Melanoma is a pigmented tumor that originates from the melanocytes; pigmented cells present throughout the body, including skin and iris. The cutaneous form is the most common type, and it represents about 5% of the skin tumors diagnosed in Brazil. Although it does not have a high incidence, it represents about 80% to 85% of all skin tumor deaths. The second most frequent type of melanoma is ocular. It represents 5% of all melanoma cases and is a potentially lethal disease, especially when it causes metastasis. The main therapeutic approach for melanomas, in general, is surgery, with resection of the cutaneous lesion or enucleation in the case of ocular melanoma. Other techniques such as adjuvant immunotherapy, palliative chemotherapy, and radiotherapy are also used. However, they have low efficacy and several side effects. Photodynamic therapy is a therapeutic modality based on the interaction of light at specific wavelength and photosensitizer, in the presence of molecular oxygen, leading the cell to death. As melanoma is a pigmented cancer, it usually does not respond well to photodynamic therapy due to the high absorption of light on the surface of the tumor, making volumetric eradication impossible. This project investigated optical strategies for the diagnosis and treatment of melanoma. For the diagnosis, it was evaluated the fluorescence lifetime technique to differentiate melanoma and normal skin. A sensitivity of 99.4%, specificity of 97.4% and accuracy of 98.4% were achieved using linear discrimination analysis. For the cutaneous melanoma treatment, PDT combined to optical clearing agents (OCAs) was investigated. Vascular and cell-target photosensitizers were evaluated combined or not to OCAs. OCA improved PDT response in all pigmented tumors treated, but the best results were achieved when a dual-photosensitizer treatment combined to OCA was performed. The treatment of conjunctival melanoma was conducted using 2-photon excitation photodynamic therapy. The advantage of this technique is the use of infrared light, in a wavelength that melanin has a low absorption, improving the light penetration into the tumor. The tumor histology shows that apoptosis was induced only at the treatment site, with no damage to the surrounding tissue. Additionally, a single TPE-PDT session could treat the entire tumor. / O melanoma é um tumor pigmentado que surge dos melanócitos, células pigmentadas presentes em todo o corpo, incluindo a pele e a íris. A forma cutânea é a mais comum e representa cerca de 5% dos tumores cutâneos diagnosticados no Brasil. Embora não tenha uma alta incidência, representa cerca de 80% a 85% de todas as mortes por tumor de pele. O segundo tipo de melanoma mais frequente é o ocular. Representa 5% de todos os casos de melanoma e é uma doença potencialmente letal, especialmente em casos de metástase. A principal abordagem terapêutica para melanomas, em geral, é a cirurgia, com ressecção da lesão cutânea ou enucleação no caso do melanoma ocular. Outras técnicas, como imunoterapia adjuvante, quimioterapia paliativa e radioterapia também são usadas, porém, apresentam baixa eficiência e muitos efeitos colaterais. A terapia fotodinâmica é uma modalidade terapêutica baseada na interação da luz em um comprimento de onda específico e um fotossensibilizador, na presença de oxigênio molecular, levando a célula à morte. Como o melanoma é um câncer pigmentado, geralmente não responde bem à terapia fotodinâmica devido à alta absorção de luz na superfície do tumor, impossibilitando a erradicação volumétrica. Este projeto investigou estratégias ópticas para o diagnóstico e tratamento do melanoma. Para o diagnóstico, foi avaliada a técnica de tempo de vida de fluorescência para distinguir melanoma de pele normal. Utilizando análise de discriminação linear, obteve-se uma sensibilidade de 99,4%, especificidade de 97,4% e precisão de 98,4%. Para o tratamento de melanoma cutâneo, a PDT combinada com clareadores ópticos (OCAs) foi investigada. Um fotossensibilizador que tem como alvo vaso sanguíneo e um fotossensibilizador de alvo celular foram avaliados combinados ou não com OCAs. OCAs são soluções hiperosmóticas que desidratam o tecido, diminuindo o espalhamento da luz e melhorando a penetração de luz em profundidade. OCA melhorou a resposta de PDT em todos os tumores melanóticos tratados, mas os melhores resultados foram obtidos quando a PDT foi realizada com a combinação dos fotossensibilizadores e clareador óptico em uma única sessão. O tratamento do melanoma conjuntival foi realizado utilizando a terapia fotodinâmica por excitação de 2 fótons (TPE-PDT). A vantagem desta técnica é o uso de luz na região do infravermelho, em um comprimento de onda que melanina tem baixa absorção, melhorando a penetração de luz no tumor. A histologia do tumor mostrou que a apoptose foi induzida apenas no local do tratamento, sem danos no tecido adjacente. Além disso, uma única sessão de TPE-PDT foi capaz de tratar todo o tumor.

