Teses / dissertações sobre o tema "Molecular magnetic resonance imaging"

Imaging leucocyte trafficking is a major goal in inflammation research, since one ofthe key features of the immune system is cell migration. Ultrasmall iron oxide nanoparticles (USPIO) are iron-based T2-enhancing magnetic resonance (MR) imaging contrast agents, which are a different class ofcontrast agent compared to the more traditional, clinically-established Tl-enhancing agents such as GadoliniumDTPA. The work presented in this project investigates different methods using both these classes ofcontrast agent for imaging different aspects ofinflammation at cellular and molecular levels. The uptake and MR imaging ofUSPIO and Gadolinium-DTPA by leucocytes in-vitro and in-vivo has been investigated. Activated endothelium is a proven surrogate for inflammatory sites. E-selectin is an adhesion molecule which is luminally expressed by activated endothelium early-on in the inflammatory process. This work has tested the hypothesis that activated endothelium can be imaged using magnetic resonance. USPIO have been conjugated with a monoclonal antibody targeting expressed Eselectin, and tested in-vitro and in-vivo using a murine model ofoxazolone-induced contact hypersensitivity inflammation in the ear. The majority ofMR studies in this project were conducted at a high MR field strengths of9.4T. The results indicate that MR imaging ofleucocyte migration and ofmolecular markers of activated endothelium are feasible. However there are limitations on the degree of success, which are presented and discussed. Targeted and cell specific imaging ofinflammatory lesions has the potential to be important investigative tool.

Magnetic Resonance Imaging (MRI) is a non-ionizing imaging modality that can provide images with excellent soft tissue contrast at high resolution. In particular, molecular and cellular MRI is a powerful imaging method that could provide a non-invasive way for assessing specific biological processes in vivo in living organisms. The ability to monitor and track biological structures and processes down to molecular and cellular level and the possibility to probe the development, survival, migration, and differentiation of cells in vivo, has opened up new ways for scientists to investigate the fundamental mechanisms of health and diseases. In this dissertation, novel applications of conventional MR contrast agents to study specific biological structures and processes are demonstrated. First, the potential of manganese enhanced MRI (MEMRI) for in vivo tract tracing and assessment of neuroarchitecture was investigated. Manganese was intracortically infused into the visual cortex along the border of the primary and secondary visual cortex and then imaged 8 and 24 hours later. A dynamic migratory path of manganese from the infusion site through the corpus callosum to the contralateral hemisphere was observed. Also, layer specific enhancement on the contralateral cortex and the connection of the visual cortex with other brain structures were shown and the results were consistent with established anatomical data. Secondly, MEMRI was performed to probe in vivo neuronal changes in the rodent brain following 72-hour rapid eye movement sleep deprivation. Significant reduction in manganese uptake was observed in the cortical and hippocampal region in the sleep deprived animals when compared to the normal group. In particular, the dentate gyrus substructure in the hippocampus exhibited the least uptake. This indicated the functional vulnerability of the hippocampus and the cortex to sleep deprivation. Lastly, in vivo tracking of endogenous neural stem and progenitor cell migration during neurogenesis in neonatal rat brain was performed by micron sized iron oxide particles (MPIO) labeling. Susceptibility weighted imaging was used for image processing to highlight the susceptibility contrast induced by the iron oxide particles. MPIO-labeled cells induced contrast was clearly enhanced in the susceptibility weighted images, particularly at day 3 after MPIO injection in which the MPIO-labeled NPCs became more dispersed in the olfactory bulb. The ventral migratory pathway of endogenous neural stem and progenitor cells, which could not be easily observed in conventional T2*W imaging, couldalsobe detected. Overall, various biological systems and processes have been successfully interrogated using MR contrast agents. Through these studies, the versatility and power of molecular and cellular MRI have been demonstrated. Looking ahead, the rapid development and combination of different molecular and cellular imaging techniques would certainly revolutionize the way we study health and diseases. In the end, this could foster our understanding of basic life sciences and hence improve the quality of healthcare.<br>published_or_final_version<br>Electrical and Electronic Engineering<br>Master<br>Master of Philosophy

Ce travail porte sur le développement de sondes peptidiques pour le suivi de la protéolyse par spectroscopie de résonance paramagnétique électronique (RPE) et pour l'imagerie in vivo par résonance magnétique rehaussée de l’effet Overhauser (OMRI). Plus précisément, ce travail étudie pour la première fois une famille d’agents d’imagerie appelée « nitroxyde à déplacement de raies spectrales » spécifique d’activités enzymatiques. L'activité protéolytique, entraînant un décalage de 5 G dans les constantes de couplages hyperfins, permet une quantification individuelle des espèces substrat et produit par RPE et une excitation sélective par OMRI. Trois substrats ont été élaborés, montrant une spécificité enzymatique pour l’élastase du neutrophile (NE) (MeO-Suc-Ala-Ala-Pro-Val-Nitroxyde &amp; Suc-Ala-Ala-Pro-Val-Nitroxyde), et pour la chymotrypsine et la cathepsine G (Suc-Ala-Ala-Pro-Phe-Nitroxyde). Les constantes enzymatiques ont montré de bonnes valeurs avec globalement, Km = 28 ± 25 µM et kcat = 19 ± 3 s-1. Ex vivo, l’utilisation des substrats NE en OMRI a révélé un contraste élevé dans les lavages broncho-alvéolaires de souris sous stimulus inflammatoire. Les rehaussements de signaux IRM sont en corrélation avec la sévérité de l’inflammation. L'irradiation à la fréquence RPE de 5425,6 MHz a permis d'accéder à la bio-distribution des substrats in vivo et pourrait ainsi servir d’outil diagnostic. Les perspectives à moyen terme de ce travail reposent sur le développement de l’OMRI à très faibles champs magnétiques en vue d’une application chez l’homme<br>This work focuses on substrate-based probes for proteolysis monitoring by Electron Paramagnetic Resonance spectroscopy (EPR) and for in vivo imaging by Overhauser-enhanced Magnetic Resonance (OMRI). More precisely, this work investigates for the first time a family of MRI agents named “line-shifting nitroxide” specific for proteolytic activities. Proteolytic action results in a shift of 5 G in EPR hyperfine coupling constants allowing individual quantification of substrate and product species by EPR and selective excitation by OMRI. Three substrates were worked out, showing enzymatic specificity for neutrophil elastase (MeO-Suc-Ala-Ala-Pro-Val-Nitroxide &amp; Suc-Ala-Ala-Pro-Val-Nitroxide), and for Chymotrypsin/Cathepsin G (Suc-Ala-Ala-Pro-Phe-Nitroxide). Enzymatic constants were remarkably good with globally Km = 28 ± 25 µM and kcat = 19 ± 3 s-1. Ex vivo, the use of NE substrates in OMRI revealed a high contrast in bronchoalveolar lavages of mice under inflammatory stimulus. MRI signal enhancements correlate with the severity of inflammation. Irradiation at the RPE frequency of 5425.6 MHz provided access to the bio-distribution of substrates in vivo and could thus serve as a diagnostic tool. The medium-term perspectives of this work are based on the development of OMRI with very low magnetic fields for human application

