Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Venenos de Bothrops consistem em uma mistura complexa de substÃncias ativas, principalmente peptÃdeos e proteÃnas, capazes de interferir com muitos processos fisiolÃgicos. Serpentes do gÃnero Bothrops sÃo responsÃveis por mais de 20 mil acidentes por ano no Brasil, que totalizam 90% de todos os acidentes ofÃdicos registrados. A Bothrops leucurus à uma serpente peÃonhenta importante que habita a regiÃo nordeste do Brasil. Os efeitos biolÃgicos devido ao envenenamento tÃm perfil semelhante ao observado em outras Bothrops, ou seja, a atividade coagulante, hemorrÃgica, fibrinolÃtica e insuficiÃncia renal in vivo. O objetivo deste estudo foi investigar os efeitos do veneno de Bothrops leucurus no sistema de perfusÃo renal e em culturas de cÃlulas tubulares renais do tipo MDCK (Madin-Darby Canine kidney). Rins isolados de ratos Wistar pesando 250 a 300g (n = 5) foram perfundidos com Krebs-Henseleit contendo 6% de albumina sÃrica bovina previamente dialisadas por 120 minutos. Os efeitos do veneno de Bothrops leucurus (VBl) (10μg/mL) foram estudados sobre o Ritmo de FiltraÃÃo Glomerular (RFG), Fluxo UrinÃrio (FU), PressÃo de PerfusÃo (PP), ResistÃncia Vascular Renal (RVR), Percentual de Transporte Tubular de SÃdio (%TNa+), de PotÃssio (%TK+) e de Cloreto (%TCl-). O veneno de B. leucurus (10 μg / mL) reduziu a PP aos 90 e 120 min e o FU aos 60 e 90 min. O ritmo de filtraÃÃo glomerular diminuiu aos 60 e 90 min. A resistÃncia vascular renal diminuiu aos 120 min. Observou-se tambÃm uma diminuiÃÃo no percentual de transporte tubular de sÃdio (% TNA+) aos 120 min e de cloreto (% TCL-) aos 60 e 90 min. CÃlulas MDCK foram cultivadas em garrafas plÃsticas a 37ÂC em atmosfera de 5% de CO2, com meio RPMI 1640 suplementado com soro bovino fetal a 10%. O tratamento com VBl causou diminuiÃÃo da viabilidade celular atà na menor concentraÃÃo testada com um IC50 de 1,25 μg /mL. Citometria de fluxo com anexina V e iodeto de propÃdio mostrou que a morte celular ocorreu predominantemente por necrose de forma concentraÃÃo dependente. Quando a apoptose foi analisada atravÃs da coloraÃÃo nuclear, foi observado aumento significativo na porcentagem de morte celular, VBl nas concentraÃÃes de 2,5 e 1,5 μg/mL induziu 6,8% e 5,8% de apoptose apÃs 24 horas de tratamento, respectivamente. O tratamento com VBl (1,25 μg/mL) levou a despolarizaÃÃo significativa do potencial de membrana mitocondrial. Nas menores concentraÃÃes avaliadas, verificamos aumento de expressÃo de genes envolvidos na morte celular por apoptose. O Ãon Ca2+ participa da sinalizaÃÃo da morte celular induzida pelo veneno de B. leucurus. Esses resultados demonstram que o veneno de B. leucurus alterou todos os parÃmetros renais avaliados na perfusÃo de rim isolado e foi citotÃxico para cultura de cÃlulas MDCK. / Bothrops venoms consist of complex mixture of active substances, mainly peptides and proteins, which interfere with many physiologic processes. Bothrops species are responsible for more than 20,000 accidents per year in Brazilâ90% of all recorded snakebites. The Bothrops leucurus is an important venomous snake that inhabits the northeast of Brazil. The biological effects due envenomation have similar profile to that observed in other Bothrops, ie coagulant activity, hemorrhagic, fibrinolytic, and renal failure in vivo. The aim of this study was to investigate the effects of the Bothrops leucurus venom in the renal perfusion system and in cultured renal tubular cells of the type MDCK (MadinâDarby Canine kidney). Isolated kidneys from Wistar rats weighing 250 to 300g (n=5) were perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin previously dialyzed for 120 minutes. The effects of Bothrops leucurus venom (VBl) (10μg/mL) were studied on the Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR) Percentage of Sodium (%TNa+), Potassium (%TK+) and Chloride (%TCl-) Tubular Transport. B. leucurus venom (10 μg/mL) reduction the PP at 90 and 120 min and UF at 60 and 90 min. The glomerular filtration rate decreased at 60 and 90 min. The renal vascular resistance decreased at 120 min. It was also observed a decrease on percentual tubular transport of sodium (%TNa+) at 120 min and of chloride (%TCl-) at 60 and 90 min. MDCK cells were grown in plastic flasks at 37 C in a humidified atmosphere of 5% CO2 â air, with RPMI 1640 medium supplemented with 10% fetal calf serum. The treatment with VBl caused decrease in cell viability to the lowest concentration tested with an CI50 of 1.25 μg/mL. Flow cytometry with annexin V and propidium iodide showed that cell death occurred predominantly by necrosis. When apoptosis was analyzed by nuclear staining, it was observed significant increase in the percentage of cell death, VBl 2,5 μg/mL induced 6,8% apoptosis and VBl 1,25 μg/mL caused 5,8% of apoptosis after 24 h of treatment. VBl treatment (1,25 μg/mL) led to significant depolarization of the mitochondrial membrane potential. We found increased expression of genes involved in cell death by apoptosis in the lower concentrations tested. The ion Ca2+ signaling participates in cell death induced by the venom of B. leucurus. These results demonstrate that the venom of B. leucurus altered all the renal parameters assessed in isolated kidney perfusion and was cytotoxic to MDCK cell culture.