Hepatit-B är en leversjukdom som orsakas av hepatit-B-virus (HBV) vilket är en kapslad DNA-virus. Kapsidprotein (Cp) har stor betydelse i virusets livscykel exempelvis DNA-replikation, interaktion med värdceller och andra virala glykoproteiner. HBV, som många andra virus, tar sig in i cellen genom att binda till cellreceptorer. Transferrinreceptor är en välkänd receptor som mögliggör virus inträde i cellen genom att binda till virusproteiner, intraktionen sker i apikala domänen i TfR. Båda Cp149, kapsidsammansättnings domänen i Cp, och apikala domänen i TfR är betydelsefulla ändamål för utveckling av antivirala läkemedel. Syftet med arbetet var att klona och uttrycka olika varianter av ett nytt modifierat Cp149, där Cp149 har modifierats med AP01 (lösliga formen av apikala dömanen),  och analysera intraktioner mellan proteinerna och viralt glykoprotein, MGP1. Modifierade proteingener klonades i plasmid (pET-11a) med hjälp av rekombinant DNA-teknik och användning av restriktionsenzymer NdeI och BamHI. Agarosgelelektrofores och DNA-sekvensering användes för att  kontrollera förekomst av eftersökta DNA-sekvenser. Nya plasmider fördes över till bakterieceller, Escherichia coli, och  proteinutrycket inducerades i bakteriecellerna genom kemiskbehandling. Framrenade proteiner från respektive provlösning analyserades med Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) och proteinernas funktion undersöktes med flödescytometri genom att besämma bindningsförmågan till MGP1, som uttrycktes på jästceller, i närvaro av TfR. Rekombinant plasmid innehållande proteingen kodande Cp149 för varianter A-D samt F lyckades att framställas. Resultatet från SDS-PAGE påvisade inga tydlyga protein-band och flödescytometri resultatet var svårt att bedömma, troligen då ytterliggare proteinupprening behövdes för att isolera kapsidproteinerna. Syftet med arbetet har erhållits delvis och fortsatt undersökningar på nya proteiner förslås. / Hepatitis B is one av the major worldwide health problems that is caused by enveloped DNA virus, Hepatitis B virus (HBV). HBV’s capsid protein (Cp) has an important role in the virus life cycle, för example DNA replication, intraction with host cells, and other viral glycoproteins. HBV, like many other viruses, enters the cells by binding to cell receptors. Transferrin receptor (TfR) is a well-known receptor that enables virus entry into the cell by binding to viral proteins. The interaction takes place in the apical domain of TfR. Both Cp149, the capsid’s composition domain of Cp, and the soluble form of the apical domain, AP01, from TfR are important builing parts to be explored as starting building blocks for the development of antiviral therapeutics. Modification of Cp149 with AP01 is an interesting combination to produce new protein-based drugs to prevent viral infections. The aim of this project was to clone and express six different variants of a AP01 modified Cp149 protein as a starting points to analyze interactions between the proteins and Machupo virus glykoprotein 1 (MGP1). All target DNA templates and plasmids (pET-11a) were digested with restriction enzymes, NdeI and BamHI, and ligated by T4 DNA ligase. Agarose gel electrophoresis and DNA sequencing were used as validation methods to confirm the presence of desired and corrected DNA sequences, respectively, during gene cloning. DNA transformation and induction of Escherichia coli cells was used to express the desired proteins. The purified proteins were validated for their binding ability to MGP1, expressed on yeast cells by flow cytometry in a competition assay with TfR. Recombinant plasmid including the expected DNA sequence encoding Cp149 for variants A-D and F was successfully produced. There was no clear detection of protein bands on Sodium dodecyl sulphate polyakrylamid gel electrophoresis (SDS-PAGE) gel and flow cytometry results were difficult to interpret due to insufficient protein purification during ammonium sulphate percipitation. The purpose of the project has been obtained partially and more studies have to be carried out to produce pure proteins that can be used for further analysis.

