abstract: Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights. / Dissertation/Thesis / Doctoral Dissertation Physics 2016

INTRODUÇÃO: Superfícies de titânio modificadas por diferentes métodos foram estudadas com base nos parâmetros físicos e químicos de caracterização superficial e sua influência no comportamento de células pré-osteoblásticas (MC3T3) in vitro. MÉTODOS: Discos de titânio comercialmente puro grau II foram submetidos a três métodos de modificação de superfície (polimento, nitretados em plasma em configuração planar e gaiola catódica). As diferentes superfícies foram caracterizadas para observar o efeito do processamento na estrutura da camada superficial, na rugosidade e molhabilidade. Ensaios de adesão e proliferação celular usando linhagens de células pré-osteoblásticas MC3T3 foram realizados para avaliar o efeito das novas superfícies no comportamento celular in vitro. RESULTADOS: Os resultados demonstraram que a nitretação em plasma na configuração de gaiola catódica produz superfícies mais rugosas (p<0,02) e com menores ângulos de contato com a água. CONCLUSÕES: A adesão celular é maior nas superfícies mais rugosas do que nas superfícies polidas (p<0,05) e reagem de modo diferente a composição química do substrato e à topografia da superfície. / PURPOSE: The aim of this study was to evaluated the physico-chemical properties of different titanium surfaces modified by means of low temperature plasma nitridind on rat osteoblast cell adhesion and proliferation. METHODS: Pure Titanium discs grade II was submitted to three different surface preparations (polishing, glow discharge plasma nitriding in planar and cathodic cage configurations). Surface parameters as roughness, wettability and chemichal composition was determined to compare influency of gas mixture on the modified surface material properties. Cellular morphology was observed by scanning electron microscopy. To evaluate the effect of the surface on cellular response, osteoblast cells (MC3T3) adhesion and proliferation was quantified and data analised by Kruskal-Wallis and Friedman statistical tests. RESULTS: plasma nitriding discs shows rougher surfaces( p<0,02) in cathodic cage configuration and lower contact angle values. MC3T3 cells attached on rough surfaces produced by cathodic cage configuration was statistically significant p<0,05 compared to polished discs. CONCLUSIONS: Glow discharge plasma nitriding improve titanium surface roughness and wettability. MC3T3 cell adhesion behavior is related to substrate chemical composition and topography.

Thesis (PhD (Science) (Viticulture and Oenology. Wine Biotechnology))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae senses change within its environment and responds through specific adaptive cellular programmes, in particular by modifying gene expression. Many adaptive changes affect the physico-chemical properties of the cell wall, and several mechanisms that specifically affect the expression levels of genes that encode for cell wall components have been described previously. Cell wall modification directly impacts on general cell wall properties and cell-cell and cell-surface interactions. Many of these properties have been directly linked to families of cell wall proteins referred to as adhesins. In particular members of the Flocculation (FLO) gene family have been shown to play a crucial role in adhesion phenotypes. Flo11p functions in a variety of phenotypes including agar invasion, plastic adhesion and the formation of pseudohyphae, “flor” and “mats”, whereas Flo1p appears to control flocculation. The regulation of FLO11 expression is well documented and is mainly controlled by the mitogen activated protein kinase (MAPK) and cyclic AMP protein kinase A (cAMP-PKA) signalling cascades. Genetic analysis shows that Mss11p acts downstream and is central to these pathways, and furthermore interacts with the cAMP-PKA component Flo8p to activate transcription. In this study we further explore additional gene targets of Flo8p and Mss11p, as well as their regulation and their impact on cell wall characteristics and associated adhesion phenotypes. Our analysis shows that Mss11p is also required for FLO1 expression, and functions together with Flo8p to control many Flo-dependent adhesion phenotypes. Genome-wide gene expression analysis further reveals that altered Mss11p levels leads to the change in the expression of various cell membrane and cell wall genes, notably AQY2 and members of the DAN and TIR gene families. Further genetic analysis indicates that adhesion phenotypes display an almost exclusive dependence on FLO gene expression. We also demonstrate that these phenotypes require