No description available.

No description available.

No description available.

No description available.

L'utilisation de champs électriques pulsés (PEF) dans les secteurs de la médecine et de la biotechnologie est devenue de plus en plus courante au cours des dernières décennies. La recherche a montré qu'en ajustant la durée du PEF, nous pouvons prédire quels effets seront observés. Alors que les PEF dans la gamme micro - milliseconde ont été utilisés pour perméabiliser la membrane cellulaire et améliorer l'absorption de médicament ou de protéine, le PEF nanoseconde (nsPEF) a démontré des effets uniques sur les organites intracellulaires. Les deux PEF et nsPEF ont démontré un potentiel thérapeutique pour une variété de pathologies humaines, y compris le traitement du cancer. Utilisant l'imagerie des cellules vivantes, cette thèse a étudié in vitro les effets de champs pulsés d'une durée de 10 ns à 10 ms sur des lignées cancéreuses (U87 glioblastome multiforme) et non cancéreuses (neurones hippocampes de souris (HT22) et cellules ovariennes du hamster chinois (CHO)). Des résultats publiés antérieurement ont démontré que les cellules cancéreuses sont plus sensibles aux champs électriques que les cellules saines. Nos résultats sont en accord avec ces résultats, dans la mesure où les cellules U87 ont subi une dépolarisation significativement plus importante de leur potentiel transmembranaire après une seule impulsion électrique à toutes les durées. Dans un ensemble d'expériences parallèles, malgré des seuils de champ électrique similaires pour la perméabilisation membranaire, les cellules U87 ont démontré une absorption significativement améliorée de YO-PRO par rapport aux autres lignées cellulaires. Bien que les cellules U87 aient subi le plus grand changement dans la dépolarisation membranaire et la perméabilisation membranaire, elles ont également montré la constante de rescellement de la membrane la plus rapide, qui était environ 30 secondes plus rapide que les autres lignées cellulaires. Pour élucider certains des mécanismes sous-jacents par lesquels les cellules U87 répondent aux champs électriques, une série d'expériences a examiné le rôle des canaux ioniques transmembranaires. Plusieurs études récentes ont rapporté que les PEF peuvent agir directement sur les canaux ioniques voltage-dépendants. En utilisant divers modulateurs de canaux ioniques pharmacologiques spécifiques et à action large, nous avons démontré que nous pouvions presque entièrement inhiber la dépolarisation membranaire induite par le champ électrique dans les cellules U87 en bloquant certains canaux cationiques. Ces résultats étaient assez spécifiques, tels que le canal de potassium de grande conductance (BK), les canaux calciques de type L et T, et le canal cationique non spécifique, TRPM8, étaient capables d'inhiber la dépolarisation tandis que le blocage d'autres canaux ioniques ne produisait aucun changement significatif. . Les travaux de cette thèse ont montré que la lignée cellulaire maligne U87 présentait