La medicina regenerativa és una disciplina que ha guanyat reconeixement en les últimes dècades pel fet que moltes malalties no són tractables amb fàrmacs convencionals. Molts grups de recerca i empreses inverteixen temps i diners en la producció de nous paradigmes per curar malalties com el Parkinson, l'artrosi o la regeneració de medul·la espinal. Aquestes propostes es basen en l'ús de teixits biomimètics per reparar òrgans danyats. En aquesta tesi es presenta un nou model experimental per estudiar la formació d'os i cartílag i eventualment la reparació d'aquests teixits. Hem utilitzat Fibroblasts Embriònics de Ratolí (MEFs) combinats amb diferents materials biomimètics per estudiar os i cartílag in vitro i in vivo. Aquests MEFs es van cultivar in vitro i in vivo en RAD16-I, un pèptid auto-ensamblable amb estructura similar a matrius extracel·lulars genèriques, amb l'objectiu d'estudiar l'evolució dels fibroblasts en aquestes dues condicions. També s'han recobert superficialment micropartícules de hidroxiapatita obtenint càrregues inorgàniques similars a l'os i biològicament actives per a utilitzar-les com a substituts d'os o cartílag. Amb la idea de millorar els recobriments superficials, hem desenvolupat una plataforma que permet dur a terme proves combinatòries amb factors de creixement i altres compostos biològicament actius. Cultius in vitro de MEFs han mostrat que quan fibroblasts embrionaris primaris de ratolí es cultiven en RAD16-I, estableixen una xarxa intercel·lular que causa una contracció cel·lular organitzada, proliferació i migració cel·lulars i culmina amb la formació d'una estructura bilateral i simètrica amb un eix central distingible. Durant aquest procés morfològic, augmenta l'expressió d'un grup de gens mesodèrmics (brachyury, Sox9, Sox5, Sox6, Runx2). L'expressió de brachyury està localitzada primer en l'eix de simetria central i després s'extén als dos costats de l'estructura. Per acabar, la formació espontània d'un teixit similar al del cartílag acompanya l'expressió de Sox9 i Runx2.L'estudi in vivo de MEFs es va fer gràcies a la tècnica de presa d'imatges no invasiva basada en bioluminiscència (BLI) que ha desenvolupat en el grup de recerca del dr. Jerónimo Blanco. Aquests experiments han mostrat que el RAD16-I és una matriu molt permissiva per a la supervivència i proliferació cel·lulars in vivo. A més, sembla que les pobres propietats mecàniques que té el RAD16-I no li suposen cap desavantatge en termes de creixement cel·lular in vivo. Finalment, hem desenvolupat diferents tipus de micropartícules de hidroxiapatita (HA) no recobertes, i recobertes mitjançant polimerització assistida per plasma. Els recobriments permeten afinar les propietats de la HA i produir partícules que satisfacin les necessitats de diferents aplicacions mèdiques en reparació d'os i cartílag. També hem desenvolupat un mètode per produir plataformes basades en plaques de 96 pous que permetin fer proves combinatòries amb compostos biològicament actius per vàries aplicacions en medicina regenerativa. En conclusió, hem aportat noves idees i eines que permetran trobar teixits regeneratius basats en l'ús de fibroblasts i materials biomimètics. / La medicina regenerativa es una disciplina que ha ganado reconocimiento en las últimas décadas porque muchas enfermedades no son tratables con fármacos convencionales. Muchos grupos de investigación y empresas invierten tiempo y dinero en la producción de nuevos paradigmas para curar enfermedades como el Parkinson, la artrosis o la regeneración de médula espinal. Estas propuestas se basan en el uso de tejidos biomiméticos para reparar órganos dañados. En esta tesis se presenta un nuevo modelo experimental para estudiar la formación de hueso y cartílago y tal vez la reparación de estos tejidos. Hemos utilizado Fibroblastos Embrionarios de Ratón (MEFs) combinados con diferentes materiales biomiméticos para estudiar hueso y cartílago in vitro e in vivo. Estos MEFs se cultivaron in vitro e in vivo en RAD16-I, un péptido auto-ensamblable con estructura similar a matrices extracelulares genéricas, con el objetivo de estudiar la evolución de los fibroblastos en estas dos condiciones. También se han recubierto superficialmente micropartículas de hidroxiapatita obteniendo cargas inorgánicas similares al hueso y biológicamente activas para utilizarlas como sustitutos de hueso o cartílago. Con la idea de mejorar los recubrimientos superficiales, hemos desarrollado una plataforma que permite llevar a cabo pruebas combinatorias con factores de crecimiento y otros compuestos biológicamente activos. Cultivos in vitro de MEFs han mostrado que cuando fibroblastos embrionarios primarios de ratón se cultivan en RAD16-I, establecen una red intercelular que