Little is known regarding the impact excessive waterborne metals, like copper (Cu), have on tropical freshwater species. Zebrafish (Dania rerio) only recently became popular as a tool for environmental monitoring, due to their fully sequenced genome. Despite this, little was known regarding the ionoregulatory physiology of zebrafish. I first examined the impact softwater acclimation has on the ionoregulatory capacity of zebrafish, and identified a high degree of phenotypic plasticity associated with changes in both gene and protein expression, which highlighted the need for proper experimental design for studies involving pre-acclimation to softwater. I then examined the acute and chronic effects of waterborne Cu exposure under the influence of both softwater conditions and the addition of protective ions (Na+ & Ca2+) . On an acute timeframe, I found that Na+ provided a greater protective effect than Ca2+ in reducing Cu uptake, which was mimicked during chronic exposure, although the transcriptional effects were not as clear cut. I found that although Na+ and Ca2+ provided protective effects from Cu accumulation, there were still tissue specific and global changes at the transcript level, as determined by microarray analysis. Furthemore, this set of experiments identified the metal- and stress- induced transcriptional effects due to Cu exposure, which is key to identifying gene endpoints of chronic Cu exposure. A final experiment went further and looked at the effects of mixed metal contamination, examining chronic waterborne Cu exposure with interactive effects of elevated dietary Fe on gene expression and tissue metal accumulation. Not only did this experiment highlight that gene expression is not necessarily reflected in protein abundance/activity, but also demonstrated that high dietary Fe can significantly alter the transcriptional expression pattern of Cu transporters in the gill, liver, and gastrointestinal tract. This thesis has made significant steps in identifying viable gene endpoints of Cu toxicity. / Thesis / Doctor of Philosophy (PhD)

While adenovirus (Ad) vectors have been shown to elicit potent antigen-specific T cell responses, the kinetics and nature of antigen-specific mucosa! and systemic T-cell responses has rarely been examined, especially following mucosal administration of Ad-based vectors. In the present studies, the phenotypic and functional characterization of antigen-specific CD8+ T cell responses following intrarectal (i.r.) vaccination with an Ad vector expressing Gallus gallus ovalbumin (OVA) was conducted. The frequencies of OVA-specific CD8+ T cells was maximal at 2 weeks post-vaccination in all tissues examined and then declined, demonstrating normal expansion and contraction kinetics. CD8+ T cells induced in the course of immunization exhibited phenotypic characteristics of effector memory T cells including up-regulation of the cell surface molecules CD43, CD44 and a low level of expression of CD127 at both local and systemic sites. While the discordance between the number of tetramer-reactive and cytokine-producing OVA-specific CD8+ T cells was observed, CD8+ T cells appeared to be fully functional in vivo. Upon secondary antigen exposure, the CD8+ T cell population expanded dramatically, particularly at the mucosa! surfaces. In addition, the CD8+ T cell response generated in the course of i.r. priming protected mice from intravaginal (i. vag.) vaccinia virus one month after immunization, thus underscoring the importance of inducing a tissue-resident effector memory T cell subset for protection against pathogens at mucosal surfaces. In developing future vaccines for mucosal diseases, the induction of a tissue-resident effector memory T cell subset should be one of the immunization objectives. Lentiviral vectors represent an attractive mode of genetic vaccination. Most commonly used, vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentiviral vectors do not efficiently infect epithelial cells from the apical side, and, therefore, are not suitable as mucosa! vaccines. In the present studies, Ebola Zaïre strain glycoprotein (EboZ)-pseudotyped lentiviral vectors, which have been previously used to deliver transgene to the lung epithelium, were delivered i.r. and evaluated as a mucosal booster vaccine. Rectal delivery of EboZ-pseudotyped lentiviral vectors expressing β-galactosidase (β-gal) had resulted in low, but detectable levels of β-gal expression 2 weeks after administration. When delivered on its own, EboZ-pseudotyped lentivirus did not prime detectable antigen-specific immune response. However, when delivered i.r. 30 days after i.r. Adβ-gal immunization, a significant enlargement (boost) of β-gal-specific CD8+ T cell responses, especially in the colonic lamina propria (LP), was observed as compared to the delivery of EboZ-pseudotyped vector encoding different transgenes or VSVG-pseudotyped lentivirus expressing β-gal. When these animals were i. vag. challenged with vaccinia virus expressing β-gal, a dramatic expansion of β-gal-specific CD8+ T cells, especially in the vaginal tract, was observed. In addition, this prime and boost strategy protected the mice from i. vag. vaccinia virus challenge. Therefore, i.r. Ad-based priming followed by i.r. EboZ-pseudotyped lentiviral boosting was an effective strategy for eliciting protective mucosal CD8+ T cell responses. / Thesis / Doctor of Philosophy (PhD)

