Les lymphomes à grandes cellules B primitifs du médiastin (PMBL) partagent des caractéristiques pathologiques avec les lymphomes diffus à grandes cellules B (DLBCL), et des caractéristiques moléculaires communes aux lymphomes de Hodgkin classiques (cHL). Le cluster oncogénique miR-17-92, localisé au niveau du chromosome 13q31, est un gène amplifié dans les DLBCL. Dans notre étude, nous avons comparé le niveau d’expression de chaque membre du clustermiR-17-92 dans une série de prélèvements de patients de 40 PMBL, 20 DLBCL et 20 cHL, et étudié les gènes cibles liés aux microARN dérégulés dans les PMBL. Nous avons montré un niveau plus élevé de miR-92a dans les PMBL que dans les DLBCL, mais pas dans les cHL. La combinaison d’une analyse in silico prédictive des cibles de miR-92a et d’une analyse transcriptomique nous a permis d’identifier FOXP1 comme la cible principale de miR-92a dans les PMBL, un résultats qui n’avait jusqu’alors pas été établi. Cette observation a été confirmée par le test 3’UTR, le niveau d’expression ARN et protéique dans les lignées cellulaires transduites. Les études in vivo sur les souris à partir des cellules transduites nous a permis de démontrer l’effet tumeur suppresseur de de miR-92a et l’effet oncogénique de FOXP1. L’expression plus élevée de miR-92a et la sous-expression de FOXP1 au niveau ARN et protéique a également été retrouvé dans les prélèvements humains de PMBL, alors que le niveau d’expression de miR-92a était bas et FOXP1 était haut dans les DLBCL. Nous en avons conclu à une régulation post-transcriptionnelle de FOXP1 par miR-92a dans les PMBL, avec une relevance clinico-pathologique pour mieux caractériser les PMBL. / Primary mediastinal large B-cell lymphoma (PMBL) shares pathological features with diffuselarge B-cell lymphoma (DLBCL), and molecular features with classical Hodgkin lymphoma (cHL). The miR-17-92 oncogenic cluster, located at chromosome 13q31, is a region that is amplified in DLBCL. Here we compared the expression of each member of the miR-17-92 oncogenic cluster insamples from 40 PMBL patients versus 20 DLBCL and 20 cHL patients, and studied the target genes linked to deregulated miRNA in PMBL. We found a higher level of miR-92a in PMBL than in DLBCL, but not in cHL. Acombination of in silico prediction and transcriptomic analyses enabled us to identify FOXP1 as a main miR-92a target gene in PMBL, a result so far not established. This was confirmed by 3’UTR, and RNA and protein expressions in transduced cell lines. In vivo studies using the transduced cell lines in mice enabled us to demonstrate a tumor suppressor effect of miR-92aand an oncogenic effect of FOXP1. The higher expression of miR-92a and the down regulation of FOXP1 mRNA and proteinwere also found in human samples of PMBL, while miR-92a expression was low and FOXP1was high in DLBCL. We concluded to a post-transcriptional regulation by miR-92a through FOXP1 targeting in PMBL, with a clinico-pathological relevance for better characterisation of PMBL.