causa una contracción celular organizada, proliferación y migración celulares y culmina con la formación de una estructura bilateral y simétrica con un eje central distinguible. Durante este proceso morfológico, aumenta la expresión de un grupo de genes mesodérmicos (brachyury, Sox9, Sox5, Sox6, Runx2). La expresión de brachyury está localizada primero en el eje de simetría central y después se extiende a los dos lados de la estructura. Para terminar, la formación espontánea de un tejido similar al del cartílago acompaña a la expresión de Sox9 y Runx2.El estudio in vivo de MEFs se hizo gracias a la técnica de toma de imágenes no invasiva basada en bioluminiscencia (BLI) que han desarrollado en el grupo de investigación del dr. Jerónimo Blanco. Estos experimentos han mostrado que el RAD16-I es una matriz muy permisiva para a la supervivencia y proliferación celulares in vivo. Además, parece que las pobres propiedades mecánicas que tiene el RAD16-I no le suponen ninguna desventaja en términos de crecimiento celular in vivo. Finalmente, hemos desarrollado diferentes tipos de micropartículas de hidroxiapatita (HA) no recubiertas, y recubiertas mediante polimerización asistida por plasma. Los recubrimientos permiten afinar las propiedades de la HA y producir partículas que satisfagan las necesidades de diferentes aplicaciones médicas en reparación de hueso y cartílago. También hemos desarrollado un método para producir plataformas basadas en placas de 96 pozos que permitan hacer pruebas combinatorias con compuestos biológicamente activos para varias aplicaciones en medicina regenerativa. En conclusión, hemos aportado nuevas ideas y herramientas que permitirán hallar tejidos regenerativos basados en el uso de fibroblastos y materiales biomiméticos. / Regenerative medicine is a discipline that has gained recognition in the last decades because many diseases are not treatable with traditional drugs. Many research groups and companies invest time and money in the production of new paradigms to cure conditions such as Parkinson's, arthrosis or spinal cord injuries. These approaches are based in the use of biomimetic tissues to replace damaged organs. In this work we present a new experimental model to study the formation of bone and cartilage and eventually to repair these tissues. We have used Mouse Embryonic Fibroblasts (MEFs) combined with different biomimetic materials to study bone and cartilage formation in vitro and in vivo. MEFs have been cultured in vitro and in vivo in RAD16-I, a synthetic self-assembling peptide with structure similar to generic extracellular matrix milieu, to study the evolution of these fibroblasts in both conditions. Also, hydroxyapatite microparticles have been surface coated to produce biologically active bone-like inorganic charges for use in cartilage or bone substitutes. In order to improve the particles' coatings, we have developed a platform that allows us to perform combinatorial testing of growth factors and other biologically active compounds. In vitro cultures of MEFs has shown that when primary mouse embryonic fibroblasts are cultured in a soft nanofiber scaffold, they establish a cellular network that causes an organized cell contraction, proliferation, and migration that ends in the formation of a symmetrically bilateral structure with a distinct central axis. A subset of mesodermal genes (brachyury, Sox9, Sox5, Sox6, Runx2) is upregulated during this morphogenetic process. The expression of brachyury was localized first at the central axis, extending then to both sides of the structure. The spontaneous formation of cartilage-like tissue mainly at the paraxial zone followed the expression of Sox9 and Runx2.In vivo study of MEFs was facilitated by a non-invasive bioluminescence imaging (BLI) technique to detect luciferase-expressing cells, developed by Dr. Blanco's research group. These experiments showed that RAD16-I is a very permissive scaffold for cell survival and proliferation in vivo. Furthermore, it seems that the poor mechanical properties of RAD16-I are no disadvantage in terms of cell growth in vivo.Finally, we have developed different types of coated and uncoated hydroxyapatite (HA) microparticles by plasma polymerization. The coatings permit to tune the properties of HA and produce particles that suit the needs of different medical applications in bone and cartilage repair. Moreover, we have developed a method to produce platforms based on 96-well plates that allow the combinatorial testing of biologically active compounds for various applications in regenerative medicine. In conclusion, we have supplied new insights and tools that will enhance the finding of new regenerative tissues based on fibroblasts and biomimetic materials.

