Optimiser le diagnostic paraclinique prédictif et non invasif du déficit en cellules souches limbiques (DCSL) : in vitro après empreinte cytologique (EC) par la recherche immunohistochimique (IH) de marqueurs pertinents et par reverse-transcription polymerase chain reaction (RT-PCR), technique de haute sensibilité ; in vivo par microscopie confocale (MCIV). Matériel et Méthodes: La preuve diagnostique de DCSL est la présence de cellules conjonctivales au sein de la cornée, la persistance de cellules cornéennes signant un DCSL partiel. L'EC a été standardisée. La spécificité de chacun des marqueurs cornéens ou conjonctivaux sélectionnés a été recherchée sur des tissus normaux avant d'aborder une étude prospective monocentrique, sur 22 yeux de 18 patients cliniquement suspects de DCSL. Les cellules épithéliales de la cornée centrale étaient recueillies par EC puis transférées sur lame. L'expression des marqueurs de différentiation conjonctivale (K7, K13, K19, MUC5AC) et cornéenne (K12) a été recherchée par IH sur les 22 EC et celle de la MUC5AC par RT-PCR sur 4 d'entre elles. Enfin, la cornée et le limbe de 7 patients ont étés analysés par MCIV. La concordance entre les techniques est évaluée. Conclusion: Ces techniques complémentaires dépendent de l'équipement du service et de l'accessibilité à un laboratoire compétent. Un organigramme décisionnel a été établi pour permettre de faire un diagnostic fiable de DCSL, les examens paracliniques étant inutiles dans certains cas. La recherche IH de kératines conjonctivales K7/K13 et/ou la détection de MUC5AC par RT-PCR sur cellules cornéennes recueillies par EC sont des techniques diagnostiques de haute valeur scientifique, tandis que l'IH K12/MUC5AC doit être abandonnée pour manque de sensibilité et celle de K3/K19 pour manque de spécificité. La MCIV, quand elle est réalisable, est une technique hautement sensible qui permet une quantification du degré de sévérité de la maladie avec une forte concordance avec les autres examens / Purpose: to optimize minimally invasive techniques for diagnosing limbal stem cell deficiency: in vitro after impression cytology (IC) by means of immunocytochemical detection of relevant markers and reverse-transcription polymerase chain reaction (RT PCR), an highly sensitive method; in vivo by confocal microscopy (IVCM). Material and Methods: Presence of conjunctival cells in central cornea was diagnosis proof of LSCD, whereas corneal epithelial cells’ remaining traduces partial LSCD. IC was labeled. Corneal or conjunctival specificity of each marker was previously assessed on healthy tissues. In a prospective case control observational study, 22 eyes of 18 patients clinically suspected of LSCD were enrolled. Epithelial cells from central cornea were collected by IC. Conjunctival (K7, K13, K19, MUC5AC) and corneal (K12) differentiation markers were assessed by immunocytochemistry on each of 22 eyes and MUC5AC RT-PCR was assessed for 4 of them. Cornea and limbus of 7 eyes were assessed by IVCM. The inter-examination agreement was determined. Conclusion: These techniques require skilled technicians and laboratory facilities. We propose a decision tree model to provide unfailing LSCD diagnosis, complementary examinations being sometimes useless. Clinical examination can lead to LSCD misdiagnosis. Immunostaining of conjunctival keratins K7 and K13 as well as MUC5AC detection by RT-PCR are highly effective methods whereas MUC5AC/K12 immuno staining are not sensible and both K3 and K19 are not specific. IVCM of great sensitivity if realizable allows quantification of LSCD severity. Combining both methods provides in every case unfailing diagnosis of LSCD and evaluation of the extent of the disease with high agreement

