The cancer antigen 125 (CA125) is a cell-surface mucin which is over-expressed by the majority of ovarian cancers. However, its biology and the role it plays in ovarian cancer is largely unknown, although other cell-surface mucins have been shown to play a role in apoptosis, cell growth and tumour immune evasion. To analyse the function of CA125 in ovarian cancer, we initially knocked down the expression of CA125 using RNA interference. Knocking down CA125 expression using in vitro transcribed short interfering RNAs (siRNAs) induced a potent cell death response, which has been well characterised in the literature as an induction of an interferon response and resulting in cell apoptosis. Subsequently, using the short hairpin RNA expression vector, pSUPER, which has been shown to knock down genes with high efficiency with reduced off-target affects, we generated stable sub-lines of the ovarian cancer cell line, OVCAR-3, which had been transfected with pSUPER constructs targeting CA125. Intriguingly, these sub-lines had a range of abnormal mitotic events and nuclear defects. However, there was no clear association with the level of CA125 knock down. This could be either due to clonal selection from the parent OVCAR-3 cell line or in addition to CA125 knock down, additional genetic changes are required to occur to favour a state of survival. Similar to the in vitro data, xenografts of the sub-clones into SCID mice generated inconclusive results as to whether CA125 knock down contributes to tumour growth, invasion and metastasis in vivo. More recently, we have been able to achieve high levels of short-term CA125 knock down using synthetic siRNAs designed to reduce off-target affects. These preliminary in vitro and in vivo experiments conducted with pSUPER sub-lines should be repeated using synthetic siRNAs to confirm the role of CA125 in this context. Given the role which the cytoplasmic tail of cell-surface mucins plays in its function, we generated a polyclonal antibody recognising the CA125 cytoplasmic tail, designated M16.1. Immunofluorescence imaging of CA125 in ovarian cancer cell lines, OVCAR-3 and PEO-1, using the OC125 extracellular domain antibody indicated cell-surface localisaton of CA125. However, in addition to the cell-surface localisation, the M16.1 antibody localised to the cell cytoplasm, indicating cleavage and release of the CA125 cytoplasmic tail into the cytosol. Additionally, M16.1 co-localised with α-tubulin at perinuclear sites and to areas resembling microtubule organising centres. However, M16.1 did not co-localise with γ-tubulin at the centrosome, indicating association with non-centrosomal microtubules. Furthermore, depolymerisation of microtubules on ice for 1 hour resulted in loss of diffuse cytoplasmic M16.1 staining but co-localisation between M16.1 and α-tubulin at non-centrosomal sites remained. Intriguingly, when microtubules were allowed to reform at 37oC in PEO-1 cells which had CA125 knocked down by synthetic siRNAs, the ability to reform radial asters was impaired, possibly indicating the CA125 cytoplasmic tail involvement in anchoring microtubules to non-centrosomal sites. Furthermore, we also cloned a portion of CA125 encompassing the cytoplasmic tail, transmembrane domain and 9 tandem repeats. When this construct was transfected into COS-1 cells, the CA125 cytoplasmic tail localised to microtubule bundles during metaphase. Mitotic involvement of the endogenous CA125 cytoplasmic tail was confrmed in OVCAR-3 and PEO-1 cells using M16.1. Given this association and also the results from the pSUPER sub-lines with CA125 knockdown, CA125 may be involved in controlling the fidelity of mitosis, which is grossly altered during tumourigenesis. More recently, it was identified that galectin-1 (Gal-1), an S-type lectin, is a ligand for CA125. Gal-1 is a potent inducer of T cell apoptosis and has been implicated as playing a major role in immune evasion for cancer cells. Consequently, we analysed the expression of CA125 and Gal-1 in ovarian cancer and confirmed the two molecules were expressed concurrently at the mRNA level by RT-PCR. Moreover, immunofluorescence studies also confirmed that CA125 and Gal-1 interacted with each other at the cell-surface of 27/87 cells, an ovarian cancer cell-line. Therefore, we hypothesised that CA125 presents Gal-1 to the immune system, which then induces T cell apoptosis and allows the tumour to escape the immune system. However, CA125 did not protect tumour cells from recognition or killing by T cells, which was shown by no differences in IFN-γ secretion or tumour lysis by cytotoxic T cells using influenza peptide pulsed pSUPER sub-lines with CA125 knockdown. The work described in this thesis suggests that CA125 plays a major role in the aetiology and progression of ovarian cancer through its actions on mitosis, microtubule organisation and immune evasion.

