to 1800 s. Preliminary observations of the particle moments suggest that the magnetic confinement of the plasma isolates erosion of the channel walls of the discharge chamber to the ring cusps locations. In addition, in contrast to the DCFT, the CCFT does not have a hollow conic plume; instead, its beam profile is similar to that of traditional Hall-effect thrusters. To supplement the efforts for optimizing longevity of the cusped-field thruster, a new diagnostic tool for erosion studies, novel to the electric propulsion community, has been implemented and has undergone preliminary validation. Ion beam analysis (IBA) allows for in-situ measurements of both composition and profile of the surfaces of the discharge region of a plasma thruster during operation. The technique has been independently tested on individual coupons with the use of the Cambridge Laboratory for Accelerator Study of Surfaces (CLASS) tandem ion accelerator. The coupons, which are composed of materials with known sputtering rates and/or are commonly used as insulator material, are exposed to helicon-generated plasma to simulate the sputtering/re-deposition found in thruster discharge region. Through comparison of ion beam analysis traces taken before and after plasma exposure, the effective erosion rates were found and validated against simulated results.
by Anthony Pang.
S.M.

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.

The objective of this PhD research was the development of novel 3D interactive medical platforms for medical image analysis, simulation and visualisation, with a focus on oncology images to support clinicians in managing the increasing amount of data provided by several medical image modalities. DoctorEye and Automatic Tumour Detector platforms were developed through constant interaction and feedback from expert clinicians, integrating a number of innovations in algorithms and methods, concerning image handling, segmentation, annotation, visualisation and plug-in technologies. DoctorEye is already being used in a related tumour modelling EC project (ContraCancrum) and offers several robust algorithms and tools for fast annotation, 3D visualisation and measurements to assist the clinician in better understanding the pathology of the brain area and define the treatment. It is free to use upon request and offers a user friendly environment for clinicians as it simplifies the implementation of complex algorithms and methods. It integrates a sophisticated, simple-to-use plug-in technology allowing researchers to add algorithms and methods (e.g. tumour growth and simulation algorithms for improving therapy planning) and interactively check the results. Apart from diagnostic and research purposes, it supports clinical training as it allows an expert clinician to evaluate a clinical delineation by different clinical users. The Automatic Tumour Detector focuses on abdominal images, which are more complex than those of the brain. It supports full automatic 3D detection of kidney pathology in real-time as well as 3D advanced visualisation and measurements. This is achieved through an innovative method implementing Templates. They contain rules and parameters for the Automatic Recognition Framework defined interactively by engineers based on clinicians’ 3D Golden Standard models. The Templates enable the automatic detection of kidneys and their possible abnormalities (tumours, stones and cysts). The system also supports the transmission of these Templates to another expert for a second opinion. Future versions of the proposed platforms could integrate even more sophisticated algorithms and tools and offer fully computer-aided identification of a variety of other organs and their dysfunctions.

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 αβ induced bacterial lysis by damaging P. gingivalis membrane. In study III, we demonstrated that PLNC8 αβ dose-dependently induces proliferation and release of growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 αβ decreased P. gingivalis-induced cytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects of anti-P. gingivalis antibodies and PLNC8 αβ in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 αβ modulated P. gingivalis-induced expression of growth factors in GECs, however, their effects were diminished when used in combination. The results of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 αβ in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol)
Ichthyophonus hoferi is a highly pathogenic histozoic parasite that has low host specificity capable of producing mass mortalities of epizootic proportions in marine commercial fish populations. Currently in Southern Africa, I. hoferi has been reported from flathead mullet (Mugil cephalus) from the Kowie lagoon and from multiple species on exhibit at the Two Oceans Aquarium. Since epizootiologists rely on accurate assessments of prevalence to establish patterns of morbidity and mortality within populations, using the most accurate diagnostic techniques for accurate assessments of infection is imperative. Currently, several diagnostic techniques have been employed to detect I. hoferi in infected fish hosts. These include macroscopic examination of tissues, microscopic examinations of wet-mount squash preparations of tissue, histological examination of tissue sections, in vitro culture of tissue explants, the polymerase chain reaction (PCR) using I. hoferi-specific primers and real-time quantitative PCR (qPCR) using I. hoferi-specific primers and a hydrolysis probe.

