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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.

Background: Repeat renal biopsies in patients with lupus nephritis (LN) are usually done to guide treatment or to establish disease chronicity. Their value is not clear from available literature. There is also no available data in Africa to guide clinicians. Methods: This was a retrospective study of patients undergoing a repeat renal biopsy between January 2003 and December 2014 from a single centre in Cape Town, South Africa. Relevant demographic, clinical and histological records of patients with repeat renal biopsies were documented. Comparison of data from 1st and 2nd renal biopsy was performed. Results: 44 patients had at least 2 biopsies done during the study period. Most patients were females (81.8%). The mean biopsy interval was 2.8± 1.8 (range 0.38 – 9.4) years. Proteinuria was the main indication for the repeat biopsy (36.1%). The glomerular filtration rate and proteinuria worsened between the two biopsies (p=0.001 and 0.019) respectively suggesting disease progression. Most patients (65.4%) with a non-proliferative class of LN at first biopsy progressed into a proliferative class whereas patients with initial proliferative LN at first biopsy (77.8%) remained as proliferative at repeat biopsy. Treatment was changed in 85% of patients at second biopsy. Conclusion: Repeat renal biopsies in patients with LN presents a useful means of assessing disease progression and provides guidance regarding modification of treatment. More studies are however required to evaluate the value of repeat biopsies and perhaps the need for protocol renal biopsies in patients with LN.

L'infection par le virus de l'hépatite B est un problème de santé publique avec 250 millions de porteurs chroniques et cela malgré l'existence d'un vaccin préventif. Les traitements actuellement utilisés sont les analogues de nucléos(t)ide et/ou l'interféron a. Bien qu'ils permettent une diminution de la charge virale, ils ne permettent pas d'éradiquer la maladie du fait de la persistance de l'ADNccc, le minichromosome de l'hépatite B. Cet ADN sert de matrice à la transcription virale, et la présence d'une seule copie permet la réactivation de l'infection. En prenant en compte la longue demi-vie des hépatocytes et de la stabilité de l'ADNccc dans leur noyau, un modèle mathématique suggère que de nombreuses années de traitement seraient nécessaires pour éliminer complètement cet ADN du foie des patients infectés chroniquement. Les techniques utilisées en routine pour la quantification de l'ADNccc ne sont pas assez sensibles pour pouvoir détecter des faibles concentrations de cet ADN, notamment dans des biopsies de patients infectés chroniquement et traités à long terme. Il est nécessaire de développer de nouvelles stratégies permettant de cibler directement l'ADNccc afin d'éliminer le virus. Ainsi les travaux de cette thèse reposent sur le développement d'une nouvelle technologie : la Droplet Digital PCR (ddPCR) pour permettre la quantification de l'ADNccc dans les biopsies de patient. Cette technique permet un gain de 2 log au niveau de la sensibilité par rapport à la qPCR, technique utilisée actuellement en routine. Elle nous a ainsi permis de constater la présence de cet ADN chez des patients traités à long terme par des analogues de nucléos(t)ides et même en présence d'interféron. La présence d'ARNpg et les expériences de ChIP ont également confirmé que l'ADNccc était encore transcriptionnellement actif. Ces résultats confirment d'autant plus la nécessité d'élaborer de nouvelles thérapeutiques pour permettre l'inactivation voire l'élimination de l'ADNccc. L'une des stratégies envisagées est le système CRISPR/Cas 9. Ainsi le dernier axe de cette thèse a été de développer ce système dans des modèles d'infection du virus de l'hépatite B. Pour vérifier l'efficacité de ce système sur le VHB, nous avons testé 8 ARN guide différents incorporer via des ribonucléoprotéines dans des cellules HepG2-NTCP. Les résultats préliminaires ont ainsi démontré que ce système pouvait réduire le pool d'ADNccc dans ces cellules et ouvre des perspectives intéressantes pour le développement de nouveaux traitements / Hepatitis B virus {HBV) is a major health problem with 250 million chronic carriers, despite the existence of a preventive vaccine. Currently the treatments used are nucleos{t)ide analogues and / or interferon a. Although they efficiently reach a decrease of the viral load, they do not allow the eradication the disease due to the persistence of the cccDNA, the minichromosome of the hepatitis B. This DNA serves as a template for the viral transcription and only a single copy suffice for the infection rebound. However, the techniques used routinely for the quantification of the cccDNA are not sensitive enough to be able to detect low concentrations of this DNA, in particular in biopsies of patients chronically infected and long term treated. ln addition, it is necessary to develop new strategies to target the cccDNA in order to clear the infection. Thus, my thesis work is based on the development of a new technology: the Droplet Digital PCR {ddPCR) to allow the quantification of cccDNA in patient biopsies. This technique allows a gain of 2 log in sensitivity compared to the qPCR technique currently used in routine. lt allowed us to see the presence of this DNA in long-term treated patients even in the presence of interferon. The presence of pgRNA and ChlP experiments also confirmed that the cccDNA was still transcriptionally active.These results confirm the requirement to develop new therapeutics to allow the inactivation or even the elimination of the cccDNA. One of the strategies envisaged is the CRlSPR / Case 9 system. Thus, the following part of this thesis was to develop this system in hepatitis B virus infection models. To reduce off-target effect we tested 8 different guide RNAs incorporated via ribonucleoproteins into HepG2- NTCP cells. Preliminary results have shown that this system can reduce the pool of cccDNA in these cells and open up the possibilities to test this model on PHH and opens interesting perspectives for the development of new treatments

