Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Le tri et le transport efficace des hydrolases acides vers le lysosome jouent un rôle critique pour la fonction des cellules. Plus de 50 maladies humaines sont dues à des mutations des enzymes lysosomales, des protéines régulant des processus-clés du transport vers le lysosome ou des enzymes effectuant des modifications posttraductionnelles importantes pour la fonction du lysosome. L’objectif de cette thèse est d’identifier des protéines et des mécanismes permettant à la cellule de réguler le transport des enzymes vers le lysosome. Nous avons formulé l’hypothèse que des protéines mutées dans des maladies lysosomales et dont les fonctions étaient inconnues pouvaient jouer un rôle dans le transport vers le lysosome. Les céroïdes-lipofuscinoses neuronales forment une famille de maladies lysosomales rares mais sont aussi les maladies neurodégénératives infantiles les plus fréquentes. Plusieurs gènes impliqués dans les NCL encodent des protéines aux fonctions inconnues. Les travaux présentés dans cette thèse ont identifié la protéine « ceroid lipofuscinosis neuronal-5 » (CLN5) qui est localisée à l’endosome et au lysosome comme élément nécessaire au recrutement et à l’activation de rab7. Rab7 est une protéine Rab-clé qui contrôle le trafic à l’endosome tardif. Cette petite GTPase est impliquée dans le recrutement de retromer, un complexe protéique qui régule le trafic de l’endosome vers l’appareil de Golgi des récepteurs de tri lysosomal comme sortilin et le récepteur du mannose-6-phosphate. Dans les cellules où CLN5 est déplété, les récepteurs de tri lysosomal sont moins recyclés plus rapidement dégradés. En utilisant des expériences de photomarquage nous avons aussi pu démontrer que Rab7 est moins activées en l’absence de CLN5. Pour exécuter leur fonction les protéines rabs doivent être recrutée à la membrane et activées par l’échange d’une molécule de GDP pour une molécule de GTP. Le recrutement des Rabs à la membrane nécessite une modification posttraductionnelle lipidique pour être facilités. En utilisant un modèle de levures nous avons démontré que l’homologue de Rab7, Ypt7 est palmitoylée. Nous avons aussi démontré que la palmitoyltransférase Swif1 est nécessaire au recrutement de Ypt7 à la membrane. Nous avons aussi remarqué que les sous- unités de retromer chez la levure sont moins recrutées lorsque les palmitoyltransférases sont déplétées. Dans les cellules de mammifères nous avons démontré que Rab7 est également palmitoylé et que cette palmitoylation est possiblement effectuée par les palmitoyltransférases DHHC1 et DHHC8. La palmitoylation de Rab7 a lieu sur les cystéines en C-terminal qui sont nécessaires au recrutement membranaire et qui auparavant étaient uniquement décrites comme prénylées. En utilisant la méthode de « click chemistry » nous avons découvert que lorsque la prénylation de Rab7 est bloquée le niveau de palmitoylation augmente. Pour caractériser l’interaction entre CLN5 et Rab7 nous avons performé des expériences afin d’établir définitivement la topologie de cette protéine. Nous avons ainsi démontré que CLN5 est une protéine hautement glycosylée qui est initialement traduite en protéine transmembranaire et subséquemment clivée par un membre de la famille des peptidase de peptide signal (SPP). Cette protéine soluble peut alors possiblement interagir avec CLN3 qui est aussi palmitoylée pour recruter et activer Rab7. Nos études suggèrent pour la première fois que CLN5 pourrait être un recruteur et un activateur de Rab7 qui agirait avec la protéine CLN3 pour séquestrer Rab7 avec les autres récepteurs palmitoylés et permettre leur recyclage vers l’appareil de Golgi. / The proper sorting and trafficking of acid hydrolases plays a critical role in the normal function of cells. Over 50 known human diseases are caused by mutations of lysosomal enzymes, of proteins that regulate key processes of transport to the lysosome or of enzymes that perform posttranslational modifications which are important for the function of the lysosome. The main objective of this thesis is to identify proteins and mechanisms that allow the cell to regulate the transport of enzymes toward the lysosome. We formulated the hypothesis that proteins mutated in lysosomal diseases and that have no known functions could play a role in transport toward the lysosome. Neuronal ceroid-lipofuscinoses form a family of lysosomal storage disorders that are very rare but are also the most frequent infantile neurodegenerative diseases. The work presented in this thesis identified ceroid-lipofuscinosis neuronal-5 (CLN5), which is located at the late-endosomal/lysosomal compartment as a necessary element for the recruitment and activation of Rab7. Rab7 is an important GTPase that controls traffic from the late-endosome to the trans-Golgi network. Rab7 has been implicated in the recruitment of the retromer complex, which