Zugl.: Tübingen, Univ., Diss., 2007

Freie Univ., Diss.--Berlin, 2000.

Thesis (Ph. D.)--University of California, San Diego, 2005. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Harvard University, 1992. / Includes bibliographical references (leaves [291]-[310]).

Engineered nanoparticles provide potential opportunities for improving current drug delivery, bioimaging and biosensing modalities. In many cases, a ligand, such as a protein, peptide or nucleic acids, is attached to the nanoparticles surface to serve as a targeting group. However, the nanoconjugation (i.e. covalently bound molecules to a nanocarrier) is not an innocuous reaction. It can change the binding affinity and interfere with the intracellular trafficking of the tethered species. The understanding of this influence to the tethered species is still lacking. Therefore, the main objective of this thesis is to investigate the effect of nanoconjugation to the biological identity of the tethered biomolecules, in terms of cellular uptake, intracellular trafficking and the ultimate biological outcomes. The Epidermal Growth Factor Receptor (EGFR) is a tyrosine kinase that regulates cell proliferation and can cause cancer if dysregulated. Continuous treatment with high doses of EGF can induce apoptosis, in EGFR overexpressing cell lines. In this thesis, Epidermal Growth Factor (EGF) was chosen as the object of investigation. Covalent attachment of EGF to gold nanoparticles (NP-EGF) was found to enhance apoptosis in EGFR overexpressing cell lines (A431, MDA-MB-468) and it is sufficient to induce apoptosis in cell lines exhibiting EGFR expression at physiological levels (HeLa). NP-EGF accumulation through the endosomal pathway was also investigated to assess the impact of nanoconjugation on the spatio-temporal distribution of NP-EGF as potential origin for the observed enhancement of apoptosis. Two orthogonal experimental approaches were applied: (1) isolation of NP-EGF containing endosomes by taking advantage of the increased density of endosomes associated with the uptake of Au NPs; (2) correlated darkfield/fluorescence imaging to map the spatial distribution of NP-EGF in endosomes as a function of time. The studies reveal that nanoconjugation prolongs the dwelling time of phosphorylated receptors in the early endosomes and that the retention of activated EGFR in the early endosomes is accompanied by an EGF mediated apoptosis at effective concentrations that do not induce apoptosis in the case of the free EGF. Investigating the nanoconjugation-enhanced EGF-induced apoptosis improves the current understanding of cell-nanomatieral interactions and provides new opportunities for overcoming apoptosis evasion by cancer cells. / 2017-01-01T00:00:00Z

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-04-20T12:59:35Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação José Vinicius da Costa.pdf: 1101072 bytes, checksum: d6c6e613c94c41c0d732eba0a88ed567 (MD5) / Made available in DSpace on 2017-04-20T12:59:35Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Dissertação José Vinicius da Costa.pdf: 1101072 bytes, checksum: d6c6e613c94c41c0d732eba0a88ed567 (MD5) Previous issue date: 2016-09-30 / Qual o retrato da evasão dos cursos na modalidade integrado do Programa Nacional de Integração da Educação Profissional à Educação Básica na Modalidade Jovens e Adultos (PROEJA) no Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso (IFMT) – Campus Cuiabá/Octayde Jorge da Silva? O objetivo desta pesquisa é realizar um diagnóstico exploratório da evasão do PROEJA no IFMT – Campus Cuiabá, entre os anos 2007 a 2015. O desenho de pesquisa combina estatística descritiva dos dados colhidos junto à instituição de interesse e entrevistas semiestruturada