Flo10p and are thus dependent on the specific balance of Flo proteins in the cell wall. The analysis of signalling deletion mutants show that regulation of FLO10 shares signalling components with FLO11, but that the two genes are differentially regulated. Unlike FLO11, FLO10 transcription also does not display an absolute requirement for Mss11p but rather for the MAPK component Ste12p. Whole genome expression analysis were also performed on strains with altered levels of Flo8p which were compared with the above mentioned transcriptome data set. This analysis shows that Flo8p and Mss11p co-regulate the FLO genes, as well as AQY2 and TIR3, but also have significant unique gene targets. The combination of transcriptome data with current information concerning transcription factor (TF) interaction networks reveals the importance of network interaction between Cin5p, Flo8p, Mga1p and Mss11p. From these data we constructed a TF interaction model in which Flo8p acts as the predominantly activating TF component, whereas Mss11p function as a target hub TF, possibly as a mediator- or polymerase II holo-enzyme component. Finally we provide a first report on “mat” formation by an industrial wine yeast strain, and show that by adjusting FLO11 expression in this strain we are able to significantly change this phenotypic behaviour. / AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae neem veranderinge in sy omgewing waar en reageer daarop deur middel van spesifieke sellulêre programme, in die besonder deur geenuitdrukking aan te pas. Verskeie aanpasbare veranderinge beïnvloed die fisieke, asook chemiese eienskappe van die selwand, en talle meganismes is al beskryf wat die uitdrukkingsvlakke beïnvloed van gene wat vir selwandkomponente kodeer. Die modifikasie van die selwand het ’n direkte impak op selwand-eienskappe, asook die sel-sel- en sel-oppervlak-interaksies. Verskeie van hierdie eienskappe word direk gekoppel aan die selwandproteïenfamilies, wat ook as adhesie-faktore bekend staan. Veral lede van die Flokkulasie (FLO) -geenfamilie het ’n noodsaaklike funksie in adhesie-fenotipes. Flo11p speel ’n rol in verskeie fenotipes, wat insluit die indringende groei van agar, plastiekaanhegting en die vorming van pseudohifes, “flor“ en “matte“, terwyl Flo1p flokkulasie beheer. Die regulering van FLO11-uitdrukking is deeglik gedokumenteerd en dit word hoofsaaklik gereguleer deur die mitogeen-geaktiveerde proteïenkinase (MAPK) en sikliese AMP-proteïenkinase A (cAMP-PKA) seintransduksiekaskades. Genetiese analises toon dat Mss11p stroom-af en sentraal tot hierdie kaskades funksioneer, en dit aktiveer transkripsie deur interaksie met die cAMP-PKA-komponent, Flop8. In hierdie studie word ’n ondersoek gedoen na addisionele teikengene van Flo8p en Mss11p, en hoe hierdie gene gereguleer word, asook hul impak op selwandeienskappe en geassosieerde adhesie-fenotipes. Ons analises toon dat Mss11p ook benodig word vir die ekspressie van FLO1 en dat dit, tesame met Flo8p, beheer uit oefen oor verskeie Flo-afhanklike fenotipes. Genoomwye geenekspressie-analises wys verder daarop dat veranderde Mss11p-vlakke lei tot die aanpassing van die ekspressie van verskeie selmembraan- en selwandgene, naamlik AQY2 asook lede van die DAN- en TIR-geenfamilies. Verdere genetiese analise dui daarop dat adhesie-fenotipes byna eksklusief afhanklik is van FLO-geenekspressie. Daar is verder getoon dat hierdie fenotipes ook Flo10p benodig en dus afhanklik is van die spesifieke balans van Floproteïene in die selwand. Die analise van seintransduksiemutante demonstreer dat FLO10 en FLO11 seintransduksie-komponente deel, maar dat hierdie gene verskillend gereguleer word. Anders as FLO11, toon FLO10 nie ’n absolute noodsaaklikheid vir Mss11p nie, maar eerder vir die MAPK-komponent, Ste12p. Totale genoomekspressie-analises is ook gedoen op gisrasse met aangepaste vlakke van Flo8p en dis vergelyk met bogenoemde transkripsiedatastel. Hierdie analise wys dat Flo8p and Mss11p die FLO-gene, asook AQY2 en TIR3, koreguleer, maar ook beduidende unieke teikengene het. Die kombinasie van transkripsiedata met huidig beskikbare informasie betreffende transkripsiefaktor (TF) -interaksienetwerke dui op die relevansie van netwerkinteraksie tussen Cin5p, Flo8p, Mga1p en Mss11p. Hiervan is daar ’n model opgestel waarin Flo8p in die meeste gevalle as die aktiverende TF-komponent optree, terwyl Mss11p as TF-teiken dien, moontlik as ’n mediator- of polimerase II holoënsiemkomponent. Laatens word daar vir die eerste keer verslag gedoen van ”mat”-vorming deur ’n industriële wyngisras en toon ons verder dat hierdie fenotipe beduidend verander word deur middel van die aanpassing van FLO11-uitdrukking.

