Objective. Albumin is an abundant and slowly-cleared plasma protein. Our laboratory previously incorporated albumin into recombinant fusion proteins to extend the plasma half-life of small proteins of potential therapeutic utility. We sought to determine if reiteration of albumin in a single polypeptide chain would further extend its half-life. Design. Hexahistidine-tagged rabbit serum albumin (RSA) with Cys 34 altered to Ala to prevent disulphide-bonded dimerization was produced in yeast (H₆-RSA-C34A), and compared to a reiterated forma of H₆-RSA-C34A containing two domains of amino acids 1-584 separated by a hexaglycine spacer. Clearance of these purified iodinated proteins was compared in rabbits. Materials and Methods. Site-directed mutagenesis employing PCR was used to alter the encoding plasmid. Proteins secreted from transformed Pichia pastoris yeast cell lines were purified using nickel-chelated affinity chromatography and radioiodinated by the Iodogen method. Labeled proteins were injected intravenously into rabbits and the residual acid-precipitable protein concentration in serial plasma samples was determined over time. Results. P. 𝘱𝘢𝘴𝘵𝘰𝘳𝘪𝘴 cells transformed with the expression plasmids secreted 140 kDa H₆-diRSA and 70 kDa H₆-RSA-C34A proteins, which were purified to apparent homogeneity. Mean terminal catabolic half-lives (±SD) were 4.9 (±0.7) and 3.0 (±0.3) days for H₆-RSA-C34A and H₆-diRSA, respectively. Then values were 9 for H₆-diRSA and 12 for H₆-RSA-C34A. conclusions. H₆-diRSA was cleared from the circulation more rapidly than H₆RSA-C34A. We hypothesized that increased catabolism of the reiterated molecule could be due to an increased rate of cellular uptake and endocytosis of H₆-diRSA due to increased avidity for cellular binding sites. Non-reiterated albumin therefore appears to be optimal as a carrier protein for small recombinant blood products. / Thesis / Master of Science (MSc)

Rapid developments in microelectronics technology have allowed for phenomenal achievements in biomedical engineering. In the past few decades, an enormous amount of researches were done in the field of medical implantable microelectronic systems. The prevalence of research in this particular field have led to the design of novel systems for in-vivo applications, for example, using microelectronic systems to replace catheterization in clinical studies of urinary incontinence. In this thesis research, we study two types of wireless modules towards our goal of wireless systems for in-vivo applications. The first system, a 2.4 GHz wireless pressure sensor system, is designed as a pressure sensing module to operate as a part of a pill imaging device published in [32]. This pressure module samples pressure data and passes them to the pill imaging capsule via a serial-port-interface (SPI). The 2.4 GHz wireless pressure system has an overall system dimension of 75.0 x 20.5 x 17.5 mm3 with a current consumption of 5 mA when operating from a 3 V supply. The pressure sensitivity of this system is observed as 1.14 cmH20/LSB (least significant bit). The second system, 125kHz RFID (radio-frequency identification) dual sensor system, is designed to explore the possibility of powering the device and transmitting data using the RFID technology. The 125kHz RFID dual sensor system has an overall system dimension of 30.0 x 15.0 x 15.0 mm3 with a current consumption of 1.5 mA while operating at 3 V. The pressure sensitivity of this system is observed as 2.93 cmH20/LSB and the temperature sensitivity is observed as 0.069 °C/LSB. And, the detections of rapid pressure changes in both systems are successful. The work performed in this thesis research has provided a cost-effective method of designing medical implantable systems using off-the-shelf components as compared to full-custom designs. In this research, it is also observed that power consumption is a major issue in medical implantable systems. Finally, the possibility of transmitting data and powering such systems using RFID technology has been verified. / Thesis / Master of Applied Science (MASc)

Aluminum is a non-essential trace element that accumulates in human bone tissue (Nayak, 2002). Its toxic effects are cumulative and result in painful forms of renal osteodystrophy, most notably a dynamic bone disease and osteomalacia, but also other forms of disease (Yokel, 2001; Cannata-Andia, 2002). Presently, histological tests of bone biopsies are the only approach for the diagnosis of aluminum-related pathologies (Malluche, 2002). Neutron Activation Analysis was proposed as an alternative method for quantifying aluminum. The Trace Element Group at McMaster University has developed an in vivo procedure for detecting aluminum levels in the bones of the hand, exploiting an accelerator-based approach. A minimum detectable limit (MDL) of 1.14mg of