No description available.

Le pronostic de certains cancers s’est considérablement amélioré avec l’arrivée sur le marché des anticorps thérapeutiques. Devant l’essor de ces nouveaux médicaments associé à l’identification de nouveaux biomarqueurs, de nouvelles perspectives émergent pour l’imagerie moléculaire in vivo. En effet, disposer de nouveaux traceurs moléculaires spécifiques de ces biomarqueurs permettra de caractériser l’hétérogénéité des cellules cancéreuses, de suivre l’expression de ces marqueurs au cours de l’évolution de la tumeur, mais également de suivre l’efficacité d’un traitement sur la régression tumorale du patient. Pour répondre à cette évolution de diagnostic moléculaire in vivo, il convient de développer de nouvelles sondes moléculaires. L'objectif de ma thèse répond à ce nouveau besoin avec l'ingénierie et le marquage d'un format d'anticorps recombinant adapté à l'imagerie in vivo : le diabody 12G4 dirigé contre le récepteur de l’hormone antimüllérienne (AMH), marqueur de certains cancers de la sphère génitale. / Prognosis of cancers dramatically improved with the development on the market of therapeutic antibodies. With the increase of these new biodrugs, associated with the identification of new biomarkers, new opportunities emerge for the in vivo molecular imaging.. Indeed, to use new molecular tracers specific of tumoral biomarkers will allow to study and characterize the cancer heterogeneity, to monitor the expression of these markers during the tumor evolution, but also to check the treatment effectiveness on patients’ tumoral regression. To answer this evolution of in vivo molecular diagnosis, it is favorable to develop new molecular probes. The aim of this thesis answers this new need with the engineering and labeling of a new recombinant antibody format suitable to in vivo imaging : the 12G4 diabody directed against the type II human receptor for the anti-Müllerian hormone, a biomarker of some cancers of the genital area.

En vue de contribuer à une meilleure compréhension des atteintes du système musculo-squelettique consécutives à une infection par un arthropod-borne-virus (arbovirus), deux arbovirus ont été étudiés : le virus du chikungunya (CHIKV) et le virus Zika (ZIKV), respectivement de la famille des Togaviridae et des Flaviviridae. Cette étude a été réalisée selon deux axes. Le premier s’intéresse aux atteintes articulaires consécutives à l’infection par le CHIKV. Nous avons mis au point un modèle d’imagerie in vivo reposant sur l’utilisation d’un virus recombinant exprimant la NanoLuciférase. Dans ce modèle, nous démontrons une persistance du signal bioluminescent dans les articulations 34 jours après infection. Par isolement des cartilages articulaires et des cellules constitutives, nous avons pu démontrer que les chondrocytes des cartilages métatarso-phalangiens sont infectés par le CHIKV de manière persistante, suggérant un rôle de réservoir de ces cellules. Les conséquences de l’infection au niveau cellulaire ont ensuite été étudiées in vitro. En utilisant des chondrocytes primaires humains, nous avons confirmé ces observations. De plus, les chondrocytes infectés produisent de nombreuses cytokines, induisant une stimulation du catabolisme du cartilage avec en particulier la synthèse de métalloprotéinases de matrice 3 et 9. De plus, l’infection par le CHIKV provoque la mort des cellules par apoptose, comme démontré par marquage TUNEL et par mesure de l’activité des caspases. Nous avons ensuite étudié les atteintes musculaires et le tropisme cellulaire du ZIKV. Dans un modèle murin, nous avons confirmé la présence de lésions musculaires, et l’utilisation de cellules musculaires primaires humaines a montré la sensibilité des myoblastes à l’infection, les myotubes étant résistants, suggérant un tropisme du ZIKV dépendant de la différenciation cellulaire. Trois souches virales ont été testées, sans relever de différences significatives en termes de cinétique d’infection, de nombre de cellules infectées et de production virale. L’infection des myoblastes entraîne la mort de ces cellules par un mécanisme caspase-indépendant. Des