Characterization of ibrutinib's effects on the morphology, proliferation, phenotype, viability, and anti-inflammatory potential of adipose-derived mesenchymal stromal cells.

Ibrutinib (IB) is a tyrosine kinase inhibitor (TKI) that has immunomodulatory action and can be used as second-line therapy for steroid-refractory or steroid-resistant chronic Graft versus Host Disease (cGVHD). Mesenchymal stromal cells (MSCs) are distributed throughout the body and their infusion has also been explored as a second-line therapeutic alternative for the treatment of cGVHD. Considering the currently unknown effects of IB on endogenous MSCs, as well as the possible combined use of IB and MSCs for cGVHD, we investigated whether adipose tissue-derived MSCs present IB-targets, as well as the consequences of treating MSCs with this drug, regarding cell viability, proliferation, phenotype, and anti-inflammatory potential. Interestingly, we show for the first time that MSCs express several IB target genes. Also of note, the treatment of such cells with this TKI elevated the levels of CD90 and CD105 surface proteins, as well as VCAM-1. Furthermore, IB-treated MSCs presented increased mRNA expression of the anti-inflammatory genes PD-L1, TSG-6, and IL-10. However, continued exposure to IB, even at low doses, compromised the viability of MSCs. These data indicate that the use of IB can stimulate an anti-inflammatory profile in MSCs, but also that a continued exposure to IB can compromise MSC viability over time.

Senescence on Dental Pulp Cells: Effects on Morphology, Migration, Proliferation, and Immune Response.

INTRODUCTION: Evidence indicates that senescence can affect essential dental pulp functions, such as defense capacity and repair, consequently affecting the successes of conservative endodontic treatments. This study aims to evaluate the effects of senescence on the morphology, migration, proliferation, and immune response of human dental pulp cells. METHODS: Cells were treated with doxorubicin to induce senescence, confirmed by ß-galactosidase staining. Morphological changes, cellular proliferation, and migration were evaluated by scanning electron microscopy, trypan blue cells, and the scratch method, respectively. Modifications in the immune response were evaluated by measuring the genes for pro-inflammatory cytokines tumor necrosis factor alpha and interleukin (IL)-6 and anti-inflammatory cytokines transforming growth factor beta 1 and IL-10 using the real time polymerase chain reaction assay. RESULTS: An increase in cell size and a decrease in the number of extensions were observed in senescent cells. A reduction in the proliferative and migratory capacity was also found in senescent cells. In addition, there was an increase in the gene expression of inflammatory cytokines tumor necrosis factor alpha and IL-6 and a decrease in the gene expression of IL-10 and transforming growth factor beta-1, suggesting an exacerbated inflammatory situation associated with immunosuppression. CONCLUSIONS: Cellular senescence is possibly a condition that affects prognoses of conservative endodontic treatments, as it affects primordial cellular functions related to this treatment.

Lipid Metabolism Modulation during SARS-CoV-2 Infection: A Spotlight on Extracellular Vesicles and Therapeutic Prospects.

Extracellular vesicles (EVs) have a significant impact on the pathophysiological processes associated with various diseases such as tumors, inflammation, and infection. They exhibit molecular, biochemical, and entry control characteristics similar to viral infections. Viruses, on the other hand, depend on host metabolic machineries to fulfill their biosynthetic requirements. Due to potential advantages such as biocompatibility, biodegradation, and efficient immune activation, EVs have emerged as potential therapeutic targets against the SARS-CoV-2 infection. Studies on COVID-19 patients have shown that they frequently have dysregulated lipid profiles, which are associated with an increased risk of severe repercussions. Lipid droplets (LDs) serve as organelles with significant roles in lipid metabolism and energy homeostasis as well as having a wide range of functions in infections. The down-modulation of lipids, such as sphingolipid ceramide and eicosanoids, or of the transcriptional factors involved in lipogenesis seem to inhibit the viral multiplication, suggesting their involvement in the virus replication and pathogenesis as well as highlighting their potential as targets for drug development. Hence, this review focuses on the role of modulation of lipid metabolism and EVs in the mechanism of immune system evasion during SARS-CoV-2 infection and explores the therapeutic potential of EVs as well as application for delivering therapeutic substances to mitigate viral infections.

Evaluation of the common pauraque (Nyctidromus albicollis) cornea using light and scanning electron microscopy.