O patógeno Trichophyton rubrum é um fungo queratinofílico e o principal agente de micoses cutâneas humanas. O processo de degradação de substratos queratinizados por dermatófitos está relacionado com a alcalinização do ambiente, o que modula a expressão e a secreção de enzimas responsáveis pela captação e utilização dos nutrientes presentes no microambiente hospedeiro. Essa modulação do pH parece determinante para o sucesso do processo infeccioso, quando o fungo se depara com o pH ácido da pele humana. A hipótese deste trabalho foi verificar se há influência do pH e da fonte nutricional na modulação da expressão de genes de T. rubrum que codificam proteínas envolvidas nos processos de autofagia (atg8 e atg15), adesão celular (sowgp) e de alguns fatores de transcrição (pacC, bZIP/cys-3 e nuc-1), e se estes processos estão relacionados à patogenicidade deste dermatófito. De modo a avaliar a modulação da expressão destes genes na patogenicidade e sobrevivência fúngica, T. rubrum foi cultivado em meios de cultura tamponados (pH 5,0 e pH 8,0) e não tamponados, contendo glicose, glicina, glicose com glicina, ou queratina como fonte nutricional. Os genes envolvidos no processo de autofagia também foram avaliados na presença do inibidor de autofagia 3-metiladenina (3-MA). Além disso, o perfil transcricional destes genes de T. rubrum foi avaliado durante a interação ex vivo com unha e pele humana. As análises demonstraram a influência concomitante da fonte nutricional e do pH ambiente na modulação da expressão dos transcritos gênicos estudados nas diferentes linhagens de T. rubrum utilizadas neste trabalho (CBS, H6 e pacC-1). Nossos resultados revelaram que o crescimento destas linhagens é maior na fonte nutricional queratina, quando comparado com as outras fontes nutricionais (glicose e glicina), havendo uma diferença na taxa de crescimento entre as linhagens neste substrato preferencial. Os genes bZIP/cys-3 e nuc-1 apresentaram perfil de expressão semelhante, mais elevada durante cultivos longos, respondendo a condições de estresse nutricional e pH alcalino, sugerindo a participação dos mesmos nos processos de crescimento e sobrevivência do fungo. Além disso, mecanismos regulatórios diferentes destes genes devem ocorrer nas três linhagens de T. rubrum em relação ao crescimento na fonte nutricional queratina. A variação transcricional do gene pacC em resposta às diferentes fontes nutricionais sugere que sua expressão depende das condições nutricionais encontradas por esse fungo, sendo o pH uma resposta secundária. Nossos resultados sugerem ainda que a via de autofagia seja importante para o desenvolvimento e sobrevivência de T. rubrum nestes substratos, e que o FT PacC atue regulando um dos pontos deste processo, visto que o gene atg15 apresenta um perfil semelhante de expressão ao gene pacC, e o gene atg8 tem perfil antagônico de expressão nas linhagens H6 e pacC-1, nas condições avaliadas neste trabalho. 3-MA inibiu a transcrição dos genes atg8 e atg15 em T. rubrum de modo especifico, e por consequência a via de macroautofagia. Os dados de expressão de sowgp sugerem que essa molécula atue durante a privação nutricional e situações de estresse pelo dermatófito, provavelmente desempenhando seu papel na progressão e manutenção do processo infeccioso, permitindo a permanência do fungo em tecidos queratinizados, e auxiliando na fixação do fungo ao epitélio humano. Desta maneira, nossos resultados fornecem informações sobre os eventos moleculares envolvidos na regulação da expressão genica durante a adaptação de T. rubrum ao pH, à variação nutricional, e interação com células e moléculas do microambiente hospedeiro. Os resultados revelam o envolvimento do processo de autofagia e de moléculas de adesão no sensoriamento e resposta a variações ambientais, e a modulação dos fatores de transcrição bZIP/Cys-3, Nuc-1 e PacC durante a adaptação de T. rubrum ao pH e à fonte nutricional podem ser importantes na regulação de moléculas necessárias para sua patogenicidade. / The pathogen Trichophyton rubrum is a keratinophylic fungi and the major agent of cutaneous mycosis in humans. The process of degradation of keratinized substrates by dermatophytes is related with environment alkalinization, which modulates the expression and secretion of the enzymes responsible for the acquisition and utilization of nutrients from the host microenvironment. This pH modulation seems to be determinant for the success of the infectious process when the fungus encounters the acidic pH of the human skin. The hypothesis of this study was to determine whether there is influence of ambient pH and nutritional source in the modulation of gene expression of T. rubrum genes coding for proteins involved in the processes of autophagy (atg8 and atg15), cellular adhesion (sowgp) and some transcription factors (pacC, bZIP/cys-3 and nuc-1), and if they are related with the pathogenicity of this dermatophyte. To evaluate the modulation of the expression of these genes in fungal pathogenicity and survival, T. rubrum was cultivated in buffered (pH 5.0 and pH8.0) or non-buffered media, containing glucose, glycine, glucose and glycine, or keratin as nutrient source. The autophagy-related genes were also analyzed in the presence of 3- methyladenine (3-MA) autophagy inhibitor. Moreover, the transcriptional profile of these genes was evaluated during T. rubrum ex vivo interaction with human nail and skin. The analyses demonstrate the simultaneous influence of the nutritional source and environment pH in the modulation of gene transcription in the different T. rubrum strains evaluated here (CBS, H6 and pacC-1). Our results revealed that growth of these strains is higher in keratin source, compared with other nutrient sources (glucose or glycine), and that they present different growth rates in this preferential substrate. The genes bZIP/cys-3 and nuc-1 presented a similar expression profile, which is higher during longer periods of growth, responding to nutritional stress and alkaline pH, suggesting their involvement in fungal growth and survival. Also, different regulatory mechanisms of these genes in these three T. rubrum strains might be implicated during keratin degradation. The transcriptional variation in the pacC gene in response to different nutritional sources, suggests that its expression is dependent on the nutritional conditions, being the ambient pH a secondary response. Furthermore, our results indicate that the autophagy pathway is important for T. rubrum survival and development during nutrient and pH adaptation, and that PacC might regulates some points of this process, since atg15 and pacC genes presented a similar expression profile, and atg8 has antagonistic expression profiles in the H6 and pacC-1 strains, in the conditions evaluated here. 3-MA inhibited the transcription of atg8 and atg15 T. rubrum genes in a specific manner, and thus inhibited the macroautophagy process. The expression profile of the sowgp gene suggests that this molecule is important during nutrient starvation and stress situation, probably having a role in the progression and maintenance of the infectious process, allowing fungal maintenance in the host keratinized tissues, contributing to fungal adherence to human epithelia. Taken together, our results provide insights into the molecular events involved in the regulation of gene expression during T. rubrum adaptation to pH, nutritional variation and interaction with the host cells and molecules. Our data reveals the involvement of the autophagy process and adhesion molecules in sensing and response of environmental changes, and the modulation of the bZIP/Cys-3, Nuc-1 and PacC transcription factors during T. rubrum adaptation to pH and nutrient source might be important in the regulation of molecules required for its pathogenicity.