une plus grande sensibilité aux champs électriques allant de 10 ns à 10 ms par rapport aux lignées cellulaires non cancéreuses étudiées. Des améliorations potentielles aux protocoles de traitement actuels ont été proposées sur la base des résultats présentés ici. / The use of pulsed electric fields (PEF) in medical and biotechnology sectors has become increasingly prevalent over the last few decades. Research has shown that by adjusting the duration of the PEF we can predict what effects will be observed. Whereas PEF in the micro-to-millisecond range have been used to permeabilize the cell membrane and enhance drug or protein uptake, nanosecond PEF (nsPEF) have demonstrated unique effects on intracellular organelles. Both PEF and nsPEF have demonstrated therapeutic potential for a variety of human pathologies, including the treatment of cancer. Using live-cell imaging, this thesis investigated, in vitro, the effects of pulsed fields ranging in duration from 10 ns to 10 ms on cancerous (U87 glioblastoma multiforme) and non-cancerous cell lines (mouse hippocampal neurons (HT22) and Chinese hamster ovary (CHO) cells). Previously published results have demonstrated that cancerous cells have a greater sensitivity to applied electric fields than healthy cells do. Our results are in agreement with these findings, insofar as the U87 cells underwent a significantly greater depolarization of their transmembrane potential following a single electric pulse at all durations. In a parallel set of experiments, despite having similar electric field thresholds for membrane permeabilization, the U87 cells demonstrated significantly enhanced YO-PRO uptake compared to the other cells lines. Although U87 cells underwent the greatest change in both membrane depolarization and membrane permeabilization, they also showed the fastest membrane resealing constant, which was approximately 30 seconds faster than other cell lines. To elucidate some of the underlying mechanisms by which U87 cells respond to electric fields, a series of experiments looked at the role of transmembrane ion channels. Several recent studies have reported that PEFs can act directly on voltage-gated ion channels. Using a variety of specific and broad acting pharmacological ion channel modulators, we demonstrated that we could almost entirely inhibit the electric field-induced membrane depolarization in U87 cells by blocking certain cationic channels. These results were quite specific, such that the big conductance potassium (BK) channel, L- and T-type calcium channels, and the non-specific cationic channel, TRPM8, were able to inhibit depolarization while blocking other ion channels produced no significant change. The work in this thesis showed that the malignant U87 cell line showed a greater sensitivity to electric fields from ranging from 10 ns – 10 ms when compared to the non-cancerous cell lines that were investigated. Potential improvements to current treatment protocols have been proposed based on the findings presented herein.