The common pauraque Nyctidromus albicollis (Gmelin, 1789) is a widespread avian species; however due to its nocturnal habits and reclusive behaviour, little is known about their vision and ecology. Most avian species are visually dependent with advanced visual systems providing high spatial resolution, on the species needs. Each ocular structure has a specific role in contributing towards high visual function, and the cornea is the first refractive structure in the visual process. However, the common pauraque cornea had not been described until the present data. Therefore, this study aims to describe the morphology and morphometry of the common pauraque cornea by means of light and scanning electron microscopy to evaluate the cross-sectional anatomy as well as the ultrastructure of the endothelial cells. Histological similarities with the cornea of other birds have been observed, but the thickness of the common pauraque cornea is much smaller than the other described corneas. A better understanding of the common pauraque cornea can help us better explain the physiology of vision and the visual requirements of this species. In turn, this will help us better understand how this species successfully interacts with its environment, and will improve our knowledge on how to interpret pathological changes in their cornea in a clinical setting.

Host defense peptides combined with MTA extract increase the repair in dental pulp cells: in vitro and ex vivo study.

Host Defense Peptides (HDPs) have, in previous studies, been demonstrating antimicrobial, anti-inflammatory, and immunomodulatory capacity, important factors in the repair process. Knowing these characteristics, this article aims to evaluate the potential of HDPs IDR1018 and DJK-6 associated with MTA extract in the repair process of human pulp cells. Antibacterial activity of HDPs, MTA and HDPs combined with MTA in Streptococcus mutans planktonic bacteria and antibiofilm activity was evaluated. Cell toxicity was assayed with MTT and cell morphology was observed by scanning electron microscopy (SEM). Proliferation and migration of pulp cells were evaluated by trypan blue and wound healing assay. Inflammatory and mineralization related genes were evaluated by qPCR (IL-6, TNFRSF, DSPP, TGF-ß). Alkaline phosphatase, phosphate quantification and alizarin red staining were also verified. The assays were performed in technical and biological triplicate (n = 9). Results were submitted for the calculation of the mean and standard deviation. Then, normality verification by Kolmogorov Smirnov test, analyzing one-way ANOVA. Analyses were considered at a 95% significance level, with a p-value < 0.05. Our study demonstrated that HDPs combined with MTA were able to reduce biofilms performed in 24 h and biofilm performed over 7 days S. mutans biofilm (p < 0.05). IDR1018 and MTA, as well as their combination, down-regulated IL-6 expression (p < 0.05). Tested materials were not cytotoxic to pulp cells. IDR1018 induced high cell proliferation and combined with MTA induced high cellular migration rates in 48 h (p < 0.05). Furthermore, the combination of IDR1018 and MTA also induced high expression levels of DSPP, ALP activity, and the production of calcification nodules. So, IDR-1018 and its combination with MTA could assist in pulp-dentine complex repair process in vitro.

Lipid droplets as multifunctional organelles related to the mechanism of evasion during mycobacterial infection.

Tuberculosis (TB) is an infectious disease caused by the bacteria of the Mycobaterium tuberculosis (Mtb) complex. The modulation of the lipid metabolism has been implicated in the immune response regulation, including the formation of lipid droplets (LD)s, LD-phagosome association and eicosanoid synthesis. Mtb, M. bovis BCG and other pathogenic mycobacteria, as well as wall components, such as LAM, can induce LDs formation in a mechanism involving surface receptors, for instance TLRs, CD36, CD14, CD11b/CD18 and others. In addition, the activation of the lipid-activated nuclear receptor PPARγ is involved in the mechanisms of LD biogenesis, as well as in the modulation of the synthesis of lipid mediators. In infected cells, LDs are sites of compartmentalized prostaglandin E2 synthesis involved in macrophage deactivation, bacterial replication and regulation of the host cytokine profile. LDs also have a function in vesicle traffic during infection. Rab7 and RILP, but not Rab5, are located on LDs of infected macrophages, suggesting that LDs and phagosomes could exchange essential proteins for phagosomal maturation, interfering in mycobacterial survival. The pharmacological inhibition of LDs biogenesis affects the bacterial replication and the synthesis of lipid mediators and cytokines, suggesting that LDs may be new targets for antimicrobial therapies. However, it is still controversial if the accumulation of LDs favors the mycobacterial survival acting as an escape mechanism, or promotes the host resistance to infection. Thus, in this mini-review we discuss recent advances in understanding the important role of LDs in the course of infections and the implications for the pathophysiology of mycobacteriosis.

