Orientador: Classius de Oliveira / Resumo: Peixes e anfíbios estão vulneráveis a diversos poluentes. O benzo[a]pireno (BaP) é um contaminante com propriedades tóxicas. Metabolizado pela enzima P450 (CYP1A1), de fase I EROD (Etoxiresorufina-O-deetilase), produz subprodutos tóxicos. Conhecida como um inibidor da CYP1A1, α-naftoflavona (aNF) impede o metabolismo do BaP e a geração de metabólitos tóxicos. A aNF é um composto exógeno e não se tem conhecimento do efeito dela em órgãos e tecidos. O fígado é a maior fonte de enzimas catalizadoras de reações de biotransformação. A suscetibilidade do órgão a danos por agentes químicos é uma conseqüência de seu papel no metabolismo. Porém, animais desenvolveram mecanismos para detectar e responder a esses produtos químicos. Melanomacrófagos (MMs) hepáticos são células que contém melanina e que estão envolvidos nesse processo, devido a sua função de detoxificação. Dessa forma, nossos objetivos foram avaliar os efeitos do BaP e/ou aNF nos MMs e efeitos genotóxicos dos compostos. Os animais foram expostos a 2mg/kg de BaP e/ou 20mg/kg de aNF por 48 horas e 7 dias. Após os experimentos o fígado passou por procedimentos histológicos para quantificação da área de melanina dos MMs e fagocitose na microscopia de luz; microscopia de fluorescência para análise do citoesqueleto dos MMs; espectrofotometria para quantificar a atividade EROD e síntese de melanina dos MMs; e análise genotóxica dos eritrócitos. Em peixes, após 7d houve diminuição da área de melanina, devido a uma inibição do BaP... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Fish and amphibians are vulnerable to various pollutants. Benzo[a]pyrene (BaP) is a contaminant with toxic properties. Metabolized by the enzyme P450 (CYP1A1) produces toxic byproducts. Known as a CYP1A1 inhibitor, α-naphtoflavone (aNF) prevents BaP metabolism and the generation of toxic metabolites. aNF is an exogenous compound and is not known for its effect on organs and tissues. The liver is the major source of catalyzing enzymes for biotransformation reactions. The susceptibility of the organ to damage by chemical agents is a consequence of its role in metabolism. However, animals have developed mechanisms to detect and respond to these chemicals. Melanomacrophages (MMs) are involved in this process due to their detoxification function. Thus, our objectives were to evaluate the effects of BaP and/or aNF on MMs and genotoxic effects of compounds. The animals were exposed to 2mg/kg BaP and/or 20mg/kg aNF for 48 hours and 7 days. After the experiments, the procedures for light microscopy, fluorescence, spectrophotometry and genotoxic analysis were followed. In fish, after 7d there was a decrease in melanin area, due to a inhibition of BaP in the melanogenic pathway; melanosome aggregation by interference in actin filaments; and increased frequency of micronucleus by the genotoxic potential of the compound. In anurans, at 48h, there was an increase in melanin production in BaP treatments, due to the antioxidant role of melanin; decreased phagocytosis by interfering with this... (Complete abstract click electronic access below) / Doutor

1. Abstract ARP2/3 complex is well studied in case of animals, it plays key roles in motility of cells and intracellular organels. It's malfunctions result in severe growth disorders and even lethality of affected cells. On the contrary, plant cells do not exhibit such dramatic phenotype of ARP2/3 complex mutations like it is by animals. It is possible that just the different life strategies of plants and animals contribute to differences in a way how animal and plant cells use their cytoskeleton, where ARP2/3 complex is it's part as well. It is highly conserved 7 protein complex from yeast to human. His main functions are creation of new "de novo" actin filaments, actin branched filaments network. Some of the parasite organisms are capable of missusing its nucleator activity to actively move inside of host cell. Because of the plant cells are sourounded by the cell wall, which give them support in creating various shapes and also hinders active movement of the whole cell body, it is likely that ARP 2/3 complex could be possibly involved in novel plant specific functions as well. If we think about the different life strategy of plants and animals we can not ignore all the things these two kingdoms have in common regarding to cytoskeleton processes. That is the need both for vesicular transport and...