Les champs récepteurs corticaux se réorganisent en réponse aux changements de l'environnement. Par exemple, suite à une lésion périphérique, les modalités sensorielles préservées gagnent de l'espace cortical au détriment de celles lésées. L'étude du cortex somatosensoriel en tonneau des rongeurs a fourni des données importantes pour la compréhension des mécanismes synaptiques à l'origine de cette réorganisation corticale. En condition normale, les neurones de chaque colonne corticale répondent préférentiellement à la stimulation d'une seule vibrisse principale ("Principal Whisker, PW"). Au contraire, suite à l'amputation de l'ensemble des vibrisses sauf une ("Single Whisker Experience, SWE"), les neurones des colonnes associées aux vibrisses amputées répondent à la stimulation de la vibrisse conservée, à l'origine du renforcement et de l'expansion des représentations corticales des vibrisses conservées. Bien que des preuves indirectes aient révélées un rôle de la potentialisation à long terme ("Long-Term Potentiation, LTP") de synapses préexistantes dans la modification des cartes corticales, probablement via une augmentation du nombre des récepteurs AMPA (AMPARs) aux synapses, un lien direct entre la LTP, la réorganisation des cartes corticales, et l'adaptation des comportements sensori-moteurs suite à une altération des entrées sensorielles n'a pas encore été démontré. L'objectif de cette thèse a donc été de mettre en évidence cette relation de façon expérimentale et en condition physiologique. Pour cela, nous avons mis au point une stratégie in vivo combinant des enregistrements électrophysiologiques, de l'imagerie biphotonique et l'analyse du comportement d'exploration chez la souris contrôle ("Full Whisker Experience, FWE) et amputée de certaines vibrisses (SWE). Nous avons d'abord confirmé que la stimulation rythmique de la PW ("Rhytmic Whisker Swtimulation, RWS") renforce les synapses excitatrices (RWS-LTP) in vivo des souris anesthésiées FWE. Au contraire des souris FWE, les neurones pyramidaux des souris SWE présentent une augmentation de l'excitabilité neuronale et une absence de RWS-LTP, indiquant ainsi que les synapses corticales associées à la vibrisse intacte ont été potentialisées en réponse au protocole SWE. Pour mieux comprendre l'implication de la RWS-LTP dans la réorganisation des cartes corticales et l'adaptation des comportements sensori-moteurs, nous avons développé une nouvelle approche pour manipuler la LTP in vivo grâce à l'immobilisation des AMPARs par des anticorps extracellulaires ("cross-linking"). En effet, notre équipe a montré précédemment que le cross-linking des AMPARs empêche la LTP in vitro. Par ailleurs, une accumulation des AMPARs au niveau post-synaptique a été démontrée in vivo par imagerie biphotonique au cours d'une stimulation RWS, suggérant un rôle de la mobilité de ces récepteurs dans cette RWS-LTP. Au cours de cette thèse, nous avons démontré que le cross-linking des AMPARs in vivo bloque également l'expression de la RWS-LTP, mais sans affecter la transmission synaptique basale, ni l'induction de la RWS-LTP, indiquant ainsi que la mobilité des AMPARs est également fondamental pour l'expression de la LTP in vivo. De façon importante, le cross-linking des AMPARs de façon chronique, au cours du SWE, permet non seulement de rétablir la RWS-LTP et l'excitabilité neuronale, et donc de bloquer la réorganisation corticale, mais aussi de modifier les capacités de récupération sensori-motrices des souris amputées. Dans l'ensemble, nos données démontrent pour la première fois un rôle critique et direct de la RWS-LTP dans le réarrangement des circuits en réponse à l'amputation de certaines vibrisses. La réorganisation des cartes corticales serait ainsi assurée par le renforcement de la transmission synaptique, et constituerait alors un mécanisme compensatoire pour optimiser le comportement sensorimoteur de l'animal lors de l'altération des entrées sensorielles. / Neuronal receptive fields in the cerebral cortex change in response to peripheral injury, with active modalities gaining cortical space at the expense of less active ones. Experiments on the mouse whisker-to-barrel cortex system provided important evidences about the synaptic mechanisms driving this cortical remapping. Under normal conditions, neurons in each barrel-column have receptive fields that are strongly tuned towards one principal whisker (PW). However, trimming all the whiskers except one (single-whisker experience, SWE) causes layer (L) 2/3 pyramidal neurons located in the deprived and spared-related columns to increase their response towards the spared input. This results in a strengthening and expansion of the spared whisker representation within the barrel sensory map. Indirect evidences suggest that these cortical alterations might depend on the activity-dependent potentiation of pre-existing excitatory synapses (LTP), likely through increased levels of postsynaptic AMPA receptors (AMPARs). However, a clear link between LTP, cortical remapping, and the adaptation of sensorimotor skills following altered sensory experience has not yet convincingly been demonstrated. Here, we combined in vivo whole-cell recordings, 2-Photon calcium imaging and a whisker-dependent behavior protocol to directly demonstrate this relationship. It has been described that rhythmic whisker stimulation potentiates cortical synapses (RWS-LTP) in vivo. An accumulation of postsynaptic AMPARs during similar sensory stimulation was also reported by imaging evidences. Our data demonstrates that this potentiation is occluded by SWE, suggesting that cortical synapses are already potentiated by this trimming protocol. This is translated into an increased neuronal excitability in the spared column and sensorimotor recovery by the spared whisker. To better understand the implication of LTP in cortical remapping, we developed a novel approach to manipulate LTP in vivo without affecting overall circuit properties. Our team showed previously that the blockage of AMPARs synaptic recruitment by extracellular antibody cross-linking prevents LTP in vitro. Here, we report that in vivo cross-linking of AMPARs blocks the expression but not the induction of RWS-LTP, suggesting that the synaptic recruitment of AMPARs is fundamental for in vivo LTP as well. Moreover, chronic AMPAR cross-linking during SWE reverts RWS-LTP occlusion and the increased neuronal excitability caused by whisker trimming. As consequence, the sensorimotor performance by the spared whisker is permanently impaired by the blockage of cortical remapping. Altogether, these evidences led us to define a critical role for synaptic LTP on circuit re-arrangement after whisker trimming. Our data shows that LTP-driven cortical remapping is a compensatory mechanism to optimize animal’s sensorimotor behavior upon altered sensory experience.

