Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

Hereditary Hemochromatosis (HH) is an inherited recessive autosomal disorder characterized by accumulation of excess iron. When iron binding proteins become saturated, concentrations of free, or non-transferrin-bound iron (NTBI) rise, a condition thought to be responsible for the adverse effects associated with HH. To investigate that disturbing iron homeostasis plays a role in free radical injury in HH, protein carbonyls were found to be 1-7 times higher in patients with HH than in controls, with the greatest increases being observed in untreated HH patients with high ferritin and >90% transferrin saturation with iron. An Unpaired t test revealed a P value of 0.0278 (P< 0.05), which is considered to be statistically significant. Our data showed a significant positive correlation (linear relationship) between the level of carbonyl content and ferritin concentration in plasma samples from patients with HH. In vitro oxidation of transferrin and ferritin standards with hydrogen peroxide and excess iron, followed by immobilized trypsin digestion (Poroszyme), high-resolution LC-MS/MS analysis (Q-TOF Ultima, Waters) and MS/MS data processing (PEAKS, Bioinformatics Solution), identified several tryptic peptides containing oxidized Met,Trp and His residues. Mapping of the oxidized ferritin residues showed them to be located on the inner face of each sub-unit, the face directed toward the ferritin core where iron is normally stored. Using the same methodology, oxidized residues were subsequently detected in ferritin and transferrin isolated from plasma samples of patients severely affected with HH. Comparing of MS/MS spectra of in vitro oxidized samples that have most fragment ion peaks in common with oxidized peptide MS/MS spectra from samples of patients with HH revealed a significant correlation between the two. These data show that elevated NTBI may be involved in oxidative modification of the iron binding proteins, ferritin and transferrin, and that such modifications may play a significant role in the pathophysiology of HH.

Orientador: Fabiana Ferreira de Souza / Resumo: O ciclo estral da vaca é composto por 2-3 ondas de crescimento folicular, no qual vários folículos são recrutados e iniciam um novo crescimento. Durante o período denominado desvio folicular, um folículo se torna dominante e os outros entram em atresia. Este processo envolve um mecanismo ainda não completamente compreendido, incluindo proteínas específico, como já estabelecido pela expressão gênica. O objetivo do presente estudo foi caracterizar as proteínas do fluído folicular a fim de identificar macromoléculas relacionadas ao desenvolvimento dos folículos de vacas zebuínas nã-gestantes. Foram colhidos os ovários de 25 vacas mestiças não-gestantes em um abatedouro. A presença do corpo lúteo foi anotada para cada ovário. O líquido folicular foi colhido utilizando-se a imersão do ovário em meio líquido e ultrassonografia. De acordo com a mensuração do diâmetro folicular, foram formados 3grupos, folículos pequenos (≤6,5mm, n=25), médios (>6,5mm a ≤9mm, n=9) e grandes (>9,0mm, n=11). Após 2 centrifugações (600xg/10 minutos e 15.000xg/30 minutos, 4ºC) o sobrenadante foi separado e utilizado para determinação da concentração de proteína total (método de Bradford). A eletroforese foi conduzida sob condições desnaturantes e redutoras, em gel de separação de poliacrilamidaà 12%. A concentração de progesterona e estradiol do líquido folicular foi determinada a fim de identificar os folículos saudáveis. As proteínas diferenciais identificadas pela eletroforese foram... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Study of possibilities of thin layers depth profiling by combined use of SIMS and ToF-LEIS methods.

"Patients with repaired tetralogy of Fallot (TOF) account for the majority of cases with late onset right ventricle (RV) failure. The current surgical approach, which includes pulmonary valve replacement/insertion (PVR), has yielded mixed results in terms of RV functional recovery. Therefore, it is of great interest for clinicians to identify parameters, which may be used to predict post-PVR outcome. Pre- and post-PVR cardiac magnetic resonance (CMR) data were obtained from 60 repaired TOF patients with consent obtained for analysis. RV ejection fraction (RVEF) change (post-PVR RVEF minus pre-PVR RVEF) was used to measure post-PVR improvement. The patients were divided into Group 1(optimal outcome) and Group 2 (poor outcome) for comparison. RV wall thickness (WT) and curvature were obtained from CMR data for statistical analysis. Using mean quarter values (one CMR slice = 4 quarters), statistically significant differences in circumferential curvature (C-curvature) and longitudinal curvature (L-curvature) at end-diastole (maximum RV volume) and WT and C-curvature at end-systole (minimum RV volume) between Group 1 and Group 2 were found. Correlations between average WT at systole and between L-curvature at diastole and the change of RVEF were statistically significant. Specifically, the correlation coefficient between average WT at systole and change of RVEF was – 0.2715, (p = 0.036) and between L-curvature at diastole and change of RVEF 0.3297 (p = 0.01). This initial study suggests that the RV longitudinal curvature and wall thickness may be used as a marker/predictor for PVR surgical outcome. "

