Expression and localization of MT1-MMP and its activating enzyme furin in the glomerular wall of short and long term diabetic rats = Expression et localisation de la MT1-MMP et de la furine dans la paroi glomérulaire de rats diabétiques

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

Subcellular localization of legionella Dot/Icm effectors

The translocation of effector proteins by the Dot/Icm type IV secretion system is central to the ability of Legionella pneumophila to persist and replicate within eukaryotic cells. The subcellular localization of translocated Dot/Icm proteins in host cells provides insight into their function. Through co-staining with host cell markers, effector proteins may be localized to specific subcellular compartments and membranes, which frequently reflects their host cell target and mechanism of action. In this chapter, we describe protocols to (1) localize effector proteins within cells by ectopic expression using green fluorescent protein fusions and (2) localize effector proteins within infected cells using epitope-tagged effector proteins and immuno-fluorescence microscopy.

Localization of a mobile robot based on odometry and natural landmarks using extended kalman filter

SANTANA, André M.; SOUZA, Anderson A. S.; BRITTO, Ricardo S.; ALSINA, Pablo J.; MEDEIROS, Adelardo A. D. Localization of a mobile robot based on odometry and natural landmarks using extended Kalman Filter. In: INTERNATIONAL CONFERENCE ON INFORMATICS IN CONTROL, AUTOMATION AND ROBOTICS, 5., 2008, Funchal, Portugal. Proceedings... Funchal, Portugal: ICINCO, 2008.

Localization of a mobile robot based on odometry and natural landmarks using extended kalman filter

SANTANA, André M.; SOUZA, Anderson A. S.; BRITTO, Ricardo S.; ALSINA, Pablo J.; MEDEIROS, Adelardo A. D. Localization of a mobile robot based on odometry and natural landmarks using extended Kalman Filter. In: INTERNATIONAL CONFERENCE ON INFORMATICS IN CONTROL, AUTOMATION AND ROBOTICS, 5., 2008, Funchal, Portugal. Proceedings... Funchal, Portugal: ICINCO, 2008.

Molecular Characterization and Localization of the NAD(P)H Oxidase Components gp91-phox and p22-phox in Endothelial Cells

The production of reactive oxygen species (ROS) within endothelial cells may have several effects, including alterations in the activity of paracrine factors, gene expression, apoptosis, and cellular injury. Recent studies indicate that a phagocyte-type NAD(P)H oxidase is a major source of endothelial ROS. In contrast to the high-output phagocytic oxidase, the endothelial enzyme has much lower biochemical activity and a different substrate specificity (NADH.NADPH). In the present study, we (1) cloned and characterized the cDNA and predicted amino acid structures of the 2 major subunits of rat coronary microvascular endothelial cell NAD(P)H oxidase, gp91-phox and p22-phox; (2) undertook a detailed comparison with phagocytic NADPH oxidase sequences; and (3) studied the subcellular location of these subunits in endothelial cells. Although these studies revealed an overall high degree of homology (.90%) between the endothelial and phagocytic oxidase subunits, the endothelial gp91-phox sequence has potentially important differences in a putative NADPH-binding domain and in putative glycosylation sites. In addition, the subcellular location of the endothelial gp91-phox and p22-phox subunits is significantly different from that reported for the neutrophil oxidase, in that they are predominantly intracellular and collocated in the vicinity of the endoplasmic reticulum. This first detailed characterization of gp91-phox and p22-phox structure and location in endothelial cells provides new data that may account, in part, for the differences in function between the phagocytic and endothelial NAD(P)H oxidases.