Ce travail fait l’objet d’une avancée scientifique dans le développement de la technique d’IRM rehaussée par l’effet Overhauser dans la souris à 0,2 T. Cette dernière repose sur le transfert de polarisation des spins électroniques saturés d’un radical libre vers les spins des protons (généralement de l’eau) voisins pour rehausser le signal RMN du proton. Notre équipe a développé cette technique pour détecter une activité protéolytique au travers de deux stratégies. La première partie de la thèse a été de détecter pour la première fois une activité protéolytique in situ dans des souris saines et in vitro sur cellules vivantes. L’efficacité du rehaussement par effet Overhauser repose sur le temps de corrélation des spins des électrons non-appariés. Un radical nitroxyde greffé à l’élastine a été utilisé comme substrat. La protéolyse de ce dernier par des élastases pancréatiques a conduit l’observation en 3D d’un rehaussement du signal RMN de plus de 10 fois dans le tube digestif de souris vivantes. De plus, des développements méthodologiques, tels que l’implémentation de la séquence TrueFISP, le sous-échantillonnage par la méthode “Keyhole”, et la reconstruction des données en 3D ont été faits. La deuxième stratégie repose sur des molécules de nitroxyde ayant l’unique propriété de pouvoir décaler leurs pics de résonance après hydrolyse. Un nitroxyde phosphorylé en position Béta pouvant être détecté à deux fréquences spécifiques différentes avant et après hydrolyse d’un groupement chimique a été synthétisé par des chimistes à Marseille. L’hydrolyse de cette macromolécule a été observée in vivo dans l’estomac de souris saines avec des rehaussements de plus de 400% et imagée en 3D avec une bonne résolution spatio-temporelle. Ainsi, une prochaine étape serait de poursuivre ce travail sur un modèle pathologique et développer cette technique à un champ magnétique plus bas<br>This work relates the continuity and advances in the implementation of the Overhauser-enhanced Magnetic Resonance Imaging technique on a 0.2 T scanner. Briefly, OMRI technique is based on polarization transfer of saturated electronic spins from free nitroxide radicals to proton spins of surrounding water molecules in the aim to drastically enhance proton NMR signal. To this technique, our research team has merged specific strategies for proteolytic activity detection. The first strategy relies on a 3D visualization of proteolytic activity happening in intact living cells or in vivo in healthy mice. With an Overhauser switch based upon changes in molecular tumbling time, high Overhauser enhancements of 10-fold were observed in the intestinal tract of mice after that elastolytic activity of our probe: the nitroxide-labeled elastin macromolecule took place. In addition, MRI developments - TrueFISP sequence implementation, undersampling Keyhole method and data reconstruction were carried out for imaging these rapid biological processes. A second exquisite strategy is also described using nitroxides with shifting resonant peaks. Here, a Beta-phosphorylated nitroxide molecule was specifically detected at two distinct frequencies: one for its substrate and the other for its product once hydrolysis took place. This hydrolysis was imaged in 3D in the stomach of living mice with Overhauser enhancements of more than 400% and with a good spatiotemporal resolution. The perspectives of this work lie on a future detection of a pathological proteolytic activity in vivo and eventually and development of very low magnetic field OMRI

One approach that has demonstrated success in the field of molecular imaging utilizes microparticles of iron oxide (MPIO) conjugated to specific antibodies and/or peptides to provide contrast effects on MRI in relation to the molecular expression of a specified target. The experimental aims of this thesis were 1) to investigate the ability of VCAM-1 and P-selectin targeted MPIO to detect the expression of VCAM-1 and P-selectin on the activated endothelium in-vitro and in-vivo in mouse models of renal and cerebral ischemia reperfusion injury, and 2) develop a novel contrast agent for imaging αvβ3-integrin expression in angiogenesis using RGD peptide conjugated MPIO (RGD-MPIO) in-vitro. MPIO (1.0 µm) were conjugated to monoclonal antibodies against VCAM-1 (VCAM-MPIO) or P-selectin (PSEL-MPIO). In vitro, MPIO bound in a dose-dependent manner to tumor necrosis factor (TNF)-alpha stimulated sEND-1 endothelial cells when conjugated to VCAM-1 (R² = 0.88, P<0.01) and P-selectin antibodies (R² = 0.93, P<0.01), reflecting molecular VCAM-1 and P-selectin mRNA and protein expression. Mice subjected to unilateral, transient (30 minutes) renal ischemia and subsequent reperfusion received intravenous VCAM-MPIO and PSEL-MPIO (4.5 mg iron/kg body weight). In ischemic kidneys, MR related contrast effects of VCAM-MPIO were 4-fold higher than unclamped kidneys (P<0.01) and 1.5-fold higher than clamped kidneys of PSEL-MPIO injected mice (P<0.05). VCAM-MPIO binding was less evident in IRI kidneys pre-treated with VCAM-1 antibody (P<0.001). VCAM-1 mRNA expression and VCAM-MPIO contrast volume were highly correlated (R² = 0.901, P<0.01), indicating that quantification of contrast volume reflected renal VCAM-1 transcription. In mice subjected to cerebral ischemia, contrast volume was 11-fold greater in animals injected with VCAM-MPIO versus control IgG-MPIO (P<0.05). Finally, S-nitroso-N-acetylpenicillamine (SNAP) stimulated HUVEC-C cells, which express αvβ3-integrin, showed 44-fold greater RGD-MPIO binding than unstimulated cells (P<0.001) and 4-fold greater RGD-MPIO binding than SNAP stimulated cells blocked with soluble RGD peptide (P<0.001) in-vitro. This thesis demonstrated that targeted MPIO exhibited contrast effects that defined and quantified the molecular expression of specific targets through the use of high-resolution MRI in in-vitro and in-vivo models of vascular inflammation.

Malgré les récents progrès thérapeutiques et les avancées dans la compréhension de la physiopathologie des maladies de la paroi artérielle - et principalement la maladie athéromateuse ainsi que l'anévrisme de l'aorte abdominal - les complications secondaires liées à leur rupture représentent toujours la première cause de mortalité dans les pays développés. L'absence de symptômes majeurs jusqu'à l'apparition d'un épisode cardiovasculaire or cérébro-vasculaire compliqué, combiné à l'absence de méthode d'imagerie précise pour la détection précoce de lésion à haut risque limite la prévention et le traitement de ces deux maladies. L'imagerie par résonance magnétique (IRM) de haute résolution est devenue ces dernières années une des modalités d'imagerie les plus prometteuses pour l'évaluation non invasive des maladies de la paroi artérielle. Outre son caractère non invasif, elle possède la capacité à détecter et caractériser les différents composants de la plaque d'athérosclérose ou encore identifier les caractéristiques de l'anévrisme de l'aorte abdominal. Particulièrement, l'imagerie moléculaire IRM utilise des agents de contraste ciblant des composants des lésions de la paroi vasculaire, ayant une forte affinité pour une activité biologique caractéristique des lésions à haut-risques. Au cours de notre travail, nous avons conçu deux études afin d'apprécier la faisabilité de l'imagerie moléculaire en IRM appliqué à la caractérisation de la plaque d'athérome vulnérable d'une part et de l'anévrisme de l'aorte abdominal susceptible de rupture d'autre part. Ainsi, nous avons montré dans une première étude que la détection de l'athérothrombose carotidienne dans un modèle animal de souris est facilitée en IRM par l'utilisation d'un agent de contraste ciblant les plaquettes activées (P975). Dans une deuxième partie, nous avons démontré la caractérisation d'un nouveau modèle animal de rupture de l'anévrisme de l'aorte abdominale au travers de l'utilisation de micelles de gadolinium spécifiques du collagène (CNA-35). Nous montrons ainsi le potentiel de ces nanoparticules pour la discrimination d'anévrismes stables et ceux susceptibles de progresser rapidement vers la rupture. L'imagerie moléculaire par IRM, fournit des informations déterminantes sur la présence de molécules clés in vivo, dans les lésions de la paroi vasculaire à haut-risques. Ces nouvelles méthodes d'imagerie devraient, à terme, faciliter l'identification des pathologies susceptibles d'induire des événements cardiovasculaires dramatiques et ainsi entraîner des complications cardiovasculaires<br>Despite the emergence of cutting edge therapeutic strategies and advances in our understanding of the pathogenesis of vascular diseases - particularly atherosclerosis and abdominal aortic aneurysm (AAA) - they remain the leading cause of morbidity/mortality in Western societies. The major absence of symptoms until the onset of a critical cardio or cerebro-vascular events, associated with the lack of an appropriate imaging modality for early detection of high-risk lesions, limits the prevention and treatment of vascular diseases. Therefore, the identification of high-risk vascular lesions prone to quickly evolve and lead to dramatic episodes may greatly decrease the morbidity and mortality they are associated with. High-resolution magnetic resonance imaging (MRI) has recently emerged as one of the most promising techniques for the non-invasive identification of disease of the vessel wall such as atherosclerosis and abdominal aortic aneurysm. Specifically, MR molecular imaging using contrast agents targeted towards molecules of interest may enable the non-invasive identification of high-risk vascular lesions. In our work, we designed two studies to investigate the feasability of MR molecular imaging applied to the characterization of vulnerable atherosclerotic plaques on one hand, and abdominal aortic aneurysms susceptible to quickly evolve and rupture on the other hand. Thus, we demonstrated in the first part of our research, the ability of an activated platetelet-targeted MR contrast agent (P975) to accurately detect thrombus formation in a mouse model of carotid thrombosis. In a second study, we used a micellar plateform functionalized with a collagen-binding protein (CNA-35) to assess the presence of collagen in an abdominal aortic aneurysm mouse model prone to rupture. We report the potential of CNA-35 micelles to discriminate between stable AAA lesions and aneurysms mat were likely to rapidly progress or rupture. MR molecular imaging, as demonstrated throughout our work offers unique in vivo insights into critical biological process at play in high-risk atherosclerotic lesions and abdominal aortic aneurysms respectively. Ultimately, such techniques may enable treatment of high-risk patients prior to lesion progression and onset of secondary complications