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19144号 / 生博第327号 / 新制||生||44(附属図書館) / 32095 / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 松田 道行, 教授 原田 慶恵, 教授 井垣 達吏 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Developmental processes involving symmetry-breaking of homogeneous cell populations into leaders and followers are found in many important contexts. Cells constrained by culture on two-dimensional scaffolds, as well as in three-dimensional shapes, appear to respond to convex curves with an increasing propensity to protrude, while concave curves in contrast appear to inhibit protrusion. This has interesting implications in terms of a potential positive feedback loop. This feedback may act in symmetry-breaking, through amplification of initial stochastic differences in cell shape, and also in collective migration, through reinforcing and directing the coherent movement of collectives. In this study, epithelial cells were cultured on two-dimensional micropatterns with variable curvatures to examine the effect of edge geometry and other variables on the likelihood of protrusions forming. This platform allowed the quantification of F-actin-based protrusions at the periphery of multicellular epithelial clusters, in segments defined by cluster edge curvature. The initial observations confirmed reports in the literature of preferential localisation of protrusions at more convex regions, and relative inhibition at more concave regions. A previously-published work has postulated a role for secreted modulators of motility, with the shape of a group of cells determining the concentration of diffusing morphogen each individual cell is exposed to. To test this hypothesis, a low-shear flow culture chamber was used to disrupt the putative gradients. Despite theoretical and empirical support for the sufficiency of the flow condition to disrupt autocrine signalling, micropatterned cells cultured under flow showed no significant differences from the control condition. These findings form the basis of a manuscript which has been accepted for publication by the Journal of Anatomy. The results of an Atomic Force Microscopy (AFM) study carried out by collaborators were suggestive of a role for cellular mechanotransduction in sensing and responding to micropattern curvature. Differential calcium channel mechanoactivation was hypothesised as being one potential mechanism underlying the response to curvature, given the known involvement of mechanosensitive ion channels in cellular responses to force and substrate stiffness, and the multiple roles of calcium in cellular motility. Artificially increasing cytosolic calcium levels with Ionomycin reduced protrusion rates at convex curves. However, treatment with BAPTA-AM to sequester intracellular calcium had no effect on protrusion rates. ROCK inhibitor, in contrast, increased protrusion rates at concave curves, and Blebbistatin increased protrusion rates globally. These results together are suggestive of differential control of myosin depending on local curvature: cyclic and driven by calcium-activation of MLCK in the convex regions (with lamellipodia undergoing protrusion-retraction cycles), versus sustained and controlled by ROCK in the concave regions (where lamellipodia are inhibited). The unexpected finding that protrusions at convex regions were resistant to the actin cytoskeleton-disrupting drug Cytochalasin D may point to a role for a tropomyosin isoform in defining the differing mechanical characteristics of the actin cytoskeleton in response to local curvature. In addition, the previously-noted lack of effect of BAPTA-AM treatment (which has been shown to interfere with dynamic microtubules) is suggestive of a role for stabilised microtubules in protrusions at convex regions. These indications of unique characteristics to the protrusions promoted by convex curvature give added support to the curvature-protrusion feedback model, and its relevance to tissue morphogenesis. In summary, this work provides evidence against a previously-published suggested mechanism for the curvature-protrusion feedback loop that is proposed to act during epithelial morphogenesis, and evidence in support of a role for a calcium-based mechanism in driving the initiation and maintenance of leader cells in migrating epithelial sheets. Further work is called for in characterising the protrusions promoted by convex curvature, and the mechanisms controlling them. This area is of significance in gaining greater understanding of tissue morphogenesis in pathogenesis and development, and of potential value in tissue engineering applications.