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

Cette thèse est consacrée à l'étude des mécanismes d'interaction laser-matière en régime femtoseconde pour l'analyse de circuits intégrés par stimulation photoélectrique non-linéaire. Cette technique permet d'accroitre la résolution pour répondre à la miniaturisation des composants électroniques. Les milieux étudiés dans ce travail sont plus particulièrement le silicium, matériau constitutif des circuits intégrés, et la silice pour le transport des impulsions laser dans une fibre optique. En effet, l'émergence de cette technique d'analyse en milieu industriel requiert l'utilisation de systèmes compacts, fiables et sécuritaires. Les simulations réalisées montrent la génération de charges dans le silicium et la propagation des impulsions dans des fibres photoniques à cœur creux identifiées pour limiter les effets non-linéaires. Des expérimentations sur composants permettent de les confronter aux simulations et de valider l'utilisation de ce type de fibres.Enfin, ce travail a permis de déterminer les paramètres optiques et laser essentiels ainsi que les technologies compatibles avec les contraintes industrielles en analyse de circuits intégrés.

No description available.

The growing demand for the Internet of Things (IoT) and Artificial Intelligence (AI) need high-speed commu-nication within short-range distances. In the Back-End-Of-Line (BEOL), Single-Mode Waveguide (SMW) with micro-mirror shows the promising application as an Optical Redistribution Layer (O-RDL) connecting photonic-chip at the interposer-level. The presented study shows the potential application of the 2-Photon-Polymerization (2PP) process for fabrication of out-of-plane coupling elements (micro-mirror) for SMW using low-loss Ormocer® hybrid polymers. This fabricated micro-mirror uses as a coupling element to connect the light from RDL to chips or for inter-layer connections at Interposer level. To evaluate the processing time, structural quality, and resolution of the printed micro-mirror, two types of lenses (63x and 25x) and Ormocer® polymers (OrmoComp and OrmoCore) were used. The optimization of the process flow for the micro-mirrors for SMW applications will be described in detail.