c-MYC est un proto-oncogène surexprimé dans les lymphomes de Burkitt et une cible importante pour le traitement de ces maladies. La protéine à domaine BTB/POZ MIZ-1 est un partenaire direct de la protéine c-MYC. En effet, c-MYC via son structure hélice-boucle-hélice se lie précisément à la séquence entre le 12e et 13e doigt de zinc de MIZ-1. Notre groupe a démontré que l’expression de la protéine MIZ-1 tronquée de son domaine POZ (MIZ-1ΔPOZ) peut ralentir la lymphomagenèse. Pourtant, l’interaction directe entre c-MYC et MIZ-1 n’est pas affectée par la délétion du domaine POZ de MIZ-1. Ceci suggère que MIZ-1 est important pour l’activité oncogénique de c-MYC. Il a été observé que l’expression de MIZ-1ΔPOZ diminue le nombre de cellules B pré-tumorales dans les modèles murins de lymphomes Eμ-Myc et Igλ-Myc. De plus, les niveaux protéiques très élevés de c-MYC dans les cellules B Eμ-Myc sont diminués lorsque MIZ-1ΔPOZ est exprimé. Toutefois, l’activité du protéasome ne semble pas être responsable de la diminution de c-MYC. Une analyse des interactions protéiques de MIZ-1 par BioID effectuée par notre groupe a montré que MIZ-1 et MIZ-1ΔPOZ interagissent avec PP1ɣ, TOX4 et PNUTS, trois protéines du complexe PTW/PP1 qui est un complexe transcriptionnel dont c-MYC fait partie. Des résultats de co-IP confirment cette interaction ce qui suggère que MIZ-1 pourrait influencer l’activité de ce complexe. De plus, une lignée cellulaire dans laquelle MIZ-1ΔPOZ est exprimé a été établie ce qui permettra d’approfondir l’étude des interactions protéiques de MIZ-1 avec le complexe PTW/PP1 et d’étudier l’effet de MIZ-1ΔPOZ sur l’activité transcriptionelle de ce complexe. Une meilleure compréhension du mécanisme d’action de MIZ-1 sur c-MYC permettra de mieux comprendre son rôle dans un contexte pathologique, ce qui pourrait permettre le design de nouvelles approches thérapeutiques. / c-MYC is a proto-oncogene overexpressed in Burkitt's lymphoma and an important target for the treatment of these diseases. The BTB/POZ domain protein MIZ-1 is direct a partner of c- MYC. Indeed, c-MYC via its helix-loop-helix structure binds precisely to the sequence between the 12th and 13th zinc finger of MIZ-1. We demonstrated that expression of MIZ-1 that lacks its POZ domain (MIZ-1ΔPOZ) can impair lymphomagenesis. However, the direct interaction between c-MYC and MIZ-1 is not affected by the deletion of the MIZ-1 POZ domain. This suggests that MIZ-1 is important for the oncogenic activity of c-MYC. We observed that expression of MIZ-1ΔPOZ decreases the number of pre-tumor B cells in lymphomas mouse models such as Eμ-Myc and Igλ-Myc. The high c-MYC protein levels observed in Eμ-Myc B cells are decreased when MIZ-1ΔPOZ is expressed. However, proteasome activity does not seem to be responsible for the decrease of c-MYC. After the analysis of MIZ-1 protein interactions by BioID, our group found that MIZ-1 and MIZ-1ΔPOZ interact with PP1ɣ, TOX4 and PNUTS, three proteins of the PTW/PP1 complex which also interact with c-MYC. Co-IP results confirmed these interactions suggesting that MIZ-1 could influence the activity of this complex. In addition, a MIZ- 1ΔPOZ cell line was established which will allow further studies of MIZ-1ΔPOZ protein interactions with the PTW/PP1 complex as well as its effect on the transcriptional activity of this complex. A better characterization of the mechanism of action of MIZ-1 on c-MYC will lead to a better understanding of its role in a pathological context, which could be the basis of new therapeutic approaches.

I detta projekt har uttrycket av Affibodymolekyler i bioreaktorer av olika volymetriska skalor jämförts för att fastställa om ett tillförlitligt samband mellan de olika bioreaktorernas prestanda kan etableras för att möjliggöra utvecklingen av en nedskalad produktionsmodell för Affibodymolekyler. Baslinjen för jämförelsen i denna studie har varit en enliters- stirred tank reactor (STR) som de andra (mindre) bioreaktorernas prestanda jämfördes med. Jämförelsen av prestanda gjordes genom odling och uttryck av sex olika Affibodymolekyler i replikat i varje bioreaktorstorlek. Prestandan i detta fall hänvisar till produktionen av Affibodymolekyler (mg/L Cellodling) under odlingen, som fastställdes genom ett protokoll för proteinrening av lösligt intracellulärt protein genom affinitetskromatografi och kvantifiering genom absorbans vid 280 nm. De sex olika Affibodymolekyler som har studerats i detta projekt hade tidigare visat sig ha olika uttrycksnivåer, och har här jämförts med varandra i de olika bioreaktorskalorna. De två olika bioreaktorstorlekarna som bedömdes var en 300 mL skakkolv med 50 mL arbetsvolym och en mikrotiterplatta (MTP) med 3 mL arbetsvolym. Dessutom innebar studien en bedömning av två system för en långsam frisättning av kolkälla i odlingarna, ett i varje nedskalad bioreaktorstorlek. Detta utfördes för att delvis efterlikna den kontrollerade fed-batch-kulturen i STR, där koltillförsel kontrollerades med hjälp av en feedprofil. Resultaten visade att proteinuttrycket av metoderna med en långsam frisättning av kolkällor överensstämde närmast med proteinuttrycket i STR. Anpassningen av dessa resultat mot proteinuttrycket hos