regulates retrograde transport of the lysosomal sorting receptor such as sortilin and the mannose-6-phosphate receptor. In the cells where CLN5 is depleted, the lysosomal sorting receptors are less recycled and degraded more rapidly. Using photolabelling assays we were also able to show that Rab7 is less activated in the absence of CLN5. To perform their function, Rab proteins have to be recruited to membranes and activated by the exchange of a GDP nucleotide for GTP. The recruitment of Rabs to membranes necessitates a lipidic posttranslational modification to raise the affinity. Using yeast as a model we demonstrated that the Rab7 homolog, Ypt7 is palmitoylated. We have also showed that the yeast palmitoyltransferase Swif1 is required for Ypt7 membrane recruitment. We have also observed that retromer subunits in yeast are less recruited when palmitoyltranferases are depleted. In mammals we have shown that Rab7 is also palmitoylated and that this palmitoylation may be done by palmitoyltransferases DHHC1 and DHHC8. The palmitoylation of Rab7 occurs on the C-terminal cysteines that are required for membrane recruitment and were previously only shown to be prenylated. By using Click chemistry we have discovered that when Rab7 prenylation is blocked the level of palmitoylation is augmented. To characterize the interaction of Rab7 and CLN5 we performed experiments to definitively establish the topology of this latter protein. Our results show that CLN5 is a heavily glycosylated protein that is initially translated as a type II transmembrane protein and subsequently cleaved by a member of the signal-peptide peptidase (SPP) family. This protein can then possibly interact with another member of the CLN family, CLN3 that is predicted to be palmitoylated to recruit and activate Rab7. Our studies establish for the first time that CLN5 is required for the recruitment and activation of Rab7 and may cooperate with the possibly palmitoylated protein CLN3 to sequester Rab7 in specific membrane domains with sorting receptors to allow their recycling toward the trans-Golgi network.

Les β2-agonistes sont couramment utilisés pour prévenir et réduire les symptômes de l'asthme et de la bronchoconstriction induite par l'exercice. Mais, pris en quantités supérieures aux doses thérapeutiques, les β2-agonistes ont un effet anabolisant qui a été clairement démontré in vivo. Un certain nombre d’acteurs sont mis en jeu dans la réponse biologique du tissu musculaire aux β2-agonistes. L’un de ces acteurs est la voie de signalisation PI3K/Akt/mTOR, voie d’initiation de la traduction, ayant un rôle majeur dans la synthèse protéique. Dans ce contexte, notre première étude avait pour objectif de déterminer la cinétique des événements moléculaires responsables de l’hypertrophie du muscle squelettique de rat après administration de formotérol pendant 1 jour (J1), 3 jours (J3) et 10 jours (J10). Nous avons montré que l’administration de formotérol induisait une hypertrophie musculaire à J3 et J10 associée à l’activation transitoire de la voie de signalisation PI3K/Akt/mTOR (J1 et J3), et à une diminution de l’expression de l’E3 ubiquitine ligase MAFbx/Atrogin-1 (J3). La voie autophagie lysosome ne semblait pas être affectée. Ainsi, l’ensemble de ces résultats suggère que l’activation de la voie PI3K/Akt/mTOR est associée à la voie ubiquitine-protéasome mais pas à la voie autophagie-lysosome. La régulation transitoire de la voie PI3K/Akt/mTOR suggère que d’autres voies de signalisation sont impliquées dans l’hypertrophie musculaire induite par le formotérol. Le 007-AM, analogue de l’AMPc, a été décrit comme pouvant stimuler la voie de signalisation PI3K/Akt/mTOR via l’activation de la protéine Epac, suggérant que le 007-AM puisse constituer une molécule de substitution à l’utilisation des β2-agonistes. Notre seconde étude avait pour but de déterminer si le 007-AM avait une action anabolisante sur le tissu musculaire, mais également de déterminer si la 007-AM était une molécule stable permettant d’envisager son usage dans un cadre pharmacologique. L’administration de 007-AM pendant 7 jours chez des souris n’engendrait pas d’hypertrophie musculaire. En revanche, in vitro sur cellules C2C12, le 007-AM activait la voie de signalisation PI3K/Akt/mTOR comme en témoignait l’augmentation de la phosphorylation des protéines rpS6 et 4E-BP1. Nos résultats montraient également que le 007-AM était instable dans le plasma alors que son produit de dégradation, le 007 était plus stable. Pris ensembles, ces résultats suggèrent qu’un traitement de 7 jours au 007-AM n’est pas suffisant pour induire une hypertrophie musculaire et que l’absence d’hypertrophie musculaire pourrait provenir de l’instabilité du 007-AM dans le plasma. Toutefois, des études supplémentaires seront nécessaires pour confirmer ces résultats / Β2-agonists are traditionally used to prevent and reduce asthma symptoms