Silencing the gip gene of Phytophthora cinnamomi by iRNA and studying the subcellular localization of GIP and NPP1 proteins

Ink Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is caused by two species: Phytophthora cinnamomi and Phytophthora cambivora, being the first one the foremost pathogen of ink disease in Portugal. P. cinnamomi is one of the most aggressive and widespread plant pathogen with nearly 1,000 host species. This oomycete causes enormous economic losses and it is responsible for the decline of many plant species in Europe and worldwide. Up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels, especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP (glucanase inhibitor protein) and NPP1 (necrosis-inducing Phytophthora protein 1) produced by P. cinnamomi during the infection. The technique of RNA interference was used to knockdown the gip gene of P. cinnamomi. Transformants obtained with the silenced gene have been used to infect C. sativa, in order to determine the effect of gene silencing on the plant phenotype. To know more about the function of GIP and NPP1 involved in the mechanism of infection, the ORF’s of gip and npp1 genes have been cloned to the pTOR-eGFP vector for a future observation of P. cinnamomi transformants with fluorescent microscopy and determination of the subcellular localization. Moreover the prediction by bioinformatics tools indicates that both GIP and NPP1 proteins are secreted. The results allow to predict the secretory destination of both GIP and NPP1 proteins and confirm RNAi as a potential alternative biological tool in the control and management of P. cinnamomi. Keywords:

Molecular identification and localization of filamentous symbiotic bacteria associated with the hydrothermal vent annelid Alvinella pompejana

Alvinella pompejana is a polychaetous annelid that inhabits high temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.

Computational resources for structural and metagenomic characterization of functions in bacteria and bacterial communities. Antimicrobial resistance, pathways for xenobiotic degradation and relationship between gut microbiome and ageing.

Prokaryotic organisms are one of the most successful forms of life, they are present in all known ecosystems. The deluge diversity of bacteria reflects their ability to colonise every environment. Also, human beings host trillions of microorganisms in their body districts, including skin, mucosae, and gut. This symbiosis is active for all other terrestrial and marine animals, as well as plants. With the term holobiont we refer, with a single word, to the systems including both the host and its symbiotic microbial species. The coevolution of bacteria within their ecological niches reflects the adaptation of both host and guest species, and it is shaped by complex interactions that are pivotal for determining the host state. Nowadays, thanks to the current sequencing technologies, Next Generation Sequencing, we have unprecedented tools for investigating the bacterial life by studying the prokaryotic genome sequences. NGS revolution has been sustained by the advancements in computational performance, in terms of speed, storage capacity, algorithm development and hardware costs decreasing following the Moore’s Law. Bioinformaticians and computational biologists design and implement ad hoc tools able to analyse high-throughput data and extract valuable biological information. Metagenomics requires the integration of life and computational sciences and it is uncovering the deluge diversity of the bacterial world. The present thesis work focuses mainly on the analysis of prokaryotic genomes under different aspects. Being supervised by two groups at the University of Bologna, the Biocomputing group and the group of Microbial Ecology of Health, I investigated three different topics: i) antimicrobial resistance, particularly with respect to missense point mutations involved in the resistant phenotype, ii) bacterial mechanisms involved in xenobiotic degradation via the computational analysis of metagenomic samples, and iii) the variation of the human gut microbiota through ageing, in elderly and longevous individuals.

Examen de la phrénologie /

Examines doctrines of Gall, Spurzheim and Broussais.

Role of the protein tyrosine phosphatase DEP-1 in Src activation and the mediation of biological cell functions of endothelial and breast cancer cells