STR gav i en linjär regression ett R2 på 99,69 % i 3 ml MTP och 97,46 % i 50 ml skakkolven. Slutsatserna som drogs var att SMFP08003 FeedPlate var den bästa kandidaten för en nedskalad modellering av uppströmsprocessen för produktion av Affibodymolekyler. Samt att faktorn att använda en fed-batch-process istället för en batch-process har en större inverkan på proteinuttrycket än skalan av processerna. / In this project the expression of Affibody® molecules in bioreactors of different volumetric scales has been compared, to determine if a reliable relation between the performance of the different bioreactors can be established to allow for the development of a scale-down Affibody® molecule production protocol. The baseline of comparison in this study has been a one litre Stirred Tank Reactor (STR) to which the other (smaller) bioreactors' performance were compared. The performance comparison was achieved by the cultivation and subsequent expression of six different Affibody® molecules in replicates in each bioreactor size. Performance in this case refers to Affibody® molecule production (mg/L culture) during the cultivation, which is assessed by a protocol of protein purification of soluble intracellular protein by affinity chromatography and quantification by 280 nm absorbance. The six different Affibody® molecules studied in this project had previously been found to have different expression levels, and were in this project compared to each other in the different bioreactor scales. The two different bioreactor sizes which were assessed were a 300 mL shake flask with 50 mL working volume and a 3 mL working volume microtiter plate (MTP). In addition, the study involved an assessment of the use of two systems for a slow carbon source release in the cultivations, one in each scale-down bioreactor size. This was performed to partly mimic the controlled feed systems in STRs. The results showed that the protein expression of the methods with a slow carbon source release corresponded most closely with the protein expression in the STR. The fit of these results onto the protein expression of the STR yielded a R2 of 99.69% in the 3 mL MTP and 97.46% in the 50 mL shake flask. The conclusions drawn were that SMFP08003 FeedPlate was the best candidate for scale-down modelling of the upstream process for production of Affibody® Molecules and that the factor of using a fed-batch process instead of a batch process has a larger impact on the protein expression than the scale of either process.

Proteins, participating in a myriad of biological function, are at the core of all cellular activities occurring within living organisms. Therapeutic proteins, hence constitute a major part of the pharmaceutical industry. The glycoprotein hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (CG) regulate various reproductive and metabolic functions in humans and hence have high therapeutic potentials. The increasing demand of recombinant proteins for therapeutic uses drives the development of better expression systems. The methylotrophic yeast Pichia pastoris, has been termed as an industrial workhorse for heterologous protein expression. However, the N-glycosylation in yeast is of the high mannose type, resulting in a reduced serum half-life of the recombinant proteins. In the current work, we have re-engineered the Pichia N-glycosylation pathway to mimic the human type of N-glycosylation. Towards this end, we abolished the yeast native N-glycosylation and introduced enzymes from various eukaryotic sources into the system. These modifications resulted in the conversion of the yeast Man9-20GlcNAc2 glycan structure to a more human like GlcNAc2Man3GlcNAc2 form on over 70 % of the heterologous expressed proteins. In order to demonstrate the application of these strains as efficient protein expression hosts, the glycoengineerd Pichia was used for large scale expression of the glycoprotein hormones, hCG and FSH. The purified recombinant hormones were found to have binding affinities and structure similar to that of the natural hormones. These recombinant hormones were also able to elicit over two fold responses in animal models compared to buffer controls and the activity was comparable to the natural counterparts. Thus, we report the generation of a glycoengineered Pichia pastoris, which can be considered as a serious contender for the expression of glycosylated proteins of therapeutic importance.

Techniques for conditional gene or protein expression are important tools in developmental biology and in the analysis of physiology and disease. On the protein level, the tunable and reversible expression of proteins can be achieved by the fusion of the protein of interest to a destabilizing domain (DD). In the absence of its specific ligand (Shield-1), the protein is degraded by the proteasome. The DD-Shield system has proven to be an excellent tool to regulate the expression of proteins of interests in mammalian systems but has not been applied in teleosts like the medaka. We present the application of the DD-Shield technique in transgenic medaka and show the ubiquitous conditional expression throughout life. Shield-1 administration to the water leads to concentration-dependent induction of a YFP reporter gene in various organs and in spermatogonia at the cellular level.