Regulatory T-Cell Enhancement, Expression of Adhesion Molecules, and Production of Anti-Inflammatory Factors Are Differentially Modulated by Spheroid-Cultured Mesenchymal Stem Cells.

The culture of mesenchymal stem cells (MSCs) as spheroids promotes a more physiological cellular behavior, as it more accurately reflects the biological microenvironment. Nevertheless, mixed results have been found regarding the immunosuppressive properties of spheroid-cultured MSCs (3D-MSCs), the mechanisms of immunoregulation of 3D-MSCs being scarcely described at this point. In the present study, we constructed spheroids from MSCs and compared their immunosuppressive potential with that of MSCs cultured in monolayer (2D-MSCs). First, we evaluated the ability of 2D-MSCs and 3D-MSCs to control the activation and proliferation of T-cells. Next, we evaluated the percentage of regulatory T-cells (Tregs) after the co-culturing of peripheral blood mononuclear cells (PBMCs) with 2D-MSCs and 3D-MSCs. Finally, we investigated the expression of adhesion molecules, as well as the expressions of several anti-inflammatory transcripts in 2D-MSCs and 3D-MSCs maintained in both inflammatory and non-inflammatory conditions. Interestingly, our data show that several anti-inflammatory genes are up-regulated in 3D-MSCs, and that these cells can control T-cell proliferation. Nevertheless, 2D-MSCs are more efficient in suppressing the immune cell proliferation. Importantly, contrary to what was observed in 3D-MSCs, the expressions of ICAM-1 and VCAM-1 are significantly upregulated in 2D-MSCs exposed to an inflammatory environment. Furthermore, only 2D-MSCs are able to promote the enhancement of Tregs. Taken together, our data clearly show that the immunosuppressive potential of MSCs is significantly impacted by their shape, and highlights the important role of cell-cell adhesion molecules for optimal MSC immunomodulatory function.

Correction: Exploratory comparisons between different anti-mitotics in clinically-used drug combination in triple negative breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.28068.].

Impact of the Extracellular Vesicles Derived From Trypanosoma cruzi: A Paradox in Host Response and Lipid Metabolism Modulation.

Chagas disease is a major public health problem, especially in the South and Central America region. Its incidence is related to poverty and presents a high rate of morbidity and mortality. The pathogenesis of Chagas disease is complex and involves many interactive pathways between the hosts and the Trypanosoma cruzi. Several factors have been implicated in parasite-host interactions, including molecules secreted by infected cells, lipid mediators and most recent, extracellular vesicles (EVs). The EVs of T. cruzi (EVsT) were reported for the first time in the epimastigote forms about 42 years ago. The EVsT are involved in paracrine communication during the infection and can have an important role in the inflammatory modulation and parasite escape mechanism. However, the mechanisms by which EVs employ their pathological effects are not yet understood. The EVsT seem to participate in the activation of macrophages via TLR2 triggering the production of cytokines and a range of other molecules, thus modulating the host immune response which promotes the parasite survival. Moreover, new insights have demonstrated that EVsT induce lipid body formation and PGE2 synthesis in macrophages. This phenomenon is followed by the inhibition of the synthesis of pro-inflammatory cytokines and antigen presentation, causing decreased parasitic molecules and allowing intracellular parasite survival. Therefore, this mini review aims to discuss the role of the EVs from T. cruzi as well as its involvement in the mechanisms that regulate the host immune response in the lipid metabolism and its significance for the Chagas disease pathophysiology.

Exploratory comparisons between different anti-mitotics in clinically-used drug combination in triple negative breast cancer.

