Thèses sur le sujet « Sfp1 »

Cell-size homeostasis requires that proliferating cells coordinate growth and cell cycle, such that each division is matched by a doubling of mass. Size homeostasis is a universal but poorly understood feature of the cell cycle control. In the unicellular budding yeast Saccharomyces cerevisiae, the coordination of division with growth occurs at Start, a short interval during late G1 phase, after which cells are committed to division. A prerequisite for the passage through Start is the attainment of a critical cell size, whose value is set by ploidy and growth conditions. The critical-size threshold maintains uniform the cell size over many generations, and under minimal nutrient conditions forces cells to accumulate the energy stores required to complete the division cycle. Nutrients modulate the critical cell-size threshold according to the proliferation rate. Generally, cells growing slowly on a poor medium pass Start at a smaller size than fast-growing cells on a rich medium. In S. cerevisiae mutants that subvert the size control process have two phenotypes: small (whi) and large (lge). The former undergo Start a smaller cell size and the latter at a larger. Moreover, a systematic determination of cell-size distributions for all yeast deletion strains identified many new potential Start regulators. Many of the genes encoding potential Start repressors are implicated in ribosome biogenesis, suggesting the existence of a link between these two seemingly disparate processes. One of the smallest whi mutant is linket to SFP1 gene deletion. sfp1Δ cells display a disproportionate effect on size relative to the change in growth rate. SFP1 gene encodes a zinc-finger protein that is a key transcriptional regulator of ribosome biogenesis whose function is required for normal yeast growth. Nuclear localization of Sfp1, requires active TORC1 and it is highly stress sensitive. In addition, Sfp1 interacts directly with and is phosphorylated by TORC1. In contrast to Sch9 kinase, a major downstream target of TORC1, TORC1 phosphorylation of Sfp1 is unaffected by either osmotic or nutritional stresses, suggesting a different mode of regulation. Significantly, Sfp1, through its transcriptional activation function, exerts a negative feedback control on TORC1 activity toward the Sch9 kinase. Sfp1 also interacts with Mrs6, a conserved Rab escort protein that in turn regulates Sfp1 nuclear localization. The Mrs6 interaction with Sfp1 and TORC1 is related to a still poorly understood connection between TOR signaling and vesicle transport. The aim of this work has been to better characterized the relationship among Sfp1, the cell size control and some signalling pathways involved in the coordination of division with growth. In order to better elucidate the role of Sfp1 as a negative regulator of Start, we analyzed the level of some of the key players of the G1 to S transition, the G1 cyclins (Cln1-3) and the Cki Sic1, in a sfp1 mutant. sfp1Δ cells are characterized by a whi phenotype, slow growth, decreased budding index elongation of G1 phase and reduction of G2/M transition. Accordingly with some aspects of this phenotype, the Cln1-2 level resulted decreased while Cln3 levels were unaffected in agreement with data reporting that the mechanism through which Sfp1 couples ribosome biogenesis to Start is independent of Cln3. Interestingly, the main effect of the SFP1 deletion is on Sic1 that resulted entirely nuclear, all linked to Clb5 and stabilized by phosphorylation on threonine 173 (Thr173). Phosphorylation that is well known to induce Sic1 accumulation by preventing its degradation. In the sfp1 mutant, Sic1 stabilization is required for both the elongation of the G1 phase and the reduction of the G2/M transition. A similar situation that involves Sic1 stabilization by phosphorylation on Thr173 but leading to a G1 arrest is observed after inhibition of TORC1 by rapamycin where Sic1 accumulates in the nucleus to avoid improper Clb5/6-Cdc28-driven DNA replication under conditions of poor nutrient availability. This parallelism is in line with the fact that Sfp1 associated with Tor1 kinase and that this binding is essential for a correct localization of TORC1 together with Sfp1 at the RP promoters. A condition of poor nutrient availability can be considered as a stress condition for a cell. Similarly, the activation of the Hog1 MAP kinase after osmotic stress also induces a cellular response where the stabilization of Sic1, always via Thr173 phosphorylation is involved. Moreover, in this context, the cellular response to SFP1 inactivation appears more similar to the response to osmotic stress than that to rapamycin. In fact, the latter induces a G1 arrest linked to a Sic1 stabilization but subsequently a decrease of Cln3 accumulation takes place; such a decrease is essential for maintaining a prolonged G1 arrest. On the contrary, after the osmotic stress Sic1 is stabilized, Cln1 and Cln2 are low, Cln3 levels are unaffected as in the mutant. In addition, the stress response do not always provoke a cell cycle arrest, but is often a slowdown of cell cycle progression, necessary for cell adaptation to new conditions. Only if the stress is too intense, cells arrest growth. Yeast cells modulate stress response via the activation of mitogen-activated protein kinases (MAPKs) which respond to different conditions such as pheromone signals (mediated by the MAPK Fus3), osmolarity (mediated by the MAPK Hog1), nutrient deprivation (mediated by the MAPK Kss1) and cell wall stress (mediated by the MAPK Slt2). Since the first three MAPKs pathways use basically the same signaling machinery, when one of the three pathways is activated, the others are suppressed (cross-talk). The stress response linked to SFP1 inactivation involves a complex cross-talk between the Hog1 and Kiss1 pathways. Both pathways are activated but only Kiss1 is phosphorylated. Kss1 is the MAP kinase that primarily functions under conditions of nutrient deprivation such as the lack of nitrogen and/or glucose in the growth medium. Under these conditions the signal mucin Msb2 regulates the activation of the filamentous growth (FG) pathway that induces the phosphorylation of Kss1, necessary to guarantee cell survival. The lack of Sfp1 is sensed by the cell as a condition of nutrient scarcity. In fact, under optimal growth conditions, Sfp1 localizes to the nucleus, where it promotes the RP and RiBi genes expression. In response to changes in nutrient availability, Sfp1 is released from RP and RiBi gene promoters and exits from the nucleus; thus, the ribosome biogenesis is down-regulated. Moreover, since Msb2 is also required for activation of the Hog1 pathway a reciprocal inhibitory loop takes place between the Hog1 and Kss1 pathways allowing stable activation of the latter. We found that once activated Kiss1 is able to stabilized Sic1. We hypothesize that the activation of the FG pathway following Sfp1 lost of function involves a glycosylation defective-like response. In fact, activation of FG pathway by inhibition of N-glycosylation combined with a specific O-glycosylation defect induces activation of both Hog1 and Kss1 pathways and only Kss1 is phosphorylated. We found that SFP1 inactivation induces some defects that are also observed following the inhibition of glycosylation such as alterations in cell wall permeability, activation of the cell wall integrity pathway and alteration in the secretory pathway. All our data indicate that not only Sfp1 is regulated by stress and nutrients (both affecting its localization), but that Sfp1 can, in turn, regulate the stress response. The linker between Sfp1 and stress response pathway is the secretory pathway. We can hypothesize that a reduction of ribosome biogenesis may induce a defect in the secretory pathway leading to the activation of Msb2 and thus of the FG pathway. The exit from the nucleus of Sfp1, necessary for the reduction of ribosome biogenesis, allows the release of the Rab GTPase that is essential to switch off the defect in the secretory pathway. Consequently, the inactivation of SFP1 induces a complex activation of the MAPKs pathway that is responsible of the regulation of different aspects that characterized the mutant. The main of these is the regulation of the G1-S transitions by the stabilization of Sic1. Finally we showed that consequently to the SFP1 inactivation (probably due to the alteration in the secretory pathway), the mutant cells are characterized by an alteration of Cytoplasmic volume/ Protein content linked to an increase in the cytoplasmic volume. This let us to speculate that growth might be composed of two elements: the Size that is the growth in cell volume and the Mass that is the increase in the protein content. Consequently, alterations of cell growth in response to changes in the environmental conditions imply a coordinate regulation of Size and Mass with the aim of maintaining their ratio constant. One of the key elements necessary to maintain this balance is Sfp1.

Sfi1 est une protéine récemment découverte qui est impliquée dans le processus de division cellulaire. Concrètement, son rôle est intimement lié à la duplication des MTOCs (centres organisateurs de microtubules) chez les organismes eucaryotes, un rôle partagé avec la centrine. Les centrines sont des calciprotéines de la superfamille EF-hand très conservées chez les eucaryotes et impliquées dans des fonctions cellulaires diverses, comme la duplication des MTOCs, la réparation de l'ADN et l'exportation nucléaire de l'ARNm. La découverte de l'interaction entre la centrine et Sfi1 nous a conduit à nous intéresser à l'étude structurale et biochimique du complexe Sfi1-centrine. Sfi1 possède plusieurs motifs de liaison aux centrines (chacun d'entre eux pouvant lier une molécule de centrine) tout le long de sa structure. Ces séquences répétées présentent des résidus conservés qui correspondent au motif classique 1-4-8 de liaison aux centrines. Ce travail est centré sur la compréhension de la liaison des molécules de centrine de manière individuelle et collective à la protéine Sfi1. Pour cela, des études structurales par RMN de l'extrémité C-terminale de la centrine 2 humaine (HsCen2) en complexe avec un peptide de 20 résidus, correspondant à la séquence répétée n°17 de Sfi1 humaine (hSfi1), ont été réalisées. Celles-ci ont permis de comprendre les bases structurales de la liaison Sfi1-centrine. En outre, des études biochimiques ont été réalisées concernant le domaine de Sfi1 de levure (ScSfi1) contenant 6 séquences répétées avec la centrine de levure (Cdc31). Ce complexe, ainsi que divers variants de Sfi1 nous ont permis d'explorer les caractéristiques de l'interaction de la centrine avec un enchainement de séquences répétées de Sfi1
Sfi1 is a recently discovered protein that is involved in cell division, with its specific role in the MTOC duplication in eukaryotes, a function also shared by centrins. Centrins are well conserved calciproteins in eukaryotes belonging to the EF-hand superfamily and they are involved in diverse cellular functions, like MTOC duplication, DNA repair and nuclear mRNA transport. The discovery of the interaction between centrins and Sfi1 led us to be interested in the structural and biochemical study of the complex Sfi1-centrin. Sfi1 has several centrin-linking motifs along its structure (each one able to link one centrin molecule). These repeated sequences have well conserved residues corresponding to the classical 1-4-8 centrin-link motif. This work is focussed on the understanding of individual and collective centrin linking to Sfi1. In order to achieve this, we have made NMR structural experiments with the C-terminus of HsCen2 complexed with a 20-residue peptide corresponding to repeat n°17 of hSfi1. These experiments have allowed us to understand the structural basis of the Sfi1-centrin binding. In parallel, biochemical studies have been made with a yeast Sfi1 (ScSfi1) domain that contains 6 repeats in a complex with yeast centrin (Cdc31). This complex, and some Sfi1 mutants made of it, have allowed us to explore the interaction characteristics of the centrin with several Sfi1 repeats

Le centrosome est le centre organisateur des microtubules dans les cellules animales, il nucléé les microtubules interphasiques ainsi que le fuseau mitotique. Les centrosomes sont produits par duplication, mécanisme rigoureusement régulé au cours du cycle cellulaire. En effet, un centrosome comporte deux centrioles qui se dupliquent une fois par cycle cellulaire. Des erreurs de duplication conduisant à plus de deux centrosomes induisent la formation de fuseaux multipolaires et provoquent des défauts de ségrégation des chromosomes. Chez la levure Schizosaccharomyces pombe, un organisme modèle pour l’étude de la division cellulaire, les homologues des centrosomes sont les SPBs (pour Spindle Pole Body). Une structure annexe spécifique liée aux SPBs est appelée demi-pont (quand les SPBs ne sont pas dupliqués) puis pont (quand elle relie les deux SPBs dupliqués). Les deux principaux composants du pont chez la levure S. pombe sont Cdc31 et Sfi1. Sfi1 est une protéine linéaire formée de répétitions en hélice α formant des sites de liaison pour la Centrine/Cdc31. Sfi1 s’assemble en réseau de molécules parallèles interagissant avec le SPB via leur domaine N-terminal. Lors de la première partie de ma thèse, j’ai démontré que Sfi1 est requis pour la duplication et la séparation des deux SPBs. Dans la première partie de ma thèse, je me suis intéressée aux fonctions de Sfi1 chez la levure. Cette étude a permis de démontrer que Sfi1 est un composant du demi-pont et qu’il est essentiel pour la duplication des SPBs et l’assemblage d’un fuseau bipolaire. De plus, nous avons déterminé que le pont est dupliqué en fin de mitose. Enfin, nous avons aussi montré que la déstabilisation du pont menant à sa rupture en mitose, dépend de la phosphorylation de Cdc31 par la kinase mitotique Cdk1. Lors de la seconde partie de ma thèse, je me suis intéressée au complexe Sfi1/Centrine dans les cellules humaines. J’ai confirmé que Sfi1 est localisée aux centrioles. De plus, j’ai montré que la déplétion de Sfi1 dans les cellules RPE1, conduit à une perte de localisation de la Centrine, suggérant soit un défaut de recrutement, soit une déstabilisation. De plus, en absence de Sfi1, les cellules RPE1 ne sont plus capables de former de cil primaire. Ce résultat suggère que Sfi1 et la Centrine sont requis pour la ciliogénèse. Enfin, j’ai aussi démontré que la déplétion deSfi1 induit un arrêt de cycle cellulaire dans les cellules non tumorales RPE1. Dans les cellules cancéreuses, HeLa, le cycle n’est pas arrêté mais j’ai pu observer une prolongation du temps de mitose. En conclusion mes travaux montrent que bien que la fonction de Sfi1/Centrin ne soit pas conservée, le complexe reste essentiel pour l’intégrité structurale et fonctionnelle du centrosome
The centrosome is the main microtubule organizing center. It nucleates and organizes interphase microtubule and contributes to the assembly of the bipolar mitotic spindle. To do so, the centrosome, present in one copy at the beginning of the cell cycle, duplicates to produce a second copy. The duplication process is tightly controlled and regulated since centrosome over-duplication can lead to multipolar mitotic spindles and promote genome instability and tumorigenesis. The duplication of the yeast centrosome, the SPB (Spindle pole body), begins with the duplication of the half bridge. This appendage is composed of Sfi1/Cdc31 complex organized in a parallel array attached to the core SPB. SPB duplication relies on the assembly of a second array of Sfi1/Cdc31, anti-parallel to the first one, creating thereby an assembly site for the new SPB. Therefore Sfi1 is essential for SPB duplication and our work defined the timing of half-bridge duplication and some of the regulatory mechanisms that favor bridge splitting to release duplicated centrosomes and allow spindle assembly at mitotic onset. Sfi1 and Cdc31/Centrins are conserved in human cells where the centrosome is composed of two centrioles surrounded by the pericentriolar material. Centrins are concentrated in the distal end of centrioles. Sfi1 has also been localized to centrioles, but its function remained unknown. Thus, we started investigating Sfi1 function in human cells. We found that Sfi1 depletion leads to a decrease in Centrin recruitment to the centrioles. It also leads to a cell cycle arrest in G1 in RPE1 cells, an event previously observed in presence of defects in centriole biogenesis. In HeLa cells where the cell cycle is not affected, Sfi1 depletion leads to a mitotic delay. Moreover, Sfi1 depletion leads to cilium assembly. To conclude, these results altogether point towards a role of human Sfi1 in centriole biogenesis