A Doença de Alzheimer (DA) é uma desordem neurodegenerativa influenciada por elementos genéticos e ambientais. O diagnóstico é baseado em parâmetros clínicos, mas sua confirmação é post-mortem, após avaliação patológica durante a autópsia. A identificação de biomarcadores baseados em tecido periférico como o sangue permitiria um diagnóstico menos invasivo e mais preciso, como também poderia servir como marcador de predisposição, conversão, progressão e resposta à tratamento. Para atingir tal objetivo, métodos de prospecção em larga escala de perfis de expressão gênica e protéica têm sido extensamente aplicados. O presente estudo se propôs a selecionar e validar potenciais candidatos a biomarcadores na DA, e também, de identificar potenciais biomarcadores pelo desenvolvimento de perfis proteômicos de plaquetas de pacientes. Dados publicados de microarray para DA foram comparados e genes com perfil de expressão similar em pelo menos dois estudos independentes foram selecionados. Foram identificados 4 genes de expressão aumentada em pacientes (HLADRB1, HLAB, CIRBP, S100A4) e 4 genes de expressão diminuída (CALM1, ATP5J2, KIF1B, FLOT1), que posteriormente foram submetidos a validação por PCR em tempo-real em amostras de RNA extraído a partir de leucócitos de sangue periférico de 20 pacientes e 20 controles idosos. Ao mesmo tempo, padronizamos e traçamos o perfil proteômico de pools de proteínas de plaquetas de pacientes do sexo feminino e masculino e seus respectivos controles idosos por Eletroforese Bidimensional (2-D). Dentre os genes analisados por meio de PCR em tempo-real, três (ATP5J2, S100A4 e KIF1B) apresentaram expressão aumentada no grupo DA em relação ao grupo controle (p >0,05). Estes genes podem estar envolvidos na resposta inflamatória periférica e indução da apoptose. A padronização do perfil proteômico por 2-DE foi bem sucedida e um conjunto de 5 proteínas diferencialmente expressas para ambos os gêneros foram obtidas, mas não foi possível a sua identificação por Espectrometria de Massas devido a massa limitada dessas proteínas em cada spot. Por ambas as técnicas foi possível identificar perfis diferentes de expressão gênica e protéica entre pacientes e controles idosos, demonstrando que sangue periférico é uma boa matriz de prospecção de biomarcadores de doenças neurodegenerativas. / Alzheimer\'s disease (AD) is a neurodegenerative disorder influenced by genetic and environmental elements. The diagnosis is based on clinical parameters, but its confirmation needs post-mortem pathologic evaluation at autopsy. The identification of biomarkers based on peripheral tissue would allow a less invasive and more accurate diagnosis, but could also serve as a marker of predisposition, conversion, progression and response to treatment. To achieve this goal, methods of exploring large-scale gene expression profiles and protein have been extensively applied. The present study proposed to select and validate potential candidate genes for biomarkers in AD, and also identify potential biomarkers from proteomic profiles of platelets of these patients. Published microarray data for AD were compared and genes with similar expression profile in at least two independent studies were selected. We identified four up-regulated genes (HLADRB1, HLAB, CIRBP, S100A4) and four down-regulated genes in patients (CALM1, ATP5J2, KIF1B, FLOT1), which were then submitted to validation by real time PCR in RNA samples extracted from peripheral blood leukocytes of 20 patients and 20 elderly controls. At the same time, standardized, and traced the proteomic profile of platelet proteins pools of female patients and male elders and their respective controls by two-dimensional electrophoresis (2-D). Among the genes analyzed by real time PCR three of them (ATP5J2, S100A4 and KIF1B) showed increased expression in the AD group compared to the control group (p> 0.05). These genes may be involved in peripheral inflammatory response and induction of apoptosis. The standardization of proteomic profile by 2-DE has been successful and a set of five differentially expressed proteins in both genders were obtained, but could not be identified by mass spectrometry due to limited mass of these proteins in each spot. For both techniques were able to identify different patterns of gene and protein expression between patients and elderly controls, demonstrating that peripheral blood is a good prospect source of biomarkers for neurodegenerative diseases.