Le virus de la mosaïque jaune du navet (TYMV) possède un génome monopartite constitué d’ARN de polarité positive codant pour trois protéines, dont seule la polyprotéine 206K est indispensable à la réplication virale.Elle subit une maturation protéolytique, générant les protéines 140K et 66K, localisées au niveau de l’enveloppe des chloroplastes, siège de la réplication virale.Adressée aux chloroplastes, la protéine 140K y recrute la 66K et se comporte comme une protéine intégrale membranaire.Le domaine d’adressage aux chloroplastes (DAC) de la protéine 140K a été défini grâce à la transfection et à des protoplastes d’Arabidopsis thaliana par différentes constructions codantpour des versions délétées de la protéine fusionnées à l’EGFP, et à leur observation en microscopie confocale. Le DAC comprend deux hélices alpha amphipathiques dont la présence a été attestée par dichroïsme circulaire. Leur nécessité pour la localisation aux chloroplastes, l’association aux membranes et la réplication virale, a été étudiée. Différents patterns de distribution subcellulaire de la protéine 140K ont été observés. Ils sont corrélés au taux d’expression de la protéine. Sa dimérisation a également été démontrée.L’implication d’autres résidus du DAC dans la localisation subcellulaire, la dimérisation et la réplication virale, a également été recherchée. / Turnip yellow mosaic virus (TYMV) is a positive single-stranded RNA virus. Among the three ORFs encoded by the TYMV genome, 206K is the only protein required for viral replication. It is cleaved into 140K and 66K, which are both present at the chloroplast envelope membrane, where viral replication takes place.The 140K protein is targeted to chloroplasts, where it recruits 66K, and behaves as an integral membrane protein. The chloroplast targeting domain (DAC) of the 140K protein was defined using Arabidopsis thaliana protoplasts transfected by various constructs encoding deleted versions of 140Kfused to EGFP and subsequent confocal microscopy. The DAC comprises two amphipathic alpha helices, as confirmed by circular dichroism. Their involvement in chloroplast localisation and membrane association has been assessed, as well as their contribution to viral replication.We observed different subcellular distribution patterns of 140K protein, which correlate with the expression level of the protein. Its capability to dimerize has also been demonstrated.The involvement of other DAC residues in subcellular localisation, dimerization and viral replication has been studied.

We present a fluid-membrane lens with two piezoelectric actuators that offer versatile, circular symmetric lens surface shaping. A wavefront-measurement-based control system ensures robustness against creeping and hysteresis effects of the piezoelectric actuators. We apply the adaptive lens to correct synthetic aberrations induced by a deformable mirror. The results suggest that the lens is able to correct spherical aberrations with standard Zernike coefficients between 0 μm and 1 μm, while operating at refractive powers up to about 4m-1. We apply the adaptive lens in a custom-built confocal microscope to allow simultaneous axial scanning and spherical aberration tuning. The confocal microscope is extended by an additional phase measurement system to include the control algorithm. To verify our approach, we use the maximum intensity and the axial FWHM of the overall confocal point spread function as figures of merit. We further discuss the ability of the adaptive lens to correct specimen-induced aberrations in a confocal microscope.

The purpose of this Master thesis is to verify migration of mesenchymal stem cells on the principle known as chemotaxis. First part of this study is focused on cell migration in order to explain the whole migration process. Next part describes various chemotaxis methods and selected studies dealing with clinical applications of mesenchymal stem cells in different medical and biomedical fields. The following step describes confocal microscopy, which is used for acquiring images of the cells. The experimental part is focused on cultivation of mesenchymal stem cells in a laboratory, which is necessary for cell vitality. Furthermore, there are designed two main experiments. Firstly there is a 2D experiment with adherent cells for chemotaxis using -Slide Chemotaxis. Secondly Transwell migration test is designed and executed. Finally, the acquired images from confocal microscope are used for image processing, which was done in Matlab R2020a programming environment. The result of this processing is evaluation of cell confluence and migration. In the end, experimental part of this study was optimized according to recommended studies. The results are summarized in the conclusion with proposal for improvements of those methods.

The Coherence Controlled Holographic Microscope (CCHM) was developed at BUT Brno for a quantitative phase imaging of living cells. Nowadays it ocurres that its imaging properties are enhanced by the use of additional modules. In the present the microscope is equipped with the epifluorescence module, which allows observation of fluorescently marked living cells. This thesis is going to follow up on the development of this module and is going to extend its options by confocal imaging. The disadvantage of current multi-channel confocal microscopes is a mechanical rotation of the Nipkow discs, which causes undesired mechanical vibrations. That is why in this thesis it is replaced by Digital Micromirror Device. With its use was developed optical system of the whole confocal model, whose correct funcion was simulated in optical CAD. The experimentally verified prototype serves to test the imaging properties. On this basis is designed an application idea of the fluorescence confocal module, which will be possible to connect to the CCHM microscope.