observations en microscopie électronique ont mis en évidence une vacuolisation du cytoplasme suite à l’infection, caractéristique d’une mort cellulaire par paraptose. Une analyse protéomique a démontré que l’infection des myoblastes par une souche asiatique conduit à une modification du protéome s’accentuant entre 24 heures et 48 heures post-infection, avec 225 protéines modulées 24 heures après infection contre 473 après 48 heures, indiquant une activation des voies de synthèse Interferon de type I et l’inhibition des capacités de synthèse des protéines. Ces résultats permettent une meilleure compréhension des atteintes du système-musculo-squelettique par les arbovirus / Musculoskeletal lesions caused by arthropod-borne-viruses (arboviruses) are invalidating for patients and remain poorly understood. In this study, we investigated the development of these manifestations after infection with two arboviruses: chikungunya virus (CHIKV) from the Togaviridae family, and Zika virus (ZIKV) from the Flaviviridae family. The first part of the study focused on arthritis following CHIKV infection. For this purpose, we developed a reporter virus expressing NanoLuciferase and performed experimental infections in a murine model. In vivo, a strong bioluminescent signal indicated viral replication and we observed persistence of the signal in the joints up to 34 days post-infection. By isolating primary murine cells from cartilage, we demonstrated the susceptibility of chondrocytes to CHIKV infection suggesting a role of reservoir of these cells. Furthermore, we investigated the consequences of the infection using in vitro models. We showed that primary human chondrocytes are also susceptible to CHIKV infection, which leads to the production of several cytokines involved in cartilage catabolism and induces apoptosis. In the second part of the study, we studied ZIKV muscular tropism and the associated lesions. Firstly, we confirmed the development of muscular lesions in a mouse model of ZIKA. Then, using human primary muscle cells we observed the infection of myoblasts but not myotubes, suggesting a differentiation-dependent tropism. We compared three viral strains and observed no significant difference in terms of replication, number of infected cells and viral production. Myoblasts infection induced a caspase-independent cells death mechanism and electronic microscope observations revealed intense vacuolization of cytoplasm, suggesting a paraptosis-like cell death. Proteomic analysis revealed that the Asian ZIKV strain modulated protein expression of infected cells with an increased effect after 48 hours. ZIKV-infection induced an important upregulation of biological processes associated with type I interferon and an inhibition of protein synthesis in the infected cells. These results provide valuable information about the mechanisms involved in the development of musculoskeletal lesions during arboviroses

Non-invasive imaging techniques, such as micro-CT or optical imaging, provide valuable information about tumour microstructure, size, volume and growth dynamics. Although histology is an approach capable of describing several of these characteristics, the invasiveness of this analysis remains a disadvantage. The main aim of this work is the methodological development of non-invasive imaging of the dynamics of tumour growth and progression. The preparation of a dual-reporter lentiviral vector enables non-invasive study of tumour growth and dissemination of metastasis. The same dual reporter will also be a part of a second vector designed as a construct for targeting mouse embryonic stem cells with aim to produce corresponding transgenic reporter mouse line. This reporter mouse line can be beneficial for future projects by providing a novel approach for studying the dynamics of tumour growth under various genetic conditions. In addition to optical imaging, this project will also include the use of micro-CT technology which, as a non-invasive approach, has the potential to provide information about the microstructure of tumour tissue in 3D that histology is not able to report.