and bronchoconstriction induced by exercise. Nevertheless, when administrated in vivo, at relatively high, far away from therapeutic doses, β2-agonists induce anabolic effects. Numerous actors are involved in biological response of the skeletal muscle, induced by β2-agonists. PI3K/Akt/mTOR signaling pathway, which initiates translation, is one of these actors. In this context, our first study aimed at determined the kinetic of molecular events responsible for skeletal muscle hypertrophy after 1 day (D1), 3 days (D3) and 10 days (D10) of formoterol administration. We have shown that formoterol administration induced skeletal muscle hypertrophy at D3 and D10 associated with a transient activation of PI3K/Akt/mTOR signaling pathway (D1 and D3), and, with a decrease in E3 ubiquitin ligase MAFbx/atrogin-1 expression (D3). The autophagy-lysosome pathway seems not to be regulated by formoterol administration. Taken together, these results suggest that PI3K/Akt/mTOR activation is temporally associated with the regulation of ubiquitin-proteasome but not the autophagy-lysosome pathway. The transient nature of the regulation of PI3K/Akt/mTOR signaling pathway also indicates that other unidentified pathways are probably activated to sustain the increase in skeletal muscle mass. Recently, 007-AM synthetic molecule has been described to stimulate PI3K/Akt/mTOR signaling pathway through Epac protein activation, suggesting that 007-AM could be an alternative to the use of β2-agonists. The purpose of our second study was to determine whether 007-AM had an anabolic action on skeletal muscle and if 007-AM was stable allowing considering its use in pharmacology. 007-AM administration for 7 days to mice does not lead to muscle hypertrophy. Nonetheless, in vitro on C2C12 cells, 007-AM activated PI3K/Akt/mTOR signaling pathway by increasing phosphorylation of rpS6 and 4E-BP1. Our results showed that contrary to 007, 007-AM was instable in plasma. Altogether, these results suggest that a 7-day 007-AM treatment is not sufficient to induce skeletal muscle hypertrophy. This lack of hypertrophy could be due to 007-AM instability in plasma. However, supplemental studies are needed to confirm these results

Ca2+ is a universal and versatile intracellular messenger, regulating a vast array of biological processes due to variations in the frequency, amplitude, spatial and temporal dynamics of Ca2+ signals. Increases in cytosolic free Ca2+ concentration ([Ca2+]c) are due to influx from either an infinite extracellular Ca2+ pool or from the more limited intracellular Ca2+ stores. Stimulation of the endogenous muscarinic (M3) receptors of human embryonic kidney (HEK) cells with carbachol results in the activation of phospholipase C (PLC) and formation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3Rs), release of Ca2+ from the endoplasmic reticulum (ER), and activation of store-operated Ca2+ entry (SOCE). Lysosomes are the core digestive compartments of the cell, but their importance as signalling organelles is also now widely appreciated. Accumulating evidence indicates that lysosomal Ca2+ is important for their physiological functions. Lysosomal Ca2+ release triggers fusion during membrane trafficking and, through calmodulin, it regulates lysosome size. Luminal Ca2+ is critical for regulation of lysosomal biogenesis and autophagy during starvation through the transcription factor, TFEB. Furthermore, aberrant lysosomal Ca2+ is associated with some lysosomal storage diseases. Lysosomes in mammalian cells have long been suggested to accumulate Ca2+ via a low-affinity Ca2+-H+ exchanger (CAX). This is consistent with evidence that dissipating the lysosomal H+ gradient increased [Ca2+]c and decreased lysosomal free [Ca2+], and with the observation that lysosomal Ca2+ uptake was followed by an increase in pHly. Furthermore, heterologous expression of Xenopus CAX in mammalian cells attenuated carbachol-evoked Ca2+ signals. However, there is no known CAX in mammalian cells, and so the identity of the lysosomal Ca2+ uptake pathway in mammalian cells is unresolved. Using mammalian cells loaded with a fluorescent Ca2+ indicator, I show that dissipating the pHly gradient pharmacologically or by siRNA-mediated knockdown of an essential subunit of the H+ pump, increases the amplitude of IP3-evoked cytosolic Ca2+ signals without affecting those evoked by SOCE. A genetically encoded low-affinity Ca2+ sensor expressed on the lysosome surface reports larger increases in [Ca2+]c than the cytosolic sensor, but only when the Ca2+ signals are evoked by IP3R rather than SOCE. Using cells expressing single IP3R subtypes, I demonstrate that each of the three IP3R subtypes can deliver Ca2+ to lysosomes. I conclude that IP3Rs release Ca2+ within near-lysosome microdomains that fuel a low-affinity lysosomal Ca2+ uptake system. The temporal relationship between the increase in pHly and reduced