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, 2016.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (pages 71-84).<br>Magnetic resonance imaging (MRI) is a powerful neuroimaging tool that allows non-invasive visualization of the brain with high spatial and temporal resolution. Research on MRI contrast agents and their application to problems in neuroscience is burgeoning, and there is particular interest in developing MRI agents that are sensitive to time varying components of neurophysiology. Relatively recent advances in biomolecular probes has demonstrated the potential and versatility of bioengineered MRI sensors for molecular imaging. However, a major limitation of these probes is the high concentration needed for imaging, which can lead to issues such as analyte buffering and toxicity, and restrict the applicability of the sensors. In this work, we explore two approaches for developing high relaxivity protein-based contrast agents to address the issues of low detectability. First, we coupled monoamine sensing with the disaggregation of superparamagnetic iron oxide nanoparticles (SPIOs). Ligand detection was imparted by integration of a monoamine sensing protein-based contrast agent derived from P450- BM3h (BM3). We demonstrated that this mechanism can produce robust signal changes of approximately 2-fold, while reducing the concentration of BM3 needed by 100-fold compared to the amount needed when only the protein is used for imaging. The second method demonstrated the feasibility of using semi-rational protein design to engineer a high relaxivity metalloprotein by tuning phenylalanine hydroxylase to bind gadolinium at high affinity. Mutations were found that increased the protein affinity by two orders of magnitude and enhanced relaxivity. The results of this thesis advance approaches for creating high relaxivity contrast agents which can be applied to the development of probes for other analytes, ultimately advancing and broadening the applicability of bioengineered probes in molecular functional neuroimaging.<br>by Vivian Hsieh.<br>Ph. D.

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2009.<br>Committee Chair: Gang Bao; Committee Member: Kurt D. Pennell; Committee Member: Mark M. Goodman; Committee Member: Xiaoping P. Hu; Committee Member: Yadong Wang. Part of the SMARTech Electronic Thesis and Dissertation Collection.

While providing non-invasive tissue detection, magnetic resonance imaging (MRI) presently possesses limited sensitivity for protein target recognition. This limitation was addressed for the target beta-galactosidase (beta-gal) by constructing three beta-gal-specific MRI probes. The probes are based on a bipartite design in which a vasoactive moiety known as a diazeniumdiolate (NONOate) is bound to a specifier, specifically galactose. Upon galactose' interaction with beta-gal, the NONOate is cleaved from galactose, and actively generates nitric oxide (NO). The released NO leads to microvascular permeability changes in surrounding tissues affecting localized T1 measurements. These changes serve as a quantitative index of beta-gal detection. The three beta-gal-specific NO-releasing probes constructed include GALPYRNONO, GALPIPNONO and a 'bi-functional' probe, which is similar to the first two but with glucose additionally incorporated so that the third probe may easily cross cellular membranes. Synthesis and characterization of this novel class of MRI probes are described in this work.<br>Keywords: non-invasive detection, magnetic resonance imaging (MRI), nitric oxide (NO), diazeniumdiolates (NONOates), NO-releasing compounds, novel MRI probes, molecular targets, protein targets, specifier, vasoactive, vasodilation, microvascular permeability, tissue localization, bipartite systems, bifunctional probes, blood-brain barrier, cell membrane trafficking, saccharide-bound NONOates, sugar diazeniumdiolates, glycosylated diazeniumdiolates, galactose, beta-galactosidase, glucose, glucose transporters, thermal & photolytic degradation, half-life optimization, Griess test, rat serum, stability.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006.<br>Dr. X. Hu, Committee Member ; Dr. Al Merrill, Committee Member ; Dr. Niren Murthy, Committee Member ; Dr. Gang Bao, Committee Chair ; Dr. Nicholas Hud, Committee Member. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2008.<br>Principal faculty advisors: Norman J. Wagner, Dept. of Chemical Engineering, University of Delaware; Eric Wickstrom, Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University. Includes bibliographical references.

Los tumores cerebrales constituyen menos del 2% de los tumores primarios, pero son uno de los peores tipos de cáncer en cuanto a “años de vida perdidos”. Los gliomas son los tumores cerebrales más prevalentes, con una esperanza de vida inferior a 15 meses para los casos de alto grado, como los glioblastomas (GBM). El método no invasivo más utlilizado para diagnóstico y seguimiento de la respuesta a la terapia en tumores cerebrales es la Resonancia Magnética (MR), en forma de imagen (MRI) y espectroscopía (MRS) o imagen espectroscópica (MRSI). Sin embargo, debido a restricciones éticas relacionadas a la participación de pacientes humanos en investigación, la optimización de los métodos de diagnóstico y seguimiento de la terapia requieren modelos preclínicos que reproduzcan las patologías humanas. Para este tipo de estudios, se utilizan principalmente modelos murinos, que pueden dividirse en modelos genéticamente modificados (GEM) con desarrollo espontáneo de tumor y los modelos de generación de tumores por inyección estereotáxica de líneas celulares. En la presente tesis, se llevó a cabo una detallada caracterización de dos colonias GEM, S100β-v-erbB/inK4a-Arf (+/-) y GFAP-V12 HA-ras B8. Se observó una baja penetrancia de desarrollo de tumores (16% y 1% respectivamente), haciendo de éstos una herramienta poco útil para estudios de respuesta a la terapia. La escasez de modelos preclínicos de grado bajo/intermedio sirvió de motivación para desarrollar un modelo de tumor glial transplantable con esas características, por disgregación de tumores provenientes de la colonia GEM. Ello nos debería permitir obtener una mayor incidencia tumoral en comparación con las colonias GEM. Se generaron gliosferas a partir de tumores GEM de grado III y se logró más de un 60% de penetrancia cuando estas células fueron inyectadas estereotácticamente en el estriado de ratones C57BL/6. Sin embargo, la aplicación de protocolos de descongelación y cultivo a éstas células ocasionó una progresión a grado IV (GBM), lo que sugiere que el modelo generado constituye, potencialmente, un modelo murino de GBM secundario. Además, este modelo transplantable fue ampliamente caracterizado por métodos de MRI/MRSI, así como por métodos de perturbación del patrón espectral con MRSI (PE-MRSI) para una posible aplicación en el desarrollo de clasificadores de respuesta a la terapia en tumores. También se llevó a cabo una evaluación genética restringida de los modelos murinos seleccionados (p.e. tumores GL261, línea celular GL261, GEM y tumores derivados de GEM), utilizando el método de secuenciación de Sanger para comprobar la presencia de mutaciones normalmente presentes en gliomas (en los genes IDH1, IDH2 y p53). Finalmente, esta tesis describe la estrategia desarrollada para estudios longitudinales de detección de respuesta temprana a la terapia/recidiva en tumores preclínicos, utilizando métodos de imagen molecular basados en MRSI. Así ratones implantados con tumores GL261 (glioblastoma) recibieron tratamiento con temozolamida (TMZ), basándonos en protocolos previamente establecidos. La detención del crecimiento tumoral (respuesta a la terapia) fue detectada por MRI. Tanto animales tratados como animales control fueron estudiados por MRSI y técnicas de reconocimiento de patrones (extracción de fuentes en sistema semi-supervisado). Las fuentes extraídas de regiones de interés fueron capaces de distinguir entre tumores GL261 proliferando activamente y tumores respondiendo a la terapia, basándose en cambios de patrón del metaboloma registrado por MRSI. Se obtuvieron mapas nosológicos codificados en tipo e intensidad de color durante y después de la terapia, lo que permitió un seguimiento de la respuesta, así como la detección de heterogeneidad intratumoral en dicha respuesta, siendo capaces de detectar la detención del crecimiento tumoral y la recidiva antes de los cambios observados en el volumen tumoral por MRI. Esta metodología fue ratificada por análisis histopatológico y cálculo de tasas de proliferación, apoptosis y de índice mitótico.<br>Brain tumours account for less than 2% of all primary tumours, but are one of the most lethal cancers when “life lost” years are considered. Gliomas are the most prevalent type with a median life expectancy below 15 months for the high grade ones, such as glioblastomas (GBM). The most common non-invasive medical technique used for tumour diagnosis and therapy monitoring of brain tumours patients is Magnetic Resonance (MR), in the form of imaging (MRI) and spectroscopy (MRS) or spectroscopic imaging (MRSI). However, due to the ethical restrictions regarding the use of human patients for research study, the improvement of diagnostic and therapy follow-up protocols requires reliable models that mimic human disease. In this regard, mainly murine models are used and can be divided into the genetically engineered model (GEM) of spontaneous tumour development and the engrafted tumour model. In this thesis, a comprehensive MR characterization of two GEM colonies, namely S100β-v-erbB / inK4a-Arf (+/-) and GFAP-V12 HA-ras B8, was carried out. A low tumour penetrance found (16% and 1%, respectively) together with stochastic onset of GEM tumours, made them impractical for use in therapy response studies. The latter and the scarcity of low/intermediate grade brain tumour preclinical models motivated us to attempt to develop a transplantable glial tumour model of low/intermediate grade by disaggregation of a tumour mass from GEM. This should allow us to obtain an increased tumour incidence rate in comparison to GEM animals. Gliospheres from a grade III GEM tumour were successfully generated and displayed more than 60% penetrance, when stereotactically injected into the striatum of C57BL/6 mice. However, the application of freezing and cell culture protocols produced a progression to grade IV GBM, which made the developed transplantable model qualify as potential secondary GBM model in mice. Additionally, this transplantable model was widely characterized using MRI/MRS methods, as well as perturbation-enhanced MRSI (PE-MRSI) for a possible application in the future in therapy strategies and development of tumour therapy response detection classifiers. A restricted genetic evaluation of selected murine tumour models (i.e. GL261 tumours, GL261 cell line, GEM and GEM-derived tumours) was carried out using the Sanger method to check for a possible presence of particular driver mutations commonly occurring in gliomas (IDH1, IDH2 and p53). Finally, the work describes the strategy followed for longitudinal therapy studies follow-up and early response/relapse detection in preclinical brain tumours, through molecular imaging methods based in MRSI. GL261 (glioblastoma) tumour bearing mice were treated with temozolomide (TMZ), based on previously established protocols. The expected transient growth arrest (response to therapy) was detected by MRI. Animals subjected to therapy and control animals were followed up by MRSI and pattern recognition techniques (semi-supervised source extraction) were applied. The sources extracted from the region of interest were able to discriminate between GL261 tumours actively proliferating and tumours responding to therapy, based on their metabolome pattern changes recorded by MRSI. Colour-coded nosological images produced throughout and after the course of therapy allowed convenient tracking of response changes and differentiated the intratumoural heterogeneity of response, hinting the growth arrest and relapse, before changes in tumour volume were observed by MRI. The methodology was validated with histopathological analysis and calculation of proliferation and apoptotic rates and mitotic index.