The effect of capsazepine and nonylphenol on cytosolic free Ca2+ concentrations ([Ca2+]i) in MDCK renal tubular cells is unclear. This study explored whether capsazepine and nonylphenol changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-selective fluorescent dye. Capsazepine at concentrations between 10 and 200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partially by 40% after removing extracellular Ca2+. Capsazepine induced Mn2+ quench of fura-2 fluorescence, indirectly implicating Ca2+ entry. Capsazepine-induced Ca2+ influx was not changed by L-type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12-myristate 13-acetate (PMA) and GF109203X]. In Ca2+-free medium, 100microM capsazepine-induced Ca2+ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin-induced Ca2+ release. Nonylphenol also increased [Ca2+]i in a concentration- dependent manner like capsazepine does. Similar response in [Ca2+]i rise can be found by inhibition of phospholipase C and using thapsigargin. Different from capasazpine, the [Ca2+]i rise was inhibited by PMA. At concentrations between 5 and 100microM, nonylphenol killed cells in a concentration-dependent manner. Collectively, in MDCK cells, capsazepine induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non-L-type Ca2+ channels. Nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other cellular storage in a phospholipase C-independent manner.

Made available in DSpace on 2018-08-01T21:35:35Z (GMT). No. of bitstreams: 1 tese_11464_Dissertação Final Guilherme.pdf: 10896079 bytes, checksum: edbcec6d32e63a6cb320b5fc5d9ca7ae (MD5) Previous issue date: 2017-07-21 / As Amebas de Vida Livre (AVL) do gênero Acanthamoeba são protozoários encontrados em todos os ambientes do mundo, e podem causar doenças como Ceratite e Encefalite Granulomatosa. Os avanços no conhecimento dessas amebas e de seu processo invasivo, da sua variabilidade fenotípica e genotípica, bem como o desenvolvimento de metodologias diagnósticas e terapêuticas, só têm sido possíveis devido a ferramenta de cultivo desses microrganismos. No entanto, seu cultivo prolongado e sua passagem em hospedeiros (ou células) podem alterar a maquinaria celular e modificar mecanismos relacionados a patogenicidade, tornando-os amebas mais ou menos virulentas. No presente trabalho, procurouse investigar características in vitro associadas à patogenicidade de Acanthamoeba castetellanii (Genótipo T4), utilizando quatro amostras de um mesmo isolado clínico, originado de raspado de córnea, a fim de investigar sua virulência. Células epiteliais da linhagem MDCK foram utilizadas para verificar o efeito citopático. Também foi avaliado o meio onde as amebas foram cultivadas (meio condicionado) visando verificar a atividade de proteases em gel e o efeito citotóxico gerado em culturas de MDCK. Foram verificadas as porcentagens de encistamento e a resposta da cultura de amebas a partir da exposição à clorexidina. Os dados obtidos nesse trabalho confirmaram o potencial patogênico das amostras, especialmente aquelas que sofreram passagem em MDCK. O meio condicionado não produziu efeito citotóxico significativo sobre a monocamada de células. O perfil zimográfico evidenciou um padrão muito parecido em todas as amostras testadas, diferenciando apenas a amostra de longo período de axenização, havendo o aumento de uma banda de aproximadamente 133 kDa. Foi verificado que a porcentagem de encistamento das amostras recém axenizadas foi maior que as de longo período de axenização, sendo que esta última amostra obteve taxas maiores de encistamento após interação com MDCK. O teste com a clorexidina demonstrou que, apesar da amostra ser oriunda de uma mesma fonte, as condições de manutenção das amostras levaram a comportamentos distintos. Neste trabalho foi observado que após nove anos de cultivo axênico, a ameba perde virulência, contudo a recupera após passagem em células, como a MDCK. Colaborando para que os estudos em Acanthamoeba avancem demonstrando a potencialidade plástica da ameba em gerir seu perfil patogênico.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Helena Zálešáková Supervisor: PharmDr. Marie Vopršálová, CSc. Title of diploma thesis: In vitro Methods for the Prediction of Blood Brain Barrier Penetration This thesis deals with the correlation between two in vitro models simulating the blood- brain barrier (HEB, hematoencephalic barrier) and their comparison in terms of practical use. These are the PAMPA (Parallel Artificial Membrane Permeability Assay) method and the MDCK (Madin-Darby Canine Kidney) cell line, which are models for potential central nervous system (CNS) penetration screening. Within this work, a set of sixteen standard drugs were measured. The procedure was similar in both methods in order to obtain information on the amount of test substances passing through the membrane from the donor portion of the plate to the acceptor. The concentration in the donor portion was measured by UV-VIS spectrophotometry. The main difference between these methods is the membrane through which the substances penetrate. In the case of PAMPA, a lipid solution that has been isolated from pig brain (PBL, polar brain lipid) is used. This lipid simulates the phospholipid membrane of the brain capillary endothelium. In the MDCK model, the membrane...