Ca2+ sequestration suggests that pHly affects the organization of the microdomain rather than the Ca2+ uptake mechanism. I show that abrogation of the lysosome H+ gradient does not acutely prevent uptake of Ca2+ into lysosomes, but disrupts junctions with the ER where the exchange of Ca2+ occurs. The dipeptide, glycyl-L-phenylalanine 2-naphthylamide (L-GPN), is much used to disrupt lysosomes and release Ca2+ from them. The mechanism is widely assumed to require cleavage of GPN by cathepsin C, causing accumulation of amino acid residues, and osmotic lysis of lysosomal membranes. I show, using LysoTracker Red and Oregon Green-dextran to report pHly, that L-GPN is effective in HEK cells lacking functional cathepsin C, following CRISPR-Cas9-mediated gene disruption. Furthermore, D-GPN, which is resistant to cleavage by cathepsin C, is as effective as L-GPN at increasing pHly, and it is similarly effective in cells with and without cathepsin C. L-GPN and D-GPN increase cytosolic pH, and the effect is similar when the lysosomal V-ATPase is inhibited with bafilomycin A1. This is not consistent with GPN releasing the acidic contents of lysosomes. I conclude that the effects of GPN on lysosomes are not mediated by cathepsin C. Both L-GPN and D-GPN evoke Ca2+ release, the response is unaffected by inhibition or knock-out of cathepsin C, but it requires Ca2+ within the ER. GPN-evoked increases in [Ca2+]c require Ca2+ within the ER, but they are not mediated by ER Ca2+ channels amplifying Ca2+ release from lysosomes. GPN increases [Ca2+]c by increasing pHcyt, which then directly stimulates Ca2+ release from the ER. I conclude that physiologically relevant increases in pHcyt stimulate Ca2+ release from the ER independent of IP3 and ryanodine receptors, and that GPN does not selectively target lysosomes. I conclude that all three IP3R subtypes selectively deliver Ca2+ to lysosomes, and that the low pH within lysosomes is required to maintain the junctions between ER and lysosomes, but not for lysosomal Ca2+ uptake. I suggest that GPN lacks the specificity required to allow selective release of Ca2+ from lysosomes.

L'X-ALD et la P-NALD sont deux maladies peroxysomales, métaboliques et neurodégénératives rares. L'X-ALD et la P-NALD résultent de déficiences respectives en ABCD1 et ACOX1. Ces deux maladies dans leurs formes sévères sont associées à des phénomènes de démyélinisation inflammatoire du SNC. Au niveau des lésions, des signes d'oxydation et une mort cellulaire sont observés. L'accumulation des AGTLC plasmatiques et tissulaires est le critère biochimique commun à ces deux maladies. Dans un premier temps, nous avons caractérisé une lignée d'oligodendrocytes murins 158N afin de l'utiliser comme modèle. Cette lignée qui présente des caractéristiques d'oligodendrocytes matures (expression des protéines de myéline MOG, MBP, PLP) possède aussi des peroxysomes fonctionnels possédant les protéines Abcd1 et Acox1. Ensuite, nous avons étudié les effets cytotoxiques et pro-oxydants des AGTLC (C24:0 et C26:0), ainsi que l'incidence de l'extinction d'Abcd1 et d'Acox1 par siRNA sur l'équilibre RedOx et la mort cellulaire. Les effets des AGTLC sur les caractéristiques biophysiques de la membrane cytoplasmique ont aussi été abordés. Par ailleurs, des marqueurs du stress oxydant ont été recherchés sur des plasmas des patients atteints de différentes formes d'X-ALD. In vitro, nous avons montré que l'accumulation d'AGTLC dans les cellules 158N induit une surproduction d'espèces radicalaires de l'oxygène et de l'azote et une perturbation des défenses anti-oxydantes (catalase, SOD, GSH). Ceci s'accompagne d'une peroxydation lipidique, d'une carbonylation des protéines et d'une dégradation de l'ADN. L'extinction d'Abcd1 et d'Acox1 par des siRNA augmente la production d'espèces radicalaires et potentialise le stress oxydant induit par les AGTLC. Sur les plasmas de patients atteints de différentes formes d'X-ALD, comparativement à des sujets sains, nous avons montré l'accumulation des produits de peroxydation lipidiques (7-hydroxycholestérols, HODEs). Le taux de ces deux produits est corrélé avec la sévérité de la maladie: CCALD>AMN>Addison>ACALD. Les AGTLC induisent aussi la mort des cellules 158N par un processus non apoptotique. Cette mort cellulaire est caractérisée par: une perturbation rapide du calcium intracellulaire, une diminution du pH, une chute du potentiel transmembranaire mitochondrial associée à des modifications structurales des mitochondries, une déstabilisation des lysosomes et une formation de figures d'autophagie. Les AGTLC perturbent aussi la fluidité membranaire. Par ailleurs, les AGTLC n'affectent pas l'expression des protéines majeures de la myéline PLP et MBP. Ces travaux ont mis en évidence un lien direct entre l'accumulation des AGTLC, le stress oxydant et l'induction de mort cellulaire faisant intervenir les lysosomes. La déficience en Abcd1 et Acox1 favorise le stress oxydant. En accord avec les résultats obtenus in vitro, la mise en évidence de marqueurs de peroxydation lipidiques dans le plasma de malades atteints d'X-ALD conforte l'hypothèse d'une intervention du stress oxydant dans cette pathologie.