Denne oppgaven tar for seg hvilke målinger som er nødvendig for å bestemme SFP-faktoren for et typisk nyere kontorbygg med mekanisk ventilasjon. Den tar for seg hvordan disse målingene kan utføres, hva slags måleutstyr som kan benyttes, og hvordan nøyaktigheten til målingene kan bestemmes.Oppgaven tar så for seg gjennomføring av målinger for å bestemme SFP-faktoren for to bygninger. Disse to bygningene er Høyskolen i Oslo (HiO) og PricewaterhouseCoopers-, eller PWC-bygget i Bjørvika i Oslo. Det gis en innføring i de to bygningenes ventilasjonsanlegg og oppbyggingen av disse samt en gjennomgang av styresystemene for ventilasjonsanleggene. Det måles på ett aggregat i hver bygning. For HiO velges aggregat 36.13 som betjener et auditorium med VAV-styring. For PWC-bygget velges aggregat 36.09 som betjener hovedsakelig cellekontorer og styres som CAV. Alle målinger blir utført ved hjelp av differansetrykkmåling over viftene. Aggregatet ved HiO klarer akkurat å komme seg ned på kravet i teknisk forskrift om 2,0 kW/m3/s ved bruk av gjennomsnittlig SFP over driftstiden, men ved personbelastning i lokalet er SFP-verdien 2,66 kW/m3/s. Aggregatet ved PWC-bygget ligger på 2,0 kW/m3/s ved full innregulert drift og klarer dermed fint forskriftskravet. Målingene viser likevel at det ville være mye energi å spare på VAV-styring av anlegget fremfor CAV.Forbedring av ventilasjonsdesign er også en del av oppgaven, og det beregnes hvordan strengere krav til maksimal lufthastighet og alternativ utforming av fordelingskammer kan redusere strømningsmotstanden i et anlegg. Dette viste seg å kunne redusere SFP-faktoren for aggregat 36.09 ved PWC-bygget til 1,52 kW/m3/s ved å ikke tillate hastigheter over 3 m/s samt å benytte en alternativ utforming av fordelingskammeret.Det presenteres forslag til forbedringer i rutiner og programvare for ventilasjonsdesign som kan redusere SFP-fakoren i fremtidige kontorbygninger, og det diskuteres hvorvidt de løsninger som fremkommer i denne oppgaven vil være økonomisk lønnsomme.Til slutt kommer anbefalinger for videre arbeid med problemstillingen.

Due to the increasing resistance demonstrated by insects to conventional insecticides, the need for compounds with novel modes of action is becoming more urgent. Also, the discovery and production of new insecticides is vital as regulations and restrictions on conventional insecticides become increasingly stringent (Casida and Quistad 1998). Research in this area requires screening of many candidate compounds which is costly and time-consuming. The goal of this research was to produce in vitro insect neurons from Sf21 insect ovarian cell lines, which could lead to new high throughput screening methods and a way to mass produce insect material for basic research. This study used a culture of Sf21 cells and a mixture of differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, Sf21 cells began to express neuronal morphology, or the production of elongated, axon-like processes within 2-3 days. Maximal differentiation occurred when in the presence of 42 μM 20-HE or 10 μM insulin. Effects were maximal on day 2 for 20-E and day 3 for insulin. Insulin was more potent at day 2 for inducing differentiation (EC₅₀ = 247 nM) than 20-HE (EC₅₀ = 13 μM). In combination, 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system (CNS) stimulant, inhibited induction of elongated processes by 20-HE and/or insulin. Caffeine was a potent inhibitor of 42 μM 20-HE, with an IC50 of 9 nM, and the inhibition was incomplete, resulting in about one quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies studies with of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined. If they are present, high throughput screening for new insecticides will be accelerated and made more economical by the utility of this method.
Master of Science

La découverte de l’ARN interférence et des micro-ARN a permis de définir un principe majeur de régulation de l’expression des gènes, et a produit de nouveaux outils pour la médecine. Chez les mammifères, l’étude des fonctions des micro-ARN a été restreinte au cytoplasme, bien qu’ils soient aussi présents dans le noyau.Cette thèse présente une série d’expériences visant à caractériser les facteurs moléculaires requis pour l’activité nucléaire des micro-ARN. Nous avons débuté ce projet en explorant les partenaires ARN-dépendant de la protéine AGO2 par immunoprécipitation et spectrométrie de masse quantitative. Parmi les interactants ARN-dépendants, nous nous sommes concentrés sur trois protéines nucléaires abondantes : SFPQ, PSPC1 et NONO qui forment la famille drosophila behavior and human splicing (DBHS). Nous avons démontré que le complexe RISC nucléoplasmique est associé aux protéines SFPQ, PSPC1 et NONO dans plusieurs lignées cellulaires murines et humaines, d’une manière qui dépend de SFPQ. Des expériences de type HITS-CLIP de la protéine AGO2 et/ou de la protéine SFPQ dans des cellules souches nous ont permis de montrer que SFPQ se lie préférentiellement aux 3’UTR longs en utilisant deux motifs spécifiques. En effet, SFPQ contrôle significativement environ 20% de l’activité de liaison de AGO2, ce qui est répercuté au niveau transcriptomique. Cependant, cette activité concerne uniquement les sites de liaison de SFPQ proches (<500 nucléotides) de AGO2. De plus, nous avons observé que cette régulation s’étend aux ARNm cytoplasmiques. Ce résultat suggère que la liaison et l’agrégation de la protéine SFPQ à l’ARN programme la structure du 3’UTR et donc les possibilités de ciblage par les miARN dans le noyau, et ceci d’une manière qui semble préservée dans le cytoplasme. Enfin, nous avons montré en particulier que l’expression de SFPQ contrôle le programme de ciblage par let-7a, et module la transition des cellules souches vers l’état différencié. Ces résultats contribuent à la diversité des mécanismes de régulation de l’activité des miARN. Dans la deuxième partie du projet, nous avons exploré les partenaires ARN-indépendant de la protéine nucléaire AGO2. Nous avons découvert que la protéine AGO2 interagit avec le complexe CCR4-NOT1 et l’exosome nucléaire d’une manière indépendante de l’ARN. Nous proposons une série d’expériences visant à confirmer ces résultats. Brièvement, l’hypothèse de travail qui semble la plus cohérente avec les données actuelles est la liaison directe de l’exosome au module CNOT2-CNOT3 du complexe CCR4-NOT1. Ce modèle permettrait d’expliquer le mécanisme d’extinction des gènes par les miARN nucléaires qui reposerait donc sur leur interaction avec les complexes CCR4-NOT1 et exosome. Son mode opératoire comprendrait des protéines de liaison à l’ARN et des micro-ARN pour sélectionner les cibles
The discovery of RNA interference and micro-RNA has unravelled a fundamental principle of gene expression regulation, and has produced new tools for medicine. In mammals, the study of micro-RNA functions have been confined to the cytoplasm, although there is a growing body of evidence about their presence in the nucleus.This thesis present a set of experiments directed towards understanding the molecular factors required for nuclear miRNA activity.We started this project by exploring the RNA-dependent interactors of AGO2 by immunoprecipitation followed by quantitative mass spectrometry analysis. We focused on three partners: SFPQ, PSPC1 and NONO which are abundant nuclear proteins and belong to the DBHS family (drosophila behavior and human splicing). We demonstrated that the nucleoplasmic RISC complex associates with DBHS proteins in multiple murine and human cell lines in an SFPQ-dependent manner.HITS-CLIP experiments of AGO2 and SFPQ proteins in mouse embryonic stem cells showed that SFPQ preferentially binds long 3’UTR using two specific motifs. Using these motifs, SFPQ significantly controls about 20% of local AGO2 binding activity and target mRNA stability as we observe by transcriptomic analysis. SFPQ mode of action appears to be local and SFPQ motifs are functional only when proximal to AGO2 binding sites in a window of 500 nucleotides.Moreover, although SFPQ is only nuclear, we observe that SFPQ has an effect on cytoplasmic mRNAs, which suggests that SFPQ binding and aggregation to mRNA in the nucleus programs the 3’UTR for miRNA targeting, in a way that appears preserved in the cytoplasm.In particular, we found that SFPQ controls let-7 targeting program and modulates embryonic stem cell differentiation into neuron cells.These results contribute to expand the diversity of miRNA regulatory mechanisms.In the second part of the project, we explored the RNA-independent interactors of AGO2. We discovered that nuclear AGO2 interacts with CCR4-NOT1 complex and the RNA exosome in an RNA-independent manner.We propose a set of experiments to confirm these results, based on a new working model. Briefly, the most likely hypothesis to explain the current data is the direct binding of RNA exosome to the CNOT2-CNOT3 module of CCR4-NOT1 complex.This model would explain the silencing mechanism of genes by nuclear miRNAs, whose activity would depend on the RISC complex interaction with CCR4-NOT1 complex and RNA exosome.Hence, the RNA exosome would use both RNA binding proteins and miRNAs to select targets for degradation, based on CCR4-NOT1 mode of action

The purpose of the research has been to develop an experimental biofilm ecocystem for the characterization of Listeria monocytogenes biofilms in a simulated food processing (SFP) environment. Individual strains of L. monocytogenes were initially surveyed for their ability to attach to surfaces and form biofilms under a variety of conditions. Five strains of L. monocytogenes were then screened for optimal cell attachment and biofilm formation. Significant differences in surface attachment and biofilm formation were observed among the different strains of L. monocytogenes. The biofilms of the five-strain mixture of L. monocytogenes were formed on surfaces that are commonly used in food processing facilities, such as stainless steel and Teflon®. The biofilms were subjected to the SFP system in sequential 24-h daily cycles. Conditions of the SFP system included: starvation, washing, rinsing, and sanitation that routinely occur in a food processing plant. Cell survival in biofilms was determined during the time course of the experiment. The susceptibility of the cells in biofilms and detached cells to different sanitizers was measured. The morphology of the cells in biofilms and the planktonic cells detached from biofilms was observed. The study indicated that the progressive resistance developed by L. monocytogenes biofilms to a sanitizer could protect the cells in biofilms from this and other sanitizing agents. The progressive resistance and cross protection was observed in biofilms, but not in detached cells. These findings could provide a basis for further research on the mechanism of progressive resistance to stresses by L. monocytogenes in biofilms under food processing conditions. The data may help to establish effective sanitation programs for food processing and related industries.

Thesis (Ph. D.)--University of Oregon, 2007.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 81-88). Also available for download via the World Wide Web; free to University of Oregon users.

A novel dual resin based, substrate feed and product removal (SFPR) strategy has been investigated to overcome the substrate and product inhibition in an industrially important Baeyer-Villiger monooxygenase catalysed bioconversion in order to enhance the productivity of the bioconversion process. The bioconversion of the ketone substrate, bicyclo[3.2.0]hept-2-en-6-one, to the lactone products, (1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one, catalysed by a recombinant whole cell biocatalyst, Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase from Acinetobacter calcoaceticus, was used as the model reaction to prove the feasibility of the novel dual resin SFPR concept. Before the application of the dual resin SFPR strategy to the Baeyer-Villiger bioconversion, adsorption of the ketone and lactone onto non-specific resins was investigated. Several resins were initially characterised at the bench scale by determining adsorption isotherms for the ketone and lactone compounds. Thereafter adsorption isotherms were generated via a high throughput resin screening (HTRS) method using both 96 wells and 24 wells microplate platforms. Comparison of the adsorption isotherm data between the bench scale and the two HTRS platforms, together with results of resin mixing in wells of the 96 wells and 24 wells microplate platforms, as investigated by high speed imaging experiments, shows that the 24 well microplate platform was the most suitable to investigate adsorption kinetics of the ketone and lactone on the resins. Based on the adsorption studies, resins Dowex® Optipore L493 and Amberlite® XAD7 were chosen to be separately used for substrate feeding in the dual resin SFPR strategy. Dowex® Optipore L493 was chosen for its high capacity of 0.21 g/gadsorbent for ketone, whereas Amberlite® XAD7 was chosen for its high selectivity of ketone over lactone compared to any other resin. Amberlite® IRC50 was chosen for lactone removal in the dual resin SFPR strategy because of its high selectivity of lactone over ketone than any other resin. To demonstrate the feasibility of the dual resin SFPR strategy, the Baeyer-Villiger bioconversion was performed in shake flasks and compared to bioconversions without the use of resins and with the use of a single resin based SFPR strategy. At an initial ketone concentration of 3g/l, both resin based strategies performed significantly better than the bioconversion performed without resins. The dual resin SFPR strategy, carried out with both types of resins free in suspension without spatial separation, also showed improvement compared to results obtained with the single resin SFPR strategy. The dual resin SFPR strategy was also performed with the spatial separation of the two resins by housing one of the resins in a porous bag. This allowed observation of the majority of lactone product adsorbed onto the Amberlite® IRC50 resin as expected based on adsorption studies. Carrying out the Baeyer-Villiger bioconversion with the implementation of the dual resin SFPR strategy in shake flasks saw an increase of productivity compared to the Baeyer-Villiger bioconversions carried out without resins by as much as 132% and in comparison with the single resin SFPR strategy by as much 10%, thus demonstrating a ‘proof of concept’ of the novel dual resin SFPR bioconversion strategy. After demonstrating a ‘proof of concept’ for the dual resin SFPR strategy, its application in a miniature stirred tank bioreactor was investigated to open the way for scale up studies. Two configurations were investigated, namely the conventional reactor system where both resins were added directly into the bioreactor, and the recycle reactor system where a column housed one of the two types of resins. Using resins with low adsorption capacities and the need of an extra resin type in a dual resin SFPR strategy, makes the recycle reactor configuration a more attractive system, however it was the conventional reactor configuration that performed better than the recycle reactor system. The L493-IRC50 combination in the conventional reactor configuration achieves a 21% greater productivity than in the recycle reactor. The dual resin SFPR strategy using the L493-IRC50 combination performed better than any other resin based SFPR strategy when carried out with both resins in the reactor. It reached a productivity of 0.85g/l/h after 2.5 hours of reaction, 5% higher than the productivity achieved with the single resin strategy in the conventional reactor configuration. The novel dual resin based SFPR strategy and the HTRS method developed in this work has the potential to be applied in any bioconversion that needs to overcome substrate and product inhibition.