The pathophysiology of atherosclerotic cardiovascular disease (CVD) is highlighted by vascular dysfunction and low-grade vascular inflammation. Furthermore, the site-specific distribution of atherosclerosis throughout the arterial vasculature is primarily determined by local hemodynamic force. Therefore, this dissertation outlines three experiments designed to investigate the role of acute mental and physical (i.e., aerobic exercise), and vascular wall shear stress (SS) on the inflammatory aspects of atherosclerosis. Chapter 2 examines the effect of acute laboratory-induced mental stress on intracellular pro-inflammatory signaling pathways in peripheral blood mononuclear cells. Chapter 3 investigates the impact of acute laboratory-induced mental stress and maximal aerobic exercise on the concentration of soluble VCAM-1 (sVCAM-1) and CX3CL1/fractalkine (sCX3CL1) in human serum. Lastly, Chapter 4 examines the role of short- (30 min) and long-term (24 hr) low-to-negative oscillating SS (LOSS) and high laminar SS (HLSS) on the expression and secretion (i.e., cleavage) of cell-membrane VCAM-1 and CX3CL1 by human umbilical vein endothelial cell cultures in vitro. Together, these experiments provide evidence that acute psychological stress, maximal aerobic exercise, and HLSS influence vascular inflammation and adhesive properties of the vessel wall. More specifically, the results from Chapter 2 provide evidence that acute mental stress promotes the immune-cell mediated synthesis of pro-inflammatory cytokines in circulation. In addition, Chapter 3 and Chapter 4 demonstrate that the elevations in blood flow and hemodynamic force associated with maximal aerobic exercise, and unidirectional high SS may have the capacity to alter the expression of endothelial-bound cellular adhesion molecules, in part by eliciting their release from the vessel wall.

L’adhésion et les propriétés mécaniques des cellules jouent un rôle crucial dans le fonctionnementcellulaire ainsi que dans l’apparition de maladies dégénératives. Pour mesurer ces quantités, nousavons développé dans ce travail un microscope opto-acoustique pour l’imagerie non-invasive de lamécanique de cellules individuelles avec une résolution sub-cellulaire. Ce microscope utilise latechnique d’acoustique picoseconde qui permet de générer et détecter optiquement des ondesacoustiques avec une large bande s’étendant jusqu’à 1 THz. Dans le but de reproduire lecomportement mécanique des cellules à des fréquences acoustiques supérieures à 10 GHz, uneétude sur des objets mous biomimétiques est menée dans une première partie. Les rigidité, viscositéet épaisseur de ces systèmes multicouches micrométriques sont caractérisées. Dans la deuxièmepartie de ce manuscrit, la technique d’acoustique picoseconde est employée pour imager le contactentre une cellule animale modèle et un biomatériau, ainsi que l’impédance acoustique de cette cellule.Un outil d’analyse nécessaire pour le traitement du signal acoustique est mis en place. Enfin, unmicroscope opto-acoustique opérationnel entre 10 et 100 GHz est présenté dans la dernière partie. Ilest basé sur un dispositif pompe-sonde asynchrone qui permet de produire des images acoustiquesen un temps court (4 pixels/min) avec une résolution axiale de l’ordre d’une dizaine de nm. Cetteapproche est comparable à une échographie mais à l’échelle cellulaire. L’étude de l’adhésion et despropriétés mécaniques de plusieurs types de cellules à différents stades de maturation est abordée.Des images topographiques des zones fines (< 50 nm) d’une cellule sont également analysées. Lemicroscope développé durant cette thèse offrira la possibilité d’explorer de nouvelles pistes derecherche dans les domaines de la biologie cellulaire et des biotechnologies. / Adhesion and mechanical properties of cells are key players in several cellular functions and areinvolved in the development of degenerative diseases. To characterize these quantities, we developedin this work an opto-acoustic microscope for the non-invasive imaging of the mechanics of individualcells with a sub-cell resolution. This microscope uses the Picosecond Ultrasonics (PU) technique thatallows optical generation and detection of acoustic waves with a large bandwidth up to 1 THz. In orderto reproduce the mechanical behaviour of cells at acoustic frequencies greater than 10 GHz, a studyof cell-mimicking micro-objects is first considered. The rigidity, viscosity and thickness of these microlayeredstructures are characterized. In the second part of this manuscript, the PU technique isapplied for imaging the contact between a simple animal cell and a biomaterial, as well as the acousticimpedance of this cell. An essential tool for analysing the acoustic signal is developed. In the thirdpart, the opto-acoustic microscope operating between 10 and 100 GHz is finally presented. It is basedon an asynchronous pump-probe setup that allows producing acoustic images within a short time (4pixels/min) and offering an axial resolution of about 10 nm. This is similar to cell ultrasonography. Thestudy of the adhesion and of the mechanical properties of different cell types at different stages of cellmaturation is then tackled. The topographic images of thin cell regions (< 50 nm) are also analysed.The microscope implemented during this thesis should offer the possibility of exploring new avenuesin the field of cellular biology.