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-01-03T13:03:15Z No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2017-01-16T16:23:49Z (GMT) No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2017-01-16T16:23:57Z (GMT) No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5) / Made available in DSpace on 2017-01-16T16:24:06Z (GMT). No. of bitstreams: 1 DissPRS.pdf: 2244710 bytes, checksum: d11e0b755d5503e93a47a8a6dc113814 (MD5) Previous issue date: 2016-10-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Titanium dioxide nanoparticles (TiO2-NP) are commonly used in many industrial activities. Consequently, the daily consumption by humans is estimated in 5.4 mg day, with an input in the environment of 4.2 mg day per person, receiving or not appropriated treatment before disposure. The cytotoxicity, genotoxicity and mutagenicity of TiO2-NP were investigated using the established fish cell line derived from zebrafish (Danio rerio) liver (i. e. ZF-L cells). Prior to the evaluation of nanoparticle’s toxic potential, a careful characterization was realized in culture medium in the presence or not of fetal bovine serum (FBS). Regarding to the characterization in terms of size was accessed using a transmission electron microscope (TEM), the agglomeration potential and surface charge were accessed by diameter light scattering (DLS) and zeta potential measurements, respectively, using a spectrophotometer. TiO2-NP in environmentally relevant concentrations were tested for cytotoxicity, genotoxicity and mutagenicity. Cell viability was accessed by four different tests, the trypan blue assay (membrane integrity), MTT reduction assay (mitochondria), neutral red retention assay (lysosomes) and finally, induction of apoptosis and necrosis. Genotoxicity was determined by observing the fragmentation of DNA by the comet assay, while mutagenicity was determined by Cytokinesis-block micronucleus technique. The characterization showed that the FBS was effective in dispersing the nanoparticles and prevent the formation of large agglomerates allowing robust responses on the real toxicity of NP. After 24 hours of treatment, there was cell membranes rupture, decreasing cell viability to 35.33%, at the highest concentration (1.0 μg mL-1). Mitochondrial metabolic activity remained unchanged, but it was possible to detect the proliferation of lysosomes, which was mainly attributed to the NP endocytosis. The induction of apoptosis was 50.4%, and necrosis was 13.9%, both in the concentration 1.0 μg mL-1 TiO2-NP. In the case of necrosis, a result 10 times greater than that presented by the negative control. Added necrosis and apoptosis indicated a decrease in cell viability to 35.7%. The comet test showed the fragmentation of the DNA, it was also possible to observe the formation of micronuclei, bridges and shoots demonstrated by the micronucleus assay. In general, this study demonstrated that TiO2-NP, after 24 hours of exposure, significantly affect cell viability and cause DNA damage, which may become irreversible. In conclusion, this study showed the cytotoxic, genotoxic and mutagenic potential of TiO2-NP for ZF-L cells. Mitochondrial and lysosome responses require further studies on the effect of TiO2-NP on these organelles. / Nanopartículas de dióxido de titânio (NP-TiO2) são comumente usadas em muitos produtos industriais. Por conseguinte, seu consumo diário por seres humanos é estimado em 5,4 mg dia-1, o que acarreta em um aporte no ambiente de 4,2 mg dia-1 por pessoa, podendo ou não receber um tratamento apropriado antes do seu despejo. Foram avaliadas a citotoxicidade, genotoxicidade e mutagenicidade de NPTiO2 para a linhagem permanente derivada de fígado de peixe-zebra (Danio rerio) (células ZF-L). Antes da avaliação do potencial tóxico das nanopartículas, foi realizada uma caracterização cuidadosa em meio de cultura com e sem a adição de soro fetal de bovino (SFB). A caracterização em termos do tamanho físico da nanopartícula (NP) foi realizada utilizando um microscópio eletrônico de transmissão (TEM); os tamanhos hidrodinâmicos dos aglomerados e as cargas de superfície foram acessados por medidas de DLS (diameter light scattering) e potencial zeta, respectivamente utilizando um espectrofotômetro. A viabilidade celular foi avaliada por três ensaios diferentes, o ensaio de exclusão do corante azul de tripano (integridade de membrana), ensaio de redução do sal MTT (atividade metabólica mitocondrial) e ensaio de retenção do corante vermelho neutro (viabilidade dos lisossomos). A indução de apoptose e necrose foi avaliada por citometria de fluxo. A genotoxicidade foi