Blocking tumor exosome release using nanovectorization systems

Exosomes are small membrane vesicles secreted by most cell types, either normal or malignant and are found in most body fluids such as saliva, plasma and breast milk. In the past decade, the interest in these vesicles has been growing more and more since it was found that besides their beneficial functions such as the removal of cellular debris and unnecessary proteins during cell maturation process, they can also interact with other cells and transfer information between them, thus helping diseases like cancer to progress. The present work intended to use gold nanoparticles as vehicles for gene silencing in an attempt to reduce the tumor-derived exosome secretion, regulated by Rab27a protein, and also aimed to compare the exosome secretion between two breast cell lines, MCF7 and MDA. Changes in RAB27A gene expression were measured by Real-time Quantitative PCR and it was revealed a decreased in RAB27A gene expression, as expected. Exosomes were isolated and purified by two different methods, ultracentrifugation and the commercial kit ExoQuick™ Solution, and further characterized using Western Blot analysis. ExoQuick™ Solution was proven to be the most efficient method for exosome isolation and it was revealed that MDA cells secrete more exosomes. Furthermore, the isolated MCF7-derived exosomes were placed together with a normal bronchial/tracheal epithelial cell line (BTEC) for an additional assay, which aimed to observe the uptake of exosomes by other cells and the exosomes’ capability of promoting cell-cell communication. This observation was made based on alterations in the expression levels of c-Myc and miR-21 genes and the fact that they both have an increased expression in BTEC cells incubated with tumor-derived exosomes when compared to control cells (without incubation with the exosomes) lead us to the conclusion that the exosome uptake and exchange of information between the exosomes and the normal cells did occurred.

Peptaibolin analogues by incorporation of α,α-dialkylglycines: synthesis and study of their membrane permeating ability

Analogues of Peptaibolin, a peptaibol with antibiotic activity, incorporating α,α-dialkylglycines (Deg, Dpg, and Ac6c) at selected positions were synthesised by MW-SPPS and fully characterized. A control analogue incorporating L-alanine was also prepared. The native peptide and the analogues were studied by fluorescence spectroscopy for their membrane permeating activity. Small unilamellar vesicles (SUVs) of egg phosphatidylcholine/ cholesterol (70:30) containing an encapsulated fluorescence probe (6-carboxyfluorescein) were used as membrane models. The assays of carboxyfluorescein release from SUVs upon peptide addition showed that Peptaibolin-Dpg and Peptaibolin-Ac6c are the most active peptides. These results indicate that the structure of the α,α-dialkylglycines is crucial for the membrane permeating ability of these Peptaibolin analogues.

Fluorescence-based approaches towards the assessment of structural and functional alterations in lysosomes and vacuoles induced by lactoferrin and acetic acid

Dissertação de mestrado em Biofísica e Bionanossistemas

Exploring the role of O-Mannosylation in the modulation of E-Cadherin function in Gastric Cancer cell line models

Dissertação de mestrado em Bioquímica Aplicada – Biomedicina

Magnetoliposomes based on manganese ferrite nanoparticles as nanocarriers for antitumor drugs

Manganese ferrite nanoparticles with a size distribution of 26 ± 7 nm (from TEM measurements) were synthesized by the coprecipitation method. The obtained nanoparticles exhibit a superparamagnetic behaviour at room temperature with a magnetic squareness of 0.016 and a coercivity field of 6.3 Oe. These nanoparticles were either entrapped in liposomes (aqueous magnetoliposomes, AMLs) or covered with a lipid bilayer, forming solid magnetoliposomes (SMLs). Both types of magnetoliposomes, exhibiting sizes below or around 150 nm, were found to be suitable for biomedical applications. Membrane fusion between magnetoliposomes (both AMLS and SMLs) and GUVs (giant unilamellar vesicles), the latter used as models of cell membranes, was confirmed by F¨orster Resonance Energy Transfer (FRET) assays, using a NBD labeled lipid as the energy donor and Nile Red or rhodamine B-DOPE as the energy acceptor. A potential antitumor thienopyridine derivative was successfully incorporated into both aqueous and solid magnetoliposomes, pointing to a promising application of these systems in oncological therapy, simultaneously as hyperthermia agents and nanocarriers for antitumor drugs.