Cette thèse concerne le développement de nanocristaux (NCs) cœur/coquille d'InP/ZnS émettant dans le proche infrarouge pour l'imagerie biologique. Dans la synthèse chimique des NCs cœur d'InP, nous avons utilisé la phosphine générée in situ comme précurseur de phosphore en combinaison avec le myristate d'indium comme précuseur d'indium et l'1-octadécène comme solvant. Les NCs obtenus sont hautement cristallins et présentent une fluorescence dans la gamme 720-750 nm, selon leur taille. La croissance d'une ou deux monocouches (coquille) de ZnS sur la surface des NCs d'InP a considérablement amélioré leur rendement quantique de fluorescence. Nous avons de plus étudié le transfert de phase de ces NCs InP/ZnS du milieu organique au milieu aqueux en utilisant diverses molécules hydrophiles contenant un groupe thiol. En particulier, nous nous sommes intéressés au transfert de phase avec des molécules zwitterioniques tels que la penicillamine et la cystéine afin d'obtenir une taille hydrodynamique compacte, et de réduire les interactions non-spécifiques en milieu biologique. Dans l'étude du transfert de phase, l'accent a été mis sur la stabilité colloïdale des NCs et sur la préservation de leur efficacité de fluorescence en milieu aqueux. La cytotoxicité des NCs InP/ZnS fonctionnalisés avec la pencillamine a été évaluée en culture cellulaire. Puis la bio-distribution de ces NCs a été étudiée dans des souris vivantes par imagerie de fluorescence grâce à leur émission dans le proche infrarouge. Pour finir, les fonctionnalisations de NCs InP/ZnS d'une part avec un peptide de pénétration cellulaire, d'autre part avec des agents de contraste IRM (complexes de gadolinium) et enfin avec un nombre contrôlé de molécules streptavidine ont été explorées, démontrant le grand intérêt de ces NCs pour l'imagerie biologique. Mots clés: phosphure d'indium, boîtes quantiques, nanocristaux, imagerie biologique de fluorescence, infrarouge, transfert de phase, fonctionnalisation de surface / This thesis concerns the development of near infrared (NIR) emitting InP/ZnS core/shell nanocrystals for biological imaging. In situ generated phosphine gas was used as the phosphorous precursor, indium myristate as the In precursor and 1-Ocadecene as the solvent to produce InP NCs with emission in the range of 720-750 nm. Growth of 1-2 monolayers of a ZnS shell on the surface of the InP NCs strongly improves their quantum yield. Next, we studied the phase transfer of the obtained InP/ZnS NCs to aqueous medium with various thiol group containing ligands. Emphasis is put on the colloidal stability and the retention of fluorescence quantum efficiency during the transfer. Zwitterionic ligands such as pencillaime and cysteine have been studied in view of their biological interest in providing compact size and reduced non-specific interaction with cells. The cytotoxicity of pencillamine capped InP/ZnS NCs has been evaluated in cell culture. The NIR emitting properties of the QDs have been exploited to their study bio-distribution in mice by fluorescence imaging. In addition, functionalisation of the InP/ZnS NCs with a cell penetrating peptide, with a MRI contrast agent (gadolinium complex) and with a controlled number of streptavidin molecules have been explored to demonstrate the large interest of InP/ZnS NCs in biology. Key words: Indium phosphide, quantum dots, near infrared fluorescence imaging, phase transfer, surface functionalisation

Micro- and nanorobots (MNRs) are small autonomous devices capable of performing complex tasks and have been demonstrated for a variety of non-invasive biomedical applications, such as tissue engineering, drug delivery or assisted fertilization. However, translating such approaches to an in vivo environment is critical. Current imaging techniques do not allow localization and tracking of single or few micromotors at high spatiotemporal resolution in deep tissue. This thesis addresses some of these limitations, by exploring the use of two optical-based techniques (IR and photoacoustic imaging (PAI)) and a combination of both US and PAI. First, we employ an IR imaging setup to visualize mobile reflective micromotors under scattering phantoms and ex vivo mouse skull tissues, without using any labels. The reflective micromotor reflects more than tenfold the light intensity of a simple particle. However, the achieved penetration depth was ca. 100 μm (when using ex vivo tissues), limiting this technique to superficial biomedical