Orientador: Jörg Kobarg / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T05:39:09Z (GMT). No. of bitstreams: 1 Cardoso_AlineMonticelli_M.pdf: 19291466 bytes, checksum: 5920430c506c4670ca04da10ad934398 (MD5) Previous issue date: 2015 / Resumo: A Stanniocalcina-1 (STC1) humana é uma glicoproteína homóloga a Stanniocalcina (STC) originalmente identificada como um hormônio regulador da homeostase de cálcio em peixes. A STC1 humana secretada atua em diferentes processos fisiológicos incluindo a angiogênese, a hipóxia e, principalmente, a carcinogênese, demonstrando assim uma atividade abrangente. Atualmente não se conhece o receptor da STC1 e pouco se sabe sobre o mecanismo de ação e de entrada nas células dessa proteína. Assim, o objetivo desse trabalho foi investigar um candidato a receptor de membrana dessa proteína, o receptor de transferrina (TfR1), uma proteína transmembrana responsável pela absorção de ferro nas células. Esse receptor é provavelmente expresso por todas as células em diferentes níveis, em destaque em células do sistema hematopoiético, em células em divisão celular e células neoplásicas. Assim, avaliou-se por citometria de fluxo o efeito do tratamento com STC1 em células não transfectadas e células transfectadas superexpressando o receptor de transferrina. Células tratadas com STC1 demonstraram um efeito semelhante ao tratamento com transferrina, um conhecido ligante desse receptor, no qual ambos diminuíram o número de células positivas para a marcação da superfície com transferrina conjugada com fluorocromo (transferrina-Alexa Fluor® 488 - Life Technologies). Em outro conjunto de experimentos de Western Blot foi demonstrado que a STC1 adicionada no sobrenadante das culturas de células é internalizada nas células e detectável no lisado celular, principalmente as células transfectadas para a superexpressão do receptor de transferrina. Complementarmente, em experimentos de localização subcelular por imunofluorescência a STC1 foi detectada em uma forma pontual e espalhada no citoplasma. Em conjunto, todos esses experimentos sugerem que STC1 e transferrina interferem na localização do receptor de transferrina na superfície celular e que possivelmente esse receptor está envolvido em mecanismos de internalização da própria STC1 / Abstract: Human Stanniocalcin 1 (STC1) is the mammalian homologue of STC, which was originally identified as a calcium-regulating hormone in bony fishes. The human secreted Stanniocalcin acts on different physiological processes, including angiogenesis, hypoxia and especially carcinogenesis, facts that demonstrate their activity is wide. Currently there are few data on the mechanism of action of this protein or how it enters the cell. Thus, the aim of this study was to investigate transferrin receptor (TfR1) as a candidate to membrane receptor protein of STC1. This receptor is a membrane protein responsible for the iron uptake in cells. This receptor is probably expressed by all cells especially by cells in division and cancer cells, but its expression level may vary. We evaluated by flow cytometry the effect of STC1 treatment in non-transfected cells and cell with TfR1 overexpression. The treatment with STC demonstrated a similar effect to treatment with transferrin, a known ligand for receptor, which decreased the number of positive cells for staining with fluorochrome (transferrin conjugated to Alexa Fluor® 488 - Life Technologies). We also demonstrated by Western Blot that STC1 added to the supernatant of cultures of cells, especially cells that overexpress transferrin receptor, is internalized into the cells and detectable in the cell lysate. Additionally, in subcellular localization experiments by immunofluorescence STC1 was detected in a timely manner and scattered in the cytoplasm. Together all this information suggests that STC1 and transferrin interferes with the localization of the transferrin receptor in the cellular surface and perhaps this receptor is involved in the mechanism of internalization of STC1 / Mestrado / Bioquimica / Mestra em Biologia Funcional e Molecular