Large area detectors for time correlated single photon counting (TCSPC) are nowadays being implemented in CMOS technology to benefit a large variety of applications including positron emission tomography (PET) and 3D laser ranging (LiDAR), exploiting the advanced timing and counting capabilities inside single chips. Single photon avalanche diodes (SPADs) and silicon photomultipliers (SiPMs) represent a great option to realise such detectors thanks to their exceptional timing resolution and the ability to be arranged into arrays. Recently, digital SiPMs (dSiPMs) have been introduced to improve the integration with CMOS technology overcoming limitations on the readout of analogue SiPMs and thus improving the photon resolution of the detector. This work presents a 14GSamples=s time-to-digital converter (TDC) to improve the throughput of dSiPM sensors commonly limited by the sampling rate of the timing/counting readout circuitry. The converter has been demonstrated on a test chip in 130nm CMOS imaging technology paired with a novel XOR-based 32 32 SPAD array single-channel detector. The overall achieved throughput equals 1GEvents=s demonstrated in a direct time-of-flight LiDAR experiment. By acquiring a number of photons significantly higher than one per laser pulse, this approach represents the first example in TCSPC of an input rate and conversion rate both higher than the excitation rate. The following part of the work presents a modelling analysis on how to match the achieved high sampling rate / throughput of the single-channel TDC to the performance of a SPAD array. The impact of a selection of dSiPM design parameters, such as photon detection efficiency, dead time and size of the SPAD cell, number of cells per single-channel, digital N-to-1 combining network and channel bandwidth, on the overall sensor throughput and the dynamic range has been characterised thanks to a computational Monte-Carlo simulator and useful equations describing each of the processes in the sensing chain. The pile-up effect, i.e. the event-loss causing non-linear distortions on the output signal, has been characterised on each element of the dSiPM and optimisations have been proposed. Event losses in the SPAD cells due to dead time, in the digital combining network due to network dead time and single-channel bandwidth have all been identified, simulated and described by analytical equations. All the results coming from the theoretical analysis have been reproduced in real dSiPM design thanks to a reconfigurable test chip realised in the same 130nm CMOS imaging technology specifically to validate the proposed theory. The manufactured test chip provides the very first direct comparison between OR-based and XOR-based single-channel dSiPM sensors highlighting the promising timing and counting performance of the newly introduced XOR-based dSiPM. Direct evidence of pile-up distortions and subsequent reduction through design optimisations are demonstrated. A recommended design flow for next generation dSiPMs is proposed at the end of the publication.

Acinetobacter baumannii est une bactérie responsable d’infections communautaires, en particulier dans les régions tropicales et chez des blessés lors de conflits armés. Or, les réservoirs extra-hospitaliers de la bactérie sont peu connus. L’objectif de ce travail était d’étudier ces réservoirs dans une zone tropicale, l’île de la Réunion, en étudiant à la fois le réservoir animal (dépistage), environnemental et humain. En parallèle, des études ont été conduites en zone tempérée (France métropolitaine), avec le dépistage des animaux domestiques. Dans un premier temps, l’identification des espèces du complexe Acinetobacter calcoaceticus-Acinetobacter baumannii par spectrométrie de masse a été perfectionnée sur l’automate VitekÒ MS. La deuxième partie du travail a permis de mettre en évidence une prévalence élevée de portage de la bactérie chez les animaux domestiques réunionnais (6,5 et 8,5%). Une prévalence inférieure a été décelée chez les animaux domestiques de métropole (2,7%). Dans tous les réservoirs étudiés, une grande diversité des souches a été retrouvée lors de l’analyse par Multi-Locus Sequence Typing, avec la mise en évidence de nouveauxSequence Types. Enfin, 2 souches productrices d’OXA-23 et OXA-24 ont pu être isolées respectivement en portage chez un patient et un animal d’élevage de l’île de la Réunion, et 2 souches productrices d’OXA-23 ont été isolées chez des animaux de compagnie de métropole. Ce travail a permis des observations intéressantes sur l’épidémiologie communautaire d’ A. baumannii. D’autres travaux restent à prévoir pour mieux comprendre les relations entre les réservoirs extra-hospitaliers, mais aussi avec le réservoir hospitalier. / Acinetobacter baumannii is a bacterium responsible for community-acquired infections, especially in tropical areas and among soldiers during warfare. However, community reservoirs of this bacterium are not very well known. The aim of this work was to study these reservoirs in a topical area, Reunion Island, by analyzing at the same time the animal (carriage), the environmental, and the human reservoirs. Studies were also performed in a temperate zone (metropolitan France), by analyzing carriage of companion animals. First, identification of species belonging to the Acinetobacter calcoaceticus - Acinetobacter baumannii complex by mass spectrometry has been improved on the VitekÒ MS automated system. In asecond part of the study, a high prevalence of A. baumannii carriage has been observed in domesticated animals of La Reunion Island (6,5 and 8,5%). A lower prevalence has been detected in metropolitan companion animals (2,7%). Within allthe reservoirs studied, a great diversity of strains isolated has been shown by Multi-Locus Sequence Typing analysis, with the dicovery of new Sequence Types. At last, 2 strains producing OXA-23 and OXA-24 have been isolated respectively in carriage in a patient and in a livestock animal of La Reunion Island, and 2 strains producing OXA-23 have been isolated in metropolitan companion animals. This work has permitted to do some interesting observations on the community epidemiology of A. baumannii. Other studies are needed to better understandrelationships between the different community reservoirs, and also with the hospital reservoir.