para testar a hipótese de que a avaliação da implementação do PROEJA no IFMT – Campus Cuiabá apontam para uma alta taxa evasão ligados a fatores próprios do contexto da prática. Os resultados encontrados são: 1) a média da evasão nos cursos modalidade integrado do PROEJA no IFMT – Campus Cuiabá foi de 77% (setenta e sete por cento) com a maior proporção do abandono ocorrendo nos três primeiros semestres dos cursos; 2) desigualdades estão sendo criadas ou reproduzidas pelo programa, na medida em que se observa um menor acesso e permanência de mulheres e a prevalência de fixação de discentes com idade menor do que 31 (trinta e um) anos; 3) a percepção dos agentes envolvidos na ponta da política em análise (gestores, professores e alunos) assinalam que a incidência da evasão estão ligados a fatores de trabalho/emprego; questões pessoais dos discentes; desnivelamento dos discentes quando acessam o PROEJA e durante o transcorrer do curso; a insuficiente capacitação dos docentes e preparação do órgão para oferecer o programa; desconhecimento da base legal e teórica do PROEJA por parte dos atores executores do programa; estrutura inadequada para realização, por parte dos discentes, de algumas atividades pedagógicas e técnicas; falta da devida adaptação da instituição ao público diferenciado do PROEJA; aparente descompromisso com as atribuições profissionais, por parte de alguns professores, que faltam as aulas que são de sua responsabilidade; o acesso ao estágio na área, tendo em vista a sua baixa remuneração; as duas greves deflagradas pela representação sindical local no período analisado. / What is the portrait of the evasion of the courses in the modality integrated of the National Program of Integration of the Professional Education to the Basic Education in the Modality Youth and Adults (hereby PROEJA) in the Federal Institute of Education, Science and Technology of Mato Grosso (hereby IFMT) – Campus Cuiabá/Octayde Jorge da Silva? The aim of this research is to make an exploratory diagnosis of the evasion of PROEJA in IFMT – Campus Cuiabá, between the years of 2007 to 2015. The research outline combines descriptive statistics of the data collected together with the institution of interest and the semi-structured interview to test the hypothesis that the evaluation of the implementation of PROEJA in IFMT – Campus Cuiabá point to a high dropout rate related to factors specific of the context practice. The results found are: 1) the average of the evasion in the courses modality integrated of the PROEJA in IFMT – Campu Cuiabá was 77% (seventy-seven per cent); 2) inequalities are being created or reproduced by the program, as can be observed a lower access or permanency of women and the prevalence of setting of students with age smaller than 31 (thirty-one) years old; 3) the perception of the involved agents in the tip of the politic in analysis (managers, teachers and students) point that the incidence of the evasion are linked to factors of work/job; personal matters of the students; unevenness of students when access the PROEJA and during the course; the insufficient capacitation of teachers and preparation of the organ to offer the program; unawareness of the legal and theoretic basis of PROEJA by the actors performers of the program; inadequate structure to the realization, by the students, of some pedagogical and technical activities; lack of the proper adaptation of the institution to the differentiated public of PROEJA; seemingly lack of commitment with the professional attribution, by some teachers, that missed the classes that were their responsibility; the access to the internship in the area, having a low payment; the two strikes triggered by the local syndical representation in the analyzed period.