The Jaagsiekte Sheep Retrovirus (JSRV), an exogenous type B/D-retrovirus with about 10-15 endogenous counterparts in all normal sheep genomes, causes Jaagsiekte (JS) or ovine pulmonary adenocarcinoma (OPA), a contagious lung cancer of sheep. This sheep lung cancer has been identified as the best natural out-bred model that can be used to study human epithelial tumours. The close similarity between the pathology of the sheep disease and Human Bronchiolo-alveolar carcinoma are highly suggestive that the human disease could have a similar aetiology and mechanism to the sheep disease. However, in the case of sheep at the time of the study there was a need for an assay that could be used to screen for infected sheep. The isolation, cloning and subsequent sequencing of the first full-length exogenous and endogenous forms of JSRV contributed greatly towards JSRV research. Until recently the diagnosis of OPA was based mostly on clinical presentation with confirmation by micro and macro examination of the affected lungs by expert pathologists. In the absence of a specific humoral response no serology-based tests were available to diagnose the disease early in live animals. Control and management of the disease was primarily by regular flock inspections and prompt culling of the suspected cases. The objective of this research project was therefore to assess and investigate the serological and molecular diagnosis of JSRV. In an attempt to develop a serology based assay three proteins were identified as candidate diagnostic antigens, the group specific antigen JSRV p26, the transmembrane and the orf-X proteins. Genes coding for all three proteins were isolated, cloned and expressed. The JSRV p26 was sufficiently purified and its potential as a diagnostic antigen was evaluated in both a Western blot and ELISA. Our studies confirmed that there were no circulating antibodies to the JSRV capsid protein. Evidence suggested that the immune response was localised to the lungs. Lung lavage samples were therefore collected from infected and normal sheep and analysed for the presence of JSRV p26 antibodies using an in-house JSp26 peroxidase conjugate in an antigen capture assay. This assay lacked sensitivity but the results indicated that there was a specific localised immune response to JSRV in the lungs of OPA affected sheep. This was confirmed with an in-house antigen capture assay that we developed. JS antigen was detected in the lung and nasal fluid of affected sheep, but not in equivalent samples from normal sheep. Three molecular assays were investigated for their sensitivity and specificity, the LTR-gag PCR, U3/LTR hemi-nested PCR and the PCR that covered the V1/V2 region. The U3/LTR hemi-nested assay was 2 logs more sensitive than the LTR-gag PCR. However, it detected the endogenous JSRV5.9A1 loci at higher concentrations. This was overcome by designing a more specific primer P3M for the first step of the U3/LTR hemi-nested PCR and the use of the AmpliTaq Gold DNA polymerase. This assay proved to be both sensitive and specific enough to screen for the infectious exogenous JSRV in peripheral blood samples from individual sheep. It is now possible to use this assay to selectively eradicate the disease from a flock through a selective culling programme. Furthermore, the assay could be made quantitative by the inclusion of concentration standards. / Thesis (M.Med.)-University of KwaZulu-Natal, 2005.

Thesis (Ph. D.)--University of Missouri--Columbia, 1998. / "May 1998." Typescript. Vita. Includes bibliographical references (leaves 325-355). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 233-247). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 233-247). Also available on the Internet.

Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia. Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CXCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation. We conclude that MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.

Made available in DSpace on 2014-10-09T12:27:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:04Z (GMT). No. of bitstreams: 0 / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Cells in living organisms are constantly experiencing a variety of mechanical cues. From the stiffness of the extra cellular matrix to its topography, not to mention the presence of shear stress and tension, the physical characteristics of the microenvironment shape the cells’ fate. A rapidly growing body of work shows that cellular responses to these stimuli constitute regulatory mechanisms in many fundamental biological functions. Substrate strains were previously shown to be sensed by cells and activate diverse biochemical signaling pathways, leading to major remodeling and reorganization of cellular structures. The majority of studies had focused on the stretching avoidance response in near-uniform strain fields. Prior to this work, the cellular responses to complex planar strain fields were largely unknown. In this thesis, we uncover various aspects of strain sensing and response by first developing a tailored lab-on-a-chip platform that mimics the non-uniformity and complexity of physiological strains. These microfluidic cell stretchers allow independent biaxial control, generate cyclic stretching profiles with biologically relevant strain and strain gradient amplitudes, and enable high resolution imaging of on-chip cell cultures. Using these microdevices, we reveal that strain gradients are potent mechanical cues by uncovering the phenomenon of cell gradient avoidance. This work establishes that the cellular mechanosensing machinery can sense and localize changes in strain amplitude, which orchestrate a coordinated cellular response. Subsequently, we investigate the effect of multiple changes in stretching directions to further explore mechanosensing subtleties. The evolution of the cellular response shed light on the interplay of the strain avoidance and the newly demonstrated strain gradient avoidance, which were found to occur on two different time scales. Finally, we extend our work to study the influence of cyclic strains on the early stages of cancer development in epithelial tissues (using MDCK-RasV12 system), which was previously largely unexplored. This work reveals that external mechanical forces impede the healthy cells’ ability to eliminate newly transformed cells and greatly promote invasive protrusions, as a result of their different mechanoresponsiveness. Overall, not only does our work reveal new insights regarding the long-range organization in population of cells, but it may also contribute to paving the way towards new approaches in cancer prevention treatments.