O patógeno Trichophyton rubrum é um fungo queratinofílico e o principal agente de micoses cutâneas humanas. O processo de degradação de substratos queratinizados por dermatófitos está relacionado com a alcalinização do ambiente, o que modula a expressão e a secreção de enzimas responsáveis pela captação e utilização dos nutrientes presentes no microambiente hospedeiro. Essa modulação do pH parece determinante para o sucesso do processo infeccioso, quando o fungo se depara com o pH ácido da pele humana. A hipótese deste trabalho foi verificar se há influência do pH e da fonte nutricional na modulação da expressão de genes de T. rubrum que codificam proteínas envolvidas nos processos de autofagia (atg8 e atg15), adesão celular (sowgp) e de alguns fatores de transcrição (pacC, bZIP/cys-3 e nuc-1), e se estes processos estão relacionados à patogenicidade deste dermatófito. De modo a avaliar a modulação da expressão destes genes na patogenicidade e sobrevivência fúngica, T. rubrum foi cultivado em meios de cultura tamponados (pH 5,0 e pH 8,0) e não tamponados, contendo glicose, glicina, glicose com glicina, ou queratina como fonte nutricional. Os genes envolvidos no processo de autofagia também foram avaliados na presença do inibidor de autofagia 3-metiladenina (3-MA). Além disso, o perfil transcricional destes genes de T. rubrum foi avaliado durante a interação ex vivo com unha e pele humana. As análises demonstraram a influência concomitante da fonte nutricional e do pH ambiente na modulação da expressão dos transcritos gênicos estudados nas diferentes linhagens de T. rubrum utilizadas neste trabalho (CBS, H6 e pacC-1). Nossos resultados revelaram que o crescimento destas linhagens é maior na fonte nutricional queratina, quando comparado com as outras fontes nutricionais (glicose e glicina), havendo uma diferença na taxa de crescimento entre as linhagens neste substrato preferencial. Os genes bZIP/cys-3 e nuc-1 apresentaram perfil de expressão semelhante, mais elevada durante cultivos longos, respondendo a condições de estresse nutricional e pH alcalino, sugerindo a participação dos mesmos nos processos de crescimento e sobrevivência do fungo. Além disso, mecanismos regulatórios diferentes destes genes devem ocorrer nas três linhagens de T. rubrum em relação ao crescimento na fonte nutricional queratina. A variação transcricional do gene pacC em resposta às diferentes fontes nutricionais sugere que sua expressão depende das condições nutricionais encontradas por esse fungo, sendo o pH uma resposta secundária. Nossos resultados sugerem ainda que a via de autofagia seja importante para o desenvolvimento e sobrevivência de T. rubrum nestes substratos, e que o FT PacC atue regulando um dos pontos deste processo, visto que o gene atg15 apresenta um perfil semelhante de expressão ao gene pacC, e o gene atg8 tem perfil antagônico de expressão nas linhagens H6 e pacC-1, nas condições avaliadas neste trabalho. 3-MA inibiu a transcrição dos genes atg8 e atg15 em T. rubrum de modo especifico, e por consequência a via de macroautofagia. Os dados de expressão de sowgp sugerem que essa molécula atue durante a privação nutricional e situações de estresse pelo dermatófito, provavelmente desempenhando seu papel na progressão e manutenção do processo infeccioso, permitindo a permanência do fungo em tecidos queratinizados, e auxiliando na fixação do fungo ao epitélio humano. Desta maneira, nossos resultados fornecem informações sobre os eventos moleculares envolvidos na regulação da expressão genica durante a adaptação de T. rubrum ao pH, à variação nutricional, e interação com células e moléculas do microambiente hospedeiro. Os resultados revelam o envolvimento do processo de autofagia e de moléculas de adesão no sensoriamento e resposta a variações ambientais, e a modulação dos fatores de transcrição bZIP/Cys-3, Nuc-1 e PacC durante a adaptação de T. rubrum ao pH e à fonte nutricional podem ser importantes na regulação de moléculas necessárias para sua patogenicidade. / The pathogen Trichophyton rubrum is a keratinophylic fungi and the major agent of cutaneous mycosis in humans. The process of degradation of keratinized substrates by dermatophytes is related with environment alkalinization, which modulates the expression and secretion of the enzymes responsible for the acquisition and utilization of nutrients from