A leptospirose é uma zoonose de distribuição mundial, com maior incidência nas regiões tropicais. As bactérias que causam a doença pertencem ao gênero Leptospira, família Leptospiracea e ordem Spirochaetales. A leptospirose é mantida na natureza pela colonização persistente dos túbulos renais proximais dos animais portadores. Uma estratégia adotada por estas espiroquetas para sobreviver à ação do sistema imune inato do hospedeiro é a capacidade que possuem de interagir com os reguladores do sistema complemento Fator H (FH) e proteína de ligação a C4b (C4BP). O sistema complemento é um componente vital da imunidade inata, uma vez que desempenha um papel crucial na defesa do hospedeiro, particularmente contra bactérias Gram-negativas. Dados recentes gerados por nosso grupo mostraram que C4BP interage com a proteína de superfície LcpA de Leptospira. Com cerca de 20 kDa, essa proteína é capaz de se ligar a C4BP purificado ou solúvel no soro de maneira dose-dependente. Uma vez ligado à proteína, C4BP permanece funcional agindo como cofator de Fator I na clivagem de C4b. O presente estudo teve como principal objetivo avaliar a interação da proteína LcpA com FH humano, principal regulador solúvel da via alternativa do sistema complemento. A proteína LcpA e suas porções N-Terminal, Intermediária e CTerminal recombinantes foram purificadas por cromatografia de afinidade a metal a partir da fração insolúvel. A interação dessas proteínas com FH foi avaliada por dois métodos distintos: ELISA e Western blot com overlay. Os resultados indicaram que a porção C-Terminal da proteína LcpA é responsável pela interação com FH. Curiosamente, C4BP também se liga a esse domínio da proteína. Uma vez que esses dois reguladores solúveis do sistema complemento interagem com o mesmo segmento da LcpA, realizaram-se, a seguir, ensaios de competição com o objetivo de avaliar se ambos compartilhariam os mesmos sítios de interação. Os dados mostraram que FH e C4BP devem se ligar a sequências distintas desta proteína. Com o objetivo de se avaliar a funcionalidade de FH ligado à LcpA, realizou-se um ensaio para investigar sua atividade de co-fator de Fator I na clivagem de C3b. Produtos de degradação de 46 kDa e 43 kDa da cadeia α' de C3b foram detectados, indicando que FH permanece funcional. Em se tratando de uma proteína com funções relacionadas ao processo de evasão ao sistema imune inato, decidiu-se realizar ensaios de desafio em modelo de hamster com a finalidade de se avaliar seu potencial imunoprotetor. Os três ensaios realizados indicaram que a proteína não é capaz de conferir proteção. Os ensaios de ELISA visando à avaliação dos títulos de anticorpos mostraram que LcpA não é imunogênica, fato que explica os resultados dos ensaios de desafio observados. Portanto, embora interaja com moléculas do hospedeiro e pareça contribuir para o processo de evasão ao sistema imune inato, essa proteína de membrana não se mostrou promissora como candidato vacinal contra leptospirose. / Leptospirosis is a zoonosis of global distribution, with higher incidence in tropical areas. The bacteria that cause the disease belong to the genus Leptospira, family Leptospiracea and order Spirochaetales. Leptospirosis is maintained in nature by persistent colonization of proximal renal tubules of carrier animals. One strategy adopted by these spirochetes to escape from host´s innate immune system is the ability to interact with the complement regulators Factor H (FH) and C4b Binding Protein (C4BP). The complement system is a vital component of the innate immune system, being crucial for host´s defense, particularly against Gram-negative bacteria. According to our recent published data, C4BP interacts with the leptospiral surface protein LcpA. This 20 kDa outer membrane protein binds both purified and serum C4BP in a dose-dependent manner. Once bound, C4BP remains functional acting as a cofactor for Factor I in the cleavage of C4b. In the present study we evaluated the interaction of LcpA with human FH, the main soluble regulator of the alternative pathway of complement. The intact protein as well as its N-terminal, intermediate and C-terminal portions were purified by metal-affinity chromatography from the insoluble pellet. The interaction of these proteins with FH was evaluated by two distinct methods: ELISA and Western blot overlay. Our results indicate that the C-terminal domain of LcpA mediates interaction with FH, and also with C4BP. Since both complement regulators interact with the same fragment of LcpA, we next performed competition assays to assess if they would share binding sites. According to our data, FH and C4BP have distinct binding sites on LcpA. Cofactor activity of FH bound to immobilized LcpA was confirmed by detecting the C3b α' chain cleavage fragments of 46 and 43 kDa upon incubation with Factor I, thus indicating that it remains functionally active. Given the LcpA´s role in host´s innate immune evasion, we also evaluated its vaccine potential in a hamster model. Data from three challenge assays indicated that the protein can not afford protection. Low ELISA antibody titers of hamsters immunized with LcpA were observed, which strongly suggests that this protein is not immunogenic. In conclusion, LcpA interacts with host´s molecules and seems to contribute to the bacterial immune evasion. Nevertheless, this outer membrane protein is not a promising vaccine candidate against leptospirosis.