Le cytosquelette représente un élément crucial dans les processus cellulaires essentiels des cellules lymphoïdes. Les différents filaments du cytosquelette et leurs modes de régulation représentent donc des cibles thérapeutiques majeures pour le développement de nouveaux composés pharmacologiques. Au cours de ce travail de thèse, nous avons mis au point une nouvelle méthode d’analyse par cytométrie en flux (CytoFRET) permettant de visualiser simultanément la dynamique de polymérisation des filaments d’actine, des microtubules et des filaments intermédiaires de vimentine dans la lignée leucémique T Jurkat. Cette méthode a été utilisée pour le criblage d’une mini-chimiothèque composée d’inhibiteurs d’enzymes impliquées dans les modifications post-traductionnelles des protéines. Nous avons ainsi identifié deux composés, le WP1130 et le b-AP15, des inhibiteurs d’enzymes de déubiquitination (DUBs), comme puissants inducteurs de la polymérisation/nucléation de l’actine. Nous avons montré que l’effet de ces inhibiteurs sur les microfilaments d’actine est consécutif à une poly-ubiquitination de la Destrine, une protéine de liaison à l’actine. Nous avons également identifié des inhibiteurs des déacétylases HDAC6 et SIRT2 comme inducteurs de la polymérisation des microtubules et de l’assemblage de la vimentine. L’effet de ces inhibiteurs a été corrélé à une acétylation directe de la tubuline mais pas de la vimentine. Ces résultats ouvrent ainsi de nouvelles perspectives à la fois fondamentale et thérapeutique sur la physiopathologie du cytosquelette des cellules lymphoïdes. / Actin, microtubules, and intermediate filaments compose three major cytoskeletal structures of vertebrate cells that are characterized by highly dynamic balances between assembly and de-assembly, underlying critical cellular processes such as mitosis, architecture and movement. Consequently, cytoskeleton dysfunctions have been implicated in several pathological situations including cell transformation and metastasis. Thus, cytoskeletal networks represent major targets for the development of novel anti-cancer and anti-metastatic therapies. However, drug development is currently limited by the availability of high-throughput screening systems allowing the simultaneous monitoring of actin, microtubules and intermediate filaments dynamics in living cells. In this work, we have developed a novel screening assay of cytoskeleton dynamics based on the simultaneous recording by flow cytometry of FRET signals produced by the variation of actin, tubulin and vimentin filaments dynamics in living cells. Our novel method was employed to screen a mini-library of drugs known for their ability to interfere with post-translationnal modifications of proteins. Interestingly, our approach revealed that compounds interfering with lysine acetylation have a dramatic impact on vimentin filaments assembly and microtubules polymerization. In addition, two inhibitors (WP1130 and b-AP15) of deubiquitinating enzymes showed increase of actin polymerization. This effect was attributed to poly-ubiquitnation of Destrin, an actin binding protein. In conclusion, our FRET multiplex flow cytometry assay represents a novel effective method for the future development of new anti-cancer therapies.

Identification of different cell types is an indispensable task of biomedical research and clinical application. During the last decades, much attention was given to molecular characterization, and many cell types can now be identified using established markers that bind to cell-specific antigens. The required staining process is a lengthy and costly treatment, which can cause alterations of cellular properties, contaminate the sample and therefore limit its subsequent use. For example, for photoreceptor transplantations, highly pure samples of photoreceptor cells are required, which can currently only be obtained using molecular labelling, rendering the resulting sample incompatible for clinical application. A promising alternative to molecular markers is the label-free identification of cells using mechanical or morphological features. Real-time deformability cytometry (RT DC) is a microfluidic technique, which allows capturing both types of information simultaneously for single cells at high-throughput. In this thesis, I present machine learning methods which allow identifying different cell types, based on bright-field images from RT DC. In particular, I introduce algorithms that are fast enough to be applied in real-time during the measurement (at >1000 cells/s), which can be used for image-based cell sorting. The performance of the algorithms is shown for the identification of rod precursor cells in retina-samples, indicating that image-based sorting based on those algorithms would allow enriching photoreceptors to a final concentration, applicable for transplantation purposes.:Contents Abstract iii Kurzfassung iv List of figures viii List of tables x 1. Introduction 1 1.1. Texture and mechanical properties: label-free markers 4 1.2. The retina, diseases and cure by photoreceptor transplantation 5 1.3. Technologies for label-free assessment of cells 8 2. Materials and Methods 10 2.1. Experimental setup 10 2.1.1. Chip design for RT DC and RT-FDC 10 2.1.2. Chip design for soRT-FDC 11 2.1.3. Chip fabrication 13 2.1.4. RT-DC, RT-FDC and soRT FDC Setup 14 2.1.5. Physics of surface acoustic wave mediated sorting 16 2.1.6. Measurement buffer (MB) for RT DC 17 2.2. Online parameters 18 2.3. Offline parameters 21 2.4. Linear mixed models (LMM) 28 2.5. Normality test using probability plots 31 2.6. Gaussian mixture model (GMM) and Bayesian information criterion (BIC) 32 2.7. Random forests 33 2.8. Confusion matrix 34 2.9. Deep learning 36 2.10. Preparation of retina samples 43 2.11. Preparation of blood samples 44 2.12. Staining of neutrophils and monocytes 45 3. Results 46 3.1. Meta-analysis of RT-DC data 46 3.1.1. Correlations of area and volume 47 3.1.2. Correlations of deformation and inertia ratio 48 3.1.3. Further screening of correlations 50 3.1.4. Shape of distributions 52 3.1.5. Discussion 54 3.2. Characterization of retina cells in RT-DC 57 3.2.1. Maturation of retina cells 57 3.2.2. Comparing retina cell types using statistical tests 61 3.2.3. Discussion 63 3.3. Classification of retina cells using supervised machine learning 66 3.3.1. The dataset 66 3.3.2. Cell classification using optimized area gating 72 3.3.3. Cell classification using random forests 74 3.3.4. Cell classification using deep neural nets 80 3.3.5. Improving DNN accuracy using image augmentation 85 3.3.6. Tuning of final models and classification performance 93 3.3.7. Visualization of model attention 98 3.3.8. Discussion 100 3.4. Software tools to train and apply deep neural nets for sorting 105 3.4.1. AIDeveloper 105 3.4.2. Sorting Software 113 3.4.3. Discussion 114 3.5. Sorting experiments 117 3.5.1. Sorting of rod precursor cells 117 3.5.2. Sorting of neutrophils 120 3.5.3. Discussion 125 4. Conclusion and outlook 128 A. Appendix 131 I. Comparison of dense and convolutional layer 131 Bibliography 133 Acronyms 148 Acknowledgements 150 Erklärung 152