L’implication des protéines tyrosines phosphatases (PTPs) dans la régulation de la signalisation et la médiation des fonctions cellulaires a été bien établie dans les dernières années. Cependant, les mécanismes moléculaires par lesquels les PTPs régulent les processus fondamentaux tels que l’angiogenèse demeurent méconnus. Il a été rapporté que l’expression de la PTP DEP-1 (Density-enhanced phosphatase 1) augmente avec la densité cellulaire et corrèle avec la déphosphorylation du récepteur VEGFR2. Cette déphosphorylation contribue à l’inhibition de contact dans les cellules endothéliales à confluence et diminue l’activité du VEGFR2 en déphosphorylant spécifiquement ses résidus catalytiques Y1054/1059. De plus, la plupart des voies de signalisation en aval du VEGFR2 sont diminuées sauf la voie Src-Gab1-AKT. DEP-1 déphosphoryle la Y529 de Src et contribue à la promotion de la survie dans les cellules endothéliales. L’objectif de cette thèse est de mieux définir le rôle de DEP-1 dans la régulation de l’activité de Src et les réponses biologiques dans les cellules endothéliales. Nous avons identifié les résidus Y1311 et Y1320 dans la queue C-terminale de DEP-1 comme sites majeurs de phosphorylation en réponse au VEGF. La phosphorylation de ces résidus est requise pour l’activation de Src et médie le remodelage des jonctions cellules-cellules dépendantes de Src. Ce remodelage induit la perméabilité, l’invasion et la formation de capillaires en réponse au VEGF. Nos résultats démontrent que la phosphorylation de DEP-1 sur résidu tyrosine est requise pour diriger la spécificité de DEP-1 vers son substrat Src. Les travaux révèlent pour la première fois un rôle positif de DEP-1 sur l’induction du programme angiogénique des cellules endothéliales. En plus de la phosphorylation sur tyrosine, DEP-1 est constitutivement phosphorylé sur la thréonine 1318 situé à proximité de la Y1320 en C-terminal. Cette localisation de la T1318 suggère que ce résidu pourrait être impliqué dans la régulation de la Y1320. En effet, nous avons observé que la T1318 de DEP-1 est phosphorylée potentiellement par CK2, et que cette phosphorylation régule la phosphorylation de DEP-1 sur tyrosine et sa capacité de lier et d’activer Src. En accord avec ces résultats, nos travaux révèlent que la surexpression du mutant DEP-1 T1318A diminue le remodelage des jonctions cellules-cellules et par conséquent la perméabilité. Nos résultats suggèrent donc que la T1318 de DEP-1 constitue un nouveau mécanisme de contrôle de la phosphorylation sur tyrosine et que ceci résulte en l’activation de Src et l’induction des fonctions biologiques des cellules endothéliales en réponse au VEGF. Suite à ces travaux dans les cellules endothéliales qui démontrent un rôle positif de DEP-1 dans la médiation des réponses angiogéniques, nous avons voulu approfondir nos connaissances sur l’implication potentielle de DEP-1 dans les cellules cancéreuses où l’activité de Src est requise pour la progression tumorale. Malgré le rôle connu de DEP-1 comme suppresseur tumoral dans différents types de cancer, nous avons émis l’hypothèse que DEP-1 pourrait promouvoir les fonctions biologiques dépendantes de Src telles que la migration et l’invasion dans les cellules cancéreuses. Ainsi, nous avons observé que l’expression de DEP-1 est plus élevée dans les lignées basales de cancer du sein qui sont plus invasives comparativement aux lignées luminales peu invasives. Dans les lignées basales, DEP-1 active Src, médie la motilité cellulaire dépendante de Src et régule la localisation des protéines impliquées dans l’organisation du cytosquelette. L’analyse d’un micro-étalage de tissu a révélé que l’expression de DEP-1 est associée avec une réduction tendencielle de survie des patients. Nos résultats proposent donc, un rôle de promoteur tumoral pour DEP-1 dans la progression du cancer du sein. Les travaux présentés dans cette thèse démontrent pour la première fois que DEP-1 peut agir comme promoteur des réponses angiogéniques et du phénotype pro-invasif des lignées basales du cancer du sein probablement du à sa capacité d’activer Src. Nos résultats suggèrent ainsi que l’expression de DEP-1 pourrait contribuer à la progression tumorale et la formation de métastases. Ces découvertes laissent donc entrevoir que DEP-1 représente une nouvelle cible thérapeutique potentielle pour contrer l’angiogenèse et le développement du cancer.