Abstract Hematopoietic cells, i.e., erythrocytes, platelets and white blood cells, differentiate from hematopoietic stem cells in a process that is similar in vertebrates. Hematopoiesis is regulated by molecules expressed by both the hematopoietic stem and progenitor cells and the surrounding microenvironments. Knowledge of these molecules is important since many of the genes involved in normal hematopoiesis are mutated in leukemia. Furthermore, this information can be utilized in more efficient isolation and expansion of hematopoietic cells in vitro. However, these molecules are not yet sufficiently characterized. Transmembrane immunoglobulin and mucin domain (TIM) genes form a known family of immunoregulators. In mammals, TIM-4 is expressed by antigen presenting cells, while TIM-1, TIM-2 and TIM-3 are expressed by T cells, in which they regulate differentiation of TH cells. The role of TIM molecules in hematopoiesis has not yet been investigated. The aim of this thesis work was to identify and analyze novel molecules involved in embryonic hematopoiesis using chicken and mouse as model organisms. This was carried out by generating a cDNA library of hematopoietic stem and progenitor cells from embryonic chicken para-aortic region. Both previously known and novel candidate genes were identified from the library. Among them, we found homologs to tim genes. Their expression and role in hematopoiesis was studied further. TIM-2 expression was shown to be tightly governed during B cell development. It is expressed by common lymphoid progenitors and highly proliferative large-pro and large pre-B cells during both fetal liver and adult bone marrow hematopoiesis. In mouse, tim-4 expression was restricted to fetal liver CD45+F4/80+ cells. Furthermore, two distinct populations were identified: F4/80hiTIM-4hi and F4/80loTIM-4lo. The results suggest that the F4/80hiTIM-4hi cells are yolk sac-derived macrophages and the F4/80loTIM-4lo cells myeloid progenitors. This work shows for the first time that TIM family molecules are expressed during hematopoiesis. TIM-2- and TIM-4 are expressed by specific cell types during hematopoietic cell development, and in the future they may be utilized as markers in isolation of hematopoietic progenitor cells. / Tiivistelmä Verisolut eli punasolut, verihiutaleet ja immuunipuolustuksessa tärkeät valkosolut kehittyvät alkion veren kantasoluista prosessissa, joka on kaikissa selkärankaisissa samankaltainen. Veren kanta- ja esisolujen sekä ympäröivän mikroympäristön tuottamat molekyylit säätelevät hematopoieesia eli verisolujen kehitystä. Näiden molekyylien tunteminen on tärkeää, sillä useat normaalia verisolujen kehitystä säätelevät geenit ovat osallisena myös verisyöpien synnyssä. Lisäksi tätä tietoa on mahdollista hyödyntää verisolujen tehokkaammassa eristämisessä ja kasvattamisessa hoitoja varten. Immuunipuolustuksen solut, kuten syöjäsolut eli makrofagit ja T-solut, ilmentävät TIM-molekyylejä (Transmembrane Immunoglobulin and Mucin). Ne toimivat immunologisen vasteen säätelyssä sekä solusyönnissä, mutta niiden roolia verisolujen kehittymisessä ei ole selvitetty aikaisemmin. Tässä väitöstutkimuksessa etsittiin uusia hematopoieesiin vaikuttavia geenejä käyttäen mallieläiminä sekä kanaa että hiirtä. Tutkimuksessa luotiin geenikirjasto kanan alkion para-aortaalisen alueen veren kanta- ja esisoluista. Kirjastosta tunnistettiin useita ennalta tiedettyjä sekä uusia verisolujen kehitykseen vaikuttavia geenejä. Tutkimuksessa analysoitiin tarkemmin kirjastosta löytyneiden TIM-geeniperheen jäsenten ilmentymistä ja roolia verisolujen kehityksessä. Tutkimuksessa osoitettiin, että TIM-2 proteiinin ilmentymistä säädellään tarkasti B-solujen kehityksen aikana. Lymfosyyttien yhteiset esisolut sekä suuret pro-B- ja pre-B-solut ilmentävät TIM-2 proteiinia B-solukehityksen aikana sekä alkion maksassa että aikuisen luuytimessä. Hiiren alkiossa tim-4 geenin ilmentyminen oli rajoittunut maksaan, jossa erottui kaksi erillistä solupopulaatiota: F4/80hiTIM-4hi ja F4/80loTIM-4lo. Tutkimuksen tulokset viittaavat siihen, että maksan F4/80hiTIM-4hi solut ovat ruskuaispussista lähtöisin olevia syöjäsoluja ja F4/80loTIM-4lo solut myeloidisen linjan esisoluja. Tämä tutkimus on ensimmäinen osoitus TIM-molekyylien ilmentymisestä kehittyvissä verisoluissa. Havaitsimme, että TIM-2 ja TIM-4-molekyylejä ekspressoidaan tietyissä soluissa verisolujen erilaistumisen aikana, joten tulevaisuudessa niitä on mahdollista käyttää merkkiproteiineina hematopoieettisten solujen esiasteita eristettäessä.