the host microenvironment. This pH modulation seems to be determinant for the success of the infectious process when the fungus encounters the acidic pH of the human skin. The hypothesis of this study was to determine whether there is influence of ambient pH and nutritional source in the modulation of gene expression of T. rubrum genes coding for proteins involved in the processes of autophagy (atg8 and atg15), cellular adhesion (sowgp) and some transcription factors (pacC, bZIP/cys-3 and nuc-1), and if they are related with the pathogenicity of this dermatophyte. To evaluate the modulation of the expression of these genes in fungal pathogenicity and survival, T. rubrum was cultivated in buffered (pH 5.0 and pH8.0) or non-buffered media, containing glucose, glycine, glucose and glycine, or keratin as nutrient source. The autophagy-related genes were also analyzed in the presence of 3- methyladenine (3-MA) autophagy inhibitor. Moreover, the transcriptional profile of these genes was evaluated during T. rubrum ex vivo interaction with human nail and skin. The analyses demonstrate the simultaneous influence of the nutritional source and environment pH in the modulation of gene transcription in the different T. rubrum strains evaluated here (CBS, H6 and pacC-1). Our results revealed that growth of these strains is higher in keratin source, compared with other nutrient sources (glucose or glycine), and that they present different growth rates in this preferential substrate. The genes bZIP/cys-3 and nuc-1 presented a similar expression profile, which is higher during longer periods of growth, responding to nutritional stress and alkaline pH, suggesting their involvement in fungal growth and survival. Also, different regulatory mechanisms of these genes in these three T. rubrum strains might be implicated during keratin degradation. The transcriptional variation in the pacC gene in response to different nutritional sources, suggests that its expression is dependent on the nutritional conditions, being the ambient pH a secondary response. Furthermore, our results indicate that the autophagy pathway is important for T. rubrum survival and development during nutrient and pH adaptation, and that PacC might regulates some points of this process, since atg15 and pacC genes presented a similar expression profile, and atg8 has antagonistic expression profiles in the H6 and pacC-1 strains, in the conditions evaluated here. 3-MA inhibited the transcription of atg8 and atg15 T. rubrum genes in a specific manner, and thus inhibited the macroautophagy process. The expression profile of the sowgp gene suggests that this molecule is important during nutrient starvation and stress situation, probably having a role in the progression and maintenance of the infectious process, allowing fungal maintenance in the host keratinized tissues, contributing to fungal adherence to human epithelia. Taken together, our results provide insights into the molecular events involved in the regulation of gene expression during T. rubrum adaptation to pH, nutritional variation and interaction with the host cells and molecules. Our data reveals the involvement of the autophagy process and adhesion molecules in sensing and response of environmental changes, and the modulation of the bZIP/Cys-3, Nuc-1 and PacC transcription factors during T. rubrum adaptation to pH and nutrient source might be important in the regulation of molecules required for its pathogenicity.

No description available.

Les trois projets que j’ai développés au cours de ma thèse s’inscrivent dans un même contexte, celui de l’étude de l’acétylcholinestérase (AChE) et des molécules apparentées à l’AChE au plan structural ou fonctionnel. Existence, identification et caractérisation du fonctionnement d’une « porte arrière » dans l’AChE. Caractérisation de la « protéine X », un récepteur non AChE de la fasciculine. Recherche d’un partenaire protéique endogène de l’AChE dans le cerveau de rat. / The three projects I developed during my thesis are related to the study of acetylcholinesterase (AChE) and of molecules related to AChE in their function or structure. Existence, identification and characterization of the functioning of a back door in AChE. Characterization of “protein X”, a non-AChE receptor for fasciculin. Search for an endogenous proteic partner of AChE in rat brain.