Study of grapevine solute transporters involved in berry quality. A biochemical and molecular approach

Tese de Doutoramento em Biologia de Plantas

Development of a water-in-oil-in-water multiple emulsion system integrating biomimetic aqueous-core lipid nanodroplets for protein entity stabilization. Part I: experimental factorial design

Lipid nanoballoons integrating multiple emulsions of the type water-in-oil-in-water enclose, at least in theory, a biomimetic aqueous-core suitable for housing hydrophilic biomolecules such as proteins, peptides and bacteriophage particles. The research effort entertained in this paper reports a full statistical 23x31 factorial design study (three variables at two levels and one variable at three levels) to optimize biomimetic aqueous-core lipid nanoballoons for housing hydrophilic protein entities. The concentrations of protein, lipophilic and hydrophilic emulsifiers, and homogenization speed were set as the four independent variables, whereas the mean particle hydrodynamic size (HS), zeta potential (ZP) and polydispersity index (PI) were set as the dependent variables. The V23x31 factorial design constructed led to optimization of the higher (+1) and lower (-1) levels, with triplicate testing for the central (0) level, thus producing thirty three experiments and leading to selection of the optimized processing parameters as 0.015% (w/w) protein entity, 0.75% (w/w) lipophilic emulsifier (soybean lecithin) and 0.50% (w/w) hydrophilic emulsifier (poloxamer 188). In the present research effort, statistical optimization and production of protein derivatives encompassing full stabilization of their three-dimensional structure, has been attempted via housing said molecular entities within biomimetic aqueous-core lipid nanoballoons integrating a multiple (W/O/W) emulsion.

Participación del tráfico y la distribución de proteínas en la polaridad y el desarrollo de procesos neuronales. Protein sorting and trafficking participation in neuronal processes polarity and development.

El estudio del tráfico intracelular en neuronas ha despertado gran interés en los últimos años, debido a que un gran número de enfermedades neurodegenerativas y neuropsiquiátricas parecen tener origen en en el transporte defectuoso de proteínas en estos tipos celulares. Mediante el uso de técnicas de biología celular y molecular, fuimos capaces de describir una de las vías que regula la fisión de las vesículas que llevan su cargo desde la última cisterna del Aparato de Golgi hacia la superficie celular en células epiteliales no polarizadas. Uno de los componentes clave de esa vía resultó ser la Proteina Kinasa D1 (PKD1), cuya actividad en el Aparato de Golgi es esencial para un normal transporte intracelular. Sorprendentemente, observamos que la PKD1 en neuronas con polaridad establecida no regula la fisión en el Golgi, pero si estaría involucrada en la selectividad y distribución (sorting) de vesículas cuyo cargo debe ser específicamente dirigido a las membranas dendríticas. El bloqueo de la actividad de la PKD1 no solamente cambia el destino final de estos cargos, que son enviados de esta forma a la membrana terminal del axón, sino que también es capaz de inducir defectos en el desarrollo y crecimiento de los procesos dendríticos a largo plazo. En este proyecto estudiaremos de que manera influye la perturbación del sorting, en ausencia de PKD1 activa y de otros componentes que la regulan, en la distribución de receptores de factores neurotróficos y de neurotransmisores glutamatérgicos, y cómo estos cambios en su distribución afectan el número, tamaño, y funcionalidad de los procesos neuronales (axones y dendritas). Estos resultados contribuirán a adquirir mayores conocimientos de los mecanismos dependientes del transporte y sorting de proteínas de membrana que participan en la regulación del crecimiento neuronal, los cuales a su vez aportarán información valiosa en la comprensión de un gran número de enfermedades neurológicas. The study of intracellular trafficking in neurons has arisen a great deal of interest in the last years, since a great number of neurodegenerative and neuropsychiatric disorders seem to be originated in abnormal protein transport in these type of cells. Using cell and molecular biology methodologies, we have been capable of describe one of the pathways that regulate the fission of vesicles that carry their cargo from the last Golgi Apparatus cisternae to the cell surface in non-polarized epithelial cells. One of the key components in this pathway is the Protein Kinase D1 (PKD1), whose activity in the Golgi Apparatus is essential for a normal intracelular transport. Surprisingly, we have observed that PKD1 does not regulate fission in neurons with established polarity, but it would be involved in vesicles' sorting at Golgi, particularly of those that carry specific dendritic cargo. Blocking PKD1 activity changes the final destination of these cargoes, which is now sent to the axons' terminal membranes, and also produces late dendritic development and growing defects. In this project we will study how sorting perturbation in absence of PKD1 and its regulators activities influences selectivity and distribution of neurotrophic and neurotransmitter receptors, and how these sorting changes affect number, size and functionality of neuronal processes (axons and dendrites). These results will help to acquire greater knowledge about transport and sorting mechanisms of neuronal growth regulatory membrane proteins. In addition, these studies will contribute with new valuable information necessary to understand numerous neurological diseases.