The master’s thesis is focused on desing high-speed ethernet switch based on circuit FPGA. The switch is able to divide one data stream, created from ethernet frames to the two data streams with half data flow.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico
Entre todos os tipos de cÃncer, o de pulmÃo (CP) à um dos mais comuns de todos os tumores malignos, apresentando aumento de 2% por ano na sua incidÃncia mundial. No Brasil, para o ano de 2014 sÃo estimados 27.330 casos novos de CP, sendo destes 16.400, em homens e 10.930 em mulheres. Neste contexto, à de fundamental importÃncia para saÃde pÃblica realizar e determinar diagnÃsticos precoces e mais precisos para detectar os estÃgios reais das doenÃas pulmonares. O auxÃlio ao diagnÃstico mostra-se importante tanto do ponto de vista clÃnico quanto em pesquisa. Dentre os fatores que contribuem para isto, pode-se citar o aumento da precisÃo do diagnÃstico do mÃdico especialista à medida que aumenta o nÃmero de informaÃÃes sobre o estado do paciente. Deste modo, certas doenÃas podem ser detectadas precocemente, aumentando as chances de cura. O tratamento inicial para esta doenÃa consiste na lobectomia. Nesse contexto, costuma-se realizar a segmentaÃÃo dos lobos pulmonares em imagens de Tomografia Computadorizada para extrair dados e auxiliar no planejamento da lobectomia. A segmentaÃÃo dos lobos a partir de imagens de TC à geralmente obtida atravÃs da detecÃÃo das fissuras pulmonares. Nesse sentido, com o intuito de obter uma segmentaÃÃo da fissura pulmonar mais eficaz e realizar um processo totalmente independente das demais estruturas presentes no exame de TC, o presente trabalho possui o objetivo de realizar a segmentaÃÃo das fissuras utilizando medidas de textura LBP e Redes Neurais Artificiais (RNA). Para a implementaÃÃo do algoritmo foi utilizado uma MLP (Multilayer Perceptron) com 60 entradas, 120 neurÃnios na camada oculta e 2 neurÃnios de saÃda. Os parÃmetros de entrada para a rede foi o histograma LBP do voxel a ser analisado. Para o treinamento da rede foi necessÃrio criar um sistema para identificaÃÃo das classes fissuras e nÃo-fissuras de forma manual, onde o usuÃrio seleciona os pixels da classe fissura e da nÃo-fissura. Para realizar as validaÃÃes do algoritmo foi criado um padrÃo-ouro que foi extraÃdo um total de 100 imagens de 5 exames do banco de dados LOLA11. Nessas imagens, as fissuras foram destacadas por 2 especialistas. A partir do padrÃo-ouro, o as imagens foram processadas pelo algoritmo e assim os resultados obtidos. Para o conjunto de imagens testadas, o classificador obteve um melhor desempenho quando o tamanho, 15x15 pixels, da janela utilizada para gerar o histograma do LBP. Para chegar atà essa definiÃÃo foram testados os tamanhos 11x11, 15x15, 17x17 e 21x21 e os resultados foram comparados utilizando as mÃtricas de Especificidade Es(\%), Coeficiente de Similaridade CS(%), Sensibilidade S(\%), distÃncia mÃdia e desvio padrÃo da distÃncia. A primeira abordagem de anÃlise dos resultados à atravÃs dos voxels}definidos como fissura no final da metodologia proposta. Para a metodologia proposta, detecÃÃo automÃtica utilizando LBP ( extit{Local Binary Pattern}) e MLP, as taxas obtidas foram CS = 96,7%, S = 69,6% e Es = 96,8% para o mÃtodo proposto antes do afinamento e CS = 99,2%, S = 3% e Es = 99,81% para o mÃtodo proposto com o afinamento no fim, considerando a incidÃncia de falsos positivos e falsos negativos. Outra abordagem utilizada na literatura para avaliaÃÃo de mÃtodos de segmentaÃÃo de fissuras à baseado na distÃncia mÃdia entre a fissura delineada pelo especialista e a fissura resultante do algoritmo proposto. Desta forma, o algoritmo proposto neste trabalho foi comparado com o algoritmo de Lassen(2013) atravÃs da abordagem da distÃncia mÃdia entre a fissura segmentada manual e a fissura segmentada de forma automÃtica. O algoritmo proposto com afinamento no final obteve uma menor distÃncia no valor de e um menor desvio padrÃo comparado com o mÃtodo de Lassen(2013). Por fim, sÃo apresentados os resultados da segmentaÃÃo automÃtica das fissuras pulmonares. A baixa incidÃncia de detecÃÃes falso negativas, juntamente com a reduÃÃo significativa de detecÃÃes falso positivas, resultam em taxa de acerto elevada. Conclui-se que a tÃcnica de segmentaÃÃo de fissuras pulmonares à um algoritmo Ãtil para segmentar fissuras pulmonares em imagens de TC, e com o potencial de integrar sistemas que auxiliem o diagnÃstico mÃdico.
Among all cancers, lung cancer (LC) is one of the most common tumors, an increase of 2% per year on its worldwide incidence. In Brazil, for the year of 2014, 27,330 new cases of LC are estimated, these being 16,400 in men and 10,930 in women. In this context, it is of fundamental importance for public health the identication on early stages of lung diseases. The diagnosis assistance shows to be important both from a clinical standpoint as in research. Among the factors contributing to this scene, one important is the increasing accuracy of diagnosis of a medical expert as you increase the number of information about the patient's condition. Thus, certain disorders might be detected early, including saving lives in some cases. The initial treatment for this disease consists of lobectomy. In this context, it is customary to perform the segmentation of lung lobes in CT images to extract data and assist in planning for lobectomy. The segmentation of the lobes from CT images is usually obtained by detection of pulmonary fissures. Thus, in order to obtain a more effective segmentation of pulmonary fissures, and perform a completely independent process from the other structures present in the CT scan, the present work has the objective to perform the fissure segmentation using LBP texture measures and Neural Networks (NN). To implement the algorithm we used one MLP with 60 inputs, 120 hidden neurons and 2 output neurons. The input parameters for the network was the LBP histogram of the voxel being analyzed. For network training, it was necessary to create a system to label the features as fissures and non-fissures manually, where the user selects the fissure pixels class. To perform the validation of the algorithm was necessary to create a "gold standard"in which it was extracted a total of 100 images from 5 exams from the dataset LOLA11, where these images were the fissures were highlighted by two experts. From the gold standard, the proposed algorithm was processed and the results were obtained. For all tested images, the classifier obtained a better performance when the size of 15x15 pixels of the window was used to generate the histogram of the LBP. To get to this definition were tested sizes of 11x11, 15x15, 17x17 and 21x21 and the results were based on metrics comaprados ACC (%), TPR (%), SPC (%) distance mean and standard deviation of the distance. The first approach to analyze the results is through the voxels defined as fissure at the end of the proposed methodology. For the proposed methodology, using automatic detection and MLP LBP before thinning, the rates were obtained ACC= 96.7 %, TPR = 69.6 % and SPC = 96.8 % and ACC = 99 2 % TPR = 3 % and SPC = 99.81 % for the proposed method with the thinning in the end, considering the incidence of false positives and false negatives. Another approach used in the literature for evaluating methods of fissure segmentation is based on the average distance between the fissure delineated by the expert and the resulting fissure through the algorithm. Thus, the algorithm proposed in this paper was compared with the algorithm Lassen et al. (2013) by the average distance between the manual segmented and the automatically segmented fissure. The proposed algorithm with the thinning in the end achieved a shorter distance average value and a lower standard deviation compared with the method of (LASSEN et al., 2013). Finally, the results obtained for automatic segmentation of lung fissures are presented. The low incidence of false negative detections detection results, together with the significant reduction in false positive detections result in a high rate of settlement. We conclude that the segmentation technique for lung fissures is a useful target for pulmonary fissures on CT images and has potential to integrate systems that help medical diagnosis.

CAVALCANTI NETO, E. SFPT neural: nova técnica de segmentação de fissuras pulmonares baseada em texturas em imagens de tomografia computadorizadas do tórax. 2014. 74 f. Dissertação (Mestrado em Engenharia de Teleinformática) – Centro de Tecnologia, Universidade Federal do Ceará, Fortaleza, 2014.
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Among all cancers, lung cancer (LC) is one of the most common tumors, an increase of 2% per year on its worldwide incidence. In Brazil, for the year of 2014, 27,330 new cases of LC are estimated, these being 16,400 in men and 10,930 in women. In this context, it is of fundamental importance for public health the identication on early stages of lung diseases. The diagnosis assistance shows to be important both from a clinical standpoint as in research. Among the factors contributing to this scene, one important is the increasing accuracy of diagnosis of a medical expert as you increase the number of information about the patient's condition. Thus, certain disorders might be detected early, including saving lives in some cases. The initial treatment for this disease consists of lobectomy. In this context, it is customary to perform the segmentation of lung lobes in CT images to extract data and assist in planning for lobectomy. The segmentation of the lobes from CT images is usually obtained by detection of pulmonary fissures. Thus, in order to obtain a more effective segmentation of pulmonary fissures, and perform a completely independent process from the other structures present in the CT scan, the present work has the objective to perform the fissure segmentation using LBP texture measures and Neural Networks (NN). To implement the algorithm we used one MLP with 60 inputs, 120 hidden neurons and 2 output neurons. The input parameters for the network was the LBP histogram of the voxel being analyzed. For network training, it was necessary to create a system to label the features as fissures and non-fissures manually, where the user selects the fissure pixels class. To perform the validation of the algorithm was necessary to create a "gold standard"in which it was extracted a total of 100 images from 5 exams from the dataset LOLA11, where these images were the fissures were highlighted by two experts. From the gold standard, the proposed algorithm was processed and the results were obtained. For all tested images, the classifier obtained a better performance when the size of 15x15 pixels of the window was used to generate the histogram of the LBP. To get to this definition were tested sizes of 11x11, 15x15, 17x17 and 21x21 and the results were based on metrics comaprados ACC (%), TPR (%), SPC (%) distance mean and standard deviation of the distance. The first approach to analyze the results is through the voxels defined as fissure at the end of the proposed methodology. For the proposed methodology, using automatic detection and MLP LBP before thinning, the rates were obtained ACC= 96.7 %, TPR = 69.6 % and SPC = 96.8 % and ACC = 99 2 % TPR = 3 % and SPC = 99.81 % for the proposed method with the thinning in the end, considering the incidence of false positives and false negatives. Another approach used in the literature for evaluating methods of fissure segmentation is based on the average distance between the fissure delineated by the expert and the resulting fissure through the algorithm. Thus, the algorithm proposed in this paper was compared with the algorithm Lassen et al. (2013) by the average distance between the manual segmented and the automatically segmented fissure. The proposed algorithm with the thinning in the end achieved a shorter distance average value and a lower standard deviation compared with the method of (LASSEN et al., 2013). Finally, the results obtained for automatic segmentation of lung fissures are presented. The low incidence of false negative detections detection results, together with the significant reduction in false positive detections result in a high rate of settlement. We conclude that the segmentation technique for lung fissures is a useful target for pulmonary fissures on CT images and has potential to integrate systems that help medical diagnosis
Entre todos os tipos de câncer, o de pulmão (CP) é um dos mais comuns de todos os tumores malignos, apresentando aumento de 2% por ano na sua incidência mundial. No Brasil, para o ano de 2014 são estimados 27.330 casos novos de CP, sendo destes 16.400, em homens e 10.930 em mulheres. Neste contexto, é de fundamental importância para saúde pública realizar e determinar diagnósticos precoces e mais precisos para detectar os estágios reais das doenças pulmonares. O auxílio ao diagnóstico mostra-se importante tanto do ponto de vista clínico quanto em pesquisa. Dentre os fatores que contribuem para isto, pode-se citar o aumento da precisão do diagnóstico do médico especialista à medida que aumenta o número de informações sobre o estado do paciente. Deste modo, certas doenças podem ser detectadas precocemente, aumentando as chances de cura. O tratamento inicial para esta doença consiste na lobectomia. Nesse contexto, costuma-se realizar a segmentação dos lobos pulmonares em imagens de Tomografia Computadorizada para extrair dados e auxiliar no planejamento da lobectomia. A segmentação dos lobos a partir de imagens de TC é geralmente obtida através da detecção das fissuras pulmonares. Nesse sentido, com o intuito de obter uma segmentação da fissura pulmonar mais eficaz e realizar um processo totalmente independente das demais estruturas presentes no exame de TC, o presente trabalho possui o objetivo de realizar a segmentação das fissuras utilizando medidas de textura LBP e Redes Neurais Artificiais (RNA). Para a implementação do algoritmo foi utilizado uma MLP (Multilayer Perceptron) com 60 entradas, 120 neurônios na camada oculta e 2 neurônios de saída. Os parâmetros de entrada para a rede foi o histograma LBP do voxel a ser analisado. Para o treinamento da rede foi necessário criar um sistema para identificação das classes fissuras e não-fissuras de forma manual, onde o usuário seleciona os pixels da classe fissura e da não-fissura. Para realizar as validações do algoritmo foi criado um padrão-ouro que foi extraído um total de 100 imagens de 5 exames do banco de dados LOLA11. Nessas imagens, as fissuras foram destacadas por 2 especialistas. A partir do padrão-ouro, o as imagens foram processadas pelo algoritmo e assim os resultados obtidos. Para o conjunto de imagens testadas, o classificador obteve um melhor desempenho quando o tamanho, 15x15 pixels, da janela utilizada para gerar o histograma do LBP. Para chegar até essa definição foram testados os tamanhos 11x11, 15x15, 17x17 e 21x21 e os resultados foram comparados utilizando as métricas de Especificidade Es(\%), Coeficiente de Similaridade CS(%), Sensibilidade S(\%), distância média e desvio padrão da distância. A primeira abordagem de análise dos resultados é através dos voxels}definidos como fissura no final da metodologia proposta. Para a metodologia proposta, detecção automática utilizando LBP ( extit{Local Binary Pattern}) e MLP, as taxas obtidas foram CS = 96,7%, S = 69,6% e Es = 96,8% para o método proposto antes do afinamento e CS = 99,2%, S = 3% e Es = 99,81% para o método proposto com o afinamento no fim, considerando a incidência de falsos positivos e falsos negativos. Outra abordagem utilizada na literatura para avaliação de métodos de segmentação de fissuras é baseado na distância média entre a fissura delineada pelo especialista e a fissura resultante do algoritmo proposto. Desta forma, o algoritmo proposto neste trabalho foi comparado com o algoritmo de Lassen(2013) através da abordagem da distância média entre a fissura segmentada manual e a fissura segmentada de forma automática. O algoritmo proposto com afinamento no final obteve uma menor distância no valor de e um menor desvio padrão comparado com o método de Lassen(2013). Por fim, são apresentados os resultados da segmentação automática das fissuras pulmonares. A baixa incidência de detecções falso negativas, juntamente com a redução significativa de detecções falso positivas, resultam em taxa de acerto elevada. Conclui-se que a técnica de segmentação de fissuras pulmonares é um algoritmo útil para segmentar fissuras pulmonares em imagens de TC, e com o potencial de integrar sistemas que auxiliem o diagnóstico médico

The Swedish People’s Party have for over one hundred years functioned as a political tool for the liberals and center-right of the Swedish-speaking minority of Finland. As a party the SPP stands out in comparison to most other European minority parties as not working for regional self-governance or independence, but instead wanting to uphold constitutional language and service rights. The SPP was a part of every coalition government in Finland from 1979 to 2015 and has held multiple ministerial posts despite only having approximately 5% of the votes. The aim of this essay has been to examine the variance in the value based political issues in the SPP to see if there is a divergence on a liberal/conservative divide within the party. With a theoretical framework based on the GAL-TAN political scale, a socially based dimension of measuring party positions, I have operationalized a liberal and conservative stance to Weberian ideal types, and by first e-mail interviewing party representatives to build a frame of issues to investigate, I have then performed an idea based content analysis by examining motions from 2009 to 2017. The results show that, even though there are a divergence between some of the proposals themselves, and in some cases, between the official party line as described in the party program, the differences are too insignificant to draw conclusions from. The conflict between the motions speaking for and against nuclear power stands out as being very even, while the motions concerning Finland joining NATO stands out as being fully for.