Regulation of SRC-3 localization and dynamics by phosphorylation during ERα-dependent transcriptional activation

Transcription is controlled by promoter-selective transcriptional factors (TFs), which bind to cis-regulatory enhancers elements, termed hormone response elements (HREs), in a specific subset of genes. Regulation by these factors involves either the recruitment of coactivators or corepressors and direct interaction with the basal transcriptional machinery (1). Hormone-activated nuclear receptors (NRs) are well characterized transcriptional factors (2) that bind to the promoters of their target genes and recruit primary and secondary coactivator proteins which possess many enzymatic activities required for gene expression (1,3,4). In the present study, using single-cell high-resolution fluorescent microscopy and high throughput microscopy (HTM) coupled to computational imaging analysis, we investigated transcriptional regulation controlled by the estrogen receptor alpha (ERalpha), in terms of large scale chromatin remodeling and interaction with the associated coactivator SRC-3 (Steroid Receptor Coactivator-3), a member of p160 family (28) primary coactivators. ERalpha is a steroid-dependent transcriptional factor (16) that belongs to the NRs superfamily (2,3) and, in response to the hormone 17-ß estradiol (E2), regulates transcription of distinct target genes involved in development, puberty, and homeostasis (8,16). ERalpha spends most of its lifetime in the nucleus and undergoes a rapid (within minutes) intranuclear redistribution following the addition of either agonist or antagonist (17,18,19). We designed a HeLa cell line (PRL-HeLa), engineered with a chromosomeintegrated reporter gene array (PRL-array) containing multicopy hormone response-binding elements for ERalpha that are derived from the physiological enhancer/promoter region of the prolactin gene. Following GFP-ER transfection of PRL-HeLa cells, we were able to observe in situ ligand dependent (i) recruitment to the array of the receptor and associated coregulators, (ii) chromatin remodeling, and (iii) direct transcriptional readout of the reporter gene. Addition of E2 causes a visible opening (decondensation) of the PRL-array, colocalization of RNA Polymerase II, and transcriptional readout of the reporter gene, detected by mRNA FISH. On the contrary, when cells were treated with an ERalpha antagonist (Tamoxifen or ICI), a dramatic condensation of the PRL-array was observed, displacement of RNA Polymerase II, and complete decreasing in the transcriptional FISH signal. All p160 family coactivators (28) colocalize with ERalpha at the PRL-array. Steroid Receptor Coactivator-3 (SRC-3/AIB1/ACTR/pCIP/RAC3/TRAM1) is a p160 family member and a known oncogenic protein (4,34). SRC-3 is regulated by a variety of posttranslational modifications, including methylation, phosphorylation, acetylation, ubiquitination and sumoylation (4,35). These events have been shown to be important for its interaction with other coactivator proteins and NRs and for its oncogenic potential (37,39). A number of extracellular signaling molecules, like steroid hormones, growth factors and cytokines, induce SRC-3 phosphorylation (40). These actions are mediated by a wide range of kinases, including extracellular-regulated kinase 1 and 2 (ERK1-2), c-Jun N-terminal kinase, p38 MAPK, and IkB kinases (IKKs) (41,42,43). Here, we report SRC-3 to be a nucleocytoplasmic shuttling protein, whose cellular localization is regulated by phosphorylation and interaction with ERalpha. Using a combination of high throughput and fluorescence microscopy, we show that both chemical inhibition (with U0126) and siRNA downregulation of the MAP/ERK1/2 kinase (MEK1/2) pathway induce a cytoplasmic shift in SRC-3 localization, whereas stimulation by EGF signaling enhances its nuclear localization by inducing phosphorylation at T24, S857, and S860, known partecipants in the regulation of SRC-3 activity (39). Accordingly, the cytoplasmic localization of a non-phosphorylatable SRC-3 mutant further supports these results. In the presence of ERalpha, U0126 also dramatically reduces: hormone-dependent colocalization of ERalpha and SRC-3 in the nucleus; formation of ER-SRC-3 coimmunoprecipitation complex in cell lysates; localization of SRC-3 at the ER-targeted prolactin promoter array (PRL-array) and transcriptional activity. Finally, we show that SRC-3 can also function as a cotransporter, facilitating the nuclear-cytoplasmic shuttling of estrogen receptor. While a wealth of studies have revealed the molecular functions of NRs and coregulators, there is a paucity of data on how these functions are spatiotemporally organized in the cellular context. Technically and conceptually, our findings have a new impact upon evaluating gene transcriptional control and mechanisms of action of gene regulators.