aluminum could be distinguished for a local dose to the hand of 48mSv (Pejovic-Milic, 2001). For the procedure to be clinically effective, the MDL should be comparable to the levels normally contained in healthy subjects (0.3-0.4 mg AI). Further refining of the method is therefore necessary. This dissertation presents an improved algorithm for data analysis, based on Spectral Decomposition. Following phantom measurements, a new MDL of(0.7±0.1)mg AI was reached for a local dose of (20±1)mSv, representing an improvement by a factor of 1.60±0.04. In addition, a time-dependent variant of this algorithm was proposed. The study also addresses the feasibility of a new data acquisition technique, the electronic rejection of the coincident events detected by the Nai(Tl) system. It is expected that the application of this technique, together with Spectral Decomposition Analysis, would provide an acceptable MDL for the method to be valuable in a clinical setting. / Thesis / Master of Science (MS)

A Espectroscopia por Ressonância Magnética Nuclear (RMN) tem oferecido inúmeras possibilidades de pesquisa nas áreas de Biologia e Medicina. Uma de sus principais aplicações é a Espectroscopia in vivo do 31P no estudo da fisiologia de músculos esqueléticos. A partir desta metodologia pode-se quantificar as concentrações dos diferentes metabólitos fosforados que participam do metabolismo energético dos músculos, bem como suas alterações em função de contrações musculares. Com o objetivo de implantar a técnica de Espectroscopia in vivo no Laboratório de RMN do IFSC/USP, introduzimos várias adaptações ao Espectrômetro de Alta Resolução em Sólidos já existentes, incluindo a confecção de sondas . A partir desta adaptação, estudamos as alterações metabólicas dos compostos fosforados presentes em músculos esqueléticos, bem como o pH intraceleular, de ratos e camundongos em função de estímulos elétricos e exercício físico intenso. / The Nuclear Magnetic Resonance (NMR) spectroscopy has been offering many possibilities of research in the areas of Biology and Medicine. One of the most important applications is the 31P in vivo Spectroscopy to study the skeletal muscle physiology. Using this methodology, it is possible to quantify different phosphorus metabolite concentrations that take part of the muscle energetic metabolism, as their variations as a function of muscle contraction. To accomplish the objective of developing the in vivo spectroscopy technique in the NMR laboratory of the IFSC/USP, many modifications to our Solid High Resolution Spectrometer were introduced and probes were constructed. Following these changes, we studied phosphorus compounds metabolic changes in the skeletal muscle and the intracellular pH of rats and mice as a function of electric stimulation and intensive running exercise.

Francisella tularensis est une bactérie intracellulaire à Gram négatif et l'un des agents les plus infectieux connu à l'heure actuelle, en particulier par voie respiratoire. L'inhalation d'une dizaine de bactéries suffit à provoquer une maladie mortelle : la tularémie pulmonaire. Sa facilité de dissémination par aérosols, ainsi que le caractère létal de cette pathologie, ont contribué à considérer F. tularensis comme une arme biologique potentielle. La tularémie pulmonaire est une infection aigue qui s'accompagne d'une réponse immunitaire inadaptée. F. tularensis infecte en premier lieu les cellules phagocytaires, notamment les macrophages. Alors que ces derniers sont des acteurs majeurs de la défense contre les agents infectieux, de nombreux mécanismes d'échappement permettent à F. tularensis d'éviter ou de résister aux réponses de l'hôte et ainsi de se multiplier et de disséminer dans l'organisme. Ainsi, après un retard initial dans la mise en place de la réponse immunitaire, la présence d'un grand nombre de bactéries et de signaux de danger libérés par les cellules infectées, conduisent au déclenchement d'une réponse inflammatoire excessive. Celle-ci se caractérise par une tempête cytokinique provoquant un recrutement massif de cellules immunitaires, en particulier de neutrophiles, dans les tissus infectés. Les dommages tissulaires associés à cette réponse inflammatoire sont en grande partie responsable de la mortalité associée aux infections pulmonaires par F. tularensis. La tularémie est actuellement traitée par antibiothérapie. Malheureusement, l'absence de symptômes spécifiques de cette maladie rend le diagnostic difficile et, par conséquent, retarde la prescription du traitement adapté. Or, l'efficacité des antibiotiques est considérablement réduite par cette administration tardive. De nouvelles stratégies thérapeutiques sont donc nécessaires pour remplacer ou compléter l'antibiothérapie. Dans ce contexte, nous avons cherché à déterminer si la modulation de la réponse inflammatoire excessive induite