Lipoides do plasma normal

1. In a series of 21 normal cases we found for fatty acids per 100 cc. of plasma an average of 332 mgm., being 314 mgm. for the male sex and 350 mgm. for the female sex. 2.- For lecithin, in four normal cases we found per 100 cc. of plasma 182 mgm. estimated by the contents is phosphorus which ranged from 6.12 to 9.0 mgrs. 3.- Cholesterol in 20 normal cases showed 172 mgm. per 100 cc. of plasma. The averages were 151 mgm. for men and 194 mgm. for women. 4.- The readings of the fractions were 2.01 for the ratio fatty acids divided by lecithins 0.90 for lecithin divided by cholesterol and 1.93 for fatty acids divided by cholesterol. 5.- On comparing the results obtained by us with those reported in foreign literature an absolute conformity is noted chiefly with the values supplied by Bloor and Horiuchi for the ratios among the various lipoid fractions. The average for Cholesterol is comparable with that obtained by Myers but is slightly under that of Bloor's. The lecithin contents found by us did not reach such high values as those supplied by foreign authors.

Immunogold Detection of L-glutamate and D-serine in Small Synaptic-Like Microvesicles in Adult Hippocampal Astrocytes.

Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (∼45 and ∼55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

The potassium impermeable apical membrane of insect epithelia: a target for development of safe pesticides

Columnar cell apical membranes (CCAM) in series with goblet cell apical membranes (GCAM) form an electroosmotic barrier separating the midgut lumen from epithelial cell cytoplasm. A unique K+ ATPase in GCAM generates three gradients across this barrier. A greater than 180 mV electrical gradient (lumen positive) drives amino acid uptake through voltage-dependent K+ symports. A greater than 1000-fold [H+] gradient (lumen alkaline) and a greater than 10-fold [K+] gradient (lumen concentrated) are adaptations to the high tannin and high K+ content, respectively, in dietary plant material. Agents which act on the apical membrane and disrupt the PD, H+, or K+ gradients are potential insecticides. Insect sensory epithelia and mammalian stria vascularis maintain similar PD and K+ gradients but would not be exposed to ingested anti-apical membrane insecticides. Following the demonstration by Sacchi et al. that Bacillus thuringiensis delta-endotoxin (Bt) induces specifically a K+ conductance increase in CCAM vesicles, we find that the K+ channel blocking agent, Ba2+, completely reverses Bt inhibition of the K+-carried short circuit current in the isolated midgut of Manduca sexta. Progress in characterizing the apical membrane includes finding that fluorosulfonylbenzoyladenosine binds specifically to certain GCAM polypeptides and that CCAM vesicles can be mass produced by Ca2+ or Mg2+ precipitation from Manduca sexta midgut.

Leishmania mexicana amazonensis: heterogeneity in 5-nucleotidase and peroxidase activities of mononuclear phagocytes during in vivo and in vitro infection

The degree of maturation of cells of the Mononuclear Phagocyte System (MPS), during in vivo and in vitro infection by Leishmania mexicana amazonenesis, was evaluated in this study. The macrophages' differentiation was assayed by cytochemical characterization at the ultrastrctural level, using two well-established markers: 5'-nucleotidase enzyme activity, for revealing the mature cells, and the peroxidase activity present in the cell granules to demonstrate immature mononuclear phagocytes. only a few mcrophages, demonstrating 5'-nucleotidase positive reaction in both the plasma membrane and within their cytoplasmic vesicles, were found scattered in the chronic inflammation at the L. m. amazonensis lesions in albino mice. However, by the peroxidase activity analysis, we were also able to demonstrate the presence of immature MPS cells, which predominate, together with parasitized vacuolated macrophages, in chronic lesions induced in this systemby L. m. amazonensis. The implications of these results on the pathogenesis of murine cutaneous leishmaniasis are discussed.