Les cortico-surrénalomes (CCS) sont considérés comme des tumeurs malignes endocriniennes rares, associées à un pronostic sombre. Les trois mécanismes moléculaires les plus fréquemment altérés dans les CCS comprennent les mutations inactivatrices du gène suppresseur de tumeur TP53,la surexpression de IGF-II et l'activation constitutive de la voie de signalisation Wnt/β-caténine. En utilisant des modèles de souris transgéniques, nous avons montré que ces altérations, même combinées, ne sont pas suffisantes pour permettre la progression maligne.Nous avons précédemment identifié l'histone méthyltransférase EZH2 comme le modificateur d'histone le plus dérégulé dans les CCS. Nous avons également montré que sa surexpression est associée à une progression tumorale et à un mauvais pronostic. Cependant, les mécanismes sous-jacents de cette agressivité sont largement inconnus. Dans cette étude, nous avons cherché à identifier les gènes cibles de EZH2 dans les CCS, qui sont soient activés, soient réprimés. Ainsi, nous avons effectué une analyse bio-informatique des données du transcriptome de trois cohortes de patients porteurs de CCS. L’analyse montre une forte corrélation entre la surexpression de EZH2 et les gènes régulés positivement, suggérant un rôle majeur d’inducteur transcriptionnel de EZH2 dans les CCS. Nous avons montré que cette activité positive repose sur une interaction entre EZH2 et E2F1, qui entraîne la surexpression de gènes impliqués dans la régulation du cycle cellulaire et la mitose tels que RRM2,PTTG1 et PRC1/ASE1. Nous avons montré que l'inhibition de RRM2 par ARN interférent ou traitement pharmacologique avec le GW8510 inhibe la croissance cellulaire, la capacité à combler les blessures, la croissance clonogénique, la migration et induit l'apoptose des cellules H295R en culture. En revanche, l'expression du facteur pro-apoptotique NOV/CCN3 est diminuée dans les CCS, ce qui est corrélé au développement de tumeurs agressives. Nos analyses moléculaires montrent que l'inhibition de EZH2 augmente l'expression de NOV/CCN3, suggérant que la surexpression de EZH2 pourrait favoriser la progression maligne des CCS en inhibant les stimulateurs de l'apoptose. Le facteur NOV a déjà été identifié comme cible négative du récepteur nucléaire SF1 dans les cellules du CCS, bien que les mécanismes moléculaires à l'origine de cette inhibition n'aient pas été identifiés. De manière intéressante, dans le cancer de la prostate, l'expression de NOV est inhibée par le récepteur des androgènes AR, grâce au recrutement de EZH2 qui pose la marque répressive H3K27me3. Nous avons pu identifier une coopération similaire entre SF1 et EZH2 pour réprimer l'expression de NOV et bloquer ainsi l'apoptose dans les CCS.Au total, ces résultats identifient SF1 et E2F1 comme deux partenaires indépendants de EZH2, induisant la répression de facteurs pro-apoptotiques et l'activation des gènes du cycle cellulaire respectivement, conduisant ainsi à l'agressivité des CCS
Adrenocortical carcinomas (ACC) are regarded as rare endocrinemalignancies associated with dismal prognosis. The three common molecularmechanisms predominantly altered in ACC include inactivating mutations of theTP53 tumor suppressor gene, overexpression of IGF-II and constitutive activationof the Wnt/β-catenin signaling pathway. Using transgenic mouse models, wehave shown that these alterations, even when combined together, were notsufficient to induce malignant progression.We previously identified the histone methyltransferase EZH2 as the mostderegulated histone modifier in ACC. We have also shown that its overexpressionis associated with tumor progression and poor prognosis. Yet, the mechanismsunderlying this aggressiveness are largely unknown. Here, we aimed to identifyEZH2 target genes in ACC, which are either activated or repressed.Thus, we conducted a bio-informatics analysis of transcriptome data fromthree cohorts of ACC patients. The analysis showed a strong correlation betweenhighly expressed EZH2 and positively regulated genes suggesting a major role of‘transcriptional inducer‘ for EZH2 in ACC. We have shown that this positiveactivity relies on an interaction between EZH2 and E2F1 that results in theupregulation of genes implicated in cell cycle regulation and mitosis such asRRM2, PTTG1 and PRC1/ASE1. We showed that Inhibition of RRM2 by RNAinterference or pharmacological treatment with GW8510 inhibits cellular growth,wound healing, clonogenic growth, migration and induces apoptosis of H295Rcells in culture.In contrast, expression of the pro-apoptotic factor NOV/CCN3 is decreasedin ACC, which is correlated with development of aggressive tumours. Ourmolecular analyses show that EZH2 inhibition increases expression ofNOV/CCN3, suggesting that EZH2 overexpression may also favour malignantprogression in ACC by inhibition of apoptosis stimulators. NOV has previouslybeen identified as a negative target of the nuclear receptor SF1 in ACC cells,although the molecular mechanisms underlying this inhibition were unidentified.Interestingly, in prostate cancer, NOV expression is inhibited by the androgenreceptor, through recruitment of EZH2 and deposition of the H3K27me3 mark.We have been able to identify a similar cooperation between SF1 and EZH2 tosuppress NOV expression and block apoptosis in ACC.Altogether, these findings identifiy SF1 and E2F1 as two independentpartners of EZH2, inducing repression of proapoptotic factors, and activation ofcell cycle genes respectively, thus leading to aggressiveness of ACC

FrzA est une protéine secrétée (sFRP) susceptible d'interférer dans le système de signalisation Wnt/Frizzled. Cette protéine, exprimée dans le système cardiovasculaire, possède un rôle modulateur de la croissance des cellules vasculaires, inducteur sur la migration des cellules endothéliales (CE) in vitro et angiogénique sur différents modèles in vivo. Le but de ce travail a été d'étudier le rôle que pouvait jouer FrzA sur la réparation vasculaire et tissulaire après infarctus du myocarde chez la souris. Nous avons utilisé différentes lignées de souris transgéniques surexprimant FrzA soit de manière ubiquitaire (promoteur CMV) soit de manière ciblée (promoteur endothéliale Tie 2 et cardiomyocytaire α-MHC). FrzA s'avère être un facteur angiogénique augmentant la densité capillaire de la zone ischémique et modulateur des processus inflammatoires permettant ainsi de réduire la taille des infarctus, d'améliorer la fonction cardiaque et de modifier différentes étapes de cicatrisation tissulaire. Ces résultats font de FrzA et de la voie Wnt/Frizzled, un système important dans le contrôle des processus de cicatrisation post infarctus chez la souris. En parallèle, ces travaux nous ont permis d'élargir nos recherches selon 2 grands axes : d'une part sur le préconditionnement ischémique, où nous démontrons pour la première fois la directe implication de la voie Wnt/Frizzled dans ce phénomène ; et, d'autre part, sur un nouveau concept de thérapie post infarctus utilisant l'association de thérapie cellulaire avec des souches médullaires et de thérapie tissulaire avec couverture ventriculaire par patch musculaire autologue
FrzA is a secreted Frizzled Related Protein (sFRP) thought to interfere with the Wnt/Frizzled pathway. This protein is expressed in the cardiovascular system, has antiproliferative effect on vascular cells, induces endothelial cells (EC) migration in vitro and has proangiogenic effects in vivo. The goal of this study was to investigate, in a mouse model, the effect of FrzA in tissular and vascular healing process after process after myocardial infection. Our strategy was based on an overexpression of FrzA in different transgenic mice construction : non specific (CMV promotor) and specific transgenic construction (endothelial Tie 2 promotor and cardiomiocyte ptomotor α-MHC) We showed that FrzA was able to be an angiogenic factor increasing capillary density in the scar and to modulate inflammatory response reducing infarct size and improving cardiac function. These results point to the importance of FrzA and the Wnt/Frizzled pathway in the healing process after myocardial infarction. In side, we have worked on 2 different directions : firstly, in the ischemic preconditioning in which, we demonstrated for the first time, the direct implication of the Wnt/Frizzled pzthway in this phenomenon ; secondly, in a new concept using, in a post myocardial infection model, the association between cellular (with bone marrow cells) and tissular therapy covering the left ventricular by an autologous muscle patch

O interesse em estudar o cultivo das células de inseto está relacionado entre outros usos a sua utilização na produção de biopesticidas. Há muitos anos os pesticidas químicos vêm contribuindo no controle de pragas na agricultura. Entretanto, o uso desses compostos prolongadamente tem resultado na seleção de insetos resistentes e em poluição ambiental. Diante disso, torna-se necessário o desenvolvimento e aprimoramento dos bioinseticidas. No Brasil, o baculovírus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) foi o principal agente de controle biológico da praga da soja Anticarsia gemmatalis. Assim, estudos que viabilizem a produção desses vírus in vitro possibilitariam uma produção mais controlada e de melhor qualidade desses biopesticidas. Neste trabalho, investigou-se a suscetibilidade à infecção por AgMNPV de diferentes linhagens celulares de Sf21 e o crescimento dessas células em diferentes sistemas: cultivos em schotts, em spinner e em biorreator, variando-se a idade do inóculo (IA) e a concentração celular inicial (X0). Constatou-se variação no perfil de infecção das linhagens, sendo as linhagens mais adequadas para a produção de bioinseticida as linhagens de Sf21 denominadas EMBRAPA, UFRN e GibcoG, uma vez que estas apresentaram mais do que 40 % das células com poliedros em cultivos em suspensão, enquanto a linhagem denominada GibcoSF teve menos de 2 % das células infectadas com poliedros. Ao se estudar o efeito do número de subcultivos na morfologia e crescimento celular, foi averiguado um aumento no diâmetro de 10 % e no volume de 26 % das células UFRN em relação às células GibcoSF. Além disso, o crescimento das células UFRN foi 49% menor do que das células GibcoSF. Quando realizado o Delineamento Composto Central Rotacional (DCCR) para se analisar o efeito da IA e a X0 na taxa de crescimento específica máxima (?max) e na concentração celular máxima (Xvmax) em cultivos em schott com células UFRN, obteve-se um modelo empírico. Quando analisadas as variáveis IA e X0 separadamente, não foram encontradas diferenças significativas para as respostas Xvmax e ?max em relação a X0. Para a IA, entretanto, obteve-se os resultados mais satisfatórios para os inóculos com IA de 72 e 96 horas: Xvmax de 5,97.106 cel/mL e 5,99.106 cel/mL, e ?max de 0,70 dia-1 e 0,63 dia-1, respectivamente. Nos cultivos em spinner com células UFRN, foi observada a formação de grumos, o que levou a Xvmax de 2,00.106 cel/mL. No cultivo em biorreator com células UFRN, foi obtido um Xvmax de 6,21.106 cel/mL, ?max de 0,70 dia-1, Qo2 na fase exponencial de 67,3 ± 3,6 .10-18 molO2/cel/s, rendimento de glicose em célula igual a 1,0.109 cel/g de glicose e um rendimento de glutamina em células de 3,0.109 cel/mL. Comprovou-se, portanto, a existência de alterações na infecção entre diferentes linhagens de Sf21; a importância do estado fisiológico da célula nos subcultivos, a ocorrência de mudanças no crescimento celular de acordo com os sistemas de cultivo e o efeito do número de subcultivos na morfologia e crescimento de células Sf21.
Investigate the cultivation of insect cells is related to its use in the production of biopesticides among others. For many years, chemical pesticides have contributed in pest control in agriculture. However, the use of these compounds for prolonged periods has resulted in the selection of resistant insects and environmental pollution. Therefore, it is necessary the development and improvement of biopesticides. In Brazil, the baculovirus Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) is the main biological control agent of the plague of soy Anticarsia gemmatalis. Thus, studies that enhance the production of these viruses in vitro would allow a more controlled production and better quality of biopesticides. In the present research, it was investigated the susceptibility of different Sf21 cell lines to infection by AgMNPV and the growth of these cells in different systems: cultivations in schotts, in spinner flasks and in bioreactor, varying the inoculum age (IA) and initial cell concentration (X0). Variation was observed in the lineage\'s infection profile. The most appropriate lineage for the production of biopesticida where the ones denominated EMBRAPA, UFRN and GibcoG, since these showed more than 40% of the infected cells with polyhedra, while the one denominated GibcoSF had less than 2% of the infected cells with polyhedra. When studying the effect of the number of subcultures in morphology and in cell growth, an increase of 10% of the diameter and 26% in the volume of the UFRN cells was observed compared to the GibcoSF cells. Moreover, the cell growth of UFRN was 49% lower than the GibcoSF\'s. When performed the Rotational Central Composite Design (RCCD) to analyze the effect of IA and X0, the maximum specific growth rate (?max) and the maximum cell concentration (Xvmax) in cultures in schott with UFRN cells, it was obtained an empirical model. When the IA and X0 were separately analysed, it was not found significant differences for Xvmax and ?max in relation to X0. For IA, however, it was achieved the most satisfactory results for inocula with IA of 72 and 96 hours: Xvmax equals to 5.97x106 cells/mL and to 5.99x106 cells/ml, and ?max of 0.70 day-1 and 0.63 day-1, respectively. Cultures in spinner with UFRN cells clumped what led to Xvmax of 2.00x106 cells/mL. In cultivation in bioreactor with UFRN cells, was reached Xvmax of 6.21x106 cells/mL, ?max of 0.70 day-1, Qo2 in the exponential phase of 67.3 ± 3.6x10-18 molO2/cell/s, glucose to the cell yield equal to 1.0x109 cell/g of glucose and glutamine to cell yield of 3.0x109 cell/g of glutamine. It was shown, therefore, the existence of the infection alterations among different lineages of Sf21, the importance of the physiological state of the cell for the subcultivation, the occurrence of changes in cell growth according to the cultivation systems and the effect of the number of subcultivation in morphology and in growth of Sf21 cells.