<p>The aims of the thesis were to</p><p>• explore whether radical prostatectomy is beneficial compared with watchful waiting in survival and disease progression</p><p>• find possible effect modifiers</p><p>• evaluate a protocol of multiple biopsies and investigate if men with previous benign prostate biopsies are a group at risk for later prostate cancer</p><p>• inquire into patients’ and clinicians’ experiences of randomization in order to find out what made this study possible to conduct, and thereby contribute to improve randomization in the future</p><p>The background material was a large randomized clinical trial, the Scandinavian Prostatic Cancer Group Study Number 4, or SPCG-4, which was open for inclusion from February 1989 through December 1999. It comprised 695 men in Sweden, Finland and Iceland who had localized prostate cancer and were randomized to either radical prostatectomy or watchful waiting. </p><p>After a mean follow-up time of 6.2 years the first analyses, according to intention-to-treat, showed that radical prostatectomy reduced disease specific mortality, risk of metastases and risk of local progression but did not statistically significantly reduce overall mortality. </p><p>The second analyses confirmed our earlier findings and furthermore, at ten years, radical prostatectomy also statistically significantly reduced overall mortality. Age appeared as an independent effect modifier that will be further investigated.</p><p>A total of 547 men, with a suspicion of prostate cancer that had undergone multiple biopsies, and whose biopsies had benign histology were later compared with the background population to evaluate whether they were a group at risk of developing prostate cancer. Within six years of follow-up, there was no increased risk of prostate cancer.</p><p>Patients as well as clinicians used individual strategies to cope with the situation. The randomizing clinician has to understand the patient’s strategy and his expectations in order to individualize the information accordingly.</p>

Work related musculoskeletal disorders are the dominating causes of reported ill-health in industrialized countries. These chronic pain conditions are one of the most costly public health problems in Europe and North America. When work related musculoskeletal disorders are considered to be of muscular origin and the trapezius muscle is affected, the common appellation is trapezius myalgia. Since little is known about the genesis or how it is maintained, it is of great importance to better understand the pathophysiology of trapezius myalgia; doing so will better enable recommendations for prevention, treatment and rehabilitation. Several hypotheses have been presented based on biochemical alterations in the muscle, suggesting increased signaling of inflammatory substances and altered metabolism. Previous research has not been able to present the comprehensive picture of the muscle in pain. Thus there is a demand for more comprehensive research regarding the biochemical milleu of the chronic trapezius muscle. Proteomic and metabolomic methods allow non-targeted simultaneous analyses of a large number of proteins and metabolites. The main emphasis in this thesis is on a proteomic method, two-dimensional differential gel electrophoresis (2D-DIGE). The method is validated to human skeletal muscle biopsy research with laboratory specific settings. In the baseline study, there were 14 metabolic, contractile, structural and regulatory proteins that differed significantly in abundance when trapezius and vastus lateralis muscles were compared. Using the validated 2D-DIGE method and the baseline study, a comparison between healthy and myalgic muscles was made. Biopsies from female cleaners with and without myalgia were compared to obtain results from women with the same type of work exposure. In the multivariate model, 28 identified unique proteins separated healthy and myalgic muscle and were grouped according to function: metabolic (n=10), contractile (n=9), regulatory (n=3), structural (n=4), and other (n=2). Finally, a second screening method, metabolomics, was introduced to analyze differences in metabolite content as a complement to and verification of the proteomic results. Gas chromatography-mass spectrometry (GC-MS) was performed on muscle interstitial fluid samples obtained with microdialysis, and differences in the abundance of extracellular metabolites were revealed.  The 2D-DIGE method is a reliable method to analyze human skeletal muscle. The outcomes of the proteomic analyses were dependant on the statistical approach. Systematic differences in protein and metabolite content were detected using a multivariate approach. Univariate analyses were used to analyze individual proteins for their significance. The significant proteins in the baseline study were predominately related to muscle fiber type which correlated with the differences in fiber type content between trapezius and vastus lateralis. The proteomic and metabolomics studies where myalgic and healthy muscles were compared provide us with new clues and new aspects regarding the pathophysiology of the myalgic muscle. Technically advanced methods employed in the thesis enabled an explorative screening of proteins of relevance for the pathophysiology of the myalgic muscle. The results of these analyses may contribute to the formulation of future hypothesis that need to be further evaluated.