Iron oxide nanoparticles are of great interest as contrast agents for research and potentially clinical molecular magnetic resonance imaging (MRI). Biochemically modifying the surface coatings of the particles with proteins and polysaccharides enhances their utility by improving cell receptor specificity, increasing uptake for cell labeling and adding therapeutic molecules. Together with the high contrast they produce in MR images, these characteristics promise an expanding role for iron oxide nanoparticles and molecular MR imaging for studying, diagnosing and treating diseases at the molecular level. However, these contrast agents produce areas of signal loss with traditional MRI sequences that are not specific to the nanoparticles and cannot easily quantify the contrast agent concentration. With the expanding role of iron oxide nanoparticles in molecular imaging, new methods are needed to produce a quantitative contrast that is specific to the iron oxide nanoparticle. This dissertation presents a new method for detecting and quantifying iron oxide nanoparticles using an adiabatic preparation pulse and the failure of the adiabatic condition for spins diffusing near the particles. In the first aim, the theoretical foundation of the work is presented, and a Monte Carlo simulation supporting the proposed mechanism of the contrast is described. Adiabatic pulse prepared imaging sequences are also developed for imaging at 3 Tesla and 9.4 Tesla to highlight the translational potential of the approach for clinical examinations and scientific research, and the linear correlation of the contrast with iron concentration ideal for quantification is presented. Further, the physical characteristics of the nanoparticles and the parameters of the MRI sequence are modified to characterize the approach. In the second aim, the contrast is characterized in more realistic phantoms and in vitro, and a method to more accurately quantify nanoparticle concentration in the presence of magnetization transfer is presented. Finally, accelerated imaging methods are implemented to acquire the adiabatic contrast in a time compatible with in vivo imaging, and the technique is evaluated in an in vivo model of quantitative iron oxide nanoparticle imaging. Together, these aims present a method using an adiabatic preparation pulse to generate an MR contrast based on the microscopic magnetic field gradients surrounding the iron oxide nanoparticles that is suitable for in vivo quantitative, molecular imaging.

Background. In the last 15 years, it has become increasingly accepted that cerebral grey matter (GM) damage in MS is evident since early MS stages and provides the best correlate of the rate of clinical progression since early disease stages. The lack of substantial inflammatory infiltrates, complement deposition and blood brain barrier damage in MS cortical lesions led to the initial suggestion that the mechanisms underlying GM and white matter (WM) pathology substantially differ and that activated microglia are the dominant effector cell population causing GM damage. Furthermore, in the last decade, post-mortem studies have revealed that the extent of meningeal inflammation associates positively with subpial cortical demyelination and disease progression, suggesting that chronic immune activity in the subarachnoid compartment plays a key role in mediating damage in the adjacent cerebral cortex of affected patients. Factors released by immune cells circulating in the CSF and/or colonizing the subarachnoid space may diffuse across the pial membrane, inducing a gradient of glia and neuronal pathological alterations, directly and/or indirectly through activated microglia. Aim of the study. To combine CSF molecular and protein analyses with advanced MRI imaging techniques, able to better identify cortical grey matter demyelination, in order to identify potential early biomarkers of GM pathology and disease progression with prognostic and predictive value and to obtain information on the biological and immunological mechanisms that link the inflammatory process of the GM and the progressive neurodegeneration Methods. Two independent cohorts of MS patients have been recruited and followed by the MS Centre of Padova. The first cohort of MS patients, composed of 35 patients and 5 controls was recruited retrospectively between February 2009 and September 2011 and followed by a detailed neurological and clinical follow-up. The second cohort of 31 patients and 13 controls was collected prospectively from January 2014 to May 2015. The patients, at their disease onset or in the very early phase of the disease, underwent a complete diagnostic work-up comprehensive of clinical evaluation, lumbar puncture and MRI evaluation inclusive of non-conventional sequences. By using the immuno-assay Bio-Plex System technique (Biorad - Bio-Plex Pro Human Chemokine panel 40-plex) we performed a protein analysis of the presence and levels of inflammatory molecules in both cohorts. In the second cohort we carried out also a gene expression analysis of the matched cellular fraction (using pre amplification real-time PCR for a panel of genes of interest). Results. CSF protein analysis of the first cohort of MS patients reveal higher levels of proinflammatory cytokines CXCL13 (p=0,00006), CCL19 (p=0,0019), CCL1 (p=0,00018), and CCL22 (0.0009) compared to controls. Protein analysis on our second cohort pointed out an important increase of CXCL13, CXCL10, CXCL11 and CCL2 in MS population compared to controls. After stratification according to GM pathology we reported an increase of CXCL13 protein levels in the MS subgroup with higher cortical demyelination. Gene expression analysis reveals a significant increase in MS patients for CD20, CD138 and LTa compared to controls supporting the hypothesis of a key role of a B-cell response and lymphoid neogenesis in MS pathology Conclusions. Combined CSF analysis and MRI analysis suggested that B cell immune response may play an important role in MS since the disease onset and correlates with the level of intrathecal inflammation and cortical pathology. A more detailed analysis of the CSF biomarkers suggested in the current study might provide, in addition to MRI optimization, a better indication of severity of disease process that characterized GM pathology and important tools in predicting/monitoring the evolution of the disease.<br>Presupposti dello studio. Studi istopatologici e neuroradiologici hanno dimostrato, soprattutto negli ultimi 15 anni come la sclerosi multipla, considerata classicamente una patologia elettiva della sostanza bianca, sia una patologia caratterizzata da un coinvolgimento, fin dalle prime fasi di malattia, anche della sostanza grigia corticale e profonda che ben correla con il quadro di disabilità fisica e cognitiva ampiamente descritto. Dal punto di vista istopatologico tale coinvolgimento è caratterizzato dalla presenza di lesioni corticali che si distinguono per una minor componente infiammatoria, per un’assenza di danno a carico della barriera ematoencefalica e per un minor deposito di fattori del complemento. Alla luce di tale descrizione sembra che i meccanismi che sottendono il danno a carico della sostanza grigia differiscano almeno in parte da quelli osservati a carico della sostanza bianca. In particolare è stata osservata un’associazione tra lesioni corticali e la presenza di infiltrati infiammatori meningei correlata ad una maggiore disabilità clinica. È stato quindi evidenziato come molecole pro-infiammatorie rilasciate dalle cellule infiammatorie residenti nelle meningi diffondano attraverso lo spazio subaracnoideo e agiscono, direttamente o indirettamente attivando la componente microgliale, sulla adiacente corteccia cerebrale determinando un gradiente di danno corticale. Obiettivi. L’obiettivo di questo studio è stato quello di verificare la presenza di possibili potenziali biomarcatori di danno corticale mediante l’applicazione combinata di: a) tecniche avanzate di analisi proteica e analisi molecolare applicate allo studio del liquido cerebro spianale; b) tecniche non convenzionali di risonanza magnetica, in grado di caratterizzare il danno a carico della sostanza grigia sia focale che diffuso. Materiali e metodi. Sono state arruolate due coorti di pazienti: la prima studiata retrospettivamente era composta da 35 pazienti e 5 controlli; la seconda arruolata nello studio con un approccio di tipo longitudinale era composta da 31 pazienti e 13 controlli. Tutti i pazienti erano caratterizzati da un esordio relativamente recente e la precedente somministrazione di terapie immunomodulanti rappresentava un criterio di esclusione. Tutti i pazienti si sono sottoposti ad un iter diagnostico completo comprensivo di valutazione clinica, esami di laboratorio, esame del liquido cerebrospinale e risonanza magnetica comprensiva di sequenze non convenzionali per verificare la presenza di lesioni della sostanza grigia. Lo studio del liquido cerebrospinale prevedeva inoltre uno studio di analisti proteica mediante immuno-assay (Bio-Plex System technique Biorad - Bio-Plex Pro Human Chemokine panel 40-plex) per lo studio di 40 citochine/chemochine e uno studio di analisi di gene expression. Risultati. L’analisi proteica del liquor nella prima coorte ha evidenziato la presenza di elevati valori di CXCL13 (p=0,00006), CCL19 (p=0,0019), CCL1 (p=0,00018), e CCL22 (0.0009) rispetto alla popolazione di controllo. L’analisi proteica della seconda coorte ha evidenziato, sempre rispetto alla popolazione di controllo, un aumento delle seguenti citochine: CXCL13, CXCL10, CXCL11 e CCL2. Dopo stratificazione in base al carico corticale abbiamo evidenziato nei pazienti con un maggior coinvolgimento della sostanza grigia un aumento dei livelli proteici di CXCL13 e una maggior espressione di CD20, CD138, CXCL13 and LTa a supporto di un ruolo della risposta infiammatoria mediata dai linfociti B. Conclusioni. L’analisi combinata di liquor e risonanza magnetica suggerisce che la risposta immunologica mediata dai linfociti B gioca un ruolo importante nella patogenesi della sclerosi multipla e che il livello di infiammazione intratecale ben correla con la patologia corticale. I risultati del nostro studio suggeriscono quindi che l’uso di biomarker liquorali potrebbero essere di supporto nella caratterizzazione della patologia corticale nella sclerosi multipla.