El objetivo de la presente investigación es determinar la naturaleza jurídica del Sistema de Franja de Precios (SFP) establecido mediante el Decreto Supremo N° 115-2001-EF, con el que se grava la importación al Perú de determinados productos agropecuarios (arroz, maíz amarillo, leche, azúcar, y sus derivados), a fin de determinar si los mismos constituyen derechos arancelarios propiamente dichos, más bien una sobretasa adicional al arancel, cuya imposición viola los compromisos asumidos en el marco de la Organización Mundial de Comercio (OMC), determinación que reviste especial importancia a partir del proceso de Celebración de Consultas con el Estado Peruano solicitado por Guatemala ante el Órgano de Solución de Diferencias de la OMA (OSD) por gravar con el SFP la importación de azúcar, y que según afirman constituye un “gravamen variable adicional” o un “precio mínimo” cuya imposición contraviene los compromisos adquiridos ante la OMC. La referida diferencia fue resuelta a favor de Guatemala, recomendándose al Perú adecuar el SFP a las obligaciones internacionales contraídas; sin embargo, teniendo en cuenta que Guatemala sólo impugnó los derechos adicionales derivados del SFP y no el sistema en sí mismo, el pronunciamiento de la OSD no evaluó la necesidad o no de su eliminación; lo que no impide que se puedan promover nuevos procesos que ataquen directamente al SFP. En ese sentido, resulta imprescindible determinar la real naturaleza del SFP y su compatibilidad con las obligaciones asumidas por el Perú ante OMC, caso contrario, mantener un SFP en contravención con las mismas, pondría en grave peligro político y económico al País. Partimos para el presente trabajo bajo la hipótesis de la naturaleza arancelaria del SFP, condición que se ve demostrada a lo largo de su desarrollo y que concluimos es la que le corresponde, por lo que su aplicación es consistente con los compromisos asumidos ante la OMC. Cabe señalar adicionalmente, que el estudio del tema es abordado desde un análisis netamente jurídico y no económico, por lo que no se incidirá en sus aspectos económicos, tales como su impacto sobre el sector agrícola nacional, ni sobre los mecanismos adoptados para la determinación de los precios piso y techo o los precios internacionales de referencia.
Tesis

Denna rapport kommer att undersöka elanvändningen på ventilationsaggregaten i R-huset på Mälardalens högskola och även undersöka om byte av fläkt och motor i ventilationsaggregat vid eventuellt hög elanvändning är ekonomisk lönsamt. R-husets byggår är 1993 och har de mest uråldriga ventilationsaggregaten av skolans fastigheter. Ett ventilationsaggregats livslängd kan uppskattas mellan 20-30 år. Enligt undersökningar finns det stora möjligheter till energieffektiviseringsåtgärder i sektorn bostäder och service där ventilationsaggregat med föråldrad teknik och komponenter kan vara miljöbovar med hög elanvändning. En avgränsning i arbetet är att ventilationsaggregatet med högst specifik fläkt effekt (SFP) ska undersökas noggrant i form av energieffektivisering. Energieffektiviseringen kommer att begränsas till luftbehandlingsaggregatets fläktar och motorer. Kartläggning genomfördes med tryckmätningar på ventilationsaggregaten i R-huset och även el mätningar. El mätningarna behövdes för att beräkna den aktuella elanvändningen för fläktarna. Resultatet av den beräknade elanvändningen för vardera fläkt visade sig vara hög. Tryck mätningarna utfördes för att få fram tryckuppsättningen för fläktarna i aggregaten och vid beräkning av till- och frånluftsflöde. Tryckuppsättningen och flöde över fläktar behövdes vid val av ny fläkt och motor. Resultatet av kartläggningen visar att det totala SFP-värdet som står för den specifika fläkteffekten är höga i jämförelse vid nyproduktion där boverkets byggregler har krav på SFP-värde under 2,0 kW/(m3,s). Högsta SFP-värdet ligger på 4,4 kW/(m3/s) och en livscykelkostnad (LCC) utfördes för detta ventilationsaggregat. LCC-kalkylen baseras på en kalkylperiod på 10 år då på befintliga fläktar och motorer bedöms ha den livslängden. Kalkylräntan är satt till 9 %, energiprisökningen 5 % och detta ger en real kalkylränta på 4 %. Elpriset är valt till det aktuella på 0,80 kr/kWh. LCC-kalkylen visar att det inte är lönsamt att byta till nya fläktar och motorer. Payback metoden gav 12 års återbetalningstid vilket är länge i jämförelse med studerad litteraturstudie. En känslighetsanalys utfördes med ett fördubblat elpris och då blev det ekonomiskt lönsamt att byta befintliga fläktar och motorer till nya.
This report will examine the electricity consumption of the ventilation units in the R building at Mälardalen University and also investigate whether replacing the fan and motor in the ventilation unit with new combined fan and engine units in the event of high electricity consumption is economic profitable. The building was built in 1993 and has the oldest ventilation units of all properties at the university. A ventilation unit’s life can be estimated to last for 20-30 years. According to studies, there are major opportunities for energy efficiency measures in the residential and service sector, where ventilation systems with outdated technology and components can be energy wasters with high electricity consumption. A limitation of this work is that the ventilation unit with the highest Specific Fan Power (SFP) should be examined carefully in terms of energy efficiency. Energy efficiency will be limited to the air handling fans and motors. Mapping was conducted with pressure measurements in the ventilation units and also electrical measurements were done. Electricity measurements are needed to calculate the current electricity consumption of the fans. The result of the estimated power consumption of each fan was found to be high. Pressure measurements were performed to obtain the pressure of the fans in the units and to calculate supply and exhaust air flow. The pressure rise and flow of the fans are needed in order to select a new fan and motor. The result of the survey shows that the total SFP value is high in comparison with the new construction where Boverkets regulations requirements for SFP value is below 2.0. The highest SFP-value is 4.4 kW / (m3 / s) and a Life Cycle Cost (LCC) calculation was performed for that ventilation unit. The LCC calculation is based on a calculation time period of 10 years, where the existing fans and motors are predicted to last for 10 years. The discount rate is set at 9%, energy with a price increase of 5% and this gives a total discount rate of 4%. Electricity prices are up to date and are 0.80 SEK / kWh. LCC calculation shows that it is not profitable to switch to new fans and motors and as payback method gave a 12-year repayment period. A sensitivity analysis was performed with a redoubled electricity price and it then became economically viable to replace existing fans and motors with new ones.

I dagens samhälle ligger stort fokus på energianvändningen för bostäder och kommersiella fastigheter. Energianvändningen beskriver inte bara en byggnads energibehov, utan också miljö- och ekonomiska aspekter. Flerbostadshus är en del av Sveriges bostad- och servicesektor, som utgör 40 % av Sveriges totala energianvändning (Henning, 2017). Ventilationssystem i flerbostadshus utgör således en betydande del av fastighetens energianvändning och måste därför ständigt effektiviseras för att tjäna ett hållbart samhälle. I nuläget är den verkliga energianvändningen för ett flertal av JM:s driftsatta FTX-aggregat (till- och frånluftssystem med värmeåtervinning) okänd. Detta på grund av att tillgängliga energiberäkningar och deklarationer redovisar byggnadens totala energianvändning uppdelat i fyra huvudkategorier; uppvärmning, komfortkyla, tappvatten och byggnadens fastighetsenergi. Energianvändningen för ventilationssystemet framgår inte specifikt från dessa beräkningar, utan delas in i kategorierna uppvärmning och byggnadens fastighetsenergi. Rapporten undersöker, under perioden mars – juni 2018, energianvändningen för totalt elva stycken FTX-aggregat i två av JM:s projekt i Stockholm, Kista Torn och Nya Kvarnen 2. Undersökningens syfte är att försöka beräkna den verkliga energianvändningen för dessa FTX-aggregat. Energiberäkningar har genomförts med värden hämtade från egna samt tidigare utförda mätningar, som jämförts med projekterade värden. Resultatet indikerar att den genomsnittliga energianvändningen för FTX-aggregaten i Kista Torn är ca 120 000 kWh/år. Den större delen av energianvändningen går åt till att värma tilluften. Undersökningen visar på att den genomsnittliga temperaturverkningsgraden är ca 7 % lägre än den projekterade verkningsgraden. De beräkningar som genomförts visar även att uppvärmningskostnaden har ökat med ca 150 000 kr/år, i jämförelse med de projekterade värdena. Det beror till stor del på den minskade temperaturverkningsgraden, men också på grund av förändrade luftflöden i aggregaten. Undersökningen i Nya Kvarnen 2 har inte kunnat göras lika omfattande. Beräkningarna som genomförts här tyder på att energianvändningen uppgår till ca 61 000 kWh/år och aggregat. Tillförlitligheten av beräkningarna störs dock av att inga egna mätningar kunde utföras, att dokumentationen var bristfällig, samt att övervakningssystemet SCADA redovisade orimliga värden. Slutsatsen för rapporten indikerar att den verkliga energianvändningen för FTX-aggregaten generellt är högre än vad som tidigare projekterats. Energianvändningen varierar markant beroende på luftflöden och temperaturverkningsgrad. Störst påverkan på energianvändningen har dock temperaturverkningsgraden. Det visade sig att temperaturverkningsgraden vara svår att bestämma, då många felkällor och faktorer påverkar framtagningen, samt att resultatet varierar beroende på vald mätmetod. För bättre kontroll på energianvändning rekommenderas fler och mer kontinuerligt genomförda mätningar. Alternativt skulle övervakningsprogrammet SCADA kunna användas i större utsträckning för beräkning av energianvändning, förutsatt att precisionen och placering av temperaturgivare förbättras.
In today's society the energy use within residential and commercial real estate is of great importance. Energy use does not only describe a building's energy needs, but also its environmental and economic aspects. Apartment buildings are a part the Swedish residential and service sector, which constitutes 40 % of Sweden's total energy use (Henning, 2017). The ventilation system in apartment buildings makes up a significant part of the property's energy use, it must therefore continuously be improved to work towards a more sustainable society. The actual energy use for several of JM's powered air assemblies is unknown today. Energy calculations and declarations show that the building's total energy use is divided into four main categories; heating, comfort cooling, tap water and property energy. The energy use of the ventilation system is not made apparent in these aforementioned calculations since it is distrubuted into two of the main categories, heating and property energy. The report examines the energy use for a total of eleven air assemblies within two of JM's projects in Stockholm, Kista Torn and Nya Kvarnen 2. The purpose of the study is to find the actual energy use of these air assemblies. Energy calculations will be carried out using values ​​taken from our own and previously performed measurements, which will be compared to expected projected data. The result indicate that the average energy use of the air assemblies in Kista Tower is approximately 120 000 kWh/year. The majority of the energy use goes to heating the supply air. The study shows that the average heat recovery efficiency is circa 7 % lower than the projected efficiency. The calculations show that the cost of heating has increased by approximately 150 000 SEK/year compared to projected data. This is largely due to the reduced heat recovery efficiency but partly due to changes in the airflow within the air assemblies. The study made in Nya Kvarnen 2 was not as extensive, although calculations of energy use have been carried out. These calculations show an energy use of approximately 61 000 kWh/year for each separate air assembly. The reliability of these numbers is considered low because no control measurements could be made. The documentation was inadequate along with the monitoring system, SCADA, which reported unrealistic values. The conclusion of the report shows that the actual energy use of the air assemblies is in general higher than expected. The energy use varies depending on the airflow and heat recovery efficiency, however the greatest impact comes from the heat recovery efficiency in the air assemblies. Heat recovery efficiency was proven difficult to determine because of the multiple sources and factors affecting the measurement. This made the results fluctuate greatly depending on the method chosen of calculating the heat recovery efficiency. To oversee the actual energy use in these air assemblies, more frequent measurements are recommended. Alternatively, the SCADA monitoring program could be used to calculate the energy use, but to do so the accuracy and placement of temperature sensors needs to be improved.