determinada pela observação da fragmentação do DNA por meio do ensaio do cometa, enquanto a mutagenicidade foi determinada pelo ensaio de micronúcleos com bloqueio da citocinese. A análise DLS mostrou que o SFB foi eficaz quanto à dispersão das nanopartículas e preveniu a formação de grandes aglomerados, o que permitiu a obtenção de respostas mais robustas relativas à toxicidade real das nanopartículas. Após 24 horas de tratamento, houve ruptura das membranas celulares diminuindo a viabilidade celular a 35,33%, na concentração mais elevada (1,0 μg mL-1). A atividade metabólica mitocondrial manteve-se inalterada, mas foi possível detectar a proliferação dos lisossomos, que foi atribuída principalmente à endocitose das NP. A indução de apoptose foi de 50,4%, e de necrose de 13,9%, ambos na concentração 1,0 μg mL-1 NP-TiO2. No caso da necrose, um resultado 10 vezes maior do que o apresentado pelo controle negativo. Necrose e apoptose somadas indicaram a diminuição da viabilidade celular a 35,7%. O teste do cometa mostrou a fragmentação do DNA, também foi possível observar a formação de micronúcleos, pontes e brotos demonstrados pelo ensaio do micronúcleo. Em geral, este estudo demonstrou que as NP-TiO2, após 24 horas de exposição, afetam significativamente a viabilidade celular e causam danos ao DNA, que podem se tornar irreversíveis. Em conclusão, este estudo mostrou o potencial citotóxico, genotóxicos e mutagênico das NP-TiO2 para células ZF-L. Respostas mitocondriais e dos lisossomos requerem novos estudos quanto ao efeito das NP-TiO2 sobre essas organelas.

Orientador: Aureo Tatsumi Yamada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-07T02:54:51Z (GMT). No. of bitstreams: 1 Copi_Cecilia_M.pdf: 12156283 bytes, checksum: 93ce542963e020dcfb1af6c2bf9d5283 (MD5) Previous issue date: 2006 / Resumo: Durante a gestação normal em primatas e em roedores observa-se o acúmulo de linfócitos natural killer no ambiente uterino (uNK). A funções comprovadas das células uNK estão relaciondas com o reconhecimento das células trofoblásticas alogênicas e produção de citocinas imunomoduladoras do ambiente uterino que garantem o sucesso da gestação. Além da produção de citocinas envolvidas na homeostase do ambiente uterino, as uNK produzem e estocam nos seus grânulos moléculas citotóxicas como a perforina e granzimas. Estas moléculas estão envolvidas na resposta imune inata das células NK, com potencial de promover a lise de células alvo. Porém, se ocorre a ativação desta atividade citolítica, ou mesmo se as uNK promovem a secreção deste arsenal de moléculas citolíticas no ambiente uterino em gestação normal, ou em casos de interrupção da gestação, não são conhecidas. Os conhecimentos nesta área não avançam pelas limitações éticas de estudar experimentalmente o ambiente uterino em gestantes humanos e a falta de modelos experimentais estabelecidos em animais laboratoriais. No presente trabalho, propos-se utilizar o modelo experimental de indução de lesões embrionárias para provocar o desequilíbrio do ambiente uterino e avaliar as alterações nas células uNK. Para tanto, foram utilizados camundongos prenhes no 9° dia de gestação que foram submetidas à procedimentos cirúrgicos de lesão do embrião. Amostras uterinas dos sítios de desenvolvimento embrionários de animais de gestação normal, dos animais com embriões lesionados nos intervalos de 10, 30 e 60 minutos póslesão, e dos animais manipulados cirurgicamente sem a lesão embrionária foram coletados e processados para avaliações através de métodos citoquímicos e imunocitoquímicos em microscopia de luz e eletrônica de transmissão. Os sítios uterinos de embriões lesionados apresentaram reação hiperêmica na região mesometrial já aos 10 minutos pós-lesão, que se acentuava nos períodos mais longos de pós-lesão. As células uNK presentes nesta região apresentaram alteração na reatividade pela citoquimica de lectina DBA e immunocitoquímica com a anti-perforina, sendo evidente a gradual redução na intensidade das reações no decurso do tempo pós-lesão. Na análise ultra-estrutural verificou-se a desestruturação dos grânulos lisossomo-secretores, sendo notória a perda do conteúdo do compartimento secretor evidenciada pela reação com o azul-cuprolínico. O compartimento lisossomal do grânulo embora sofra também uma desestruturação pronunciada, mantêm a reatividade para anti-catepsina D, sugerindo a preservação de parte da sua funcionalidade. A perda do conteúdo do compartimento secretor, notadamente a perforina e os proteoglicanos no período de 10 minutos pós-lesão, sugere a degranulação das uNK e portanto a ativação da atividade citotóxica destas células, numa resposta aguda frente ao desequilíbrio do ambiente uterino afetado pela lesão embrionária. Porém, não foi constatado qualquer indício morfológico da ocorrência da degranulação através do mecanismo exocitose-símile ou ainda, o efeito da ação citolítica sobre as células