This thesis investigates the restricted rotational and translational dynamics of water molecules as they interact with collagen fibre networks. Knowledge of the relationship between these motions and the magnetic resonance proton signal can be used to determine collagen fibre organisation in anisotropic, collagen-rich tissues. Both experimental magnetic resonance techniques and theoretical computer simulations were used to probe water-collagen interactions. The results of this work allow for a more comprehensive analysis of collagen organisation, and changes to this organisation as a result of biomechanical load, in collagen-rich tissues using magnetic resonance imaging.

L'athérosclérose, à l’origine de la plupart des maladies cardiovasculaires telles que l'infarctus du myocarde ou l'AVC, est la principale cause de décès dans le monde. Les cliniciens ont donc besoin de techniques d'imagerie fiables pour identifier les patients «vulnérables» porteur d’athérome à haut risque d'occlusion thrombotique. Cette pathologie est une maladie inflammatoire qui implique beaucoup d'acteurs cellulaires et moléculaires, parmi lesquels les cellules endothéliales et immunitaires, les lipoprotéines, les cellules apoptotiques et les plaquettes. L'imagerie moléculaire visant à détecter ces acteurs avant la survenue d'événements cardiovasculaires dramatiques est en plein essor. Des anticorps humains (HuAbs) sélectionnés par phage-display pour reconnaître des biomarqueurs de la pathologies ont ici proposés comme ligands servant à fonctionnaliser des vecteurs pour l'imagerie IRM, de fluorescence ou TEP. Les HuAbs ont été modifiés, en introduisant soit des Cystéines soit un site de reconnaissance pour la Sortase, afin de développer un greffage site-spécifique. Les agents ciblant ont été validés in vitro et ex vivo sur des coupes d'athérome de modèles animaux. Des résultats prometteurs ontété obtenus en injectant dans des souris ApoE-/- l’anticorps antiplaquettaire TEG4, apportant ainsi de nouvelles connaissances sur la biologie de l'athérome et la preuve de concept d'une possible détection des plaques à haut risque riches en plaquettes. Des améliorations sont en cours pour développer des agents de contraste multi-fonctionnalisés avec des HuAbs et permettant de réaliser une imagerie moléculaire multimodale de l'athérosclérose facilement transposable en clinique<br>Because cardiovascular diseases are the leading cause of death in the world, providing clinicians with reliable and straightforward imaging techniques to identify "vulnerable" patients from the general population appears like the Holy Grail of the cardiovascular field. Atherosclerosis, identified as the underlying condition for most acute cardiovascular events, is characterized by the constitution of a lipidrich atheroma plaque, driven both by excess cholesterol and inflammation, which eventual rupture triggers clotting into the blood flow. It involves a wealth of cellular and molecular actors, which are so many potential markers for molecular imaging, aiming at deciphering how to warn clinicians about the possible occurrence of myocardial infarction or stroke. Here, human antibodies (HuAbs) selected by phage-display for their recognition of over-expressed biomarkers of the pathology are proposed as targeting ligands. They were further engineered for site-specific grafting, either by introducing Cysteine or Sortase recognition tags, and used to target contrast agents for MRI, fluorescence, or PET imaging. In vitro and ex vivo validation studies were carried out on atheroma sections of animal models. In vivo studies in the ApoE-/- mouse model were realized with the anti-platelet TEG4 HuAb using MRI, which provided insights on the biological relevance and feasibility to detect platelets-rich, high-risk atheroma plaques. The development of contrast agents useful in multi-modality imaging, and multi-functionalized with HuAbs is underway. It should serve as an accurate molecular imaging method for atherosclerosis, further more easily translated into the clinical arena