Etude des propriétés dynamiques de la sécrétion régulée des vésicules glutamatergiques des astrocytes

RESUME GRAND PUBLICLe cerveau est composé de différents types cellulaires, dont les neurones et les astrocytes. Faute de moyens pour les observer, les astrocytes sont très longtemps restés dans l'ombre alors que les neurones, bénéficiant des outils ad hoc pour être stimulés et étudiés, ont fait l'objet de toutes les attentions. Le développement de l'imagerie cellulaire et des outils fluorescents ont permis d'observer ces cellules non électriquement excitables et d'obtenir des informations qui laissent penser que ces cellules sont loin d'être passives et participent activement au fonctionnement cérébral. Cette participation au fonctionnement cérébral se fait en partie par le biais de la libération de substances neuro-actives (appellées gliotransmetteurs) que les astrocytes libèrent à proximité des synapses permettant ainsi de moduler le fonctionnement neuronal. Cette libération de gliotransmetteurs est principalement causée par l'activité neuronale que les astrocytes sont capables de sentir. Néanmoins, nous savons encore peu de chose sur les propriétés précises de la libération des gliotransmetteurs. Comprendre les propriétés spatio-temporelles de cette libération est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information cérébrale. En utilisant des outils fluorescents récemment développés et en combinant différentes techniques d'imagerie cellulaire, nous avons pu obtenir des informations très précises sur la libération de ces gliotransmetteurs par les astrocytes. Nous avons ainsi confirmé que cette libération était un processus très rapide et qu'elle était contrôlée par des augmentations de calcium locales et rapides. Nous avons également décrit une organisation complexe de la machinerie supportant la libération des gliotransmetteurs. Cette organisation complexe semble être à la base de la libération extrêmement rapide des gliotransmetteurs. Cette rapidité de libération et cette complexité structurelle semblent indiquer que les astrocytes sont des cellules particulièrement adaptées à une communication rapide et qu'elles peuvent, au même titre que les neurones dont elles seraient les partenaires légitimes, participer à la transmission et à l'intégration de l'information cérébrale.RESUMEDe petites vésicules, les « SLMVs » ou « Synaptic Like MicroVesicles », exprimant des transporteurs vésiculaires du glutamate (VGluTs) et libérant du glutamate par exocytose régulée, ont récemment été décrites dans les astrocytes en culture et in situ. Néanmoins, nous savons peu de chose sur les propriétés précises de la sécrétion de ces SLMVs. Contrairement aux neurones, le couplage stimulussécrétion des astrocytes n'est pas basé sur l'ouverture des canaux calciques membranaires mais nécessite l'intervention de seconds messagers et la libération du calcium par le reticulum endoplasmique (RE). Comprendre les propriétés spatio-temporelles de la sécrétion astrocytaire est essentiel pour comprendre le mode de communication de ces cellules et leur implication dans la transmission de l'information cérébrale. Nous avons utilisé des outils fluorescents récemment développés pour étudier le recyclage des vésicules synaptiques glutamatergiques comme les colorants styryles et la pHluorin afin de pouvoir suivre la sécrétion des SLMVs à l'échelle de la cellule mais également à l'échelle des évènements. L'utilisation combinée de l'épifluorescence et de la fluorescence à onde évanescente nous a permis d'obtenir une résolution temporelle et spatiale sans précédent. Ainsi avons-nous confirmé que la sécrétion régulée des astrocytes était un processus très rapide (de l'ordre de quelques centaines de millisecondes). Nous avons découvert que cette sécrétion est contrôlée par des augmentations de calcium locales et rapides. Nous avons également décrit des compartiments cytosoliques délimités par le RE à proximité de la membrane plasmique et contenant les SLMVs. Cette organisation semble être à la base du couplage rapide entre l'activation des GPCRs et la sécrétion. L'existence de compartiments subcellulaires indépendants permettant de contenir les messagers intracellulaires et de limiter leur diffusion semble compenser de manière efficace la nonexcitabilité électrique des astrocytes. Par ailleurs, l'existence des différents pools de vésicules recrutés séquentiellement et fusionnant selon des modalités distinctes ainsi que l'existence de mécanismes permettant le renouvellement de ces pools lors de la stimulation suggèrent que les astrocytes peuvent faire face à une stimulation soutenue de leur sécrétion. Ces données suggèrent que la libération de gliotransmetteurs par exocytose régulée n'est pas seulement une propriété des astrocytes en culture mais bien le résultat d'une forte spécialisation de ces cellules pour la sécrétion. La rapidité de cette sécrétion donne aux astrocytes toutes les compétences pour pouvoir intervenir de manière active dans la transmission et l'intégration de l'information.ABSTRACTRecently, astrocytic synaptic like microvesicles (SLMVs), that express vesicular glutamate transporters (VGluTs) and are able to release glutamate by Ca2+-dependent regulated exocytosis, have been described both in tissue and in cultured astrocytes. Nevertheless, little is known about the specific properties of regulated secretion in astrocytes. Important differences may exist between astrocytic and neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca2+ from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We took advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses like styryl dyes and pHluorin in order to follow exocytosis and endocytosis of SLMVs at the level of the entire cell or at the level of single event. We combined epifluorescence and total internal reflection fluorescence imaging to investigate, with unprecedented temporal and spatial resolution, the events underlying the stimulus-secretion in astrocytes. We confirmed that exo-endocytosis process in astrocytes proceeds with a time course on the millisecond time scale. We discovered that SLMVs exocytosis is controlled by local and fast Ca2+ elevations; indeed submicrometer cytosolic compartments delimited by endoplasmic reticulum (ER) tubuli reaching beneath the plasma membrane and containing SLMVs. Such complex organization seems to support the fast stimulus-secretion coupling reported here. Independent subcellular compartments formed by ER, SLMVs and plasma membrane containing intracellular messengers and limiting their diffusion seem to compensate efficiently the non-electrical excitability of astrocytes. Moreover, the existence of two pools of SLMVs which are sequentially recruited suggests a compensatory mechanisms allowing the refill of SLMVs and supporting exocytosis process over a wide range of multiple stimuli. These data suggest that regulated secretion is not only a feature of cultured astrocytes but results from a strong specialization of these cells. The rapidity of secretion demonstrates that astrocytes are able to actively participate in brain information transmission and processing.