Triple-negative breast cancer (TNBC) constitutes a very aggressive type of breast cancer with few options of cytotoxic chemotherapy available for them. A chemotherapy regimen comprising of doxorubicin hydrochloride and cyclophosphamide, followed by paclitaxel, known as AC-T, is approved for usage as an adjuvant treatment for this type of breast cancer. In this study we aimed to elucidate the role of KIF11 in TNBC progression throughout its inhibition by two synthetic small molecules containing the DHPM core (dihydropyrimidin-2(1H)-ones or -thiones), with the hypothesis that these inhibitors could be an interesting option of antimitotic drug used alone or as adjuvant therapy in association with AC. For this purpose, we evaluated the efficacy of DHPMs used as monotherapy or in combination with doxorubicin and cyclophosphamide, in Balbc-nude mice bearing breast cancer induced by MDA-MB-231, having AC-T as positive control. Our data provide extensive evidence to demonstrate that KIF11 inhibitors showed pronounced antitumor activity, acting in key points of tumorigenesis and cancer progression in in vivo xenograft model of triple negative breast cancer, like down-regulation of KIF11 and ALDH1-A1. Moreover, they didn't show the classic peripheral neuropathy characterized by impaired mobility, as it is common with paclitaxel use. These results suggest that the use of a MAP inhibitor in breast cancer regimen treatment could be a promising strategy to keep antitumoral activity reducing the side effects.

From cow manure to bioactive carbon dots: a light-up probe for bioimaging investigations, glucose detection and potential immunotherapy agent for melanoma skin cancer.

Bioactive carbon dots (C-dots) with ca. 4 nm were successfully produced with singular photophysical properties, low-toxicity and interesting immunological response. The optical properties of the C-dots were investigated and the "light-up" behaviour enabled them to be explored in glucose detection and bioimaging experiments (mitochondrial selective probe). C-dots were not selective to the tumour region and several fluorescent spots were visualized spread on animal bodies. The histology investigations showed that cancer-bearing mice treated with C-dots presented a large number of regions with necrosis and inflammatory infiltrates, which were not identified for cancer-bearing mice without the treatment. These results suggested that C-dots have the potential to be explored as an immune therapy agent for melanoma skin cancer.

Rhamnolipids production from sucrose by engineered Saccharomyces cerevisiae.

Biosurfactants are biological tensioactive agents that can be used in the cosmetic and food industries. Rhamnolipids are glycolipid biosurfactants naturally produced by Pseudomonas aeruginosa and are composed of one or two rhamnose molecules linked to beta-hydroxy fatty acid chains. These compounds are green alternatives to petrochemical surfactants, but their large-scale production is still in its infancy, hindered due to pathogenicity of natural producer, high substrate and purification costs and low yields and productivities. This study, for the first time, aimed at producing mono-rhamnolipids from sucrose by recombinant GRAS Saccharomyces cerevisiae strains. Six enzymes from P. aeruginosa involved in mono-rhamnolipid biosynthesis were functionally expressed in the yeast. Furthermore, its SUC2 invertase gene was disrupted and a sucrose phosphorylase gene from Pelomonas saccharophila was also expressed to reduce the pathway's overall energy requirement. Two strains were constructed aiming to produce mono-rhamnolipids and the pathway's intermediate dTDP-L-rhamnose. Production of both molecules was analyzed by confocal microscopy and mass spectrometry, respectively. These strains displayed, for the first time as a proof of concept, the potential of production of these molecules by a GRAS eukaryotic microorganism from an inexpensive substrate. These constructs show the potential to further improve rhamnolipids production in a yeast-based industrial bioprocess.

Correction to: The role of pparγ and autophagy in ros production, lipid droplets biogenesis and its involvement with colorectal cancer cells modulation.

[This corrects the article DOI: 10.1186/s12935-017-0451-5.].

The role of pparγ and autophagy in ros production, lipid droplets biogenesis and its involvement with colorectal cancer cells modulation.

BACKGROUND: In cancer cells, autophagy can act as both tumor suppressor, when autophagic event eliminates cellular contends which exceeds the cellular capacity of regenerate promoting cell death, and as a pro-survival agent removing defective organelles and proteins and helping well-established tumors to maintain an accelerated metabolic state while still dealing with harsh conditions, such as inflammation. Many pathways can coordinate the autophagic process and one of them involves the transcription factors called PPARs, which also regulate cellular differentiation, proliferation and survival. The PPARγ activation and autophagy initiation seems to be interrelated in a variety of cell types. METHODS: Caco-2 cells were submitted to treatment with autophagy and PPARγ modulators and the relationship between both pathways was determined by western blotting and confocal microscopy. The effects of such modulations on Caco-2 cells, such as lipid bodies biogenesis, cell death, proliferation, cell cycle, ROS production and cancer stem cells profiling were analyzed by flow cytometry. RESULTS: PPARγ and autophagy pathways seem to be overlap in Caco-2 cells, modulating each other in different ways and determining the lipid bodies biogenesis. In general, inhibition of autophagy by 3-MA leaded to reduced cell proliferation, cell cycle arrest and, ultimately, cell death by apoptosis. In agreement with these results, ROS production was increased in 3-MA treated cells. Autophagy also seems to play an important role in cancer stem cells profiling. Rapamycin and 3-MA induced epithelial and mesenchymal phenotypes, respectively. CONCLUSIONS: This study helps to elucidate in which way the induction or inhibition of these pathways regulate each other and affect cellular properties, such as ROS production, lipid bodies biogenesis and cell survive. We also consolidate autophagy as a key factor for colorectal cancer cells survival in vitro, pointing out a potential side effect of autophagic inhibition as a therapeutic application for this disease and demonstrate a novel regulation of PPARγ expression by inhibition of PI3K III.