As Anemias Hemolíticas compreendem um grupo de doenças em que há redução acentuada na sobrevivência dos glóbulos vermelhos circulantes e a medula óssea não é capaz de compensação, mesmo aumentando sua produção, o que causa anemia desde os primeiros anos da vida da pessoa. Dentre as doenças deste grupo, a Anemia Falciforme (SCA) e a Esferocitose Hereditária (HS) destacam-se por se tratarem de enfermidades com defeitos genéticos intrínsecos das células vermelhas (RBCs) que geram complicações multissistêmicas agudas e crônicas em seus portadores. Por vias patofisiológicas distintas, reticulócitos e respectivas hemácias defeituosas de tais doenças, falciformes e esferócitos, são continuamente aprisionados e fagocitados no baço, importante órgão de destruição de células velhas e/ou defeituosas via hemólise extravascular, o que leva progressivamente à disfunção e eventual perda da função esplênica. O objetivo desse trabalho é avaliar o papel do baço em relação à habilidade e ao fenótipo adesivos de reticulócitos (ret) e eritrócitos (erit) em pacientes com SCA e HS, com e sem função esplênica preservada. Amostras de sangue de 37 pacientes (22 SCA and 15 HS) com função esplênica e 19 pacientes (13 SCA e 6 HS) sem ela foram avaliadas. Ainda, sangue de 22 crianças com SCA foi coletado em estudo longitudinal dos 6 e 29 meses de vida. Todas as amostras de sangue foram analisadas quanto à função esplênica (Contagem de células PIT e de corpúsculos de Howell-Jolly - HJB), quanto ao perfil imunofenotípico celular (em % e em média de intensidade de fluorescência - MFI) e quanto à habilidade de adesão das células vermelhas à laminina e à linhagem celular endotelial HMEC-1. A análise da transição da perda de função esplênica demonstrou que a mesma se intensificou a partir dos 3 anos de idade (PIT: r=0,8; p<0,0001; HJB: r=0,7; p<0.0001). Quanto à imunofenotipagem celular, a contagem PIT se correlacionou positivamente, principalmente com os marcadores CD147 (%ret: r=0,6; p<0,0001; MFIret: r=0,6; p<0,0001; %erit: r=0,7; p<0,0001; MFIerit: r=0,6; p<0,0001), LuBCAM (%ret: r=0,5; p=0,004; MFIret: r=0,6; p<0,0001; %erit: r=0,6; p<0,0003; MFIerit: r=0,4; p<0,004) and CD58 (%ret: r=0,4; p=0,006; MFIret: r=0,5; p<0,0013; %erit: r=0,4; p<0,009; MFIerit: r=0,6; p<0,0001). Na comparação entre ausência ou presença do baço, a perda de sua função exerceu influência no aumento da expressão de adesão de RBCs em SCA, principalmente CD147 (%ret: p=0,002; MFIret: p=0,003; %erit: p<0,0001; MFIerit: p=0,005), LuBCAM (%ret: p=0,0001; MFIret: p<0,0001; %erit: p<0,0001; MFIerit: p<0,0001) e CD58 (%ret: p=0,007; MFIret: p=0,006; %erit: p=0,003; MFIerit: p=0,0004), embora a adesão celular tenha diminuído em pacientes HS esplenectomizados. Na comparação entre as doenças, pacientes HS com o baço apresentaram maior freqüência de adesão celular em relação aos SCA, notavelmente em relação ao LuBCAM (%ret: p=0,0008; MFIret: p=0,03; %erit: p<0,0001; MFIerit: p=0,0002), CD58 (%ret: p=0,0009; %erit: p=0,003) e CD44 (%ret: p=0,009; %erit: p<0,003). No entanto, as amostras SCA sem função esplênica tiveram maior expressão de adesão celular para CD147 (%ret: p=0,006; MFIret: p=0,02; %erit: p=0,02), LuBCAM (%ret: p=0,004; MFIret: p<0,0001), CD36 (%ret: p=0,0002; MFIret: p=0,01), CD242 (%ret: p=0,0008; %erit: p=0,05) e CD49d (%ret: p=0,04). Em relação ao Ensaio de Adesão in vitro, na ausência de baço, os RBCs SCA apresentaram maior adesividade à laminina do que os RBCs SCA com função esplênica preservadaem todas as taxas de fluxo de tensão de cisalhamento empregadas (0,5 dyne/cm2: p=0,01; 1 dyne/cm2: p=0,02; 2 dynes/cm2: p=0,03; 3 dynes/cm2: p=0,03; 5 dynes/cm2: p=0,04 e 7 dynes/cm2: p=0,03). Especialmente, reticulócitos de pacientes sem baço apresentaram maior adesividade à HMEC-1 em baixas tensões de cisalhamento (1 dyne/cm2) em ambas as doenças (SCA: p=0,03; HS: p=0,03). Por fim, reticulócitos apresentaram maior habilidade adesiva à células endoteliais em indivíduos SCA do que em pacientes HS, com (0,5 dyne/cm2: p=0,04; 1 dyne/cm2: p=0,03) ou sem baço (0,5 dyne/cm2: p=0,02; 2 dynes/cm2: p=0,01; 3 dynes/cm2: p=0,03; 5 dynes/cm2: p=0,02 e 7 dynes/cm2: p=0,03). Nossos resultados indicam que embora pertençam ao grupo de Anemias Hemolíticas, as patofisiologias e evoluções clínicas distintas de SCA e de HS levam a padrões imunofenotípicos diferentes de expressão da adesão celular. Na SCA, a ausência de função esplênica teria direta relação com o aumento do fenótipo pró-adesivo e com a adesividade de RBCs SCA, o que traz sérias consequências clínicas aos pacientes, enquanto na HS sem baço, de maneira geral, os eventos de adesão celular são minimizados, embora ainda apresentem reticulócitos e