Introdução: Uma elevada incidência de tumores adrenocorticais pediátricos e de adultos é observada nas regiões sul e sudeste do Brasil. Hiperexpressão dos genes IGF1R, SF1 e FGFR4 tem sido descrita em tumores adrenocorticais. Apesar de hiperexpressão ser um evento comum em diversas neoplasias, ainda não são claros os mecanismos moleculares que seriam responsáveis por essa falha na regulação da expressão. Objetivos: Determinar o número de cópias dos genes IGF1R, SF1 e FGFR4 em tumores adrenocorticais diagnosticados em crianças e adultos. Adicionalmente correlacionaremos os dados de expressão gênica e/ou protéica de IGF1R, SF1 e FGFR4 com o diagnóstico histológico e evolutivo dos tumores adrenocorticais. Pacientes e métodos: Sessenta e quatro pacientes com tumores adrenocorticais foram selecionados para o estudo. Todos os pacientes foram submetidos à avaliação clínica e tratamento cirúrgico. Oito glândulas adrenais normais obtidas em cirurgias renais ou autópsias foram utilizadas como controles. DNA genômico extraído dos tecidos normais e tumorais da glândula suprarrenal foram utilizados como substrato nas reações de multiplex ligation-dependent probe amplification (MLPA) com o intuito de se determinar o número de cópias dos genes IGF1R, SF1 e FGFR4. PCR em tempo real (SYBR Green) foi realizado para confirmar os dados de MLPA para os genes IGF1R e SF1. Resultados: Amplificação do gene IGF1R foi detectada por MLPA e confirmada por PCR em tempo real SYBR Green em apenas um carcinoma adrenocortical. Adicionalmente, amplificação gênica de outros loci (IGFBP3, FGFR4 e NSD1) bem como de sondas controles foi observada, sugerindo uma condição aneuplóide neste tumor maligno. Amplificação de SF1 foi detectada em 10 tumores adrenocorticais (8 pediátricos e 2 de adultos). Os valores de expressão gênica foram significantemente maiores em tumores associados com amplificação gênica quando comparados com tumores sem amplificação. Além disso, imunorreatividade para SF-1 foi detectada nos tumores com aumento no número de cópias. Doze amplificações do locus FGFR4 (3 pediátricos e 9 de adultos) foram demonstradas por MLPA. A amplificação do locus FGFR4 e hiperexpressão deste gene foram significantemente mais relacionados a carcinomas. Conclusões: Amplificação do gene IGF1R é um evento raro nos tumores adrenocorticais pediátricos e de adultos. A hiperexpressão de IGF1R em tumores adrenocorticais pediátricos não foi secundária à amplificação gênica. Amplificação do gene SF1 foi evidenciada predominantemente em tumores adrenocorticais pediátricos e se correlacionou com hiperexpressão gênica e protéica. Amplificação do locus FGFR4 foi demonstrada predominantemente em tumores adrenocorticais malignos de adultos. Amplificação de oncogenes representa um mecanismo molecular relevante na tumorigênese adrenocortical
Introduction: A high incidence of adrenocortical tumors in children and adults has been observed in Southern and Southeastern regions of Brazil. Overexpression of IGF1R, SF1 and FGFR4 genes have been described in adrenocortical tumors. Despite of overexpression be a common event in several neoplasias, the molecular mechanism implicated in this upregulation remains unknown. Objectives: To determine the copy number of IGF1R, SF1 and FGFR4 genes in pediatric and adult adrenocortical tumors. Additionally, correlate with IGF1R, SF1 and FGFR4 gene and/or protein expression data as well as with the histological diagnosis and evolution of the adrenocortical tumors. Patients and methods: Sixty and four patients with adrenocortical tumors were selected for this study. All patients were submitted to clinical evaluation and surgical treatment. Eight normal adrenal glands obtained in renal surgery or autopsies were used as controls. The MLPA reactions were performed with the DNA extracted from adrenal gland tissues in order to determine the copy number of IGF1R, SF1 and FGFR4 genes. SYBR Green real-time PCR was carried out to confirm MLPA data for IGF1R and SF1 genes. Results: IGF1R amplification was detected by MLPA and confirmed by SYBR green real-time PCR in only one adrenocortical carcinoma. Additionally, other loci amplification was detected (IGFBP3, FGFR4 and NSD1) as well as for control probes, suggesting aneuploidy in this malignant tumor. SF1 amplifications were shown in 10 adrenocortical tumors (8 from children and 2 from adults). The SF1 mRNA levels were significantly higher in adrenocortical tumors associated with increased SF1 gene copies when compared with adrenocortical tumors without gene amplification. Moreover, all adrenocortical tumors with SF1 gene amplification showed a strong SF1 staining. Twelve FGFR4 locus amplifications (3 from children and 9 from adults) were demonstrated by MLPA. FGFR4 locus amplification and overexpression of this gene were significantly more related to carcinomas. Conclusions: IGF1R amplification is a rare event in adrenocortical tumors and it was not responsible for the IGF1R overexpression of pediatric and adult adrenocortical tumors. SF1 gene amplification was detected predominantly in pediatric adrenocortical tumors and was associated with gene and protein overexpression. FGFR4 locus amplification was demonstrated mainly in adult maligant adrenocortical tumors. FGFR4 amplification and upregulation were more associated to adrenocortical carcinomas. Oncogenes amplification represents an important molecular mechanism in adrenocortical tumorigenesis

Humans are continuously confronted by the threat of fungal infection. Estimates put the number of individuals infected by superficial fungal disease at about 1.7 billion. Additionally there are a significant proportion of invasive infections which are difficult to treat and lead to an estimated 1.5 million deaths each year. Aspergillus fumigatus is an opportunistic, fungal pathogen which can lead to a variety of disease manifestations, generally termed aspergillosis and account for more than 200,000 life threatening infections annually with mortality rates of up to 95%. The occurrence of fungal disease has increased significantly due to the expansion of the immune deficient population, particularly those who receive immunosuppressive therapies. This combined with the problems surrounding current therapeutic options for fungal disease such as a rise in the incidence of antifungal resistance, adverse side effects in patients and drug-drug interactions has led to an urgent need to discover and develop an innovative class of antifungal agents. This thesis will address this requirement by validating a potential drug target, PptA, for combating A. fumigatus infection and explore methods to identify novel antifungal target identification. PptA is a Sfp-type 4′-Phosphopantetheinyl transferase required for transfer and covalent tethering of 4’-phosphopantetheine from coenzyme A to a conserved serine residue within a peptidyl carrier domain of a protein substrate. This project outlines the many critical roles PptA plays within A. fumigatus, such as; involvement in the production of many virulence factors as well as the biosynthesis of the essential amino acid, lysine. Furthermore, it is demonstrated that PptA is vital for secondary metabolite production in A. fumigatus, growth in iron limiting conditions and virulence. Finally, the design of a high-throughput screening assay capable of identifying inhibitors of PptA enzymatic activity further demonstrate the suitability of this target. It is the combined effect of all these characteristics that makes PptA such a suitable candidate for an antifungal target. Additionally two validation methods are included which can be used to identify novel drug targets. The first method utilises chemically induced haploinsufficiency profiling, a technique which has been proven successful for drug target identification and determining mode of action of drugs in S. cerevisiae and C. albicans. The project has shown this technology can be successfully used in A. fumigatus. Furthermore, a new technique for in vitro parallel fitness screening using next generation sequencing was validated with a library of phosphatase deletion mutants. Bioinformatic analysis against strains exhibiting severe fitness defects allowed the identification of three phosphatases that have the potential of becoming successful antifungal drug targets.

Dans ce travail, nous avons proposé deux algorithmes qui nous ont permis de régler complètement le problème de l'évaluation et de l'optimisation des performances. Pour cela nous avons fourni une méthode hiérarchique pour diminuer le temps d'analyse et obtenir les meilleures solutions. Nous avons découpé les sous-réseaux (ils s'appellent BUC) en utilisant la matrice d'incidence, fondée sur les ressources partagées des machines, et analysé BUC par le dépliage du réseau de Petri. Cette méthode d'analyse résout le problème de l'explosion combinatoire qui est apparu lors de l'analyse du réseau de Petri. Nous avons appliqué cette méthode à l'analyse de problème d'ordonnancement cyclique en SFPM. Finalement nous pouvons dire que cette approche est facile à comprendre, elle permet de calculer facilement les solutions.

Das duktale Adenokarzinom des Pankreas (PDAC) hat aufgrund seines aggressiven Wachstums, seiner frühen Metastasierung und seiner fehlenden Frühsymptome eine sehr schlechte Prognose und ist daher die vierthäufigste Krebstodesursache bei Männern und Frauen in Deutschland. Ein weiteres Charakteristikum von PDAC ist die starke desmoplas-tische Reaktion. Eine Funktion für stromale pankreatische Sternzellen (PSCs) bei der Förderung des Wachstums, der Proliferation und der Metastasierung des Tumors konnte bereits nachgewiesen werden. Für die Beantwortung der Frage, welche molekularen Ursachen einen funktionellen Zusammenhang zwischen Tumoraggressivität und Stroma-zellen herstellen könnten, wurden bereits im Vorfeld dieser Arbeit differentielle Genex-pressionsanalysen durchgeführt. Dabei fiel u.a. auf, dass das sezernierte Glykoprotein secreted Frizzled-related protein 1 (SFRP1), im Vergleich zum entsprechenden nicht-tumorigenen Pankreasgewebe, im Stroma von PDAC-Patienten transkriptionell herunterreguliert war. SFRP1 ist bereits bekannt als Tumorsuppressorgen. In den meisten Fällen wird seine Wirkung auf die Hemmung des β-Catenin-abhängigen (kanonischen) Wnt-Signalweges zurückgeführt. Es wurde bereits sehr häufig beobachtet, dass SFRP1 durch eine Hypermethylierung seiner Promotorregion in einer Vielzahl von humanen Tumoren, darunter auch PDAC sowie PDAC-Vorstufen, herunterreguliert ist. Mit dem Abschalten der SFRP1-Expression wurden erhöhte Proliferation sowie verringerte Apoptose bei den betroffenen Zellen, Merkmale des aktivierten kanonischen Wnt-Signalweges, festgestellt. SFRP1 ist jedoch auch in der Lage nichtkanonische Wnt-Signalwege zu modulieren. Das Ziel dieser von der Deutschen Gesellschaft für Forschung geförderten Arbeit war es, die stromale SFRP1-Expression auf Proteinebene in Proben von PDAC-Patienten mit der von Patienten mit chronischer Pankreatitis zu vergleichen bzw. mit deren Überleben zu korrelieren. Ein möglicher Unterschied sollte mithilfe eines Zellkultur-modells auf einen funktionellen Effekt hin untersucht werden. Dafür sollten stabil und regulierbar SFRP1-überexprimierende Zelllinien aus PDAC- bzw. PSC-Zellen für die Einbindung in Migrationsassays etabliert werden. Für die Ergründung der Mechanismen, die zur Herunterregulierung der stromalen SFRP1-Expression führen könnten, sollte der Methylierungstatus der SFRP1-Promotorregion in PSC- sowie vergleichsweise in PDAC-Zellen mittels methylierungsspezifischer PCR und Bisulfitsequenzierung analysiert werden. Desweiteren sollten diese Zellen mit Hilfe eines Reportergenassays auf eine Mikro-RNA-bedingte Modulation der SFRP1-Expression hin untersucht werden. Die immunhistochemische SFRP1-Färbung von PDAC-Patientenproben auf Tissue-Microarrays (TMAs) ergab eine signifikante Reduktion der stromalen SFRP1-Färbung im Vergleich zur entsprechenden Expression im Stroma von Patienten mit chronischer Pankreatitis (CP). Die Ergebnisse der differentiellen Genexpressionsanalyse konnten also auf Proteinexpressionsebene bestätigt werden. Bei der Korrelation der stromalen SFRP1-Färbung mit dem Überleben der entsprechenden Patienten mit R1-Resektionsstatus zeigte sich ein leichter Überlebensvorteil für die Patienten mit positiver SFRP1-Färbung. Bei der Analyse der SFRP1-Expression auf RNA- und Proteinebene in Zellkulturmodellen von PDAC zeigte sich, dass zwei von vier PDAC-Zelllinien sowie die PSCs und normale Pankreasgangzellen SFRP1-RNA exprimierten. Auch bei anderen untersuchten Tumor- oder murinen PDAC-Zelllinien war das Verhältnis zwischen Linien mit SFRP1-RNA und Linien ohne SFRP1-RNA eher gemischt. Keine dieser Zelllinien jedoch exprimierte SFRP1-Protein bis auf eine murine Fibroblastenzelllinie (3T3). Um zu ergründen, wodurch die Diskrepanz zwischen SFRP1-RNA- und fehlender -Proteinexpression zustande kam, wurden Methylierungsanalysen durchgeführt. Dabei ergaben sich individuelle Methylierungsmuster für die verschiedenen DNAs, die eine fehlende Proteinexpression bei nur einem Teil der Zelllinien erklären würden. Daher wurde eine weitere Möglichkeit der Genexpressionsregulation in eukaryontischen Zellen als Ursache in Betracht gezogen, die Hemmung der Translation von SFRP1-mRNA in Protein durch miRNAs. Hierbei konnte mittels Reportergenassays nachgewiesen werden, dass miRNAs in PSCs und PDAC-Zellen eine kleine Stelle in der 3’-untranslatierten Region sowie Stellen in der kodierenden Sequenz von SFRP1 erkennen, daran binden und translational herunterregulieren konnten. Da keine endogene Proteinexpression festgestellt werden konnte, wurden drei PDAC-Zelllinien (PANC-1, AsPC1 und MIA PaCa-2) sowie eine PSC-Zelllinie (Klon2.2) für die stabile Transfektion mit humanem SFRP1 auf einem regulierbaren Vektor ausgewählt. Mithilfe der Lipofektion gelang es jedoch nur bei MIA PaCa-2 und Klon2.2-Zellen, stabile Einzelzellklone anzuziehen, jedoch mit relativ variablen SFRP1-Expressionen und -Regulierbarkeiten. Um noch mehr PDAC-Zelllinien in funktionellen Tests untersuchen zu können, wurden alle vier Zelllinien noch einmal mit einer weiteren Gentransfermethode, der retroviralen Transduktion, in stabil SFRP1-exprimierende Zelllinien umgewandelt. Um zu untersuchen, inwiefern das in den Patienten verlorengegangene SFRP1 das migratorische Verhalten von PDAC- und PSC-Zellen beeinflussen könnte, wurden Scratch- und Invasions- (Migrations) -Assays mit diesen Zellen durchgeführt und mit den Ergebnissen der entsprechenden Leerkontrollen verglichen. Dabei ergab sich für MIA PaCa-2 sowohl mit den durch Lipofektion als auch mit den durch retrovirale Transduktion generierten Klonen/Zelllinien ein signifikant hemmender Einfluss der SFRP1-Überexpression auf das Migrationsverhalten im Scratch-Assay. PANC-1-Zellen schienen mit Lipofektions-SFRP1-Überständen signifikant schlechter durch Membranen zu migrieren und zeigten ebenfalls eine signifikante Migrationshemmung als retroviral transduzierte Zelllinie in Scatch- und Invasionsassay mit endogener SFRP1-Expression. Für PSCs zeigte sich eine SFRP1-abhängige Migrationshemmung im Scratch-Assay und in den Invasionsassays (mit endogener SFRP1-Überexpression und mit SFRP1-Anwesenheit im Überstand), allerdings nur mit Lipofektionsklonen. Es konnte also ein hemmender Einfluss von SFRP1 auf die Migration von PDAC- und PSC-Zellen nachgewiesen werden. Dieser würde theoretisch wegfallen, wenn SFRP1, wie im Tumorstroma von PDAC-Patienten nachgewiesen, herunterreguliert werden würde. Bei der Untersuchung eines Einflusses von SFRP1 auf die Expression weiterer Gene ergab sich, dass SFRP1 bei PDAC-Zellen keine Hemmung des kanonischen Wnt-Signalweges verursacht. Vielmehr scheint seine Überexpression einen positiven Effekt auf die Expression von Mitgliedern des nichtkanonischen planaren Zellpolaritätssignalweges (PCP) zu haben. Die Schlussfolgerung aus diesen Ergebnissen ist, dass ein Wegfall von SFRP1 im Stroma von PDAC in sehr frühen Phasen (und auch in den Tumorzellen selbst) durch miRNAs oder, im Falle der Tumorzellen, durch Hypermethylierung ausgelöst werden könnte. Sezernierte, tumorsupprimierende, parakrine Signale aus dem Stroma und autokrine Signale von den Tumorzellen selbst würden damit wegfallen und die planare Zellpolarität wäre nicht mehr aufrechterhaltbar. Damit würden sich die Polarität und die Migrationsbereitschaft der Tumorzellen so verändern, dass sie ungerichtet wandern könnten, wodurch das Risiko zur Metastasierung zunehmen könnte. Auf diese Weise könnte die stromale Herunterregulierung von SFRP1 bei der Aufrechterhaltung und Progression des PDAC eine Rolle spielen.