Introdução: As leucoencefalopatias constituem um vasto grupo de doenças que desafiam a equipe médica. Dentre elas, a doença da Substância Branca Evanescente, é uma das mais comuns, com destaque nos últimos anos pelo significativo avanço na caracterização de suas bases clínicas, moleculares e de imagem. O fenótipo típico tem início dos 2 aos 6 anos, marcado por declínio neurológico crônico e progressivo, com episódios de deterioração desencadeados por trauma ou infecção, que podem levar a coma e até óbito. Essa é uma desordem genética, autossômica recessiva, relacionada a mutações nos genes que codificam o fator iniciador de tradução dos eucariontes 2B (eIF2B), complexo responsável por coordenar a tradução do RNA em proteína. As características típicas na Ressonância Magnética (RM) encefálica, com padrão de acometimento difuso da substância branca e degeneração cística, constituem a forma de diagnóstico mais acessível em associação com os dados clínicos. Objetivo: Caracterizar os casos de doença da Substância Branca Evanescente em relação ao aspecto de imagem, correlação com achados clínicos e moleculares, além de avaliação evolutiva da neuroimagem e comparação com dados da literatura. Método: Delineamento do tipo prospectivo histórico por meio de prontuários e imagens de RM de encéfalo de 13 pacientes com diagnóstico molecular de doença da Substância Branca Evanescente do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - HCFMRPUSP - do período de 2006 a 2016. Resultados: Os pacientes eram na maioria do sexo feminino, todos brancos, com idade média de início dos sintomas aos 10 anos. Todas as mutações foram no gene EIF2B5, sendo prevalente c.338G> A (p.Arg113His). Trauma ou infecção como desencadeante foi descrito em 38,4%. O sintoma mais frequente foi ataxia (100%). Falência ovariana afetou metade das mulheres. Todos os exames de RM mostraram comprometimento da substância branca profunda, poupando relativamente a subcortical, com preferência frontoparietal (84,6%). Todas apresentaram lesões no corpo caloso e envolvimento cerebelar. Atrofia óptica acometeu 46,1%. Espectroscopia de prótons mostrou redução dos valores de Nacetil-aspartato e pico de lactato. O seguimento em imagem evidenciou evolução das lesões na substância branca e da atrofia, com maior acometimento da alta convexidade e dilatação ventricular, porém sem colapso do parênquima. Concomitantemente, o seguimento clínico mostrou piora neurológica progressiva e desfecho desfavorável em 12 dos 13 pacientes. Conclusões: Este é um dos estudos brasileiros com maior casuística de pacientes com diagnóstico molecular de Doença da Substância Branca Evanescente. Embora sendo uma doença multifacetada, os dados epidemiológicos, clínicos e de imagem encontrados foram semelhantes aos classicamente descritos na literatura para outras populações. Ressalta-se, ainda, extrema importância da RM de encéfalo para diagnóstico, evolução e triagem genética dessa desordem.<br>Introduction: Leukoencephalopathies constitute a vast group of differential diagnoses that challenge the medical team. Among them, Vanishing White Matter (VWM) Disease is one of the most common, and has been highlighted nowadays for significant progress in the characterization of its clinical, molecular and imaging basis. The typical phenotype begins at 2 to 6 years old, marked by chronic and progressive neurological decline, with episodes of deterioration triggered by trauma or infection that can lead to coma and even death. This is a genetic, autosomal recessive disorder related to mutations in the genes that encode the eukaryotic translation initiator factor 2B (eIF2B), a complex responsible for coordinating the translation of RNA into protein. The typical features in brain Magnetic Resonance Imaging (MRI), with a pattern of diffuse white matter involvement and cystic degeneration, are the most accessible form of diagnosis in association with clinical data. Purpose: to characterize VWM disease cases in relation to brain MRI appearance, clinical and molecular correlation and evolution over the time, comparing with data already described. Methods: A prospective historical design was performed using a review of medical records and brain MRI images of 13 patients from Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - HCFMRPUSP - with imaging and molecular diagnostic of VWM disease during the period from 2006 to 2016. Results: The patients were mostly female, all white, with a mean age of symptoms onset at 10 years old. All mutations were in the EIF2B5 gene, the most prevalent of them was c.338G> A (p.Arg113His). Trauma or infection as a trigger was described in 38.4%. The most frequent symptom was ataxia (100%). Ovarian failure affected half of women. All MRI showed deep white matter impairment, in a less prominent degree in the subcortical region, with frontoparietal preference (84.6%). All had lesions in the corpus callosum and cerebellar white matter involvement. Optic atrophy affected 46.1%. Proton spectroscopy showed a reduction in NAA values and a lactate peak. Image follow-up revealed white matter lesions and atrophy progression, with ventricular dilatation, however without parenchymal collapse. Concomitantly, there was progressive neurological worsening and unfavorable outcome in 12 of the 13 patients. Conclusion: This is one of the Brazilian studies with the largest number of patients with molecular diagnosis of VWM Disease. Although it is a multifaceted disease, the epidemiological, clinical and imaging data found were similar to those classically described in the literature for other populations. The importance of brain MRI for the diagnosis, evolution and genetic screening of this disorder is also highlighted.

A substituição de cardiomiócitos na vida pós-natal tem sido um dos maiores desafios da medicina regenerativa. O conceito de que os cardiomiócitos proliferam ativamente durante o desenvolvimento, mas perdem completamente esse potencial logo após o nascimento, foi recentemente questionado quando as primeiras evidências mostraram a existência de mecanismos endógenos de regeneração cardíaca em camundongos com um dia de vida. Nós avaliamos esse fenômeno em ratos de um dia de vida (P1) e investigamos o impacto da regeneração inicial na perfusão tecidual em longo prazo e a função cardíaca global em resposta ao stress. A homogeneidade da cirurgia de ressecção apical foi comprovada através do exame de ressonância magnética (MRI) e demonstramos que os ratos P1 apresentaram evidências de neoformação de cardiomiócitos a partir da marcação de Troponina I e Conexina 43 na àrea da lesão 21 dias após a cirurgia de ressecção, enquanto os ratos de sete dias de idade (P7) apresentaram a substituição do tecido principalmente por deposição de colágeno. De maneira interessate, as células recém-formadas apresentaram uma aparente falta de alinhamento uniforme nos ratos P1, e a hipoperfusão do tecido cardíaco foi detectada para ambos os grupos de pós-ressecção 21 e aos 60 dias do exame de SPECT. A função cardíaca basal direta aos 60 dias apresentou-se preservada em todos os grupos, enquanto sob estresse hemodinâmico, o grau de mudança na LVDEP, Volume Sitólico e Trabalho Sistólico indicaram função cardíaca diminuída nos ratos P7. Além disso, a relação pressão-volume diastólica final e o aumento da deposição de colágeno intersticial no P7 são consistentes com o aumento da rigidez da câmara. Coletivamente, nós mostramos que o potencial regenerativo com ausência de remodelamento cardíaco adverso é restrito aos ratos P1. Em seguida, procurou-se avaliar os mecanismos moleculares que regulam esse fenômeno através da combinação de ferramentas exploratórias. Embora tenha sido descrito anteriormente que o sistema imunológico não é totalmente maduro ao nascimento, o sequenciamento do RNA total de corações de ratos sham-operados, P1 e P7 mostrou que o procedimento cirúrgico foi suficiente para ativar algumas vias ligadas à resposta inflamatória e considerando as subpopulações de macrófagos pró (M1) e anti-inflamatórios (M2), sugerimos que o perfil de macrófagos anti-inflamatórios (M2) infiltrados no coração de ratos P1 são diferentes das células adultas pró-fibróticas regulares. Os meios condicionados M1 e M2 elevaram a taxa de proliferação de cardiomiócitos em condições de normóxia, mas somente o M2 apresentou resposta proliferativa em hipóxia e preveniu a diferenciação-induzida de fibroblastos cardíacos por menor expressão ?SMA. Por membranas array de citocinas, 15 citocinas apresentaram-se comuns aos dois meios condicionados, mas apenas 4 citocinas, sendo elas IL-4, IL-1?, IL-6 e Fractalkine, foram exclusivas ao meio condicionado M2, e que poderiam ser possíveis candidatos aos efeitos regenerativos encontrados. Nesse sentido, experimentos futuros fazem-se necessários a fim de explorar os efeitos dessas citocinas e desenvolver novas estratégias terapêuticas<br>The replacement of cardiomyocytes in postnatal life has proven to be one of the biggest challenges in regenerative medicine. The concept that cardiomyocytes proliferate actively during development but cease completely right after birth has been recently questioned when first evidences showed the existence of endogenous mechanisms of cardiac regeneration in one-day-old mice. We sought to evaluate this phenomenon in one-day-old rats (P1) and to assess the impact of the early regenerative process on long-term tissue perfusion and overall cardiac function in response to stress. We confirmed the successful apical resection surgery through magnetic resonance imaging (MRI) and that P1 heart was associated with evidence of cardiomyocytes neoformation as indicated by Troponin I and Connexin 43 expression at 21 days postresection, while in seven-day-old rats (P7) mainly scar tissue replacement ensued. Interestingly, there was an apparent lack of uniform alignment of newly formed cells in P1, and cardiac tissue hypoperfusion has been detected for both groups at 21 postresection and at 60 days through SPECT scanning. Direct basal cardiac function at 60 days, was preserved in all groups, whereas under hemodynamic stress the degree of change on LVDEP, Stroke Volume and Stroke Work indicated diminished overall cardiac function in P7. Furthermore, the End-Diastolic Pressure-Volume relationship and increased interstitial collagen deposition in P7 is consistent with increased chamber stiffness. Collectively, we showed that regenerative potential with slight collagen deposition is restricted to P1 rats. Then we sought to evaluate the molecular mechanisms that regulate this phenomenon through explorative tools. Although it has been previously described that the immune system is not fully mature at birth, total RNA sequenced from sham-operated, P1 and P7 heart rats showed that surgery is sufficient to activate inflammatory pathways, and considering pro (M1) and anti-inflammatory (M2) macrophages subpopulations, we suggested that invaded macrophages in resected P1 hearts are different from the traditional pro-fibrotic M2-like adult cells. Conditioned M1 and M2 medium elevated cardiomyocytes proliferative rate under basal conditions, but only M2 produced the same effect in cardiomyocytes under hypoxia and prevented myofibroblasts-induced differentiation through ?SMA intensity expression. Membrane array for cytokines showed 15 common cytokines for both M1 and M2 conditioned medium, but only 4, as IL-4, IL-1?, IL-6 and Fractalkine, were M2 exclusive and possible candidates to the regenerative potential. Additional experiments are needed to further explore these cytokines and to maybe develop new therapeutic strategies