Content is focused on fluorescence lifetime imaging techniques. Fluorescence lifetime is computed from data acquired with using of Leica TCS SP8X confocal microscope sequential scanning. Algorithms and software for the computation, imaging and analysis of fluorescence lifetime is presented. Software is allowing both 2D and 3D imaging of fluorescence lifetime. Techniques are used for fluorescence lifetime imaging of mesenchymal cells and fibroblasts tainted with SPIO-Rhodamin complex.

This master’s thesis includes an overview of profile method of surface texture evaluation according to ISO standards and VDA automotive standards, as well as an overview of areal method according to ISO 25178 standard series, which includes a proposal for improvement of czech terminology. Suitability of application of selected measurement methods for measurement of a portfolio of automotive parts was evaluated based on measurements carried out using a contact profiler, a coherence scanning interferometer and a confocal microscope. Further recommendations for practice in surface texture metrology were also formulated. A regression analysis of the relationship between values of selected profile surface texture parameters and their areal equivalents was also performed. Its results may aid the spread of areal surface texture evaluation in the industry.

The present Master thesis focuses on evalution of CO2 concentration and irradiance on selected leaf anatomical parameters of European Beech (Common Beech) Fagus sylvatica L.. The process of photosynthesis is remarkably determined by numerous extrenal factors, among them by atmoshperic CO2 concentration and irradiance and is closely correlated with leaf anatomical parameters. One of these most important anatomical parameters affecting the net assimilation rate is an internal leaf surface, which corresponds to mesophyll area avialable for gas exhcange. Experimental material of the study was sampled from the leaves of juvenile trees of F. sylvatica planted in 2005 and growing under ambient (390 ppm, AC) and elevated (700 ppm, EC) CO2 concentrations on the experimental site of the Global Change Research Center AS CR at Bílý Kříž in the Beskydy Mountains. Sun and shade leaves were sampled from trees of both CO2 treatments in two seasons 3 years apart (2009 and 2012). To determine leaf anatomical parameters, the stereological methods were applied, which yield unbiased estimation of measured parameters, particularly the Fakir method for internal leaf surface determination. The EC effect was observed on the leaves sampled in 2009 only in the decrease of proportion of intercellular spaces in mesophyll. In...

<p>The extracellular matrix (ECM) plays a significant role in defining the mechanical properties of biological tissues. The proteins, proteoglycans, and glycosaminoglycans that constitute the ECM are arranged into highly organized structures (<i>e.g.</i> fibrils and networks). Cellular behavior is affected by the stiffness of the microenvironment and influenced by the composition and organization of the ECM. Mechanosensing of ECM stiffness by cells occurs at the fibrillar (mesoscale) level between the single molecule (microscale) and the bulk tissue (macroscale) levels. However, the mechanical behavior of ECM proteins at the mesoscale are not well defined. Thus, better understanding of the ECM building blocks responsible for functional tissue assembly is critical in order to recapitulate <i>in vivo</i> conditions. There is a need for the mechanical characterization of the ECM networks formed by proteins synthesized <i>in vivo</i> while in their native configuration. </p> <p>To address this gap, my goals highlighted in this dissertation were to develop appropriate experimental and computational methodologies and investigate the 3D organization and mechanical behavior of ECM networks <i>in situ</i>. The ECM of developing mouse tissues was used as a model system, taking advantage of the low-density networks present at this stage. First, we established a novel decellularization technique that enhanced the visualization of ECM networks in soft embryonic tissues. Based on this technique, we then quantified tissue-dependent strain of immunostained ECM networks <i>in situ</i>. Next, we developed mesoscale and macroscale testing systems to evaluate ECM networks under tension. Our systems were used to investigate tendon mechanics as a function of development, calculating tangent moduli from stress - strain plots. Similarly, we characterized ECM network deformation while uniaxially loading embryonic tissues, since this testing modality is ideal for fibril and network mechanics. Taken together, this information can facilitate the fabrication of physiologically relevant scaffolds for regenerative medicine by establishing mechanical guidelines for microenvironments facilitate functional tissue assembly.</p>

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