Die Fusion von Membranen erfordert die Verschmelzung von zwei Phospholipiddoppel-schichten, wobei dies über dieselben Zwischenschritte abzulaufen scheint. Eine lokale Störung (‚Stalk’) stellt eine erste Verbindung der äußeren Membranhälften dar, die anschließend lateral expandiert und ein Hemifusionsdiaphragma (HD) bildet. Das Öffnen einer Fusionspore im HD führt zur vollständigen Fusion. Mittels konfokaler Mikroskopie wurde die Fusion von Giant unilamellar vesicles (GUVs) mit negativ geladenen Lipiden und transmembranen (TM) Peptiden in Anwesenheit von zweiwertigen Kationen beobachtet, wobei die Peptide bei der HD Entstehung völlig verdrängt wurden. Eine detaillierte Analyse zeigte, dass es sich bei diesem Mikrometer-großen Bereich um ein HD handelt, dessen Größe von der Lipidzusammensetzung und Peptidkonzentration in den GUVs abhängt. Laterale Lipiddomänen gelten als entscheidend für Signal- und Sortierungsprozesse in der Zelle. Liquid ordered (Lo) Domänen in Modellsystemen wie GUVs ähneln den mit Sphingo-lipiden und Cholesterol angereicherten biologischen Raft-Domänen, allerdings scheinen Membraneigenschaften wie die Lipidpackung sich von biologischen Membranen zu unterscheiden. In diesem Zusammenhang wird die Sortierung des TM-verankerten Hemag-glutinin (HA) des Influenzavirus und von lipidverankerten Ras-Proteinen in GUVs wie auch in abgelösten Plasmamembran-Ausstülpungen (GPMVs) untersucht. HA Protein und TM-Pepitde von HA wurden ausschließlich (GUVs) bzw. vorwiegend (GPMVs) in der liquid disordered (Ld) Domäne gefunden. K-Ras wurde inmitten der Ld detektiert, während N-Ras zur Lo/Ld Grenzlinie diffundierte. Diese Ergebnisse werden im Zusammenhang mit den Unterschieden der Lipidpackung innerhalb der verschiedenen membranverankerten Systeme diskutiert. Es ist wahrscheinlich, dass die Bildung, Größe und Stabilität sowie die physikalischen Eigenschaften der Lipiddomänen in biologischen Membranen stark von Protein-Lipid-Wechsel-wirkungen beeinflusst werden. / Membrane fusion is ubiquitous in life and requires remodelling of two phospholipid bilayers. Fusion likely proceeds through similar sequential intermediates. A stalk between the contacting leaflets forms and radially expands into a hemifusion diaphragm (HD) wherein finally a fusion pore opens up. Direct experimental verification of this key structure is difficult due to its transient nature. Confocal microscopy was used to visualize the fusion of giant unilamellar vesicles (GUVs) comprising negatively charged phosphatidylserine and fluorescent transmembrane (TM) entities in the presence of divalent cations. A complete displacement of TM peptides preceded full fusion. This is consistent with HD formation. Detailed analysis provided proof that the micrometer sized structures are in fact HDs. HD size is dependent on lipid composition and peptide concentration. Lateral lipid domain formation is believed to be essential for sorting and signalling processes in the cell. Liquid ordered (Lo) domains in model systems like GUVs resemble biological rafts enriched in sphingolipids and cholesterol, but their physical properties seem distinct from biological membranes as judged by e.g. lipid order and packing. In this context the sorting of TM anchored influenza virus hemagglutinin (HA) and different lipid anchored Ras proteins is studied in GUVs and giant plasma membrane derived vesicles (GPMVs). Authentic HA or the TM domain peptides were sorted exclusively (GUVs) or predominantly (GPMVs) to the liquid disordered (Ld) domains. Whereas K-Ras was found in the bulk Ld domains, N-Ras diffuses to the Lo/Ld interface. These results are discussed with respect to differences in lipid packing in the different membrane systems and regarding the membrane anchors and their hydrophobic matching. The results suggest that the formation, size and stability as well as the physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