Distinct patterns of yeast cell morphology and host responses induced by representative strains of Paracoccidioides brasiliensis (Pb18) and Paracoccidioides lutzii (Pb01).

Paracoccidioidomycosis (PCM) is a systemic mycosis, widespread in Latin America. PCM is a granulomatous disease characterized by a polymorphism of lesions depending on the pathogen's virulence, the immune status of the host and its genetic susceptibility. The thermodimorphic fungus Paracoccidioides brasiliensis was considered the only etiologic agent of PCM, yet recent works have shown significant genetic diversity among different strains of P. brasiliensis. Therefore, it has been proposed for a new species within the Paracoccidioides genus, named Paracoccidioides lutzii. To better understand the fungus-host interactions elicited by strains Pb01 and Pb18 as key representatives of P. lutzii and P. brasiliensis, respectively, we carried out studies to investigate differences in morphology, induced immune response, virulence and pathology between these two Paracoccidioides species. Our results demonstrate distinct patterns of host-parasite interaction and pathology caused by Pb18 and Pb01. These results open up new fronts for NEW: clinical studies, which may result in significant consequences for the diagnosis and treatment of PCM. Considering that our results cannot be extended to all strains of both species, more studies about the virulence among Paracoccioides must be explored in the future.

Unveiling the Trypanosoma cruzi Nuclear Proteome.

Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions.

Collagen-based silver nanoparticles for biological applications: synthesis and characterization.

BACKGROUND: Type I collagen is an abundant natural polymer with several applications in medicine as matrix to regenerate tissues. Silver nanoparticles is an important nanotechnology material with many utilities in some areas such as medicine, biology and chemistry. The present study focused on the synthesis of silver nanoparticles (AgNPs) stabilized with type I collagen (AgNPcol) to build a nanomaterial with biological utility. Three formulations of AgNPcol were physicochemical characterized, antibacterial activity in vitro and cell viability assays were analyzed. AgNPcol was characterized by means of the following: ultraviolet-visible spectroscopy, dynamic light scattering analysis, Fourier transform infrared spectroscopy, atomic absorption analysis, transmission electron microscopy and of X-ray diffraction analysis. RESULTS: All AgNPcol showed spherical and positive zeta potential. The AgNPcol at a molar ratio of 1:6 showed better characteristics, smaller hydrodynamic diameter (64.34 ± 16.05) and polydispersity index (0.40 ± 0.05), and higher absorbance and silver reduction efficiency (0.645 mM), when compared with the particles prepared in other mixing ratios. Furthermore, these particles showed antimicrobial activity against both Staphylococcus aureus and Escherichia coli and no toxicity to the cells at the examined concentrations. CONCLUSIONS: The resulted particles exhibited favorable characteristics, including the spherical shape, diameter between 64.34 nm and 81.76 nm, positive zeta potential, antibacterial activity, and non-toxicity to the tested cells (OSCC).

Mast cell function and death in Trypanosoma cruzi infection.