par F. tularensis pouvait être bénéfique pour l'hôte infecté et ainsi être utilisée comme thérapie accessoire. L'objectif de mon travail de thèse était d'évaluer les propriétés anti-inflammatoires et le potentiel bénéfice thérapeutique du mannodendrimère 3T (M3T), un composé synthétisé par l'équipe de J. Nigou, dans un modèle murin d'infection pulmonaire par F. tularensis. Le M3T, conçu pour mimer les propriétés anti-inflammatoires d'un glycolipide de la paroi de Mycobacterium tuberculosis, a précédemment montré un effet inhibiteur sur la production de cytokines pro-inflammatoires et le recrutement de neutrophiles dans un modèle murin d'inflammation pulmonaire aigue induite par le LPS. La souche F. novicida, provoquant chez la souris une pathologie similaire à une infection pulmonaire par F. tularensis, a été utilisée comme souche de substitution dans ces travaux. In vitro, nous avons montré que le M3T inhibe la production de cytokines pro-inflammatoires induite par F. novicida dans des macrophages et cellules dendritiques humaines. D'un point de vue mécanistique, l'ensemble des données suggère que le M3T inhibe la réponse inflammatoire induite par F. novicida via le récepteur TLR2, en activant une voie de signalisation dépendante du récepteur DC-SIGN. In vivo, le M3T a été administré par injection intraveineuse 6 h post-infection, puis quotidiennement pendant 3 jours, en combinaison avec un traitement antibiotique sous-optimal. / Francisella tularensis is an intracellular Gram negative bacterium and the causative agent of tularemia. It is one of the most infectious agents known to date. Infection by the respiratory route leads to the deadly pulmonary form of tularemia. For these reasons, F. tularensis has been considered for years as a potential biological weapon. Pulmonary tularemia is characterized by an acute infection and a defect in immune responses. Particularly, the innate immune system plays a central role in F. tularensis infection and pathology. Macrophages, key cells of the innate immune system, are the main target for F. tularensis. This bacterium has evolved many strategies to escape host defenses that allow it to replicate within the cells and then disseminate into the whole organism. At this systemic stage, bacteria, along with alarm signals from infected cells, are recognized by innate immune receptors, triggering an inappropriate inflammatory response. The latter is characterized by a cytokine storm leading to a massive recruitment of immune cells, particularly neutrophils, in infected tissue. Tissue damages caused by this inflammation are a major cause of mortality associated with F. tularensis infections. Today, the treatment of tularemia is based on antibiotherapy. However, no specific symptoms can be assigned to pulmonary tularemia making its diagnosis difficult. This delays the administration of an appropriate antibiotiotherapy whose efficacy is therefore decreased. Thus, new therapeutic strategies are needed to replace or complement antibiotics. In this context, we investigated whether reducing the excessive inflammation induced by F. tularensis could be beneficial for the host and be considered as an adjunctive host- directed therapy. The aim of my work was to evaluate the anti-inflammatory properties and the therapeutic potential of mannodendrimer 3T (M3T), a synthetic coumpond designed in the team, in a mouse model of pulmonary infection by F. tularensis. M3T was previously designed to mimic the anti-inflammatory traits of a specific glycolipid from Mycobacterium tuberculosis. It was previously shown to inhibit the production of pro-inflammatory cytokines and neutrophils recruitment in a mouse model of LPS-induced pulmonary inflammation. Here, we used F. novicida as a surrogate for F. tularensis since it induces an identical inflammatory pathology. In vitro, M3T was found to inhibit the production of pro-inflammatory cytokines in human macrophages and dendritic cells infected by F. novicida. M3T modulates inflammatory response triggered by F. novicida via TLR2 most likely by the activation of a DC-SIGN-dependant pathway. In vivo, M3T was administered 6 h post-infection and then, daily for 3 days, by intraveinous injection and combined with a suboptimal antibiotic. This combination increases the survival rate of mice infected with F. novicida as compared to mice treated with antibiotic alone. M3T treatment has no impact on bacterial burden but seems to reduce tissue damages as observed by histological analyses of lungs, liver and spleen of infected mice. Altogether, our data demonstrate that M3T administration provides a therapeutic benefit in a mouse model of pulmonary infection by F. novicida. On a more general perspective our results suggest that targeting inflammation can be considered as an adjunctive treatment in acute pulmonary infections.