This project examined the role of the cytoskeleton in regulatory mechanisms of the amiloride-sensitive Na⁺ channels in isolated frog skin epithelium. The epithelium from ventral frog skin is a model tissue which has proved significant in our understanding of the basic principles involved in water and Na⁺ homeostasis. In particular, this project examines ways in which local (non-hormonal) and hormonal regulatory mechanisms adjust the Na⁺ permeability of apical membranes of frog skin epithelium. Both mechanisms contain factors that are known to increase the apical membrane Na⁺ permeability mainly by increases in the number of open channels. The origin of these new open channels is unknown but, it is postulated that they could arise either by activation of quiescent channels already present in the apical membrane, or by recruitment of channels from cytoplasmic stores. Regarding the latter hypothesis, we also examined the idea that the cytoskeleton might somehow be involved in the insertion of Na⁺ channels within vesicles, into the apical membrane. This is based on the fact that the cytoskeleton is involved in a similar mechanism whereby, in the toad urinary bladder, anti-diuretic hormone (ADH) causes the insertion of aggregates with water channels. Much current interest focuses on the role of the cytoskeleton in the regulation of epithelial Na⁺ channels. To test this hypothesis, we used noise analysis to examine the effects of disrupting the cytoskeleton, on two different mechanisms which bring about changes in open channel densities. The mechanisms are: (1) lowering mucosal Na⁺ concentration (non-hormonal), and (2) addition of arginine-vasopressin (A VP) (hormonal). Non-hormonal, autoregulatory changes in apical membrane Na⁺ conductance were examined by investigating the effects of reducing the mucosal Na⁺ concentration. Our results showed that lowering the mucosal Na⁺ concentration induced large increases in the open channel density in order to stabilise the transport rate. In addition, we observed an average 55-60% increase in the open channel probability, which implies that in epithelium from Rana fuscigula, changes of channel open probability are also an important mechanism in the autoregulation of channel densities in response to a reduction in mucosal Na⁺. The hormonal control of Na⁺ channels by A VP has been intensively studied by noise analysis and the patch clamp. Our results confirmed previous reports that A VP increases the Na⁺ transport rate by increasing the number of open Na⁺ channels, primarily through large changes in the total number of channels, without a significant change in open probability. Regarding the role of the cytoskeleton in regulation of Na⁺ channels and/or its possible role in control of inserting putative vesicles with Na⁺ channels, we studied the effects of disrupting the cytoskeleton on the two regulatory mechanisms. Disrupting microtubules with colchicine had no, or very little effect on either of the regulatory mechanisms. On the other hand, the integrity of the microfilaments was very important for the autoregulatory changes in the number of open channels. After cytochalasin B treatment, lowering the mucosal Na⁺ concentration did not result in the usual compensatory changes in channel densities. There was no prior evidence that cytochalasin B had any actual effect on the F-actin network in the frog skin epithelium. Accordingly, modified cytochemical techniques were designed to demonstrate and localise F-actin in the epithelial granular cells. The direct immunofluorescent method proved useful, but did not allow sufficient resolution to examine the changes to different populations of actin in the cells. We then modified an immunogold method to suit our conditions, and the results demonstrated the localisation of different pools of F-actin and showed the effects of the cytochalasin B and vasopressin.

Diabetes has gained growing attendance as one of the key non communicable diseases (NCD) with the World Health Organization identifying it as the focus of the World Health Day 2016. It is reported that more than 420 million people suffer from diabetes, a number predicted to rise in the coming years. This report forms part of a broader, long term focus project that aims to establish a systems approach to type 2 diabetes (T2D), the variant that accounts for more than 90% of reported diabetes cases. The broader project objectives are to identify possible biomarkers for the onset and the progression of T2D as a precursor to enable potential future approaches to delay onset, or even reverse disease states, via active bio-compounds and/or establishment of beneficial nutritional patterns. The 6-month master’s work reported here is sub-project that focused specifically on cell level vesicle trafficking processes. These processes are believed to be crucial in understanding the formation amyloid plaques, which compromise or kill the insulin secreting beta cells. Up until now, there has been a lack of appropriate experimental techniques to directly observe this process in live cells.  Hence we have developed 2 new techniques: (i)               a method of imaging the actin and tubulin network reorganization during exocytosis of the insulin containing granules while exploring novel ways of characterizing the network. (ii)             a method of imaging the granules themselves and using particle tracking microrheology to analyze their movement patterns during stimulation with glucose. These new techniques open the door to follow up experiments which would allow development of a cell scale mathematical model or simulation correlating short term glucose dynamics to risk of amyloid plaque formation and T2D.