En raison de l'utilisation croissante de nanomatériaux dans divers procédés industriels, le nombre de salariés potentiellement exposés ne cesse d’augmenter, sans que pour autant les propriétés toxicologiques de ces agents chimiques ne soient parfaitement connues. Comme des nanoparticules (NP) peuvent être mises en suspension dans les environnements de travail, l'inhalation représente la principale voie d'exposition professionnelle. Ainsi, l’évaluation des dangers associés à l’exposition à des aérosols nanostructurés nécessite de réaliser des études de toxicologie expérimentale par inhalation, en utilisant des modèles animaux. Dans ce travail, les propriétés toxicologiques pulmonaires d’un aérosol nanostructuré de dioxyde de titane (TiO2) ont été étudiées à court et long termes, en combinant des analyses toxicologiques conventionnelles (analyses du lavage broncho-alvéolaire (LBA), histopathologie des poumons et des ganglions lymphatiques); et de criblage moléculaire à haut contenu (analyses de transcriptomique et de protéomique). Des rats Fischer 344 ont été exposés par inhalation oro-nasale, à un aérosol nanostructuré de TiO2 à 10 mg / m3 ; 6 heures par jour, 5 jours par semaine pendant 4 semaines. Des échantillons biologiques ont été prélevés immédiatement et jusqu'à 180 jours suivant la fin de l'exposition. L'exposition à l'aérsosol nanostructuré de TiO2 a entraîné une importante réponse inflammatoire pulmonaire aiguë. Cette réponse était caractérisée par un influx de granulocytes neutrophiles, la présence de macrophages chargés en particules au niveau alvéolaire, la surexpression de gènes et de protéines impliqués dans les réponses inflammatoires et immunitaires, les cascades du complément et de la coagulation, le stress oxydant. Certains gènes surexprimés étaient également impliqués dans les lésions de l'ADN et la fibrose; et certaines protéines surexprimées étaient associées au protéasome et à l'organisation du cytosquelette. Dans le surnageant du LBA, l’augmentation du niveau d'histones et d'autres protéines associées aux pièges extracellulaires des neutrophiles (Neutrophilic Extracellular Trap, NET) suggère la libération de ces pièges extracellulaires dans l'espace alvéolaire. Cette libération possible de NET se produit dans un contexte inflammatoire mais en l'absence de changements histopathologiques significatifs. Ce processus inattendu n’a fait l’objet que de très peu d’études en lien avec une exposition à des nanomatériaux. Six mois après la fin de l'exposition (réponse à long terme), l'inflammation a diminué et s’accompagnait d’une baisse de la charge pulmonaire de titane (un marqueur fiable de la charge pulmonaire en nanoparticules de TiO2), mais de nombreux gènes et protéines étaient différentiellemment exprimés. Les conséquences physiopathologiques des changements rapportés ici ne sont pas entièrement connues, mais ces résultats devraient susciter des interrogations quant aux effets pulmonaires à long terme des NP inhalées biopersistantes de faible toxicité comme le TiO2. En conclusion, ce travail montre qu'il existe une bonne relation entre les changements cytologiques et histopathologiques d'une part et les modifications des profils d'expression de gènes et de protéines d'autre part. Cependant, dans certains cas, la transcriptomique et la protéomique pourraient être plus sensibles que les méthodes conventionnelles pour identifier de nouvelles propriétés toxicologiques, ou pour mieux comprendre les mécanismes moléculaires sous-jacents de la toxicité des produits chimiques. Notre étude avec d'autres travaux de la littérature pourraient également être utiles pour identifier des biomarqueurs d’exposition aux nanomatériaux ou prédire leurs effets nocifs à long terme / Due to the growing use of nanomaterials in various industrial processes, the number of workers potentially exposed is increasing even though the toxicological properties of these compounds are not completely known. Since nanoparticles (NP) may get aerosolized, inhalation represents their main route of occupational exposure. Then, inhalation studies of nanomaterial toxicity in animal models appear to be the most relevant approach to assess their hazards. In this work, we studied the short and long term pulmonary toxicological properties of inhaled titanium dioxide (TiO2) nanostructured aerosol (NSA), using conventional (broncho-alveolar lavage (BAL) analyses, lung and lymph nodes histopathology); and high content molecular toxicological approaches (transcriptomics and proteomics analyses). Fischer 344 rats were exposed to 10 mg/m3 of TiO2 nanostructured aerosol by nose-only inhalation, 6h/day, 5 days/week for 4 weeks. Biological samples were collected immediately and up to 180 post-exposure days. Exposure to TiO2 NSA resulted in a strong acute pulmonary inflammation. This response was characterized by a neutrophil influx, the presence of particle-laden macrophages in the alveolar lumen, as well as overexpression of genes and proteins involved in inflammatory and immune responses, complement and coagulation cascades, oxidative stress. Some overexpressed genes were also involved in DNA damage and fibrosis; and some overexpressed proteins in proteasome and cytoskeleton organization. In the BAL supernatant, the increased level of histones and other