Expression and localization pattern of ABCA1 in diverse human placental primary cells and tissues

The ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol, phospholipids, and other lipophilic molecules across cellular membranes. Recent data provide evidence that ABCA1 plays an important role in placental function but the exact cellular sites of ABCA1 action in the placenta remain controversial. To clarify this issue, we analyzed the cellular and subcellular localization of ABCA1 with immunocytochemistry, immunofluorescence and subsequent confocal or immunofluorescence microscopy in different types of isolated primary placenta cells: cytotrophoblast cells, amnion epithelial cells, villous macrophages (Hofbauer cells), and mesenchymal cells isolated from chorionic membrane and placental villi. After 12 h of cultivation, primary cytotrophoblast cells showed intensive membrane and cytoplasmic staining for ABCA1. After 24 h, with progressive syncytium formation, ABCA1 staining intensity was markedly reduced and ABCA1 was dispersed in the cytoplasm of the forming syncytial layer. In amnion epithelial cells, placental macrophages and mesenchymal cells, ABCA1 was predominantly localized at the cell membrane and cytoplasmic compartments partially corresponding to the endoplasmic reticulum. In these cell types, the ABCA1 staining intensity was not dependent on the cultivation time. In conclusion, ABCA1 shows marked expression levels in diverse placental cell types. The multitopic localization of ABCA1 in diverse human placental cells not all directly involved in materno-fetal exchange suggests that this protein may not only participate in transplacental lipid transport but could have additional regulatory functions.

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

CELLULAR AND DEVELOPMENTAL FUNCTIONS OF THE XENOPUS ARVCF-CATENIN:KAZRIN COMPLEX

Xenopus ARVCF (xARVCF), a member of p120-catenin subfamily, binds cadherin cytoplasmic domains to enhance cadherin metabolic stability, or when dissociated, modulates Rho-family GTPases. We previously found that xARVCF binds directly to Xenopus KazrinA (xKazrinA), a widely expressed, conserved protein that bears little homology to established protein families. xKazrinA is also known to influence keratinocyte proliferation-differentiation and cytoskeletal activity. In my study, I first evaluated the expression pattern of endogenous Kazrin RNA and protein in Xenopus embryogenesis as well as in adult tissues. We then collaboratively predicted the helical structure of Kazrin’s coiled-coil domain, and I obtained evidence of Kazrin’s dimerization/oligomerization. In considering the intracellular localization of the xARVCF-catenin:xKazrin complex, I did not resolve xKazrinA in a larger ternary complex with cadherin, nor did I detect its co-precipitation with core desmosomal components. Instead, screening revealed that xKazrinA binds spectrin. This suggested a potential means by which xKazrinA localizes to cell-cell junctions, and indeed, biochemical assays confirmed a ternary xARVCF:xKazrinA:xβ2-spectrin complex. Functionally, I demonstrated that xKazrin stabilizes cadherins by negatively modulating the RhoA small-GTPase. I further revealed that xKazrinA binds to p190B RhoGAP (an inhibitor of RhoA), and enhances p190B’s association with xARVCF. Supporting their functional interaction in vivo, Xenopus embryos depleted of xKazrin exhibited ectodermal shedding, a phenotype that could be rescued with exogenous xARVCF. Cell shedding appeared to be caused by RhoA activation, which consequently altered actin organization and cadherin function. Indeed, I was capable of rescuing Kazrin depletion with ectopic expression of p190B RhoGAP. In addition, I obtained evidence that xARVCF and xKazrin participate in craniofacial development, with effects observed upon the neural crest. Finally, I found that xKazrinA associates further with delta-catenin and p0071-catenin, but not with p120-catenin, suggesting that Kazrin interacts selectively with additional members of the p120-catenin sub-family. Taken together, my study supports Kazrin’s essential role in development, and reveals KazrinA’s biochemical and functional association with ARVCF-catenin, spectrin and p190B RhoGAP.