No description available.

O fluido multifásico complexo (suco de açaí) tem uma forte interação com membranas poliméricas ou cerâmicas de microfiltração e a formação de incrustação depende da composição e das condições de dinâmica de fluidos. Neste estudo experimental foi investigada a influência dos dois principais ácidos graxos presentes no açaí, ácido oleico e ácido palmítico, em misturas com água e no processo de microfiltração tangencial com membranas cerâmicas de alumina com um tamanho de poro nominal de 0,2 \'mü\'m. Mediu-se o fluxo de permeado em função do tempo, nas pressões transmembranas de 300 kPa, 400 kPa e 500 kPa. Para o fluxo da corrente de alimentação foram encontrados valores de Reynolds numa faixa de 9500 a 31000. Cada amostra de misturas de água/ácido oleico, água/ácido palmítico e água/ácidos oleico e palmítico, foi estudada em três séries de ensaios realizados durante 180 minutos e 72 minutos para a mistura água/ácido palmítico, a temperatura em 25 ºC. Analisou-se as incrustações resultantes e as fortes interações fluido/membrana utilizando o modelo de resistência em série e imagens tomadas por microscopia eletrônica de varredura (MEV). Os melhores resultados de permeado encontrados para a mistura de água/ácido oleico foram para Re = 33000, no entanto, resultados satisfatórios foram encontrados para Re = 20000. Agora para a água/ácido palmítico foram encontrados para Re = 20000. Os melhores resultados de permeado para a mistura água/ácidos oleico e palmítico foram para Re = 31000. De acordo com os resultados das resistências, a mistura água/ácido causa um bloqueio dos poros da membrana, resultando em uma maior diminuição do fluxo transmembrana. A limpeza foi eficiente para reduzir a resistência associada com a polarização. / The complex fluid multiphase (açaí juice) has a strong interaction with polymeric or ceramic membranes for microfiltration fouling and its formation depends on the fluid composition and fluid dynamics conditions. In this experimental study was investigated the influence of two major fatty acids present in açaí, oleic acid and palmitic acid in mixtures with water and in the process of crossflow microfiltration with ceramic membranes. In the separation process is used alumina ceramic membrane with a nominal pore size of 0.2 micrometers. The permeate flux was measured in function of time using the 300 kPa, 400 kPa and 500 kPa for the transmembrane pressure. The flow of feed stream and its respective value of Reynolds were in range of: 8900-3300. For each sample of mixtures oleic acid/water and palmitic acid/water and palmitic acid, oleic acid/ water, three series of experiments were conducted for 180 minutes and 72 minutes for mixture palmitic acid/water at temperature in 25 Celcius. For analyze of fouling resulting from strong interactions fluid/membrane was used the model of resistance in series and images taken via scanning electron microscopy (SEM). The best results for mixing oleic acid/water were to Re = 33000, however, satisfactory results were found for Re = 20000. Now for the palmitic acid/water were found to Re = 20000. For mixture palmitic acid, oleic acid/ water were found to Re = 31000. According to the results of the resistances, the mixture oleic acid/water cause a blockage of the pores of the membrane resulting in a greater decrease of the transmembrane flow. The cleaning is efficient for reducing the resistance associated with the polarization.

O mecanismo responsável pela exposição da fosfatidilserina (PS) nas membranas celulares não está bem definido. Uma atividade dependente de ATP está envolvida, provavelmente uma ATPase tipo-P. ATPases tipo P são uma família de proteínas transmembranares envolvidas no transporte de metais, íons e fosfolipídios através da membrana plasmática. As P4 ATPases translocam aminofosfolipidios (APTLs) como a PS durante a apoptose. No entanto, o sentido do transporte de PS pela APLT não está claramente definido. Os macrófagos reconhecem a PS exposta na superfície das células apoptóticas, o que inibe sua capacidade microbicida. Formas promastigotas e amastigotas de Leishmania ssp. sofrem apoptose, porém a exposição de PS na superfície dos promastigotas sempre leva à morte, enquanto que nos amastigotas não está necessariamente associada à morte e permite a internalização desses protozoários e sua sobrevivência no macrófago. Esse trabalho teve como objetivo a caracterização molecular da APLT de L. (L.) amazonensis e a avaliação de seu papel na exposição de PS nesse parasita. / The mechanism responsible for phosphatidylserine (PS) exposure in biological membranes is still an open subject. An ATP-dependent activity is involved, probably a Type P- ATPase. Type P ATPases are a family of transmembrane proteins involved in the transport of metals, ions and phospholipids across plasma membrane. P4 ATPases mediate phospholipid transport (APLT) as PS during the process of cell death by apoptosis. However, the direction (inwards or outwards) of this translocation has not been defined. Macrophages recognize exposed PS on the surface of apoptotic cells, what inhibits their microbicidal capacity. Promastigotes and amastigotes of Leishmania ssp. die by apoptosis, but PS exposure on promastigotes always leads to apoptosis, whereas PS exposure by amastigotes is not necessarily associated to death and allows their internalization and survival in the macrophage. This work aimed to characterize APLT from L. (L.) amazonensis and to evaluate its role in PS exposure in this parasite.