applications. In this regard, PAI plays a role that combines the advantages of US such as penetration depth and real-time imaging with the molecular specificity of optics. For the first time, in this thesis, this method is evaluated for dynamic process monitoring, in particular for tracking single micromotor in real-time below ~1 cm deep phantom and ex vivo tissue. However, the precise function control of MNRs in living organisms, demand the combination of both anatomical and functional imaging methods. Therefore, in the end, we report the use of a hybrid US and PA system for the real-time tracking of magnetically driven micromotors (single and swarms) in phantoms, ex vivo, and in vivo (in mice bladder and uterus), envisioning their application for targeted drug-delivery. This achievement is of great importance and opens the possibilities to employ medical micromotors in a living organism and perform a medical task while being externally controlled and monitored.:ABSTRACT 1 1 INTRODUCTION 5 1.1 Motivation 5 1.2 Background 7 1.2.1 Microrobotics 7 1.2.2 Medical Imaging 9 1.3 Objectives and Structure of Thesis 12 2 FUNDAMENTALS 15 2.1 Optical Imaging 15 2.1.1. Reflection-based Imaging 17 2.1.2. Fluorescence-based Imaging 18 2.1.1 Light-Tissue Interaction 20 2.2 Photoacoustic Imaging 23 2.2.1 Theory 23 2.2.2 Implementation 25 2.3 Ultrasound Imaging 26 2.3.1 Theory 26 2.3.2 Implementation 28 3 MATERIALS AND METHODS 30 3.1 Fabrication of Magnetic Micropropellers 30 3.1.1 3D Laser Lithography of Polymeric Resin 30 3.1.2 Self-assembly of SiO2 Particles 31 3.1.3 Electron Beam Evaporation 32 3.1.4 Surface Functionalization 33 3.2 Fabrication of Phantom Tissue and Microfluidic Channels 34 3.2.1 Fabrication of PDMS-Glycerol Phantom 34 3.2.2 Fabrication of Agarose Phantom 35 3.2.3 Phantom based on Ex vivo Tissues (Chicken Breast and Mice Skull) 36 3.2.4 Microfluidic Channel Platform 37 3.3 Sample Characterization 38 3.3.1 Optical Microscopy 38 3.3.2 Scanning Electron Microscopy 38 3.4 Magnetic Actuation 39 3.4.1 Magnetic Force 39 3.4.2 Magnetic Torque 39 3.5 Ethic Statement for Mice Experiments 41 4 OPTICAL IMAGING OF MICROROBOTS 42 4.1 Concept of Reflective Micromotors 42 4.2 Fabrication of Reflective Micromotors 44 4.3 IR Imaging Actuation Setup 45 4.4 Actuation and Propulsion Performance below Phantom 47 4.5 Actuation and Propulsion Performance below Ex Vivo Skull Tissue 50 4.6 Actuation and Propulsion Performance in Blood 51 5 PHOTOACOUSTIC IMAGING OF MICROROBOTS 55 5.1 Absorbers for Deep Tissue Imaging 55 5.2 Absorber Micromotor Design and Fabrication 56 5.3 Photoacoustic Imaging Setup 58 5.4 Actuation Performance below Phantom Tissue 60 5.5 Actuation Performance below Ex Vivo Tissue 65 6 HYBRID ULTRASOUND AND PHOTOACOUSTIC IMAGING 67 6.1 Hybrid Ultrasound/Photoacoustic System 68 6.2 Fabrication and Characterization of Micromotors 69 6.3 Actuation and Propulsion Performance below Phantom 69 6.4 Actuation and Propulsion Performance below Ex Vivo Tissues 71 6.5 Actuation and Propulsion Performance in Mice 72 6.5.1 Swimming of Micromotors in Bladder 72 6.5.2 Actuation of Micromotors in Uterus 74 6.5.3 3D Multispectral Imaging 76 6.5.4 Towards Targeted Drug Delivery 77 7 SUMMARY AND PERSPECTIVES 80 7.1 Summary 80 7.2 Future Perspectives 83 7.2.1 Contrats Enhancing Labels 84 7.2.2 Novel Imaging Concepts 85 8 REFERENCES 88 9 APPENDIX 105 List of Figures 105 List of Tables 107 Abbreviations 108 List of Publications 109 Acknowledgements 110 Selbstständigkeitserklärung 111 Curriculum Vitae 112

Microglia and brain-resident macrophages are the sentinel immune cells of the central nervous system (CNS), and are ideally situated to respond to any damage to the brain parenchyma or vasculature. Circulating leukocytes are generally excluded from the CNS environment under homeostatic conditions but can gain access to this region in diseases that disrupt immune system function and blood-brain barrier integrity. Although these diverse immune cells exhibit properties that may engender them to be well-suited to resolve microcirculatory insults, their relative contributions to the recanalization of capillary rupture in the cortex, known as cerebral microbleeds (CMBs), has yet to be described. CMBs are particularly concerning in conditions, such as diabetes mellitus (DM), in which these insults occur more frequently and potentially underlie the onset and progression of cognitive decline. Using in vivo 2-photon microscopy and confocal