In this study, we collected 18 plasma samples from NPC patients, and 10 plasma samples from healthy person for plasma proteomic analysis. We classified these samples into 3 groups: treatment, pretreatment and recurrent. Two-dimensional electrophoresis (2-DE) and MALDI-TOF were performed followed by comparative and statistic analysis. In conclusions, we totally identified 30 proteins in Nasopharyngeal carcinoma (NPC) plasma, and found 10 proteins with expression level down regulated (p<0.001). These proteins were characterized to be Serotransferrin, Vitamin D-binding protein (VDB), alpha-1 antitrypsin (AAT), Haptoglobin, Apolipoprotein B fragment, Syntaxin-7, Apolipoprotein, A-I, PRO1779, Transthyrin, MDN1 protein, respectively. Consequently, we established a protocol to remove high abundant proteins (e.g., albumin, immunoglobin etc.) in plasma. We are especially interested in ATT and VDB. Western blotting assay was performed to confirm ATT and VDB expression. Furthermore, the quantity of ATT and VDB were measured by ELISA to obtain the threshold value of these proteins. Finally, we want to realize the relationship between these down regulation proteins and clinical parameters in NPC malignancy and tumor progression. Since there are few protein expression research of NPC in clinical studies, our works will provide insights in NPC studies for tumor progression with potential to elevate treatment efficiency.

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L’oxygénation d’un tissu ou d’une cellule résulte d’un équilibre entre la disponibilité en oxygène et sa consommation. Un arrêt de la circulation ou des variations de la pression partielle en oxygène sont responsables d’une réduction de l’apport en oxygène induisant une réponse adaptative.L’objectif de ce travail a été de caractériser l’hypoxie de l’échelle cellulaire à tissulaire par la complémentarité de deux techniques d’imagerie par spectrométrie de masse (ISM) : La spectrométrie de masse à ionisation secondaire (SIMS) et l’ionisation/désorption par laser assistée par matrice (MALDI). L’ISM fournit la détection, l’identification et la distribution d’une variété d’espèces moléculaires endogènes et exogènes directement sur tissu sans marquage. Afin de caractériser l’hypoxie, un modèle in vitro de culture cellulaire en trois dimensions (sphéroïde) et un modèle in vivo d’accident vasculaire cérébral ont été utilisés.L’imagerie TOF-SIMS nous a permis de voir que la disponibilité réduite de l’oxygène au centre des sphéroides induit de profonds changements métaboliques. L’imagerie MALDI-TOF, quant à elle, a permis de visualiser la pharmacocinétique de différents traitements dans des sphéroides traités.Concernant l’étude sur l’accident vasculaire cérébral, l’imagerie SIMS et MALDI nous ont fourni une signature moléculaire de l’hypoxie tissulaire, apportant de nouvelles connaissances sur les changements physiopathologiques induits par la lésion tissulaire.La complémentarité de ces deux techniques d’imagerie permet donc une réelle synergie pour l’étude de l’hypoxie dans différents modèles. / Tissue or cells oxygenation results from a balance between oxygen availability and consumption. This availability is determined by the amount of oxygen carried by the blood irrigating the tissue and its diffusion capacity through the cell membranes. The interruption of blood flow or variations in the oxygen partial pressure are responsible for a reduction of oxygen intake that induces an adaptive response.The aim of my work is to characterize the hypoxia from cellular to tissue-level via the complementarity of two mass spectrometry imaging (MSI) methods: the Secondary Ion Mass Spectrometry (SIMS) and Matrix-Assisted Laser Desorption and Ionization (MALDI). MSI has the potential to provide detection, identification and distribution of a variety of different endogenous and exogenous molecular species directly from the tissue without labelling. Here we combine them to characterize hypoxia in vitro on a 3D cell culture system (spheroid) and in vivo using ischemic rat model.We have shown via TOF-SIMS imaging that reduced availability of oxygen to the center of spheroids induces profound metabolic changes. MALDI-TOF imaging helped to visualize the pharmacokinetics of different treatments in treated spheroids.Concerning the ischemic stroke, MSI provides a molecular signature of hypoxia in tissue, which could bring new insights into the pathological changes induced by the tissue injury.The complementarity of these two imaging techniques allows real synergy for the study of hypoxia in different models.