circunjacentes às uNK ativadas / Abstract: In the uterine environment during normal pregnancy of primates and rodents is seen the accumulation of natural killer lymphocites (uNK). The confirmed functions of uNK cells are related to recognition of allogeneic trophoblast cells and production of immunomodulating cytokines of uterine environment that assure the successful pregnancy. Besides the production of cytokines related to homeostasis of uterine environment, the uNK produce and store citotoxic molecules of perforin and granzymes in their granules. These molecules are involved in the innate immune response of NK cells with potential to promote lysis of target cells. However, whether the lytic activity is triggered or even the uNK promotes the secretion of these sets Df cytolitic molecules into the uterus of normal pregnancy or in the miscarriages are not known. The advances of knowledge in this field are slow down due to ethical limitation of experimental studies with human pregnant uterus and lack of well established experimental models in laboratory animal. In the present work it was proposed to use the experimental model of induction of embryo lesions to promote the unbalance of uterine environment and evaluated the changes in uNK cells. Pregnant mice on 9° gestational day were submitted to surgical procedure of embryo lesion and uterine samples from these embryo lesioned sites were collected after 10, 30 and 60 minutes, as did the normal pregnant and sham operated animais. The samples were processed for cytochemical and immunocytochemical evaluations in light and transmission electron microscopy. The embryo lesioned uterine sites showed hyperemic reaction in the mesometrial region as soon as 10 min after lesion which increased as increased the time lapse after lesion. The uNK cells found in these regions showed reactivity changes with DBA lectin cytochemistry and with anti-perforin immunocytochemistry, being noticed the gradual reduction on reaction intensities on course of time after lesion. In the ultrastructural analysis were seen the disruptions of secretory-Iysosomes granules with notorious lost of secretory compartment contents as were evidenced by cuprolinic blue cytochemical reaction. In spite of structural organization of lysosome compartment of the granules was also affected it was maintained the anti-cathepsin D positive reaction which suggest the preservation of part of its functionality. The secretory compartment contents lost, notably the perforin and proteoglycans at 10 min after embryo lesion suggest degranulations of uNK and therefore an acute response of these cells by activation of citotoxic activity, under effects of unbalance of uterine environment affected by embryo lesion. However, it was not detected any morphological evidence of degranulation by exocytosis-like mechanism or even, the cytolitic effect on neighbor cells around the activated uNK cells / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural

Orientador: Áureo Tatsumi Yamada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T04:02:31Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_D.pdf: 2840118 bytes, checksum: 470a2cf0b7d63e81fd8f48aba5af44fc (MD5) Previous issue date: 2012 / Resumo: Células Natural Killer uterina (uNK) produzem moléculas angiogênicas e citocinas críticas ao sucesso da gestação , assim como proteínas citolíticas relacionadas à resposta imune inata. Contudo, se as capacidades angiogênicas e citolíticas são provenientes de diferentes subpopulações de células uNK não é conhecido; da mesma forma, estes fenótipos ainda não são estabelecidos. Assim, a proposta inicial deste trabalho foi avaliar...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Angiogenic and cytokine molecules produced by uterine natural killer (uNK) cells are critical for successful pregnancy. Cytolytic proteins are also express by uNK cells. However, it is unknown whether the angiogenic and cytolytic capacities are from different uNK subsets, or the same cells. Thus, we initially proposed to evaluate...Note: The complete abstract is available with the full electronic document / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