Although the roles of mast cells (MCs) are essential in many inflammatory and fibrotic diseases, their role in Trypanosoma cruzi-induced cardiomyopathy is unexplored. In this study, we treated infected CBA mice with cromolyn, an MC stabilizer, and observed much greater parasitemia and interferon-γ levels, higher mortality, myocarditis, and cardiac damage. Although these data show that MCs are important in controlling acute infection, we observed MC apoptosis in the cardiac tissue and peritoneal cavity of untreated mice. In the heart, pericardial mucosal MC die, perhaps because of reduced amounts of local stem cell factor. Using RT-PCR in purified cardiac MCs, we observed that infection induced transcription of P2X(7) receptor and Fas, two molecules reportedly involved in cell death and inflammatory regulation. In gld/gld mice (FasL(-/-)), apoptosis of cardiac, but not peritoneal, MCs was decreased. Conversely, infection of P2X(7)(-/-) mice led to reduced peritoneal, but not cardiac, MC death. These data illustrate the immunomodulatory role played by MCs in T. cruzi infection and the complexity of molecular interactions that control inflammatory pathways in different tissues and compartments.

A silencing suppressor protein (NSs) of a tospovirus enhances baculovirus replication in permissive and semipermissive insect cell lines.

The nonstructural protein (NSs) of the Tomato spotted wilt virus (TSWV) has been identified as an RNAi suppressor in plant cells. A recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) designated vAcNSs, containing the NSs gene under the control of the viral polyhedrin (polh) gene promoter, was constructed and the effects of NSs in permissive, semipermissive and nonpermissive insect cells to vAcNSs infection were evaluated. vAcNSs produced more budded virus when compared to wild type in semipermissive cells. Co-infection of vAcNSs with wild type baculoviruses clearly enhanced polyhedra production in all host cells. Confocal microscopy analysis showed that NSs accumulated in abundance in the cytoplasm of permissive and semipermissive cells. In contrast, high amounts of NSs were detected in the nuclei of nonpermissive cells. Co-infection of vAcNSs with a recombinant AcMNPV containing the enhanced green fluorescent protein (egfp) gene, significantly increased EGFP expression in semipermissive cells and in Anticarsia gemmatalis-hemocytes. Absence of small RNA molecules of egfp transcripts in this cell line and in a permissive cell line indicates the suppression of gene silencing activity. On the other hand, vAcNSs was not able to suppress RNAi in a nonpermissive cell line. Our data showed that NSs protein of TSWV facilitates baculovirus replication in different lepidopteran cell lines, and these results indicate that NSs could play a similar role during TSWV-infection in its thrips vector.

Trypanosoma cruzi: contribuição ao estudo da endocitose dependente e independente de clatrina em formas epimastigotas / Trypanosoma cruzi: contribution to the study of the dependent and independent endocytosis of clatrina in epimastigote forms

Endocitose em células eucarióticas é o processo de incorporação de macromoléculas por diferentes vias, com diversas proteínas associadas. Este processo ocorre através do brotamento de vesículas na membrana plasmática e endereçamento destas vesículas a compartimentos endossomais no citoplasma. Os tripanossomatídeos são protozoários flagelados patogênicos de grande importância médica e veterinária que apresentam diferentes formas adaptativas aolongo de seu ciclo de vida. Estes parasitas estão estruturalmente organizados dentro de um arcabouço de microtúbulos subpeliculares, o que dificulta invaginações de membrana na maior parte do corpo celular. Entretanto, este arcabouço é descontinuado na região da bolsa flagelar, de onde o flagelo emerge do corpo. Formas epimastigotas do Trypanosoma cruzi apresentam além da bolsa flagelar, uma segunda invaginação de membrana, o citóstoma/citofaringe. Esta estrutura é sustentada por microtúbulos especializados que participam ativamente no processo de endocitose. O objetivo desta tese foi investigar as vias endocíticas presentes nos dois sítios competentes para a captação de nutrientes (bolsa flagelar e citóstoma) de epimastigotas de T. cruzi. Após incubação dos parasitas a 28(graus) C com albumina, transferrina e LDL conjugadas a ouro coloidal e posterior processamento para microscopia eletrônica de transmissão (MET), vesículas endocíticas revestidas contendo albumina foram observadas brotando da bolsa flagelar. Através da análise do conteúdo protéico dos protozoários foi detectada a expressão de clatrina, confirmada por citometria de fluxo e análise in silico da base de dados genômicos do T. cruzi. Por microscopia confocal localizou-se a clatrina na região compreendida entre o núcleo e a bolsa flagelar dos parasitas. Como transferrina foi visualizada em vesículas sem revestimento localizadas majoritariamente no citóstoma, frações de membrana detergente-resistentes foram purificadas por fracionamento celular e o...