[ES] La Tomografía por Emisión de Positrones (PET) es una de las técnicas más importantes en la medicina de diagnóstico actual y la más representativa en el campo de la Imagen Molecular. Esta modalidad de imagen es capaz de producir información funcional única, que permite la visualización en detalle, cuantificación y conocimiento de una variedad de enfermedades y patologías. Áreas como la oncología, neurología o la cardiología, entre otras, se han beneficiado en gran medida de esta técnica. A pesar de que un elevado número de avances han ocurrido durante el desarrollo del PET, existen otros que son de gran interés para futuras investigaciones. Uno de los principales pilares actualmente en PET, tanto en investigación como en desarrollo, es la obtención de la información del tiempo de vuelo (TOF) de los rayos gamma detectados. Cuando esto ocurre, aumenta la sensibilidad efectiva del PET, mejorando la calidad señal-ruido de las imágenes. Sin embargo, la obtención precisa de la marca temporal de los rayos gamma es un reto que requiere, además de técnicas y métodos específicos, compromisos entre coste y rendimiento. Una de las características que siempre se ve afectada es la resolución espacial. Como discutiremos, la resolución espacial está directamente relacionada con el tipo de centellador y, por lo tanto, con el coste del sistema y su complejidad. En esta tesis, motivada por los conocidos beneficios en imagen clínica de una medida precisa del tiempo y de la posición de los rayos gamma, proponemos configuraciones de detectores TOF- PET novedosos capaces de proveer de ambas características. Sugerimos el uso de lo que se conoce como métodos de "light-sharing", tanto basado en cristales monolíticos como pixelados de tamaño diferente al del fotosensor. Estas propuestas hacen que la resolución espacial sea muy alta. Sin embargo, sus capacidades temporales han sido muy poco abordadas hasta ahora. En esta tesis, a través de varios artículos revisados, pretendemos mostrar los retos encontrados en esta dirección, proponer determinadas configuraciones y, además, indagar en los límites temporales de éstas. Hemos puesto un gran énfasis en estudiar y analizar las distribuciones de la luz centellante, así como su impacto en la determinación temporal. Hasta nuestro conocimiento, este es el primer trabajo en el que se estudia la relación de la determinación temporal y la distribución de luz de centelleo, en particular usando SiPM analógicos y ASICs. Esperamos que esta tesis motive y permita otros muchos trabajos orientados en nuevos diseños, útiles para instrumentación PET, así como referencia para otros trabajos. Esta tesis esta organizada como se describe a continuación. Hay una introducción compuesta por tres capítulos donde se resumen los conocimientos sobre imagen PET, y especialmente aquellos relacionados con la técnica TOF-PET. Algunos trabajos recientes, pero aún no publicados se muestran también, con el objetivo de corroborar ciertas ideas. En la segunda parte se incluyen las cuatro contribuciones que el candidato sugiere para el compendio de artículos.<br>[CA] La Tomografia per Emissió de Positrons (PET) és una de les tècniques més importants en la medicina de diagnòstic actual i la més representativa en el camp de la Imatge Molecular. Esta modalitat d'imatge és capaç de produir informació funcional única, que permet la visualització en detall, quantificació i coneixement d'una varietat de malalties i patologies. Àrees com l'oncologia, neurologia o la cardiologia, entre altres, s'han beneficiat en gran manera d'aquesta tècnica. Tot i que un elevat nombre d'avanços han ocorregut durant el desenvolupament del PET, hi ha altres que són de gran interés per a futures investigacions. Un dels principals pilars actuals en PET, tant en investigació com en desenvolupament, és l'obtenció de la informació del temps de vol (TOF en anglès) dels raigs gamma detectats. Quan açò ocorre, augmenta la sensibilitat efectiva del PET, millorant la qualitat senyal-soroll de les imatges. No obstant això, l'obtenció precisa de la marca temporal dels raigs gamma és un repte que requerix, a més de tècniques i mètodes específics, compromisos entre cost i rendiment. Una de les característiques que sempre es veu afectada és la resolució espacial. Com discutirem, la resolució espacial està directament relacionada amb el tipus de centellador, i per tant, amb el cost del sistema i la seua complexitat. En aquesta tesi, motivada pels coneguts beneficis en imatge clínica d'una mesura precisa del temps i de la posició dels raigs gamma, proposem nouves configuracions de detectors TOF-PET capaços de proveir d'ambduess característiques. Suggerim l'ús del que es coneix com a mètodes de "light-sharing", tant basat en cristalls monolítics com pixelats de diferent tamany del fotosensor. Aquestes propostes fan que la resolució espacial siga molt alta. No obstant això, les seues capacitats temporals han sigut molt poc abordades fins ara. En aquesta tesi, a través de diversos articles revisats, pretenem mostrar els reptes trobats en aquesta direcció, proposar determinades configuracions i, a més, indagar en els límits temporals d'aquestes. Hem posat un gran èmfasi a estudiar i analitzar les distribucions de la llum centellejant, així com el seu impacte en la determinació temporal. Fins al nostre coneixement, aquest és el primer treball en què s'estudia la relació de la determinació temporal i la distribució de llum de centelleig, en particular utilitzant SiPM analògics i ASICs. Esperem que aquesta tesi motive i permeta molts altres treballs orientats en nous dissenys, útils per a instrumentació PET, així com referència per a altres treballs. Aquesta tesi esta organitzada com es descriu a continuació. Hi ha una introducció composta per tres capítols on es resumeixen els coneixements sobre imatge PET i, especialmente, aquells relacionats amb la tècnica TOF-PET. Alguns treballs recents, però encara no publicats es mostren també, amb l'objectiu de corroborar certes idees. La segona part de la tesi conté els quatre articles revisats que el candidat suggereix.<br>[EN] Positron Emission Tomography (PET) is one of the greatest tools of modern diagnostic medicine and the most representative in the field of molecular imaging. This imaging modality, is capable of providing a unique type of functional information which permits a deep visualization, quantification and understanding of a variety of diseases and pathologies. Areas like oncology, neurology, or cardiology, among others, have been well benefited by this technique. Although numerous important advances have already been achieved in PET, some other individual aspects still seem to have a great potential for further investigation. One of the main trends in modern PET research and development, is based in the extrapolation of the Time- Of-Flight (TOF) information from the gamma-ray detectors. In such case, an increase in the effective sensitivity of PET is accomplished, resulting in an improved image signal-to-noise ratio. However, the direction towards a precise decoding of the photons time arrival is a challenging task that requires, besides specific approaches and techniques, tradeoffs between cost and performance. A performance characteristic very habitually compromised in TOF-PET detector configurations is the spatial resolution. As it will be discussed, this feature is directly related to the scintillation materials and types, and consequently, with system cost and complexity. In this thesis, motivated by the well-known benefits in clinical imaging of a precise time and spatial resolution, we propose novel TOF-PET detector configurations capable of inferring both characteristics. Our suggestions are based in light sharing approaches, either using monolithic detectors or crystal arrays with different pixel-to-photosensor sizes. These approaches, make it possible to reach a precise impact position determination. However, their TOF capabilities have not yet been explored in depth. In the present thesis, through a series of peer-reviewed publications we attempt to demonstrate the challenges encountered in these kinds of configurations, propose specific approaches improving their performance and eventually reveal their limits in terms of timing. High emphasis is given in analyzing and studying the scintillation light distributions and their impact to the timing determination. To the best of our knowledge, this is one of the first works in which such detailed study of the relation between light distribution and timing capabilities is carried out, especially when using analog SiPMs and ASICs. Hopefully, this thesis will motivate and enable many other novel design concepts, useful in PET instrumentation as well as it will serve as a helpful reference for similar attempts. The present PhD thesis is organized as follows. There is an introduction part composed by three detailed sections. We attempt to summarize here some of the knowledge related to PET imaging and especially with the technique of TOF-PET. Some very recent but still unpublished results are also presented and included in this part, aiming to support statements and theories. The second part of this thesis lists the four peer-reviewed papers that the candidate is including.<br>This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 695536). It has also been supported by the Spanish Ministerio de Economía, Industria y Competitividad under Grants No. FIS2014-62341-EXP and TEC2016-79884-C2-1-R. Efthymios Lamprou has also been supported by Generalitat Valenciana under grant agreement GRISOLIAP-2018-026.<br>Lamprou, E. (2021). Development and Performance Evaluation of High Resolution TOF-PET Detectors Suitable for Novel PET Scanners [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/162991<br>TESIS