Bacterial keratitis and bacterial endophthalmitis are two of the most devastating sight threatening eye infections. It is currently unclear whether the organisms isolated from these infections represent specialised members of these species or whether all strains are equally likely to cause infection. One method of differentiating strains genotypically is by a typing technique known as multilocus sequence typing (MLST). Using this technique, a better understanding of the molecular epidemiology of eye infections can be achieved. Propionibacterium acnes (P. acnes) is an important anaerobic organism causing several types of eye infections. Although it is now beginning to be recognised as a serious opportunistic pathogen, few studies have been done to investigate the population biology of P. acnes at the molecular level. Our continuing inability to distinguish between strains of P. acnes means that we still do not fully understand how antibiotic-resistant strains spread, nor whether certain strains, or clonal complexes, of P. acnes are associated with certain infections. These are key issues that can now be understood with our development of an MLST system for P. acnes. A diverse culture collection of 125 P. acnes isolates have been analysed using the MLST scheme developed. Sequence analysis shows that there are phylogenetically distinct groups within P. acnes and identified a novel cluster not previously described. Analysis of recombination using several methods suggests that frequent recombination occurs within these subgroups. There appears to be no association between these subgroups and clinical manifestation of P. acnes infection or geographical location. The P. acnes MLST scheme was validated against 16S rRNA gene and complete recA gene typing as well as immunofluorescence microscopy (IFM) and random amplification of polymorphic DNA (RAPD) analysis. P. acnes is a slow growing organism and is difficult to culture from ocular samples. A real-time PCR assay was developed in order to overcome the low culture positive rate and delay associated with conventional culture methods. Primers targeting one of the seven housekeeping genes used in the MLST scheme (gmk), specific for P. acnes were selected. The real-time PCR assay was both specific to P. acnes and highly sensitive. Pseudomonas aeruginosa (P. aeruginosa) is another important organism in the development of serious eye infections, and is the commonest cause of contact lens related microbial keratitis. Infections with this organism can lead to rapid deterioration of vision and possible blindness. A previously developed MLST scheme has been applied to 117 eye isolates from around the UK and China. This typing data was compared to 166 P. aeruginosa isolates from other clinical and environmental sources. Overall, MLST data supports previous findings that P. aeruginosa has a nonclonal population with epidemic clones. Sequence analysis showed that eye isolates do not cluster away from isolates from other clinical infection sites.

Lithium-ion batteries age over their lifetime of operation through various complex degradation mechanisms. In order to maximise battery performance and lifetime, the industrial standard method of measuring the state of health (SOH) by capacity and power fade is no longer good enough. In this thesis, Differential Thermal Voltammetry (DTV) is introduced as a new in-situ diagnosis/prognosis tool that is capable of a more sophisticated diagnosis of SOH, and is application ready with minimal installation costs. DTV uses only cell voltage and surface temperature measurements to infer detailed SOH information. The method was validated through various accelerated ageing experiments carried out on commercial lithium-ion pouch cells as well as through industrial collaborations. DTV was demonstrated for use as a diagnostic tool on a cell level for tracking battery degradation throughout its operation, for screening 2nd life batteries to estimate the degradation state/history, and for estimating the state-of-charge (SOC) of lithium iron phosphate cells during partial charge/discharges. In anticipation of an EV application, the technique was tested for its capabilities in a battery pack. DTV was able to correctly identify aged cells within a pack in a quantitative manner validating its capability embedded within a battery management system (BMS). Through an industrial collaboration, DTV demonstrated its use as a prognosis tool for forecasting cell failure in a commercial BMS mounted on a high-power battery pack used in a motorsport application. The concept of adaptive operation through DTV analysis was explored which resulted in a slower rate of degradation. Given the application opportunities validated through experiments, this research aims to provide an alternative tool for battery diagnosis/prognosis in a real-world application.