eritrócitos adesivos circulantes após a esplenectomia. / Hemolytic Anemias comprise a group of diseases in which there is marked reduction in the survival of circulating erythrocytes and the bone marrow is not capable of compensation, even by increasing its production, which causes anemia from the first years of the person\'s life on. Among the diseases of this group, Sickle Cell Anemia (SCA) and Hereditary Spherocytosis (HS) stand out for being diseases with intrinsic genetic defects of red blood cells (RBCs) that generate acute and chronic multisystemic complications in their patients. By distinct pathophysiological pathways, reticulocytes and these disease\'s respective defective erythrocytes, sickle and spheroid ones, are continuously trapped and phagocytosed in the spleen, important organ of destruction of old and/or defective cells via extravascular hemolysis, which progressively leads to dysfunction and eventual loss of splenic function. The objective of this study was to evaluate the role of the spleen in relation to the reticulocyte (ret) and erythrocyte (eryt) adhesive ability and adhesion phenotype in patients with SCA and HS, with and without preserved splenic function. Blood samples from 37 patients (22 SCA and 15 HS) with splenic function and 19 patients (13 SCA and 6 HS) without it were evaluated. Still, blood from 22 children with SCA was collected in a longitudinal study from 6 to 29 months of age. All blood samples were analyzed for splenic function [pitted cells (PIT) and Howell-Jolly bodies (HJB) counting], for the cellular immunophenotypic profile (in % and in mean fluorescence intensity - MFI) and for the adhesive ability of RBCs to laminin and to endothelial cell line HMEC-1. Analysis of the splenic function loss transition showed that it intensified from 3 years of age on (PIT: r=0.8, p<0.0001; HJB: r=0.7, p<0.0001). Regarding the cellular immunophenotyping, PIT count correlated positively, mainly with CD147 markers (%ret: r=0.6, p<0.0001; MFIret: r=0.6, p<0.0001; %eryt: r=0.7, p<0.0001; MFIeryt: r=0.6, p<0.0001), LuBCAM (%ret: r=0.5, p=0.004; MFIret: r=0.6, p<0.0001; %eryt: r=0.6, p<0.0003; MFIeryt: r=0.4, p<0.004) and CD58 (%ret: r=0.4, p=0.006; MFIret: r=0.5, p<0.0013; %eryt: r=0.4, p<0.009; MFIeryt: r=0.6, p<0.0001). In the comparison between spleen absence or presence, the loss of its function exerted influence on the increase of RBCs adhesion expression in SCA, mainly on CD147 (%ret: p=0.002; MFIret: p=0.003; %eryt: p<0.0001; MFIeryt: p=0.005), LuBCAM (%ret: p=0.0001; MFIret: p<0.0001; %eryt: p<0.0001; MFIeryt: p<0.0001) e CD58 (%ret: p=0.007; MFIret: p=0.006; %eryt: p=0.003; MFIeryt: p=0.0004), although cell adhesion has been decreased in splenectomized HS patients. In the comparison between diseases, HS patients with spleen showed higher cell adhesion frequency compared to SCA, notably in relation to LuBCAM (%ret: p=0.0008; MFIret: p=0.03; %eryt: p<0.0001; MFIeryt: p=0.0002), CD58 (%ret: p=0.0009; %eryt: p=0.003) and CD44 (%ret: p=0.009; %eryt: p<0.003). However, SCA samples without splenic function had higher cell adhesion expression for CD147 (%ret: p=0.006; MFIret: p=0.02; %eryt: p=0.02), LuBCAM (%ret: p=0.004; MFIret: p<0.0001), CD36 (%ret: p=0.0002; MFIret: p=0.01), CD242 (%ret: p=0.0008; %eryt: p=0.05) and CD49d (%ret: p=0.04). Concerning the in vitro Adhesion Assay, in the spleen absence, SCA RBCs showed greater adhesiveness to laminin than SCA RBCs with preserved splenic function did at all shear stress flow rates applied (0.5 dyne/cm2: p=0.01, 1 dyne/cm2: p=0.02, 2 dynes/cm2: p=0.03, 3 dynes/cm2: p=0.03, 5 dynes/cm2: p=0.04 and 7 dynes/cm2:p=0.03). Especially, reticulocytes from patients without spleen showed higher adhesiveness to HMEC-1 at low shear stresses (1 dyne/cm2) in both diseases (SCA: p=0.03; HS: p=0.03). Finally, reticulocytes showed greater adhesion ability to endothelial cells in SCA subjects than in HS patients, with (0.5 dyne/cm2: p=0.04 and 1 dyne/cm2: p=0.03) or without spleen (0.5 dyne/cm2: p=0.02, 2 dynes/cm2: p=0.01, 3 dynes/cm2: p=0.03, 5 dynes/cm2: p=0.02 and 7 dynes/cm2: p=0.03). Our results indicate that although both diseases belong to the Hemolytic Anemias group, SCA and HS distinct pathophysiologies and clinical evolution lead to different immunophenotypic patterns of cell adhesion expression. In SCA, the absence of splenic function may have a direct relation with the increase of SCA RBCs proadhesive phenotype and adhesiveness, which brings serious clinical consequences to the patients, whereas in HS without spleen, in general, cellular adhesion events are minimized, although they still present adhesive circulating reticulocytes and erythrocytes after splenectomy.