A Espectroscopia por Ressonância Magnética Nuclear (RMN) tem oferecido inúmeras possibilidades de pesquisa nas áreas de Biologia e Medicina. Uma de sus principais aplicações é a Espectroscopia in vivo do 31P no estudo da fisiologia de músculos esqueléticos. A partir desta metodologia pode-se quantificar as concentrações dos diferentes metabólitos fosforados que participam do metabolismo energético dos músculos, bem como suas alterações em função de contrações musculares. Com o objetivo de implantar a técnica de Espectroscopia in vivo no Laboratório de RMN do IFSC/USP, introduzimos várias adaptações ao Espectrômetro de Alta Resolução em Sólidos já existentes, incluindo a confecção de sondas . A partir desta adaptação, estudamos as alterações metabólicas dos compostos fosforados presentes em músculos esqueléticos, bem como o pH intraceleular, de ratos e camundongos em função de estímulos elétricos e exercício físico intenso. / The Nuclear Magnetic Resonance (NMR) spectroscopy has been offering many possibilities of research in the areas of Biology and Medicine. One of the most important applications is the 31P in vivo Spectroscopy to study the skeletal muscle physiology. Using this methodology, it is possible to quantify different phosphorus metabolite concentrations that take part of the muscle energetic metabolism, as their variations as a function of muscle contraction. To accomplish the objective of developing the in vivo spectroscopy technique in the NMR laboratory of the IFSC/USP, many modifications to our Solid High Resolution Spectrometer were introduced and probes were constructed. Following these changes, we studied phosphorus compounds metabolic changes in the skeletal muscle and the intracellular pH of rats and mice as a function of electric stimulation and intensive running exercise.

Introduction 52Mn (t½ =5.59 d, β+ = 29.6%, Eβmax = 0.58 MeV) has great potential as a long lived PET isotope for use in cell tracking studies, observation of immunologic response to disease states, or as an alternative to manganese-based MRI contrast agents. Its favorable max positron energy leads to superb imaging resolution, comparable to that of 18F.[1] Manganese is naturally taken up by cells via a multitude of pathways including the divalent metal transporter (DMT1), ZIP8, transferrin receptors (TfR), store-operated Ca2+ channels (SOC-Ca2+), and ionotropic glutamate receptor Ca2+ channels (GluR).[2] These natural transport mechanisms make 52Mn an attractive isotope for applications necessitating non-perturbative cell uptake. In particular, cell tracking is critical to the development and translation of stem cell therapies in regenerative medicine. Alternative-ly, 52Mn could be used in immunotherapy techniques such as adoptive cellular therapy (ACT) to evaluate the ability of external immune cells to reach their intended target. Material and Methods 52Mn was produced by natCr(p,x)52Mn using 16 MeV protons. The average thick target production yield was 0.23 mCi/µA-h with less than 0.25% co-production of 54Mn. Small amounts of 51Cr were observed in the target, but were absent from the radiochemically separated product. Target construction consisted of a water jet cooled chromium disc (3/4” diameter, 0.4” thick). Targets were purchased from Kamis Inc, and are 99.95% pure. Targets withstood beam currents of 30 µA with no visible aberration. Chromium targets were etched by concentrated HCl following bombardment. Mn2+ ions were extracted from 9M HCl to 0.8M trioctylamine in cyclohexane leaving the bulk chromium in the aqueous phase. After isolating the organic phase, 0.001M NH4OH was used to back-extract the Mn2+ ions to aqueous phase. This purification cycle was conducted a total of three times for each 52Mn production. Results and Conclusion For a starting bulk chromium mass of 456 ± 1 mg, a post-separation chromium mass of 5.35 ± 0.04 ng was measured by microwave plasma atomic emission spectrometry (MP-AES). This mass reduction corresponds to an average separation factor of 440 for a single purification cycle. Each purification cycle had a 52Mn recovery efficiency of 73 ± 7 % (n = 6), resulting in an overall separation efficiency of approximately 35 %. These efficiencies and separation factors agree reasonably well with the work conducted by Lahiri et. al.[3] Prior to use, the product was passed through a C-18 Sep-Pak to remove any residual organic phase. After four target irradiations and etchings, some pitting became noticeable on the target face. These have not yet compromised the o-ring seal with the target deplater, but it is possible that target replacement after every 6–9 52Mn productions will be necessary moving forward. Following the successful separation of 52Mn from chromium, in vitro experiments were conducted to demonstrate the uptake of 52Mn by human stem cells and mouse tumor cells. A linear uptake response was observed as a function of the amount of activity exposed to the cells for both cell models. These experiments have shown great promise for 52Mn as a long-lived PET isotope in cell tracking studies. Details will be presented.