Mestrado em Engenharia Eletrotécnica e Telecomunicações
Due to the increased need for more bandwidth, access networks are evolving constantly. At the moment, GPON networks are the main choice in Europe, but due to the increased traffic in recent years, developing a new option for access networks defined NGPON2 as one of the main focuses in telecommunication research. In this document the current PON networks and the possible NGPON2 candidates will be studied. The main focus will be the possibility of designing a WDM-PON network while maintaining the main GPON architecture already implemented. By using SFPs, an example will be shown where a normal GPON transmission will be converted to another wavelength in order to show a possible implementation of a WDM network.
Com o aumento da necessidade de largura de banda nos últimos anos as redes de acesso encontram-se em constante evolução. Sendo que as redes GPON são de momento a principal escolha num âmbito europeu, devido ao aumento de tráfego nos últimos anos, a necessidade de uma nova tecnologia para redes de acesso NGPON2 é um dos principais focos de investigação em telecomunicações. Neste documento irá ser realizado um estudo das actuais redes PON e as possíveis opções para uma rede NGPON2. Maior ênfase será dado à possibilidade de realizar uma rede WDM-PON utilizando como base a arquitectura GPON já implementada. . Utilizando SFPs irá ser demonstrado como é possível converter uma transmissão GPON para outros comprimentos de onda com o objectivo de criar uma rede WDM.

Energieffektiviseringar behövs för att klara klimatmålen. Syftet med detta arbete har därför varit att undersöka ventilationen i förskolebyggnaden Rymden i Eskilstuna kommun. Fokus i arbetet har legat på att göra en analys på energibesparingar där variabelt luftflöde, VAV-system och konstant luftflöde, CAV-system jämförs i tre olika scenarier. Metodiken för att lösa arbetets frågeställningar har varit genom informationsinsamling, beräkningsprogram, intervju samt platsbesök. Informationsinsamling har varit i form av vetenskapliga tidskrifter samt arbeten som behandlar det aktuella problemområdet. Olika programvaror har använts, dessa är MagiCAD, IDA ICE, Sektionsdata 4.21 samt Microsoft Excel. För ökad förståelse för den aktuella byggnaden har platsbesök gjorts på förskolebyggnaden samt en intervju med den projektöransvarige för ventilationen i förskolan. Resultatet visar att den mest energieffektiva systemlösningen är en roterande värmeväxlare med VAV-styrning i kombination med en korsströmsvärmeväxlare för köksavdelningen. VAV-styrning med en sådan systemlösning har en total årlig energianvändning på 20 684 kWh, medan CAV-systemet med samma systemlösning använder 30 900 kWh. Ekonomisk analys visar däremot att CAV-systemet med samma systemlösning är mest lönsam. Den totala livscykelkostnaden, LCC ligger på 2 386 857 kr för CAV-systemet vid en kalkyltid på 30 år, i jämförelse med 2 420 117 kr för VAV-systemet. För att VAV-systemet skall vara lönsam, visar känslighetsanalysen att kalkylräntan måste sjunka från 5 % till 2,56 % eller energiprisutvecklingen öka med 2,44 % årligen eller en sänkning av den årliga underhållskostnaden för VAV-styrning med 2 164 kr. Övrig känslighetsanalys visar att vid nederbörd då personer stannar inomhus är det fördel för VAV-systemet, då skillnaden i total LCC-kostnad sjunker med 1 758 kr. Vid 74 % av personnärvaro minskar LCC-skillnaden mellan systemen från 39 240 kr till 26 371 kr, alltså utgör detta även en fördel för VAV-systemet. Slutsatsen som dras för förskolebyggnaden Framtiden är att större energibesparingar kan göras med ett VAV-system, men ett CAV-system är bättre ur en ekonomisk synpunkt. Känslighetsanalysen visar dock att små förändringar behövs för att VAV-systemet skall bli ekonomiskt lönsamt. Andra faktorer som påverkar valet mellan VAV- och CAV-system är exempelvis en minskning av personnärvaron relativt till det dimensionerade. En sådan minskning utgör en fördel för VAV-systemet. Detta på grund av att med ett CAV-system överventileras byggnaden. Även högre specifik fläkteffekt har en stor betydelse i valet, då mer energiåtgång till fläktarna leder till större energibesparing för VAV-system. En högre temperaturverkningsgrad för luftbehandlingsaggregat medför däremot en fördel för CAV-system.
This thesis work has been done in collaboration with Sweco Systems in Eskilstuna, Sweden. The purpose with this work is to investigate the ventilation in a preschool in Eskilstuna. The focus of the work is to analyze the potential energy savings of using a variable air volume system instead of a constant air volume system. An analysis is also made to investigate the economical profitability of three different scenarios. The method used to solve the problem formulation has been through gathering information, in form of scientific journals in the current problem area. Information has also been gathered through an interview and a site visit. Various software has been used in this thesis work for calculations, such as MagiCAD, IDA ICE, Sektionsdata 4.21 and Microsoft Excel. The result shows that the most energy efficient solution is a rotary heat exchanger with variable air volume control in combination with a cross-flow heat exchanger for the kitchen section. However, the most profitable solution from an economical point of view is the same system, but with constant air volume control. Sensitivity analysis shows that for a profitable variable air volume system, either the interest rate has to be lowered from 5 % to 2,56 %, the energy price needs to increase by 2,44 % yearly. Or maintenance cost for the variable air volume system needs to be lowered by 2 164 kr. The conclusion of this thesis work is that energy savings can be done with a variable air volume system. However from an economical point of view, the constant air volume is better than variable air volume for the investigated preschool. The sensitivity analysis shows that small changes are needed to make a variable air volume system profitable. The choice between the systems are influenced by certain factors. A decrease of the attendance than the dimensioned are an advantage for variable air volume systems. This is due to the fact that a constant air volume system ventilates more than needed. A higher specific fan power number are also of great importance in the choice, as more energy consumption by the fans leads to greater energy savings for variable air volume systems.

Orientador: Ana Paula de Souza Pardo
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-27T07:52:53Z (GMT). No. of bitstreams: 1 Portinho_Danielle_D.pdf: 3218538 bytes, checksum: 09966eb0179669b7c0b2ceee93e7f695 (MD5) Previous issue date: 2015
Resumo: A associação entre inflamação e câncer vem sendo há tempo estudada, havendo atualmente fortes evidências que mostram o microambiente inflamatório como fator de risco para tumorigênese. Dentre os tipos de câncer mais frequentes, o câncer de boca ocupa a sexta colocação como maior causa de óbitos em todo o mundo. Os fatores que levam ao aparecimento do câncer oral são variados, entretanto existe dependência de um acúmulo de mutações as quais levam a alterações genéticas e epigenéticas das células afetadas, sendo que estas alterações influenciam diretamente no prognóstico da doença. Nos últimos anos, o estudo das alterações epigenéticas relacionadas aos diversos tipos de câncers vem se destacando, havendo marcadores epigenéticos já descritos para diferentes tipos de tumores. Dentre as alterações epigenéticas mais estudadas está a metilação do DNA que acontecem normalmente em regiões com grandes concentrações de citocina que precedem guanina (CpG). Estas regiões ricas em dinucleotídeos CpGs são denominadas ilhas CG. Ilhas CG são frequentes em regiões promotoras dos genes, sendo a metilação um mecanismo de controle do nível transcricional. Ilhas CGs metiladas na região promotora são esperadas em genes pouco expressos, podendo haver até mesmo o completo silenciamento da expressão do gene. No caso de genes supressores de tumor, pode provocar o aparecimento e o descontrole no crescimento tumoral. Assim, o objetivo deste estudo foi analisar o padrão de metilação do DNA em sequências LINE1 e nos genes supressores tumorais: SFRP1, SFRP2 e TP73, em amostras de gengiva coletadas de pacientes, pacientes com periodontite crônica e pacientes diagnosticados com carcioma espinocelular (CEC). A metilação do DNA foi analisada utilizando-se a metodologia COmbined Bisulfite Restriction Analysis (COBRA) em tecidos homogeneizados e microdissecados. A análise de expressão gênica dos genes foi realizada pela técnica qPCR amostras de tecido gengival e CEC. Os resultados obtidos pelas análises COBRA evidenciaram um desequilíbrio no estado de metilação todo DNA anto nos tecidos inflamados quanto nos tumorais. Entretanto, as regiões CpG analisadas não demonstraram estar diretamente relacionadas com o nível de expressão gênica. Adicionalmente, os resultados das análises da metilação global (LINE1) do tecido homogeneizado em comparação com as amostras microdissecadas indicam que os dados obtidos podem ser contraditórios aos reais podendo interferir no prognóstico do paciente. Esta contradição possivelmente se deve a contaminação do tecido tumoral por tecidos adjacentes a ele. As análises estatísticas dos resultados foram realizadas pelos testes Kruskall-Wallis e correlação de Pearson ao nível de significância de 5%. Alterações no padrão de metilação do DNA dos genes estudados são encontradas tanto em tecidos inflamados quanto em amostras de câncer bucal. A metodologia de escolha para análise destes tecidos pode influenciar nos resultados obtidos, gerando falsos negativos e consequentemente comprometendo o tratamento da doença
Abstract: The association between inflammation and cancer has always been sought by researchers. Nowadays, there are evidences of the role of the inflammatory microenvironment in tumorigenesis. Among the most common types of cancer, oral cancer is the sixth type that causes more deaths worldwide. The factors that lead to the development of an oral cancer are varied, depending on an accumulation of mutations that lead to genetic and epigenetic alterations of the affected cells, and these changes directly influence the prognosis of the disease. In recent years epigenetic have been widely studied in this field of researchs, and several genes have been described as markers for different types of tumors. Among the most studied epigenetic changes there is the DNA methylation in areas with large concentrations of the cytokine that precedes guanine (CpG), these regions are called CpG islands. GpC islands are fairly common in the promoter regions of the genes, being the methylation a control mechanism of gene level transcripts and it can even silence the expression of a gene. In the case of tumor suppressor genes this silencing can lead to uncontrolled tumor growth. The aim of this study was to analyse the DNA methylation status of LINE1 sequences and specific genes (SFRP1, SFRP2 e TP73) from human tissue collected from gingival normal tissue from patients underwent aesthetic surgery, patients with cronic periodontitis and patients with oral squamous cell carcinoma (OSCC). The extracted DNA was analysed by Combined Bisulfite Restriction Analysis (COBRA) submitting the DNA to bisulfite conversion by MethylSEQrBisulfiteConversion Kit from homogenized and microdissected tissues. Through qPCR technique, samples from gingival tissue and OSCC were subjected to RNA expression analysis of candidate genes. The results of COBRA analysis of genes exhibited an imbalance in methylation state of inflamed and tumor tissues. However, the CpG regions analyzed have not shown to be directly related to the level of expression of these genes. The results of the analysis of the global methylation (LINE1) of the homogenized tissue compared with microdissected samples showed that the obtained data may be contradictory to the real one and may interfere in the prognosis of the patient. This inconsistency is probably due to contamination of tumor tissue by adjacent tissues. Statistical analysis of results was performed by Kruskal-Wallis test and Pearson correlation at the 5% confidence level. Changes in DNA methylation pattern of the genes studied are found in both inflamed tissues as oral cancer samples. The choice of methodology for the analysis of these tissues can influence results, and consequently generating false negative results affecting the treatment of disease
Doutorado
Histologia e Embriologia
Doutora em Biologia Buco-Dental

Les miARN sont de petits ARN non codant dont la taille varie entre 21-24 nucléotides. Ils jouent un rôle de régulateurs post-transcriptionnels en utilisant leur complémentarité de séquence avec l’ARN messager (ARNm) cible afin d’induire sa répression. Grâce à la protéine Argonaute 2 (Ago2) dans laquelle les miARN sont incorporés formant ainsi le complexe miRISC, des cofacteurs sont recrutés afin d’induire la dégradation ou le blocage de la traduction de l’ARNm cible. Initialement connus pour réguler leurs cibles dans le cytoplasme, les miARN sont de plus en plus décrits comme étant des régulateurs de l’expression génique au niveau nucléaire. Dans ce travail, nous avons démontré la présence, au sein du noyau, d’un nouveau mécanisme de régulation post-transcriptionel par les miARN dont les facteurs majeurs sont Sfpq et Pspc1
Micro-RNA, nuclear regulation, gene silencing, SfpqThere is a growing body of evidence about the presence and the activity of the miRISC in the nucleus of mammalian cells. Here we show by quantitative proteomic analysis that Ago2 interacts with the complex formed by Sfpq, Pspc1 and NonO in a RNA-dependent fashion. Sfpq mediates the interaction between miRISC with Pspc1 and NonO in the nucleoplasm. By HITS-CLIP coupled with transcriptomic analysis, we demonstrated that Sfpq specifically controls the downregulation of a subset of crucial let-7a-target mRNAs in stem cells, including Lin28a, Prtg, and Igf2bp1. Sfpq directly binds to specific sequence in the 3'UTR to promote the recruitment of selected nucleoplasmic miRNAs and triggers the decay, as we show for Lin28a mRNA. These results extend the miRNA-mediated post-transcriptional gene silencing into the nucleus and indicate that a dual strategy