Tegumental Ca-stimulated adenosine triphosphatase activity in adult Schistosoma mansoni worms

A Ca-stimulated ATPase activity (pH 9.5) associated with the tegumental membrane enriched (TME) fraction of Schistosoma mansoni adults was partially inhibited by NAP-taurine or by increasing concentrations of chlorpromazine; endogenous calmodulin was found associated with the TME fraction. A similar activity (pH 8.6) was histochemically visualized whithin the tegument of fixed worms on the cytoplasmic leaflet of both the doubel surface membrane and the basement membrane; this reaction was inhibited by 1 µM chloropromazine and it was also observed on the inner side of double membrane vesicles present in the TME fraction. No ATPase activity could be seen at alkaline pH with added Mg or Na/K ions. Without ATP, the addition of external Ca to the fixed worms induced the appearance of lead precipitates on the tegumental discoid bodies; this reaction was inhibited by molybdate and not by chlorpromazine. The intrategumentary regulation of calcium by the systems described and the possible use of phenothiazines against schistosimes are discussed.

International Society of Urological Pathology (ISUP) Consensus Conference on Handling and Staging of Radical Prostatectomy Specimens. Working group 1: specimen handling.

The 2009 International Society of Urological Pathology Consensus Conference in Boston made recommendations regarding the standardization of pathology reporting of radical prostatectomy specimens. Issues relating to the handling and processing of radical prostatectomy specimens were coordinated by working group 1. Most uropathologists followed similar procedures for fixation of radical prostatectomy specimens, with 51% of respondents transporting tissue in formalin. There was also consensus that the prostate weight without the seminal vesicles should be recorded. There was consensus that the surface of the prostate should be painted. It was agreed that both the prostate apex and base should be examined by the cone method with sagittal sectioning of the tissue sample. There was consensus that the gland should be fully fixed before sectioning. Both partial and complete embedding of prostates was considered to be acceptable as long as the method of partial embedding is stated. No consensus was determined regarding the necessity of weighing and measuring the length of the seminal vesicles, the preparation of whole mounts rather than standardized blocks and the methodology for sampling of fresh tissue for research purposes, and it was agreed that these should be left to the discretion of the working pathologist.