Beeinflussung der Ex-vivo-Chemoresponse von Plattenepithelkarzinomen der Kopf-Hals-Region auf Cisplatin und Docetaxel durch 5-Fluorurazil

Les alphavirus arthritogènes de la rivière Ross (RRV) et du chikungunya (CHIKV) sont des arbovirus à l’origine de maladies inflammatoires musculosquelettiques chez l'homme. Ils sont largement distribués dans le monde et provoquent périodiquement des épidémies explosives. Les principaux signes cliniques lors d’une infection par un alphavirus arthritogène sont les myalgies, polyarthrites et arthralgies intenses pouvant persister plusieurs mois après l'infection. Les mécanismes de développement de l’infection et des manifestations persistantes sont peu connus. Pour étudier la pathogenèse de l'infection par RRV, nous avons généré un virus recombinant exprimant une nouvelle luciférase brillante et brillante. Nous avons montré que les monocytes humains, malgré une faible susceptibilité à l'infection in vitro par RRV, étaient capables de maintenir une réplication virale jusqu'à 45 jours post infection indiquant leur rôle potentiel dans les formes chroniques. Grâce un modèle expérimental de l’infection par RRV, nous avons suivi les phases aiguë et chronique de la maladie in vivo. Nous avons montré que les cinétiques de réplication du virus recombinant étaient proches de celles du virus parental. Nous avons également observé un tropisme musculaire et articulaire et une corrélation entre le signal bioluminescent et la charge virale confirmant ainsi la relevance de ce modèle. En étudiant la dissémination virale, nous avons montré que le Bindarit, une molécule anti-inflammatoire diminuant le développement de la maladie dans le modèle murin, induit une plus grande réplication dans le tissu cardiaque. Enfin, nous avons pu observer une réplication virale dans les tissus musculaires durant la phase chronique de la maladie et avons montré le rôle de la dose inoculée dans le développement de la persistance virale. Suite à un traitement immunosuppresseur, nous avons observé une légère augmentation du signal bioluminescent indiquant un contrôle de la réplication virale persistante par la réponse immunitaire adaptative. Ce nouveau modèle d’imagerie in vivo permet un suivi en temps réel de la dissémination virale permettant des études de pathogenèse et l'évaluation de stratégies thérapeutiques. / Ross River virus (RRV) and chikungunya virus (CHIKV) are mosquito-transmitted viruses that cause musculoskeletal inflammatory diseases in humans. They are widely distributed and periodically cause explosive epidemics. After infection with RRV, patients experience fever, maculopapular rash, myalgia and intense pain in the peripheral joints. Approximately 30% of patients develop a chronic form of the disease with myalgia and poly-arthralgia persisting for months to years after infection. The mechanisms underlying these persistent symptoms remain unclear. To study the dynamics and pathogenesis of RRV infection in vitro and in living animals, we generated a recombinant virus expressing a novel small and bright luciferase. First we showed that human monocytes, despite a low susceptibility to RRV infection, were able to maintain viral replication in vitro up to 45 days post infection. Then, using a murine model of RRV infection, we monitored the acute and chronic phases of the disease. We observed near native replication kinetics and a muscular/articular tropism after infection with our recombinant virus. Moreover, the bioluminescent signal correlated with the viral load further confirming the relevance of this new imaging model. After monitoring of the viral dissemination in live mice, we showed that Bindarit, an anti-inflammatory molecule known to prevent the development of the alphaviral disease in a mouse model, induces a higher replication in the cardiac tissue; thereby indicating that caution must be used before treatment of patients. We were also able to observe viral replication in the muscles during the chronic stage of the disease when using a low inoculation dose. Finally, following an immunosuppressive treatment, we observed a slight increase in the bioluminescent signal indicating a control of remnant viral replication by the adaptive immune response. This new model provides a non-invasive real-time assessment of viral replication and dissemination allowing pathogenesis studies and therapeutic strategies evaluation.