Theoretical Framework Professional Identity is currently receiving increased attention in the field of teacher education (Korthagen, 2004; Alsup 2005; Korthagen, Verkuyl, 2007). Korthagen (2004, Korthagen & Verkuyl, 2007) considers the professional identity as a central dimension in search of the essence of the “good teacher”. Alsup (2005) thinks that forming a professional identity is a central process of becoming an effective teacher. Teachers need a teacher education that provides them with opportunities to develop a satisfactory professional identity: they have to take care of themselves first, in order to better take care of others later. For this, teacher educators must bring issues of identity into the methods class and must talk to preservice teachers about the difficulty of professional identity development (Alsup, 2005). Taking a discursive constructionist perspective (Holstein, Gubrium, 2008), we think that in telling and sharing their narratives, teachers and students construct and perform their identities. To narrate one’s own history is a process that involves the “reflection on”, the selection and the adaptation of the events so as to render them meaningful for those, who narrate and try to persuade those who listen, of their significance. To recount history is therefore an important job of identity construction (Watson, 2006). The deeper understanding of themselves as teachers leads to an auto-transformation that represents a form of professional empowerment (Zembylas, 2003). In this light, professional identity is a narrative and discursive construction that takes place within institutions, with the help of the narrative resources available in certain settings (Holstein, Gubrium, 2000; Søreide, 2007). This construction continues as an ongoing process of positioning in the discourse (Davies, Harré, 1990). As Harré and van Langenhove (1999) highlight: the individual emerges through the process of social interaction, not as a more or less unchanged endproduct but as one who is shaped and reshaped through a variety of discursive practices in which they participate. Aim The aim of the research is to explore how teacher identities can be constructed through narrative in the online educational context of discussion. Specifically, it examines which narrative resources the participants use, and how they position themselves within the discussions, engaging in the ongoing process of identity construction. Methodology Participants. 13 in-service teachers with at least 4 years’ experience as teachers, enrolled in the online workshop during the second year of the Teacher Education University Course. Activities. Teacher-students were required to work individually or in 2 groups of 7 and 6 participants, for about three months on an asynchronous website. They were engaged in two kinds of activity: an individual activity in which they made a self presentation, defined the qualities of a “good teacher” and recounted their professional choice; a group activity where they discussed their stories and experiences as teachers. Data collection and analysis. The data comprised all the writings of the two groups: a) the texts that students wrote individually; b) the sequences of web-forum messages. With reference to Positioning theory (Harré, van Langenhove, 1999) we conducted a discourse analysis using the qualitative data analysis software AtlasTi: Findings In the 4 corpora of analysis, the participants made reference to a multitude of subjective positions, i.e. to emphasize different aspects of the understanding of oneself and one’s profession. Through analysis of the processes of identification and distancing from such subjective positions, brought into existence by the participants in the course of the narrations and interactions, have been emerging the identities that would seem to have greater significance for the teaching participants in the laboratory. Five identity constructs have been characterized: - the teacher in relation, strong identity construct, emerging from the narrations of the participants of both groups A and B, both in the individual phase and in group. It is the identity of the teacher engaged in an articulate series of relations with the various members of the scholastic reality. - the maternal teacher, less articulate and complex construct than the precedent, but emerging, in a less diffused way in the writing of the participants; it is the identity of the teacher who sees the educational relationship as “taking care” of the children, who represent the compass of their didactic action. - the teaching professional, an identity that takes shape in a clearly more defined way defined and in the two bodies of individual texts; it is an identity characterized by multiple dimensions, of which most important and common to the two groups, are those tied to the ability to collaborate and motivate the students. - the teacher in crisis, that emerges from the narrations shared by group A (of the two, that composed by the teachers with more years of service), and in particular by the body of the forum; it is the identity of the teacher in danger of burnout, tired and worn out, from their own working activity. - the teacher-person, defined by the minimum number of different subjective positions, but emerging with particular “urgency” and need to show oneself. It is the identity that takes form in the fluctuating, unstable and ambiguous space that is created between their personal identity and their professionalism. To all these identities, some participants have made reference in some way, from which, in some way, they were distanced. The individual construction process of the professional identity has been come about as a process of positioning and negotiation between the various possible identities emerging in the discursive context. It was added to verify how the emergent identities have space to overlap one another. The possibility of understanding the processes and the structures through which such constructions happen and in which images, beliefs, practices and reflections are included represents an important potentiality and an instrument of work for those who are engaged in the field of teacher training like those of scholastic policy and administration. In this sense the research throws light on possible training models for teachers in service. Throwing light on the resources and construction of narrative processes of the professional identity of the teacher in service, the search has shown other important implications. In the first place, the results confirm the idea already expressed by many investigators and widely discussed in the previous chapters of this work, that it does not make sense to propose to teachers in training a pre-manufactured and universal model of professional identity to adhere to since the professional identity construct is flexible, continuously constructed and reconstructed. In the second place they contribute to offer us uno glance, albeit limited, but also always meaningful, at today's scholastic reality, allowing the emergence of theoretical approaches, diffused beliefs, meaningful practices, that they represent the dominant value systems in the present school institution, or better, in daily scholastic life experienced by the participants. A reading of the data from the strictly more pedagogical point of view leads to finding that both the activities proposed in the on line laboratory, individual and group, can be considered meaningful since they have allowed the participants to make explicit and explore, at narrative level, one’s own professional and personal life in the search for a greater knowledge of oneself as teachers, is highlighted the value of the collective dimension of the forum as a place/tool most adequate for the carrying out of processes oriented towards a deeper knowledge of one’s own professional identity. In this general context is revealed that the search results have shown a kind of evolution of the two communities in the passage between the narration activity individual to that shared in the forum. The narrative sharing leads the participants to abandon some positions, probably the less meaningful for them, and to focus on a smaller number of aspects mainly shared. The interaction in the forum - sometimes enriched by oppositive type dynamics that have contributed to put into doubt little explored constructs, affirmations taken for granted - thus favours a greater elaboration of constructs and therefore a better exploration than any subjective position and definition of the identity construct.
In ambito di ricerca di formazione degli insegnanti un crescente interesse è dimostrato nei confronti del tema dell’identità professionale. Korthagen (2004, Korthagen & Verkuyl, 2007) considera l’identità professionale come dimensione particolarmente rilevante nella ricerca dell’essenza del “buon insegnante”. Alsup (2005) ritiene che formare un’identità professionale sia un processo centrale per assumere pienamente l’habitus d’insegnante. Gli insegnanti perciò necessitano di una formazione che offra loro opportunità per sviluppare soddisfacenti identità professionali: essi devono prendersi cura di sé, prima di poter meglio farsi carico degli altri. Per questo i formatori degli insegnanti dovrebbero porre le tematiche relative all’identità professionale al centro delle proprie proposte formative. Essi dovrebbero inoltre fare chiarezza con i formandi, sulle difficoltà che comporta raggiungere una soddisfacente e consapevole identità professionale. Assumendo una prospettiva discorsiva costruzionista (Holstein, Gubrium, 2008), si ritiene che raccontando e condividendo le proprie storie, gli insegnanti costruiscano e mostrino le loro identità. Narrare la propria storia è un processo che coinvolge la “riflessione su”, la selezione e l’adattamento degli eventi in maniera da renderli significativi per colui che narra e per cercare di persuadere della loro significatività colui che ascolta. Raccontare storie è perciò un importante lavoro di costruzione identitaria (Watson, 2006). La comprensione più profonda di sé come insegnanti conduce ad un’auto-trasformazione che rappresenta una forma di empowerment professionale (Zembylas, 2003). In questa prospettiva, l’identità risulta un costrutto narrativo e discorsivo che prende forma all’interno delle istituzioni, attraverso le risorse narrative disponibili in certi setting. (Holstein, Gubrium, 2000; Søreide, 2007). Questa costruzione si realizza come continuo processo di posizionamento nel discorso (Davies, Harré, 1990). Harré e van Langenhove (1999) sottolineano che l’individuo emerge, attraverso il processo d’interazione sociale, non come prodotto più o meno stabile, ma come costruito e ricostruito attraverso una varietà di pratiche discorsive alle quali egli partecipa. L’obiettivo della ricerca è quello di esplorare come l’identità professionale può essere costruita attraverso l’uso delle risorse narrative disponibili nel contesto di discussione on line. Specificamente si vogliono esaminare quali risorse narrative usano i partecipanti e come essi si posizionano all’interno del contesto, mentre sono impegnati nel processo di costruzione identitaria professionale. I partecipanti alla ricerca sono stati 13 insegnanti di scuola primaria o dell’infanzia, frequentanti il laboratorio on line di Tecnologie dell’istruzione nell’ambito del CL in Scienze della Formazione Primaria dell’Università di Padova. I soggetti sono stati divisi in 2 gruppi individuati in base all’età di servizio: il gruppo A formato dalle insegnanti con 8-10 anni di servizio nella scuola (insegnanti esperti) e il gruppo B formato dalle insegnanti con 4-5 anni di servizio nella scuola (insegnanti qualificati). I partecipanti hanno interagito fra di loro esclusivamente on line, in modalità testuale e asincrona, utilizzando una piattaforma specificamente realizzata. Le attività, che si sono svolte durante un lasso di tempo di circa tre mesi e sono state suddivise in una fase individuale, durante le quale essi hanno prodotto una presentazione di sé e descritto la propria visione del “buon insegnante”, e una di gruppo in cui hanno condiviso la propria scelta e i propri vissuti professionali. I dati hanno compreso gli scritti dei due gruppi: a) i testi che ciascuno studente ha scritto individualmente; b) le sequenze dei messaggi in forum. Utilizzando la Positioning Theory (Harré, van Langenhove, 1999), è stata condotta un’analisi del discorso con l’ausilio del software di analisi qualitative AtlasTi: Nei 4 corpora d’analisi le partecipanti hanno fatto riferimento a una moltitudine di posizioni soggettive, a sottolineare i differenti aspetti della comprensione di sé e del proprio lavoro. Attraverso l’analisi dei processi d'identificazione e distanziamento dalle posizioni soggettive espresse dai pari, sono andate emergendo le identità che sembrano avere avuto maggiore significatività per le insegnanti partecipanti al laboratorio. Sono stati individuati 5 costrutti identitari: - l’insegnante in relazione, costrutto identitario forte, emergente dalle narrazioni delle partecipanti di entrambi i gruppi A e B, sia in fase individuale, che in quella di gruppo. E’ l’identità dell’insegnante impegnato in una serie articolate di relazioni con i diversi componenti della realtà scolastica; - l’insegnante materna, costrutto meno articolato e complesso del precedente, ma emergente in modo diffuso negli scritti delle partecipanti; è l’identità dell’insegnante che intende il rapporto educativo come un “prendersi cura” dei bambini, i quali rappresentano la bussola della sua azione didattica; - l’insegnante professionista, un’identità che prende forma in modo nettamente più chiaro e definito nei due corpora dei testi individuali; è un’identità caratterizzata da molteplici dimensioni, di cui le più rilevanti e comuni ai due gruppi, sono quelle legate alla capacità di collaborare e di motivare gli allievi; - l’insegnante in crisi, che emerge dalle narrazioni condivise del gruppo A (fra i due, quello composto dagli insegnanti con più anni di servizio), e in particolare dal corpus del forum; è l’identità dell’insegnante in pericolo di burnout, stanco e logorato dalla propria attività lavorativa; - l’insegnante-persona, definita da numero minimo di diverse posizioni soggettive, ma emergente con particolare “urgenza” e bisogno di manifestarsi. E’ l’identità che prende forma nello spazio fluttuante, instabile e ambiguo che si crea fra la propria identità personale e la professionalità. A tutte queste identità, ciascuna partecipante ha fatto in qualche modo riferimento, da tutte vi si è, in qualche modo, distanziata. Il processo di costruzione individuale dell’identità professionale si è realizzato come processo di posizionamento e di negoziazione fra le varie possibili identità emergenti nel contesto discorsivo. Si è giunti inoltre a verificare come le identità emergenti abbiano spazi di sovrapposizione l’una all’altra. La possibilità di capire i processi e le strutture attraverso le quali tali costruzioni avvengono e quali immagini, credenze, pratiche, riflessioni vi siano comprese rappresenta un’importante potenzialità e uno strumento di lavoro per coloro che sono impegnati nel campo della formazione degli insegnanti, così come in quello dell’amministrazione e della politica scolastiche. In tal senso la ricerca getta luce su possibili modelli formativi per gli insegnanti in servizio. Focalizzando sulle risorse e sui processi di costruzione narrativa dell’identità professionale dell’insegnante in servizio, la ricerca ha mostrato altre importanti implicazioni. I risultati confermano, innanzitutto, l’idea, già espressa da molti ricercatori e ampiamente discussa nei precedenti capitoli di questo lavoro, che non abbia senso proporre agli insegnanti in formazione un modello preconfezionato e universale di identità professionale cui aderire, poiché il costrutto identitario professionale risulta flessibile, continuamente costruito e ricostruito, sempre situato. In secondo luogo essi contribuiscono ad offrirci uno squarcio, seppure limitato, ma pur sempre significativo, sull’odierna realtà scolastica, permettendo l’emergere di approcci teorici, credenze diffuse, pratiche, significati, che rappresentano i sistemi valoriali dominanti nell’attuale istituzione scuola, o meglio, nelle quotidianità scolastiche vissute dai partecipanti alla ricerca. Una lettura dei dati dal punto di vista più strettamente pedagogico conduce a rilevare che entrambe le attività proposte nel laboratorio on line, quella individuale e quella di gruppo, possono essere considerate significative poiché hanno permesso alle partecipanti di esplicitare ed esplorare, a livello narrativo, i propri vissuti professionali e personali in una crescente consapevolezza di sé come insegnanti. Va messo in luce il valore strategico del forum come luogo/strumento collettivo privilegiato alla realizzazione di processi orientati ad una più profonda consapevolezza della propria identità professionale. Va rilevato inoltre che i risultati della ricerca hanno mostrato una sorta di evoluzione delle due comunità (gruppi A e B) nel passaggio fra l’attività di narrazione individuale a quella condivisa nel forum. La condivisione narrativa conduce le partecipanti ad abbandonare alcune posizioni, probabilmente le meno significative per loro, e a focalizzarsi su un numero minore di aspetti maggiormente condivisi. L’interazione in forum - arricchita a volte da dinamiche di tipo oppositivo che hanno contribuito a mettere in discussione costrutti poco esplorati, affermazioni date per scontate - favorisce invece una maggiore elaborazione dei costrutti e perciò una migliore esplorazione di ciascuna posizione soggettiva e definizione del costrutto identitario.

Tato diplomová práce pojednává o zmírnění bitové chybovosti bezkabelového optického spoje s užitím principu reciprocity aplikovaného na komunikační kanál, spolu s možností kódování přenášených dat. V této práci je implementováno LDPC a Reed-Solomonovo kódování pro jejich vyhovující vlastnosti. Zbytková rámcová chybovost je vypočtena a k dispozici jako výstup systému, který je implementovaný v hradlovém poli (FPGA).