The body of literature on the impact of emulsion particle size on oxidation rates is unclear. This could be because emulsions are typically polydisperse and the oxidation rate of individual droplets is impossible to discern. Flow cytometry is a technique for studying individual cells and their subpopulations using fluorescence technologies. It is possible that individual emulsion droplets could also be characterized by flow cytometry as a novel approach for studying lipid oxidation. Typical emulsion droplets are too small to be visualized by flow cytometer, so emulsions were prepared to have droplets > 2 μm; weighting agent and xanthan gum were added to minimize creaming during storage. A radical-sensitive lipid-soluble fluorescence probe (BODIPY665/676) was added to the lipid used to prepare the emulsion so that the susceptibility of individual emulsion droplets could be determined. The results showed that in a polydisperse emulsion system, small droplets were oxidized faster than large droplets. Using mixtures of emulsions with and without prooxidants, it was possible to see the transfer of prooxidants between droplets, a process that is influenced by surfactant and salt concentrations. For example, surfactants micelles can transfer prooxidants to neighboring non-oxidized droplets and cause fluorescence loss when surfactant concentration was higher than critical micelle concentration (CMC). Transfer of prooxidants was promoted by adding NaCl and free fatty acid which could be attributed to the lower CMC. This study showed the potential for applying flow cytometry on oxidation of individual emulsion droplets.

This thesis focuses on autofluorescence of flavins in gram-negative bacteria Cupriavidus necator H16 and its mutant strain PHB-4. The main methods used were fluorescence microscopy and flow cytometry. To confirm the presence of flavins, excitation and emission spectra of the bacterial suspension were measured, which were compared with flavin standards. In the part of testing cells without stress response, the autofluorescence of bacteria in PBS buffer and cell suspensions stained with fluorescence probe BODIPY 493/503 was measured. The ratio of short fluorescence lifetime to long autofluorescence lifetime, and its dependence on fluorescence probe was compared with previous conditions. Autofluorescence of the supernatant was measured; it was found that the relative amplitude of long lifetime was multiple times higher than in the cell. In the part devoted to the stress response, this thesis was focused on the amount of dissolved oxygen in the production medium and the effect on bacterial autofluorescence. Then differently concentrated hydrogen peroxide was used, the best results were obtained from the concentration of 100 mM in media. For comparison a combination of hydrogen peroxide with ferro-ammonium sulphate was used, but there was no big difference. Sodium azide and antimycin A were selected as substances that directly influence on bacterial respiratory chain. Both compounds affected change in the ratio of the relative amplitudes, but the distribution of these lifetimes and the autofluorescence change over time was affected only by sodium azide.

This diploma thesis deals with the possibilities of stress resistance analysis of the Cupriavidus necator H16 and PHB-4 bacterial cells by spectroscopic methods and by testing the suitability of acridine orange as a viable dye. Based on research in literature, suitable analytical methods have been proposed, namely flow cytometer and fluorescence microscope. The first part of the experimental work was focused on the fluorescence microscope, which confirmed the basic character of acridine orange. Three stress factors, 50% and 70% ethanol, and acidic pH (pH = 1) were selected for viability monitoring. The bacteria fluoresced with green color after exposure to ethanol and red spots were found next to the cells, indicating their loss of integrity. In an acidic environment, the bacteria fluoresced red because of a partial DNA breakdown. The results were verified by the combination of propidium iodide with SYTO9 and the acridine orange suitability proved to be useful in this method. Image records were processed using image analysis. In the second part, acridine orange was used to monitor fluorescence using a flow cytometer. The result of the measurement was fluorescence expressed as histograms for individual channels, where fluorescence was characterized by median and mean intensity. By comparing the methods used, the acridine orange appears to be a more suitable fluorescent dye for the microscope than for a flow cytometer in which it was more difficult to obtain cell viability information. In the last part of the experimental work interesting photophysical properties of acridine orange were investigated.