The aims of the thesis were to • explore whether radical prostatectomy is beneficial compared with watchful waiting in survival and disease progression • find possible effect modifiers • evaluate a protocol of multiple biopsies and investigate if men with previous benign prostate biopsies are a group at risk for later prostate cancer • inquire into patients’ and clinicians’ experiences of randomization in order to find out what made this study possible to conduct, and thereby contribute to improve randomization in the future The background material was a large randomized clinical trial, the Scandinavian Prostatic Cancer Group Study Number 4, or SPCG-4, which was open for inclusion from February 1989 through December 1999. It comprised 695 men in Sweden, Finland and Iceland who had localized prostate cancer and were randomized to either radical prostatectomy or watchful waiting. After a mean follow-up time of 6.2 years the first analyses, according to intention-to-treat, showed that radical prostatectomy reduced disease specific mortality, risk of metastases and risk of local progression but did not statistically significantly reduce overall mortality. The second analyses confirmed our earlier findings and furthermore, at ten years, radical prostatectomy also statistically significantly reduced overall mortality. Age appeared as an independent effect modifier that will be further investigated. A total of 547 men, with a suspicion of prostate cancer that had undergone multiple biopsies, and whose biopsies had benign histology were later compared with the background population to evaluate whether they were a group at risk of developing prostate cancer. Within six years of follow-up, there was no increased risk of prostate cancer. Patients as well as clinicians used individual strategies to cope with the situation. The randomizing clinician has to understand the patient’s strategy and his expectations in order to individualize the information accordingly.

Le cancer de la prostate est devenu depuis deux décennies le cancer le plus fréquent de l’homme dans le monde et représente la troisième cause de décès par cancer chez l’homme en France. La biopsie de prostate est l’examen de confirmation diagnostique standard de ce cancer. Elle permet, par des prélèvements systématisés ou dirigés vers une cible, l’analyse anatomopathologique du tissu prostatique. Guidée par échographie transrectale, l’objectif de la biopsie est de réaliser une cartographie prostatique avec une série de prélèvements multiples bien distribués dans le volume de la prostate. Cependant, la réalisation de biopsies de prostate présente de nombreuses difficultés pour le clinicien car le geste demande une représentation 3D d’un organe très déformable et mobile. Ainsi l’accès à une cartographie précise des biopsies effectuées a un intérêt pour l’amélioration de la qualité des biopsies, la traçabilité des biopsies et l’apprentissage du geste.En partant de ce constat clinique, l’objectif de cette thèse est la conception d’un système de suivi pour la navigation et le guidage de la biopsie de prostate écho-guidée par voie rectale compatible avec une application clinique. Notre approche se base sur une méthode de recalage 2D-3D rigide, qui met en correspondance une image 2D échographique et un volume échographique de référence acquis en début de procédure. Le recalage 2D-3D d’images échographiques est complexe parce que l’information hors-plan n’est pas présente dans l’image 2D et les images échographiques sont très bruitées. Nous avons développé une méthode, dite hybride, qui consiste en la mise en correspondance de caractéristiques de l’image à partir d’une distance basée sur les intensités de pixels/voxels et sur une proximité euclidienne. La précision et robustesse de