neutrophilic extracellular trap (NET) -associated proteins suggests the release of these traps in the alveolar space. This possible NET release occurs in an inflammatory context but in the absence of significant histopathological changes. Very few studies reported this unexpected process related to exposure to nanomaterials. Six months after the end of exposure (long-term response), inflammation had decreased in line with the decrease of titanium lung burden (a surrogate for TiO2 pulmonary deposition), but many genes and proteins remained differentially expressed. The physiopathological consequences of the molecular changes reported here are not fully known, but these results should raise concern about the long-term pulmonary effects of inhaled low toxicity NP such as TiO2. Altogether, this work shows that there is a good relationship between cytological and histopathological changes in one hand and gene as well as protein expression profile modifications in the other hand. However, in some cases transcriptomics or proteomics could be more sensitive than conventional methods to identify new toxicological properties or to better understand the underlying molecular mechanisms of chemicals toxicity. Our study along with others could also be helpful to identify biomarkers of exposure or predict the long-term adverse effects of nanomaterials

A esquistossomose é a mais importante das doenças helmínticas humanas em termos de morbidez e mortalidade. A proteína Sm14 de Schistosoma mansoni, que pertence à família de proteínas ligadoras de ácidos graxos (fatty acid-binding proteins, FABPs) (Moser et al., 1991), mostrou um bom nível de proteção (65%) contra a esquistossomose em animais experimentais (Tendler et al., 1996). No presente trabalho foram desenvolvidos sistemas de expressão que possibilitará a produção da proteína Sm14 em larga escala em E.coli. Com o intuito de conhecer a estrutura do gene da proteína Sm14, foi clonado um fragmento de DNA genômico de S. mansoni que contém a seqüência codificante da proteína Sm14. Como os outros membros da família gênica das FABP, o gene para a proteína Sm14 contém quatro \"exons\" separados por três \"introns\" de 674, 585 e 42 bp. Esta é a primeira descrição da estrutura gênica de um membro das FABP correspondente a um helminto. A Sm14 é uma proteína que pode ser potencialmente usada como vacina. Estudamos a existência de polimorfismo em duas linhagens de S. mansoni endêmicas do Brasil: LE e BH. Para a análise de polimorfismo, a ORF correspondente à proteína Sm14 foi amplificada por RT-PCR do RNA total de vermes adultos de S. mansoni. Os produtos de amplificação independentes foram clonados no vetor pGEM-T e seqüenciados. As análises de seqüências mostraram duas isoformas principais para a proteína Sm14: Sm14-M20, com seqüência idêntica a proteína Sm14 previamente reportada para a linhagem de Puerto Rico de S. mansoni (Moser et AL., 1991), e Sm14-T20, onde o códon da Met20 (ATG) mudou para o códon de Thr (ACG) (polimorfismo M20T). Dois clones mostraram uma deleção de seqüência de aminoácidos correspondente ao \"exon\" 3 inteiro (clones ΔExon3), gerada por \"splicing\" alternativo. As outras trocas observadas acontecem em posições onde os aminoácidos são menos conservados e estão representados apenas por um único clone que podem ter sido obtidas por mutagênese na PCR. A metionina correspondente à posição 20 na Sm14 é altamente conservada nas FABP dos mais diversos organismos,e não se tem nenhuma outra proteína com treonina nesta posição. Para o estudo da estrutura e função destas isoformas, os cDNAs correspondentes foram subclonados no vetor pAE (desenvolvido no nosso laboratório), assim como o mutante M20A (Sm14-A20) construído para efeitos de comparação. A estabilidade e estrutura das proteínas recombinantes purificadas foram caracterizadas por dicroísmo circular (CD). A comparação da estrutura e termoestabilidade mostrou que as formas Sm14-T20 e Sm14-A20 são menos termoestáveis do que a Sm14-M20 (um ΔTm de aproximadamente 10°C). Porém, todas as formas de Sm14 foram capazes de ligar o DAUDA [ácido 11-(dansylamino) undecanoico] com a mesma afinidade. Para poder diferenciar as propriedades de ligação de ácidos graxos pelas isoformas, experiências de competição do deslocamento do DAUDA por ácidos graxos naturais, foram realizadas. A partir destes dados podemos assumir que a forma Sm14-M20 liga melhor todos os ácidos graxos naturais testados do que a forma Sm14-T20. Porém esta forma mantém a capacidade de ligar ácidos graxos, ao contrario do mutante Sm14-A20. Pode-se deduzir como resultado destas experiências que a proteína Sm14-M20 é mais estável e liga com maior afinidade os ácidos graxos naturais do que a forma Sm14-T20. Pelo visto, a proteína Sm14-T20 tem menos estrutura-β, porém, mantém a capacidade de ligar moléculas hidrofóbicas. Ainda é desconhecido o papel funcional do polimorfismo da proteína Sm14 no metabolismo dos vermes de S. mansoni. Problemas de estabilidade da proteína Sm14 recombinante, durante seu transporte e armazenamento, comprometem sua viabilidade como vacina. Com o intuito de melhorar a estabilidade desta proteína, foi feita uma mutagênese no único resíduo de cisteína presente na Sm14 na posição 62. Este resíduo é responsável pela formação de dímeros, o que é relacionado a