No description available.

This paper describes technology and process development for a hybrid lithography approach pairing UV-lithography for planar single mode waveguides with 2-photon-polymerization direct-laser-writing for out-of-plane coupling mirrors. Improvements to multi-layer direct patterning of OrmoCore/-Clad material system using UV-lithography are presented. Near square core cross sections are achieved. Minimum alignment accuracy at ≈ 3 μm is observed. Cut-back measurement on single mode waveguides shows attenuation of 0.64 dB cm −1 and 1.5 dB cm −1 at 1310 nm and 1550 nm respectively. Up to 2.5-times increase of shear-strength after thermal exposure up to 300 ◦ C is found using shear tests and compared for various surface treatments. Mechanical compatibility to reflow soldering is derived. An extensive study on the pattering of ORMOCER® using 2-photon-polymerization is performed. Flat 45 ◦ -micro mirrors with sub-10 μm dimensions are 3D-printed both in OrmoCore and OrmoComp. Outlook to further research on hybrid lithography integration approach is given.

This paper describes design and advances in process development for UV-lithography of planar single mode waveguides with openings for out-of-plane coupling µ-mirrors. Improvements to multi-layer direct patterning of OrmoCore/-Clad material system using UV-lithography are presented. Near square core cross sections are achieved. However, non uniformity across 4” wafer is shown due to varying proximity and UV-intensity. Openings in full stack with steep sidewalls without residual layer are patterned. Reduction in stack thickness for very small exposure doses due to inhibition even under inert atmosphere is shown. 45° -µ-mirrors are integrated in these openings to manufacture a U-link via a single mode waveguide and two adjacent micro-mirrors. Optical characterization of U-link demonstrates the feasibility of hybrid lithography approach. However, non-uniformity of core cross-section leads to cross coupling of planar waveguides. Outlook to further research on UV-lithography of multi-layer waveguide stack and alignment with µ-mirror printing is given.

Background: Diabetic kidney disease is the leading cause of end-stage renal disease. Administration of ACE inhibitors or/and SGLT2 inhibitors show renoprotective effects in diabetic and other kidney diseases. The underlying renoprotective mechanisms of SGLT2 inhibition, especially in combination with ACE inhibition, are incompletely understood. We used longitudinal intravital microscopy to directly elucidate glomerular hemodynamics on a single nephron level in response to the ACE inhibitor enalapril or/and the SGLT2 inhibitor empagliflozin. Methods: Five weeks after the induction of diabetes by streptozotocin, male C57BL/6 mice were treated with enalapril, empagliflozin, enalapril/empagliflozin or placebo for 3 days. To identify hemodynamic regulation mechanisms, longitudinal intravital multiphoton microscopy was employed to measure single nephron glomerular filtration rate (snGFR) and afferent/efferent arteriole width. Results: Diabetic mice presented a significant hyperfiltration. Compared to placebo treatment, snGFR was reduced in response to enalapril, empagliflozin, or enalapril/empagliflozin administration under diabetic conditions. While enalapril treatment caused significant dilation of the efferent arteriole (12.55 ± 1.46 µm vs. control 11.92 ± 1.04 µm, p < 0.05), empagliflozin led to a decreased afferent arteriole diameter (11.19 ± 2.55 µm vs. control 12.35 ± 1.32 µm, p < 0.05) in diabetic mice. Unexpectedly under diabetic conditions, the combined treatment with enalapril/empagliflozin had no effects on both afferent and efferent arteriole diameter change. Conclusion: SGLT2 inhibition, besides ACE inhibition, is an essential hemodynamic regulator of glomerular filtration during diabetes mellitus. Nevertheless, additional mechanisms—independent from hemodynamic regulation—are involved in the nephroprotective effects especially of the combination therapy and should be further explored in future studies.