L’X-ALD et la P-NALD sont deux maladies peroxysomales, métaboliques et neurodégénératives rares. L'X-ALD et la P-NALD résultent de déficiences respectives en ABCD1 et ACOX1. Ces deux maladies dans leurs formes sévères sont associées à des phénomènes de démyélinisation inflammatoire du SNC. Au niveau des lésions, des signes d'oxydation et une mort cellulaire sont observés. L’accumulation des AGTLC plasmatiques et tissulaires est le critère biochimique commun à ces deux maladies. Dans un premier temps, nous avons caractérisé une lignée d'oligodendrocytes murins 158N afin de l'utiliser comme modèle. Cette lignée qui présente des caractéristiques d'oligodendrocytes matures (expression des protéines de myéline MOG, MBP, PLP) possède aussi des peroxysomes fonctionnels possédant les protéines Abcd1 et Acox1. Ensuite, nous avons étudié les effets cytotoxiques et pro-oxydants des AGTLC (C24:0 et C26:0), ainsi que l’incidence de l’extinction d’Abcd1 et d’Acox1 par siRNA sur l'équilibre RedOx et la mort cellulaire. Les effets des AGTLC sur les caractéristiques biophysiques de la membrane cytoplasmique ont aussi été abordés. Par ailleurs, des marqueurs du stress oxydant ont été recherchés sur des plasmas des patients atteints de différentes formes d’X-ALD. In vitro, nous avons montré que l’accumulation d'AGTLC dans les cellules 158N induit une surproduction d'espèces radicalaires de l'oxygène et de l'azote et une perturbation des défenses anti-oxydantes (catalase, SOD, GSH). Ceci s'accompagne d'une peroxydation lipidique, d'une carbonylation des protéines et d'une dégradation de l'ADN. L'extinction d'Abcd1 et d'Acox1 par des siRNA augmente la production d'espèces radicalaires et potentialise le stress oxydant induit par les AGTLC. Sur les plasmas de patients atteints de différentes formes d’X-ALD, comparativement à des sujets sains, nous avons montré l’accumulation des produits de peroxydation lipidiques (7-hydroxycholestérols, HODEs). Le taux de ces deux produits est corrélé avec la sévérité de la maladie: CCALD>AMN>Addison>ACALD. Les AGTLC induisent aussi la mort des cellules 158N par un processus non apoptotique. Cette mort cellulaire est caractérisée par: une perturbation rapide du calcium intracellulaire, une diminution du pH, une chute du potentiel transmembranaire mitochondrial associée à des modifications structurales des mitochondries, une déstabilisation des lysosomes et une formation de figures d'autophagie. Les AGTLC perturbent aussi la fluidité membranaire. Par ailleurs, les AGTLC n'affectent pas l'expression des protéines majeures de la myéline PLP et MBP. Ces travaux ont mis en évidence un lien direct entre l'accumulation des AGTLC, le stress oxydant et l'induction de mort cellulaire faisant intervenir les lysosomes. La déficience en Abcd1 et Acox1 favorise le stress oxydant. En accord avec les résultats obtenus in vitro, la mise en évidence de marqueurs de peroxydation lipidiques dans le plasma de malades atteints d'X-ALD conforte l'hypothèse d'une intervention du stress oxydant dans cette pathologie. / X-ALD and P-NALD are two rare, peroxisomal metabolic and neurodegenerative diseases. ABCD1 and ACOX1 are known to be responsible for X-ALD and P-NALD, respectively. The actively demyelinating lesions in CNS, exhibited signs of oxidative stress and cell death. The accumulation of VLCFA in plasma and tissue is the biochemical common hallmark to both diseases. First, we characterized a murine oligodendrocytes cell line 158N to use it as a model. This 158N cell line which has characteristics of mature oligodendrocytes (expression of myelin proteins MOG, MBP, PLP), has also functional peroxisomes with Abcd1 and Acox1 proteins. Then, we studied the cytotoxic and pro-oxidative effects of VLCFA (C24: 0 and C26: 0), and the effects of in vitro silencing of the Abcd1 and Acox1 genes by siRNA on the redox balance and cell death. Effects of VLCFA on the biophysical characteristics of cytoplasmic membrane were also evaluated. Moreover, markers of oxidative stress were researched on plasma of patients with different forms of X-ALD. In vitro, we showed that the accumulation of VLCFA on 158N cells induced overproduction of reactive oxygen and nitrogen species and a disruption of antioxidant defense systems (catalase, SOD, GSH). This was accompanied by lipid peroxidation, protein carbonylation and degradation of DNA. The extinction of Abcd1 and Acox1 by siRNA increased the production of radical species and potentialized the oxidative stress induced by VLCFA. On plasma of patients with different forms X-ALD, compared to healthy subjects, we showed an accumulation of lipid peroxidation products (7-hydroxycholesterol, HODEs). The rate of these two products is correlated with the severity of the disease: CCALD> AMN> Addison> ACALD. The VLCFA also induce cell death on 158N by a non-apoptotic process. This cell death is characterized by: a rapid increased of intracellular Ca2+ level, pH decrease, a loss of mitochondrial transmembrane potential associated with structural changes of mitochondria, a destabilization of lysosomes, and formation of autophagic vacuoles. The VLCFA also disrupt the membrane fluidity. Furthermore, VLCFA do not affect the expression of myelin major proteins PLP and MBP. This work highlighted a direct link between VLCFA accumulation, oxidative stress and induction of cell death involving lysosomes. Abcd1 and Acox1 deficiency promotes oxidative stress. In agreement with results obtained in vitro, the detection of markers of lipid peroxidation in the plasma of X-ALD patients favors the hypothesis of an involvement of oxidative stress in this pathology.