estabilização da perda de estrutura-β e precipitação da proteína. Esta cisteína foi trocada por serina (C62S) e por valina (C62V) por mutagênese sítio dirigida, resultando nas proteínas Sm14-M20S62 e Sm14-M20V62. As formas mutantes não apresentaram maior termoestabilidade, mas a renaturação após o aquecimento a 80°C atingiu quase 100%, diferentemente das proteínas com Cys62. As proteínas com o resíduo de cisteina trocado foram as únicas formas que conservaram a estrutura de β-barril após 3 meses de armazenamento a 4°C, como mostram as análises de dicroísmo circular, sendo a forma mais estável a proteína Sm14-M20V62. Após estes estudos, a isoforma Sm14-M20 com a mutação C62V (Sm14-M20V62) mostrou-se como a melhor alternativa ao antígeno Sm14-T20 usado até agora como modelo de vacina experimental para S. mansoni. Esta indicação deve ser confirmada em ensaios de imunização e posterior desafio com cercárias de S. mansoni. / The schistosomiasis is the most important human helmintic disease in terms of morbidity and mortality. The Sm14 protein of Schistosoma mansoni belongs to the family of fatty acid-binding proteins (FABPs) (Moser et aI. , 1991) and showed a good protection level as vaccine antigen against the schistosomiasis in experimental animals (Tendler et al., 1996). In the present work were developed systems for the expression of Sm14 protein that will facilitate its large scale production in E.coli.. In order to know the gene structure of the Sm14 protein, we amplified by PCR a genomic DNA fragment of S. mansoni that contains the coding sequence for the Sm14 protein. As the other members of the FABP family, the Sm14 gene contains four exons separated by three introns of 674,585 and 42 bp, respectively. This is the first detailed description of the genomic structure for a member of FABPs corresponding to a helmint. We also studied the existence of polymorphisms within two Brazilian endemic strains of S.mansoni: LE and BH. For the polymorphism analysis, the ORF corresponding to the Sm14 protein was amplified by RT-PCR from total RNA of S. mansoni adult worms. The independent amplified products were cloned into pGEM-T vector and sequenced. The sequence analyses showed two main isoforms: Sm14-M20, with identical sequence to that previously reported Sm14 protein from the Puerto Rican strain of S. mansoni (Moser et al., 1991), and Sm14-T20, where the codon for Met20 (ATG) was changed for the Thr codon (ACG) (M20T polymorphism). Two clones showed the same amino acid sequence deletion corresponding to the whole third exon (ΔExon3 clones), generated by alternative splicing. The other observed changes occurred in positions where the amino acids were less conserved and were just represented by only one clone that could be obtained by PCR mutagenesis. The methionine corresponding to the position 20 in Sm14 is highly conserved among FABPs and no other related protein has threonin in this position. To study the structure and function of these amino acid in the isoforms, the corresponding cDNAs were subcloned in to the pAE vector (developed in our laboratory), as well as the mutant M20A (Sm14-A20). The stability and structure of the purified recombinant proteins were characterized by circular dicroism (CD). The comparison of their structure and thermo stability showed that the forms Sm14-T20 and Sm14-A20 are less thermostable than Sm14-M20 (ΔTm around 10ºC). However, all of the Sm14 forms were capable to bind the DAUDA [11- (dansylamine) undecanoic acid] with similar affinities. To differentiate the fatty acid binding properties of Sm14 isoforms, displacement experiments of DAUDA with natural fatty acid were performed. From these data we can assume that the Sm14-M20 form binds better than the Sm14-T20 and Sm14-A20 forms of all natural fatty acid assayed. This suggests that the Sm14-20 protein is most stable and binds better the natural fatty acids than the Sm14-T20 form. Although the Sm14-T20 protein has less structure, it maintains the capacity to bind fatty acids. It is still unknown the functional role of this Sm14 protein polymorphism in the metabolism of S. mansoni worms. Stability problems of the recombinant Sm14 protein during its transport and storage, could hamper its use as vaccine. With the aim to improve the stability of this protein, it was made a mutagenese at the unique cysteine residue present in Sm14 at the position 62. This residue is responsible for the dimer formation and is related the loss of the terciary structure and precipitation of the protein. This cysteine was changed by serine (C62S) and for valine (C62V) by site directed mutagenesis, resulting in the proteins Sm14-M20S62 and Sm14-M20V62. The mutant forms did not present a higher thermal stability but the renaturation after heating at 80°C almost reached 100%, in contrast to Sm14 proteins with Cys62. These mutants conserved the β-barrel structure after 3 months of storage at 4°C, in contrast to proteins with Cys62, as shown by circular dicroism analyses. After these studies, the Sm14-M20 isoform with the C62V mutation (Sm14-M20V62) was considered the best alternative to the antigen Sm14-T20 used up to now as the model for an experimental vaccine for S. mansoni. This indication should be confirmed by immunization and posterior challenge with S. mansoni cercaria.