Les processus de neuro-inflammation jouent un rôle majeur dans la maladie d'Alzheimer (MA). Des études génétiques récentes démontrent cette association entre neuro-inflammation et MA et impliquent notamment un gène, TREM2, qui code pour un récepteur exprimé à la surface de la microglie. La tauopathie est une lésion caractéristique de la MA. Elle se traduit par l’hyperphosphorylation et l’agrégation intraneuronale de la protéine Tau. Les travaux sur le rôle de TREM2 dans le développement de la pathologie Tau sont peu nombreux et donnent des résultats contradictoiresAinsi, l’objectif de ma thèse est d’étudier le rôle de la neuroinflammation et de TREM2 dans la progression de la tauopathie, dans deux modèles différents. Le premier est obtenu par injection stéréotaxique de vecteurs AAV dans la couche CA1 de l’hippocampe de souris déficientes ou non en TREM2. Ces vecteurs entrainent la surexpression de différentes formes de la protéine Tau humaine et permettent de récapituler les différents stades de la tauopathie.Nous avons en parallèle utilisé un modèle transgénique plus progressif de tauopathie, la souris THY-Tau22, afin d’étudier l’influence du stade de la pathologie dans l’effet provoqué par une déficience en TREM2 sur l’évolution de la pathologie. Notre étude a mis en évidence la toxicité des formes solubles de Tau dans le modèle AAV par rapport à ses formes agrégées. Le modèle transgénique THY-Tau22 nous a permis de mettre en évidence une augmentation des lésions tauopathiques dans les souris déficientes en TREM2 par rapport aux souris qui ne le sont pas, uniquement à un stade avancé. Cela suggère que, à l’instar des modèles amyloïdes, l’effet de la déficience en TREM2 sur le décours de la tauopathie est différent en fonction du stade considéré. / Neuroinflammation processes appear to play a major role in Alzheimer's disease (AD). Recent genetic studies support this correlation between neuroinflammation and AD and include a gene, TREM2, expressed on microglial surface. Tauopathy is a characteristic lesion of AD. It results in hyperphosphorylation and intraneuronal aggregation of Tau protein. In the literature, only few articles describe the role of TREM2 in the development of Tau pathology, and they report contradictory results. We therefore do not know for sure whether a deficiency in TREM2 has a deleterious effect or not on tauopathy. Thus, the goal of my thesis is to study the role of neuroinflammation and TREM2 in the progression of tauopathy, in two different models. The first is obtained by stereotaxic injection of AAV vectors into the CA1 layer of the hippocampus of TREM2-deficient or non-deficient mice. These vectors lead to the overexpression of different forms of the human tau protein, thus making it possible to recapitulate the different tauopathy stages.In parallel, we used a more progressive trangenic model of tauopathy, the THY-Tau22 mouse, to study the influence of TREM2 deficiency at different stage of the pathology. Our study demonstrated the toxicity of Tau soluble forms in the AAV model compared to its aggregated forms. The THY-Tau22 transgenic model allowed us to demonstrate an increase in tauopathic lesions in TREM2 deficient mice compared to wild type mice, at late stage only. This suggests that, similar to amyloid models, the effect of TREM2 deficiency on the course of tauopathy is influenced by the stage of the disease.