Cell competition is a form of cell-cell signalling that results in the elimination of less fit cells from a tissue by their fitter counterparts. I take advantage of an established in vitro model of cell competition using Madin-Darby canine kidney (MDCK) cells to shed insight into the molecular basis of cell competition in epithelial cells. In this system, silencing of the tumour suppressor scribble (scribKD) results in a 'loser' phenotype whereby scribKD cells are specifically eliminated from the monolayer by surrounding wild-type cells. More specifically, scribKD cells are compacted into tight clones through activation of a directed, collective migration in the wild-type population: scribKD are 'mechanical losers' and delaminate and die due to an intrinsic hypersensitivity to high cell density. Remarkably, p53 activation is both necessary and sufficient for this mechanical loser cell status. I first investigate the role of E-, N-, and P-cadherin in the directed migration between scribKD and wild-type cells and in scribKD cell loser status. I show that differential expression of E-cadherin between scribKD losers and wild-type winners is required but not sufficient for directed migration and has no impact on loser cell status. I also show that elevation of neither E-cadherin nor N-cadherin is sufficient to induce directed migration or loser status, but that P-cadherin may play a role in both. I next focus on translating findings about the molecular details of competition from the scribKD set-up into a system where p53 differences alone drive the formation and elimination of mechanical losers. I show that the ROCK - P-p38 - p53 pathway activated in response to mechanical compaction in scribKD cells is conserved in p53-driven losers. In the latter part of my thesis, I characterise the directed migration observed during MDCK competition by drawing parallels to canonical leader-follower migration. Canonical leader cells emerge when epithelial sheets are wounded and, by becoming migratory, drive collective cell migration of follower cells, which results in wound closure. It was not known what confers the leader cell fate. I show that p53 and its effector p21 (and potentially other cyclin-dependent kinase inhibitors) are the key drivers of leader cell migration. I demonstrate that p53-induced leaders use the same molecular pathways that have been shown to drive leader cell migration during wound healing and, in fact, p53 and p21 are also elevated in leaders generated by wounding. Importantly, I establish that p53 activity drives efficient wound closure. Lastly, I show that leader cells are often eliminated by cell competition in the final stages of wound closure, as their elevated p53 mediates their hypersensitivity to density. The model incorporating these data proposes that cellular damage during wounding generates cells with elevated p53, which become leaders and drive wound healing, but these are then cleared once the wound is closed because their high p53 levels cause them to become mechanical losers.

Abstract The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+-sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 mM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+-induced [Ca2+]i increase by 67%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+-free medium, the Hg2+-induced [Ca2+]i increase was nearly abolished by pretreatment with 1 mM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+-induced Ca2+ release was not altered by inhibition of phospholiase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 mM Hg2+ did not alter cell proliferation rate, but 10 mM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.

Anillin is a highly conserved multi-domain cytoskeletal protein that provides a spatial and temporal scaffold for contractile ring proteins to ensure successful cytokinesis. We have looked at the temporal order of anillin and septin recruitment to the cleavage furrow using time-lapse microscopy and found that anillin localizes to the furrow in early anaphase while septins appear there later in an anillin-dependent manner. We also characterized the effect of anillin depletion in different cell lines and observed that septins and myosin delocalize in the absence of anillin in Tet-ON HeLa, AD293 and ARPE-19 cells but not in wild type HeLa cells. Asymmetric furrow formation was also investigated using the epithelial cell model: MDCK cells. Depletion of anillin and SEPT9 in MDCK cells was achieved using lentivirus shRNA constructs and this revealed that anillin or SEPT9 depletion did not affect asymmetric cytokinesis, although localization of SEPT 9 was affected by anillin depletion.

Anillin is a highly conserved multi-domain cytoskeletal protein that provides a spatial and temporal scaffold for contractile ring proteins to ensure successful cytokinesis. We have looked at the temporal order of anillin and septin recruitment to the cleavage furrow using time-lapse microscopy and found that anillin localizes to the furrow in early anaphase while septins appear there later in an anillin-dependent manner. We also characterized the effect of anillin depletion in different cell lines and observed that septins and myosin delocalize in the absence of anillin in Tet-ON HeLa, AD293 and ARPE-19 cells but not in wild type HeLa cells. Asymmetric furrow formation was also investigated using the epithelial cell model: MDCK cells. Depletion of anillin and SEPT9 in MDCK cells was achieved using lentivirus shRNA constructs and this revealed that anillin or SEPT9 depletion did not affect asymmetric cytokinesis, although localization of SEPT 9 was affected by anillin depletion.