Adolescent depression is a major public health problem, which is associated with educational problems, long-term psychiatric illness and suicide. One major source of stress during adolescence is peer rejection. In this thesis, I investigate the nature of the relationship between peer rejection and adolescent depression. In a review of longitudinal and experimental studies, I describe a bi-directional relationship between peer rejection and depressive symptoms. I then outline how genetic, cognitive and neural vulnerability may modify the effects of peer rejection on adolescent depression. Finally, I introduce five empirical chapters which test these hypotheses using different methodological approaches. The first study is a molecular genetic analysis of a sample of adolescents with and without a diagnosis of mood disorder. I report an interaction between diagnostic group, environmental stress (though not peer rejection specifically) and 5HTTLPR genotype on symptoms of anxiety, which supports the role of genetic factors in modifying the relationship between environmental stress and adolescent mood disorder. The second study is a behavioural study of negative attention biases in a typically developing sample of adolescents. I report a negative attention bias in adolescents with low (versus high) self-esteem. Although the data do not support a causal role for attention biases in adolescent depression, such biased cognitions could also moderate responses to peer rejection, maintaining affective symptoms. A final set of three fMRI datasets investigates how neural circuitry may influence depressed adolescents’ responses to peer rejection at three distinct stages: i) expectation of peer feedback, ii) the receipt of peer rejection, iii) emotion regulation of peer rejection. Data show distinct behavioural and neural differences between depressed patients and healthy controls during expectation and reappraisal of peer rejection, although heightened emotional reactivity immediately following the receipt of peer rejection did not differentiate behavioural or neural responses in adolescents with and without depression.

Die vorliegende Arbeit beschäftigt sich mit der Bildgebung magnetisch markierter Stammzellen im ZNS mittels Magnetresonanztomographie. Dazu wurden Stammzellen mit Eisenoxidnanopartikeln (VSOP, very small superparamagnetic iron-oxide particles) in vitro effizient und ohne zusätzliche Lipofektionsagenzien magnetisch markiert. Es zeigte sich keine wesentliche Beeinflussung der Vitalität, Proliferation und Differenzierungsfähigkeit sämtlicher untersuchter Zellpopulationen. Zur Evaluierung der Grenzen der zellulären MR-Bildgebung wurde das Detektionslimit magnetisch markierter embryonaler Stammzellen in vivo nach intrastriataler Injektion im Gehirn der Ratte untersucht. Es ließen sich bei einer Feldstärke von 17,6 T weniger als 100 magnetisch markierte Zellen sicher vom Hirnparenchym abgrenzen. Die histologische Korrelation bestätigte den zellulären Ursprung der beobachteten T2*-Hypointensitäten. In einem Rattenmodel des Morbus Parkinson konnte eine spezifische Detektion der intrastriatal injizierten magnetisch markierten embryonalen Stammzellen über einen Zeitraum von 6 Monaten erreicht werden. Es konnte keine signifikante Migration der Zellen festgestellt werden, jedoch fanden sich große interindividuelle Unterschiede in ihrer räumlichen Verteilung. In der histologische Analyse stellten sich auch sechs Monate nach der Transplantation im Bereich des Stichkanals eisenoxidmarkierte Stammzellen dar. In einem Mausmodell der cerebralen Ischämie wurde erstmals die Anreicherung systemisch injizierter magnetisch markierter mononukleärer Zellen kernspintomographisch erfasst. 24 - 48 h nach der Injektion magnetisch markierter Zellen stellten sich T2*-gewichtete Signalhypointensitäten im Randbereich der Ischämie dar. Insgesamt zeigte sich in dieser Studie die zelluläre Magnetresonanztomographie zu einem nicht-invasiven Nachweis einer geringen Anzahl magnetisch markierter Zellen über einen langen Zeitraum mit hoher Sensitivität in der Lage.<br>This thesis is dealing with the imaging of magnetically labeled stem cells in the CNS using magnetic resonance imaging (MRI). Stem cells were efficiently magnetically labeled with very small superparamagnetic iron-oxide particles (VSOP), without any lipofection agents. No significant impact on vitality, proliferation and ability to differentiate could be observed after the magnetic labeling of all cell populations investigated. Magnetically labeled embryonic stem cells were injected into the striatum of rats to evaluate their detection limit by MRI. At field strengths of 17.6 T, less than 100 cells could be discriminated from the brain parenchyma as T2*-weighted hypointensities. Histology proved the cellular origin of MRI-signal changes. In a rat model of Parkinsons’s Disease, magnetically labeled embryonic stem cells could be detected by MRI after intrastriatal injection for a time period of more than 6 months. No significant migration of transplanted cells could be observed, however significant inter-individual differences concerning the spatial distribution of cells could be found. Histologically, transplanted iron-oxide-labeled cells could still be detected in the vicinity of the injection tract six months after transplantation. In a mouse model of cerebral ischemia, the enrichment of systemically injected magnetically labeled mononuclear cells was detected non-invasively by MRI. 24 to 48 hours after injection of magnetically labeled cells, T2*-weighted hypointense signal changes could be observed in the border zone of the ischemia. Over all, this study showed that cellular MRI is capable of the sensitive non-invasive detection of small numbers of magnetically labeled cells over a long period of time.

Magnetic resonance imaging (MRI) can be used to image diffusion in liquids, such as water in brain structures. Molecular diffusion can be isotropic or anisotropic, depending on the fluid's environment, and can therefore be characterized by a scalar, D, or by a tensor, D, in the respective cases. For anisotropic environments, the eigenvector of D corresponding to the largest eigenvalue indicates the preferred direction of diffusion.<br>This thesis describes the design and implementation of diffusion tensor imaging on a clinical MRI system. An acquisition sequence was designed and post-processing software developed to create diffusion trace images, scalar anisotropy maps, and anisotropy vector maps. A number of practical imaging problems were addressed and solved, including optimization of sequence parameters, accounting for flow effects, and dealing with eddy currents, patient motion, and ghosting. Experimental validation of the sequence was performed by calculating the trace of the diffusion tensor measured in various isotropic liquids. The results agreed very well with the quantitative values found in the literature, and the scalar anisotropy index was also found to be correct in isotropic phantoms. Anisotropy maps, showing the preferred direction of diffusion, were generated in human brain in vivo. These showed the expected white matter tracts in the corpus callosum.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Nuclear Engineering, 2000.<br>Some research performed with the Harvard-M.I.T. Division of Health Sciences and Technology.<br>Includes bibliographical references (leaves 132-140).<br>Functional MRI (fMRI) detects the signal associated with neuronal activation, and has been widely used to map brain functions. Locations of neuronal activation are localized and distributed throughout the brain, however, conventional encoding methods based on k-space acquisition have limited spatial selectivity. To improve it, we propose an adaptive fMRI method using non-Fourier, spatially selective RF encoding. This method follows a strategy of zooming into the locations of activation by progressively eliminating the regions that do not show any apparent activation. In this thesis, the conceptual design and implementation of adaptive fMRI are pursued under the hypothesis that the method may provide a more efficient means to localize functional activities with increased spatial or temporal resolution. The difference between functional detection and mapping is defined, and the multi- resolution approach for functional detection is examined using theoretical models simulating variations in both in-plane and through-plane resolution. We justify the multi-resolution approach experimentally using BOLD CNR as a quantitative measure and compare results to those obtained using theoretical models. We conclude that there is an optimal spatial resolution to obtain maximum detection; when the resolution matches the size of the functional activation. We demonstrated on a conventional 1.5-Tesla system that RF encoding provides a simple means for monitoring irregularly distributed slices throughout the brain without encoding the whole volume. We also show the potential for increased signal-to-noise ratio with Hadamard encoding as well as reduction of the in-flow effect with unique design of excitation pulses.<br>(cont.) RF encoding was further applied in the implementation of real-time adaptive fMRI method, where we can zoom into the user-defined regions interactively. In order to do so, real-time pulse prescription and data processing capabilities were combined with RF encoding. Our specific implementation consisted of five scan stages tailored to identify the volume of interest, and to increase temporal resolution (from 7.2 to 3.2 seconds) and spatial resolution (from 10 mm to 2.5-mm slice thickness). We successfully demonstrated the principle of the multi- resolution adaptive fMRI method in volunteers performing simple sensorimotor paradigms for simultaneous activation of primary motor as well as cerebellar areas.<br>by Seung-Schik Yoo.<br>Ph.D.