l’algorithme ont été évaluées sur des données de 20 patients acquises durant des procédures de biopsies de prostate dans deux centres hospitaliers français. Nous avons montré que l’amélioration de la TRE avant et après recalage est statistiquement significative. Près de 75% des TRE obtenues étaient inférieures à 5mm, qui est le seuil clinique de significativité des tumeurs.Par la suite, nous avons mis en application la méthode de recalage dans le cadre du suivi. Une expérimentation sur fantôme prostatique intégrant une sonde équipée d’un capteur de localisation est réalisée pour déterminer la faisabilité et l’apport en termes de précision pour la navigation pour la biopsie de prostate. Les premiers résultats obtenus par notre méthode de suivi montrent une preuve de concept approfondie pour une application clinique. L’information donnée par le capteur inertiel est difficile à exploiter de manière optimale. De nouvelles expérimentations dans des conditions plus favorables devraient être réalisées. Les premiers résultats du suivi sont malgré tout prometteurs dans l’objectif d’une application clinique.La perspective principale pour la méthode de suivi est son intégration dans la plateforme MIRAS, (commercialisée par Koelis) pour continuer l’évaluation. L’ajustement de la méthode et l’amélioration des temps de calcul sont les deux axes majeurs à approfondir pour faire naı̂tre un premier prototype de guidage temps-réel pour la biopsie de prostate. La collaboration de longue date entre les cliniciens et chercheurs de ce projet est un atout essentiel pour la future validation clinique. / Prostate cancer is the most common cancer world-wide for males and the second leading cause of cancer death in France. Prostate biopsy procedures, performed to obtain and analyze tissue samples of the gland, are required for diagnosis. The clinical standard protocol is currently performed under UltraSound (US) control following a systematic protocol. Unfortunately, prostate cancer diagnosis is complicated by the lack of image quality and the low intrinsic contrast between tumor and non-tumor on US images. Conventional biopsies are performed under Transrectal UltraSound (TRUS) guidance. Precisely localizing the biopsy sites is challenging because the gland has a symmetric shape and because the prostate moves and is deformed by the patient motion and the TRUS probe pressure.This work aims at designing a system for navigated prostate biopsies combining a low-cost tracking system and a 2D-3D US registration method. To achieve this objective, our approach is based on hybrid 2D-3D ultrasound (US) rigid registration method for navigated prostate biopsy that enables continuous localization of the biopsy trajectory during the exam.Accuracy and robusteness was evaluated on a significant patient data set recorded in routine uncontrolled conditions from two different hospitals. The results show that 75% of the cases with error less than 5mm, which is clinically acceptable.Thereafter, we developed experiments to evaluate the tracking. The method was tested in a prostate phantom and a probe tracking by a inertial sensor. It was shown that it can do a better localization than and inertial measurement unit. Those first results obtained by our tracking method have established a proof of concept for a future clinical application. We highlighted that the sensor data are complex to exploit in optimal conditions. Additional experiments sould be performed in more realistic conditions.The method adjustment and the computing time-enhancement are the two main approaches to develop to create a first prototype of real-time tracking for navigated prostate biopsy. The long-standing cooperation between clinicians and researchers is an essential asset for a future clinical validation.