The burden of osteoporosis and low bone mass is unrelenting, affecting over 50% of the U.S. population over the age of 50. In a similar reach but different clinical realm, nearly 40% of all men and women will be diagnosed with cancer at some point during their lifetimes. The impact of both of these diseases is compounded by the limited knowledge of cellular mechanisms and the insufficiency of effective treatment options. At the microscopic level of the cell cytoskeleton, increasing evidence has led researchers to further explore microtubules (MTs) and MT-based structures, such as primary cilia, as potential keys to unlocking improved treatment options. However, the way in which microtubules regulate the processes giving rise to these diseases remains a critical gap in knowledge. The works outlined here aimed to elucidate mechanisms that may be used to combat diseases attacking the skeletal and immune systems. In order to characterize the influence of primary cilia with respect to osteoclast differentiation, we implemented a series of treatments to an immortalized macrophage cell line: cilia lengthening (using Fenoldopam) and mechanical stimulation (using oscillatory fluid flow). The results were analyzed by a combination of immunocytochemistry and quantitative PCR. Our first result showed definitively that while osteoclasts do not possess primary cilia, their macrophage precursors do. We also discovered that these macrophage primary cilia are dynamic and can be modulated; cells whose cilia had been lengthened showed a significant decrease in osteoclast formation, indicating that macrophage cilia resorption may be a necessary step for osteoclast differentiation to occur. Combined with findings from previous studies, there is increasing evidence that the primary cilium, as a therapeutic target for bone diseases, may offer a dual beneficial approach to both promote bone formation and downregulate osteoclast activity. We then explored the possibility of directional MT translocation during T-cell activation being linked to Rho GTPases, which regulate actin polymerization. WASp and WAVE2, known to have functional roles in T-cell activation, were identified as primary candidates. In order to investigate this relationship, we implemented a stepwise micropatterning procedure by which PDMS was used to transfer local areas of activation (presenting fluorescently-tagged antibodies against CD3 and CD28) which, upon T-cell receptor (TCR) triggering, could mimic immune synapse (IS) formation. We showed that, although there was no correlation between the spatial organization of MTs and WASp, MTs and WAVE2 location were highly correlated, providing strong evidence for a link between these two systems. In addition, MT disruption via nocodazole resulted in a significant decrease in T-cell activation and mechanosensing capabilities. Given the role of WAVE2 in promoting cell spreading and adhesion during IS formation, this result provides additional evidence that this cytoskeletal filament is in fact connected to proteins involved in actin nucleation and elongation. We anticipate the work in Aim 1 to help reveal a previously unexplored therapeutic target for osteoporosis, a disease that currently has no clinical manifestations prior to a fracture event. Further investigation has the potential to contribute to diagnosis and prevention techniques, as well as new treatments. Similarly, given the emergence of adoptive T-cell immunotherapy for immune-related disorders, the findings of Aim 2 will advance our understanding of both the biological and mechanical influence of the cytoskeleton and motivate microtubules as one component of a more comprehensive armamentarium of treatment approaches.

Metastatic cancer is more dangerous and difficult to treat than pre-metastatic cancer. Ninety percent of cancer-related deaths are caused by metastatic cancer. When cells go through metastases, they go through changes that allow them to break away from the primary tumor and invade secondary tissues. These changes, in lipid membrane composition and cellular glycocalyx, make the cell more resistant to therapeutics. Actin cytoskeleton contractility plays a major role in these changes, as increased contractility has been linked to upregulation of phosphoinositides and production of glycoproteins. Light induced molecular adsorption of proteins (LIMAP) was used to control the actin arrangement and cell shape in order to mimic and study metastatic cells. Negatively charged proteins electrostatically adhere to the surface in order to create patterns for the cells to stick. Neutravidin was conjugated to poly(glutamic acid) to improve attachment to the surface. We observed differences in cell shape and phosphoinositide behavior based on LIMAP patterning. Additionally, expression of key glycoproteins related to cancer metastasis increased with increased actin contractility. The actin cytoskeleton was the main driver of changes to the cell membrane and glycocalyx.