This thesis focuses on fluorescent analysis of viability and PHA content in bacterial cultures, the main methods of investigation were flow cytometry and fluorescent microscopy. In order to determine viability of C. necator H16, several viability probes were tested, nevertheless, only BacLightTM kit and propidium iodide can be used to estimate portion of viable and live bacterial cell in samples. Further, Acridine orange was used to monitor physiological state of bacterial culture and two hydrophobic probes, Nile Red and BODIPY 493/503, were used to investigate PHA content in bacterial cells. Application of BODIPY 493/503 seems to be promising since this probe does not require permeabilization of bacteria cells and it can be used along with propidium iodide. Furthermore, several fluorophores were tested in the microscopic part. In was found that concentrations used in cytometric analyses were too high for microscopic use. Emission from the SYTO9 fluorophore is seen mainly in the green channel but because of the high concentration some emission was visible in the red channel. Cells stained with BODIPY 493/503 had very high fluorescence intensities when the stain concentration was 10 . At the same time, negative amplitudes of fluorescence were measured in both strains of C. necator, but in case of C. necator H16 that amplitude was much more pronounced. In this strain surprising high concentration of BODIPY stain was observed on the surface of PHB granules. Anisotropy of the fluorophore was nearing 0 which is very surprising.

The main drivers of microevolution in the genus Sorbus are interspecific hybridisation and polyploidy. The fate of new hybrid and polyploid taxa is determined by their mode of reproduction. Especially apomixis could be very advantageous for these new taxa. The S. aria agg. (subg. Aria) plays an important role within the genus since its members are involved in all hybridisation events and thereby is responsible for the substantial part of variation of the genus. Flow cytometry, molecular markers and multivariate morphological analyses were employed to evaluate the processes generating the variability in the S. aria group. Three ploidy levels were detected among species from subg. Aria in the Czech Republic. All of them could be found in the South Moravia, whereas only tetraploids occur in the Bohemia region. Moreover, most of the Czech taxa (5 out of 7) grow also only in the South Moravia which is therefore considered as a centre of diversity of the genus Sorbus in the Czech Republic. Flow cytometry seed screen revealed 7 modes of reproduction among the individuals from S. aria agg. A wide range of sexual and apomictic types of reproduction including reduced and unreduced gametes was detected. All of the diploid individuals are completely sexual. Among polyploid taxa, most of the species are...

Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...

FTIR spectroscopy as molecular fingerprint has been used to assess macromolecular and ele-mental stoichiometry as well as growth rates of phytoplankton cells. Chemometric models have been developed to extract quantitative information from FTIR spectra to reveal macro-molecular composition (of proteins, carbohydrates and lipids), C:N ratio, and growth potential. In this study, we tested these chemometric models based on lab-cultured algal species in mon-itoring changes of phytoplankton community structure in a hypertrophic lake (Lake Auensee, Leipzig, Germany), where a seasonal succession of spring green algal bloom followed by cya-nobacterial dominance in summer can be commonly observed. Our results demonstrated that green algae reacted to environmental changes such as nitrogen limitation (due to imbalanced nitrogen and phosphorus supply) with restricted growth by changing carbon allocation from protein synthesis to storage carbohydrates and/or lipids, and increased C:N ratio. By contrast, cyanobacteria proliferated under nitrogen limiting conditions. Furthermore, the FTIR-based growth potential of green alga matched well with the population biomass determined by the Chl-a concentration. However, the predicted growth potential based on FTIR spectroscopy cannot describe the realistic growth development of cyanobacteria in this lake. These results revealed that green algae and cyanobacteria have different strategies of C-allocation stoichi-ometry and growth patterns in response to environmental changes. These taxon-specific re-sponses may explain at a molecular level why green algae bloomed in the spring under condi-tions with sufficient nutrient, lower pH and lower water temperature; while cyanobacteria overgrew green algae and dominated in the summer under conditions with limited nutrient availability, higher pH and higher water temperature. In addition, the applicability of these chemometric models for predicting field cyanobacterial growth is of limited value. This may be attributed to other special adaptation properties of cyanobacterial species under stress growth conditions. We used flow cytometry to isolate functional algal groups from the water samples. Despite some drawbacks of the flow cytometry combined FTIR spectroscopy tech-nique, this method provides prospects of monitoring water quality and early warning of harmful algal blooms.