Increased milking frequency (IMF) in early lactation is a time and cost-effective farm management practice to enhance profit in the dairy industry. The process involves milking cows more often in early lactation alone. On farms that milk twice daily (2X), early lactation cows are milked four times daily (4X) for 21 d postpartum. Cows produce significantly more milk during this timeframe and continue to have increased milk yield (MY) when returned to 2X milking for the remainder of lactation. The objective of this dissertation was to discover the physiological processes of early lactation IMF that cause increased MY throughout lactation. All studies involved unilateral frequent milking (UFM) with 2X and 4X udder halves for 21 d in early lactation. The first study manipulated milking interval (MI), or the time between milkings, during early lactation IMF. Cows were either milked on an even MI every 6 h or unevenly on a 9:3:9:3 h MI. Unevenly milked cows produced more milk on the final day of 4X treatment, but there was no significant difference in the increased MY carry-over effect between MI groups. Therefore, farmers can utilize any MI that fits their schedule and still achieve significantly enhanced profits. The second study aimed to infer the metabolic mechanisms of early lactation IMF that increase MY by comparing it to bovine somatotropin (bST). Cows that underwent early lactation IMF received bST at 80 DIM. Both IMF and bST treatments significantly enhanced MY, but there was no interaction or synergistic effect between treatments. We surmised that IMF and bST cause increased MY through different metabolic mechanisms since IMF functions locally and bST operates systemically. The final study analyzed mammary tissue from 2X and 4X udder halves on the final day of UFM treatment. The mechanism in which IMF enhanced MY involved increased protein levels of signal transducer and activator of transcription (STAT5), activated and total protein kinase B (Akt), and total extracellular signal-regulated kinase 1 and 2 (ERK1/2) and reduced protein levels of total mammalian target of rapamycin (mTOR) and total mitogen activated protein kinase (MAPK) in 4X udder halves compared to 2X. / Doctor of Philosophy / Increased milking frequency in early lactation is the process of milking cows more often for the first 3 weeks after calving. Cows not only produce more milk during this timeframe but continue to have elevated milk yield throughout lactation. This phenomenon is called the increased milk yield carry-over effect. This dissertation aimed to further enhance the increased milk yield carry-over effect of early lactation increased milking frequency. All studies utilized unilateral frequent milking with left udder halves milked twice daily and right udder halves milked four-times daily. The first study compared even and uneven milking intervals alongside early lactation increased milking frequency. The right udder halves of the even groups were milked every 6 hours. Cows in the uneven milking interval group were milked on a 9:3:9:3 hour interval. The uneven milking interval group produced more milk than even milking interval group on the final day of milking frequency treatment. However, there was no difference in milk yield between groups at any other time point. All cows had an increased milk yield carry-over effect throughout 300 days of lactation. In the second study, cows received bovine somatotropin in mid-lactation to observe possible synergistic effects in enhancing milk yield after early lactation increased milking frequency. Both the bovine somatotropin and increased milking frequency increased milk yield, but there was no synergistic effect when the two treatments were combined. The final study analyzed proteins within mammary tissue after 3 weeks of unilateral frequent milking. Udder halves milked four-times daily had significantly elevated activated and total STAT5, activated and total Akt, and total ERK1/2. Udder halves milked twice daily had elevated total MAPK and total mTOR. These findings helped to understand the metabolic functioning of increased milking frequency in early lactation that causes a persistent increase in milk yield throughout lactation.

Le Cancer, l’une des pathologies les plus fréquentes au sein de la population, possède encore actuellement un taux de morbi-mortalité important tous sexes confondus, et ce malgré les importants progrès diagnostiques et thérapeutiques réalisés. D’un point de vue diagnostique, dans une approche dite de « Biologie de systèmes », en complément de l’étude anatomo- pathologique qui reste la référence, de nouvelles techniques ont été développées pour la caractérisation de profils métaboliques (Métabolomique) d’échantillons tissulaires pathologiques ou non, parmi lesquelles la Spectroscopie RMN HRMAS. Après un bref rappel théorique et avoir dressé le bilan des applications de cette technique en Cancérologie, nous avons exposé les différentes étapes du protocole à mettre en place afin d’envisager son implémentation dans un cadre hospitalier. L’ensemble des résultats présentés permettent d’envisager l’utilisation de cette technique en pratique clinique courante. Il faut néanmoins valider la robustesse des modèles statistiques élaborés et confirmer ces résultats sur de plus grandes cohortes d’échantillons. Des développements technologiques, analytiques et statistiques sont également nécessaires. / Cancer, one of the most frequent pathologies among the population, has still an important morbidity-mortality rate all sex confounded, despite the important diagnostical and therapeutical progresses achieved. From a diagnostical point of view, in a so called “Systems Biology approach”, as a complement of the gold standard histopathological study, some new techniques have been developed for the characterization of metabolic profiles (Metabolomics) of tissular samples pathological or not, among which HRMAS NMR Spectroscopy. After some brief theoretical considerations and after reporting the applications of this technique in Cancerology, we exposed the different steps of the protocol to design in order to consider its implementation in a hospital set up. All the results presented allow considering the use of this technique in a clinical routine. Nevertheless, it is necessary to validate the robustness of the statistical models built and to confirm these results on much larger cohorts of samples. Some technical, analytical and statistical developments are also needed.