Doctor Scientiae / The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC. The second publication addresses and reports on the nanosized detail of BEC monolayer morphology utilizing high-resolution scanning electron microscopy (HR-SEM) and published the first descriptions of the extrusion of a basement membrane from developing in vitro BECs. Moreover, we categorized and discussed two types of nanotubule (NT) development specific for the establishment of the BEC monolayers. NTs can occur via nanovesicle extrusion onto the BEC membrane surfaces, which fuse, forming tunneling NTs (TUNTs) between adjacent BECs. Furthermore, cytoplasmic extensions of BEC membrane leading edges give rise to tethering NT (TENTs), which result in overlapping regions across the PC spaces, resulting in PC occlusion. BEC NT communication is illuminated in a third publication utilizing immunofluorescence microscopy, which reports on the molecular, cytoskeletal elements governing NT formation. This study shows, for the first time, f-actin and α-tubulin cytoskeletal proteins extending between the soma of the cells and NT cytoskeletal structures within an in vitro BBB model. Thereafter, the effects depolymerizing agents, Cytochalasin D and Nocodazole, were investigated on f-actin and α-tubulin cytoskeletal protein generation,functionality of NT morphology, cell division and permeability. For the first time, we show that f-actin possesses an additional function, key to tight junction, plaque protein organization. Moreover, it facilitates TENT formation, essential for cytoplasmic projection across PC spaces. Conversely, α-tubulin facilitates known functions: (i) transportation, (ii) cytokinesis, (iii) cellular division, and (iv) possesses a novel function as the molecular cytoskeletal backbone of TENTs, which facilitates BBB impermeability. A critical review evaluates past literature, in light of the current findings emanating from this study. The review critiques the concept of BEC cilia, which have been reported in the literature, comprised of tubulin and actin, but at low-resolution. In the light of our novel observations, nowhere in transmission electron microscopy do we observe cilia on the BECs, we postulate that NTs have been misnamed and mischaracterized as cilia. The thesis endeavors to elucidate the complexity of BEC nanostructures by examining the emerging role of the nanoscopic landscape of BBB development and the changing nature of BEC morphology, NT formation and associated cytoarchitectural underpinnings governing NT morphology. The research study attempts to, with a view to create new avenues for treating brain pathology, revolutionize our interpretation of barrier-genesis on a nanoscale.

The hypothesis that irreversible ischemic injury is related to sub-sarcolemmal blebbing and an inherent osmotic fragility of the blebs was tested by subjecting isolated control and ischemically preconditioned (IPC) or calyculin A (CalA)-pretreated (protected) rabbit cardiomyocytes to ischemic pelleting followed by resuspension in 340, 170 or 85 mosmol medium containing trypan blue. At time points from 0-240 min, osmotic fragility was assessed by the percentage of trypan blue permeable cells. Membrane blebs were visualized with India ink preparations. Bleb formation, following acute hypo-osmotic swelling, developed by 75 min and increased with longer periods of ischemia. Osmotic fragility developed only after 75 min. Cells resuspended in 340 mosmol media did not form blebs and largely retained the ability to exclude trypan blue, even after 240 min ischemia. Although the latent tendency for osmotic blebbing preceded the development of osmotic fragility, most osmotically fragile cells became permeable without evident sarcolemmal bleb formation. The onset of osmotic fragility was delayed in protected cells, but protection did not reduce the bleb formation. It is concluded that blebbing and osmotic fragility are independent manifestations of ischemic injury. The principal locus of irreversible ischemic injury and the protection provided by IPC may lie within the sarcolemma rather than at sarcolemmal attachments to underlying adherens junctions.

No description available.

Classically considered as a soluble system of enzymes, glycolysis does not conform to the known function and subcellular microcompartmentation pattern. Certain glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) could be found at different cellular locations in animal cells, where it exhibited its non-glycolytic activities. Determination of the subcellular localization of two cytosolic GAPDH isoforms from Arabidopsis thaliana (GapC1 and GapC2), fused to Fluorescent Proteins (FP), was investigated in the transiently transformed mesophyll protoplasts, using confocal Laser Scanning Microscopy. Apart from its cytosolic distribution, the nuclear compartmentation of GapC:FP was observed in this study, as well as its punctuate or aggregate-like localization. Part of the GapC:FP foci were observed as mitochondria-associated. A further yeast two-hybrid screen with both GapC isoforms as baits allowed to identify the mitochondrial porin (VDAC3; At5g15090) as a protein-protein interaction partner. Further tests with one-on-one yeast two-hybrid and Bimolecular Fluorescence Complementation (BiFC) assays showed that the detected binding between plant VDAC3 and GapC in yeast cells was false positive. Interestingly, aldolase interacted with VDAC3, as well as with GapC in plant protoplasts, using the BiFC method. On the other hand, no such interaction could be detected in the one-on-one yeast two-hybrid assay. Thus, a model of indirect mitochondrial association of GapC via aldolase that binds directly to mitochondrial porin is proposed to occur only upon certain cellular conditions. Attempts to show the binding of Arabidopsis GAPDH to the actin cytoskeleton in vivo failed, whereas in vitro cosedimentation assays demonstrated that the fully active, recombinant glycolytic enzyme binds to rabbit F-actin. Moreover, is the presence of the GapC cofactor NAD and a reducing agent (DTT), the enzyme might exhibit an actin-bundling activity.