Cette thèse se fonde sur les résultats d’une enquête sur les représentations de la déviance fiscale construites par l’opinion publique en France et donne une réponse nouvelle à la question du consentement à l’impôt. Mêlant les méthodes quantitatives et qualitatives et se situant au carrefour de la science politique, de la sociologie, de la philosophie et de la psychologie, cette enquête révèle la manière dont la considération des représentations de la déviance fiscale peut conduire à la construction du civisme fiscal. En partant de l’influence des valeurs et des normes sous-jacentes aux représentations des évitements légaux et illégaux de l’impôt sur le comportement du contribuables, cette thèse propose une réflexion sur la nécessité d’un dépassement de l’opposition binaire entre le légal et l’illégal imposée par les réponses administratives et judiciaires aux évitements de l’impôt. Pour être efficace, la lutte contre l’« insécurité fiscale », pensée comme une voie de sortie possible de la crise économique, doit se construire dans et par la réappropriation citoyenne de la question fiscale et dans la défense d’une conception particulière du juste soutenue par un renouvellement du contrat social fiscal. / This dissertation is based on the results from a survey on the representations of tax deviance constructed by public opinion in France and gives a novel answer to the question of tax compliance. This research, situated at the crossroads between political science, sociology, philosophy and psychology, uses both quantitative and qualitative methods to reveal how the consideration of tax deviance representations can lead to the construction of fiscal civism. Looking first at the influence of values and norms which underlie the representations of legal and illegal tax avoidance on taxpayers’ behavior, this dissertation offers a reflection on the need to move beyond the binary opposition between the legal and illegal imposed by the administrative and judicial responses to tax avoidance. In order to be efficient, the fight against “fiscal insecurity”, considered as a possible way out of the economic crisis, must construct itself in and by the civic reappropriation of the fiscal question and in the defense of a particular conception of fairness supported by a renewal of the fiscal social contract.

L’étude de la réalité de la criminalité démontre que celle-ci a essentiellement un but économique. L’aspect financier peut aussi être un moyen significatif, notamment dans le cas du terrorisme. Pourtant, la réponse à la criminalité ne prend que très peu en compte la réalité économique de la criminalité et est tournée, de façon historique, vers la sanction physique. Le droit pénal concurrençant le droit fiscal, y compris sur des infractions de nature économique. Partant de ce constat, il devient nécessaire de faire évoluer la réponse à la criminalité afin de mieux répondre à cette réalité. Parmi les moyens déjà existants, le droit fiscal représente de nombreux avantages, notamment par le fait qu’il est conceptuellement tourné vers la lutte contre les flux financiers illicites, que ceux-ci soient ou non d’origine criminelle. Si le corpus juridique lié à la lutte contre la criminalité est peu tourné vers le droit fiscal, les quelques cas présents démontrent que celui-ci a un apport significatif, notamment par les moyens dont dispose l’administration fiscale (même s’ils sont sous-utilisés) en matière de recherche d’informations relatives aux flux financiers. La lutte contre la criminalité gagnerait aussi en efficacité si le paradigme de la réponse pénale était changé. Il faudra pour cela adopter une approche économique à l’instar de l’approche et des moyens mis en œuvre dans le cas de la fraude fiscale. Que ce soit en recherchant des sanctions économiques et non plus pénale ou en traitant l’environnement criminel comme un tout. / The study of the reality of crime shows that it has essentially an economic purpose. The financial aspect can also be a significant one, especially in the case of terrorism. However, the response to crime takes very little account of the economic reality of the crime and is, historically, based on criminal punishment. The tax law is in concurrence with the criminal law. In regard to this observation, it becomes necessary to change the response to crime in order to better meet this reality. Among the existing means, the tax law is representing several advantages, including the fact that it is conceptually directed to fight illicit financial flows, whether they are of criminal origin or not. If the body of law related to the fight against crime is not tax law oriented, the few existing cases show that it has a significant contribution, including the capacity of the tax administration (even though it is under-used) in research on information flows. The fight against crime could become much more effective if the paradigm of criminal law had changed. This change could be achieved by adopting an economic approach, like the approach and means used in the case of tax evasion. Whether seeking economic sanctions rather than criminal, or dealing with the criminal environment as a whole.