imaging, here I highlight the compromised repair of CMBs in a mouse model of type 1 DM and characterize the robust, heterogeneous macrophage response to these insults. Specifically, 20% of damaged capillaries were eliminated from the circulation in the diabetic cortex and chronic insulin treatment failed to prevent this microvascular loss. Administration of interferon-α or interferon-γ neutralizing antibodies to dampen inflammatory signalling, or dexamethasone to reduce global inflammation, also failed to improve repair rates of damaged microvessels in diabetic mice. In contrast, CMBs in nondiabetic mice repaired without exception. Interestingly, depletion of CNS macrophages using the colony stimulating factor-1 receptor antagonist PLX5622 resulted in microvascular elimination in nondiabetic mice. Given the robust depletion of brain macrophage populations with this treatment, at first these data suggested that these cells were necessary for microvascular repair since their elimination produced vessel loss. However, by parsing the data I identified that microvessels repaired in all cases where macrophages were not identified at the CMB; when CX3CR1+ aggregate was localized to the injury, ~20% of microvessels were eliminated. These findings show that CNS macrophages are not required for microvascular repair following CMB. Immunofluorescent co-labelling of various microglial and macrophage markers within the diabetic CMB milieu revealed a novel population of Mac2+/TMEM119- cells, distinct from homeostatic TMEM119+ microglia. These cells reliably localized to CMBs that failed to repair and rarely associated with vessels that recanalized; Mac2+/TMEM119- cells were not found within nondiabetic CMBs. Treatment of diabetic mice with clodronate liposomes (CLR) to deplete circulating phagocytic leukocytes prevented aggregation of Mac2+/TMEM119- cells to CMBs and improved capillary repair rates. The efficacy of CLR in excluding these cells from the CMB aggregate, coincident with eradication of monocytes from circulation, indicated that these cells likely arose from the periphery. In vivo 2-photon imaging revealed significant increases in lipofuscin at the site of diabetic CMBs relative to the nondiabetic context; other phagocytic markers including CD68 and TREM2 were also upregulated. Mac2+/TMEM119- cells showed elevated lipofuscin content relative to homeostatic microglia; their association with CMBs may thus signal an increase in phagocytosis that contributes to capillary pruning. Taken together, these data identify a novel Mac2+/TMEM119- macrophage associated with pathological microvascular elimination following CMB in the diabetic neocortex. These findings highlight the diversity of immune cell responses to CNS injury and provide insights into the cellular mechanisms of capillary pruning. Furthermore, these advances in our understanding of the regulation of microvascular elimination in the diabetic brain may have clinical implications for patients with DM as they provide evidence for putative adjuvant anti-inflammatory treatments, such as CLR, in mitigating cerebrovascular pathology. / Graduate / 2022-05-06

No description available.

Prostate cancer is one of the most common human malignancies and, consequently it is critical to develop appropriate diagnostic and therapeutic tools. Glutamate carboxypeptidase II (GCPII) is currently being considered one of the most important prostate cancer markers due to its tissue- specific expression. Whereas in healthy prostatic tissue the expression levels of GCPII are low, the transformation into the tumor is associated with the substantial increase of GCPII expression, with the highest levels observed in androgen-independent metastatic tumors. GCPII is thus considered a promising marker for early phase as well as advanced metastatic stages of prostate cancer. Current research is focused on the development of highly sensitive and specific reagents that allow detection of small amounts of GCPII, for example in early stages of cancer. Antibody derivatives are promising molecules for this purpose because they have high affinity and specificity and minimum negative side effects. Protein engineering is a prefered approach for preparation of various antibody molecules that differ in size, binding properties, stability, solubility, and production means. Different types of derivatives are being developed for medical needs such as in vitro diagnosis, therapy, and in vivo imagingSmall molecular...