Made available in DSpace on 2014-10-09T12:27:15Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:57:04Z (GMT). No. of bitstreams: 0 / A terapia gênica tem sido empregada em estudos pré-clínicos e clínicos, com o intuito de amenizar ou curar uma doença. Vetores retrovirais são uma ferramenta de transferência gênica largamente utilizada. Vetores bicistrônicos são uma alternativa interessante para o tratamento de doenças complexas. Na construção de um vetor bicistrônico pode-se empregar várias estratégias dentre elas a utilização da sequência IRES. A endostatina, fragmento do colágeno XVIII, tem sido muito utilizada na terapia anti-angiogênica devido sua ação inibitória no crescimento de células endoteliais. A imunoterapia tem sido utilizada como tratamento coadjuvante de tumores. Dentre as citocinas utilizadas, a interleucina-2 promovendo a proliferação de linfócitos T, tem sido utilizada em diversos estudos pré-clínicos e clínicos. O objetivo deste projeto foi construir e caracterizar in vitro um vetor retroviral bicistrônico codificando endostatina e interleucina-2 utlizando a sequência IRES. A construção do vetor foi realizada em três etapas, sendo comprovada a construção final por análise de restrição e seqüenciamento. Células de empacotamento foram transfectadas com o vetor, e posteriormente realizada a transdução na célula alvo. A endostatina e a interleucina-2 foram determinadas por Dot blot, seguido de análise da expressão por RT-PCR e ensaio de atividade. O vetor construído apresentou altos níveis de titulação viral, variando de 4.20x105 a 1.53x106UFC/mL. A determinação da endostatina e da interleucina-2 variaram entre 1.08 a 2.08g/106cels.24h e 0.66 a 0.89μg/106cels.24h, respectivamente. A expressão da endostatina no clone NIH3T3-pLend-IRES-IL2SN foi 2 vezes superior á apresentada pelo clone NIH3T3-pLend-IRES-IL2SN. A endostatina produzida promoveu uma inibição da proliferação de 40% das células endoteliais; e a interleucina-2 promoveu uma proliferação de 10.6% de linfócitos CD4 e 8.9% de CD8. Desta forma, a construção obtida neste trabalho representa uma excelente ferramenta para estudos da biologia celular do câncer e novas estratégias terapêuticas. / Dissertacao (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Os papilomavírus humanos (HPVs) oncogênicos são importantes agentes na etiologia do câncer ginecológico e atualmente tem sido relacionados também a algumas lesões cancerizáveis e a alguns tipos de cânceres de boca. Com o objetivo de avaliar a relação entre os HPVs e um tipo de lesão cancerizável de boca que acomete os lábios chamada queilite actínica crônica (QAC), foram avaliados e considerados aptos para a pesquisa 29 pacientes portadores de QAC. A reação em cadeia pela polimerase (PCR) foi utilizada para detectar a presença do HPV em amostras de tecido fresco, provenientes de lábios doentes onde todos os casos apresentaram resultados negativos. A QAC ocorreu em 100% nos indivíduos da raça branca, em 19 homens e 10 mulheres e na idade média de 56,14 anos. Foram avaliados também os aspectos clínicos e histológicos da QAC sendo encontrados 14 casos de atipia epitelial discreta (48,27%), 10 casos de atipia epitelial moderada (34,49%) e 5 casos de atipia epitelial severa (17,24%). Através de análise estatística concluímos que clinicamente a presença de áreas leucoplásicas e o tempo de evolução da lesão superior a 5 anos estão diretamente relacionados aos casos de atipias epiteliais mais graves. O hábito de fumar e de beber parecem contribuir, mas não obtiveram resultados estatisticamente significativos ao aparecimento da QAC. / The oncogenic human papillomaviruses (HPVs) are important agents in the etiology of gynecological cancer and have been recently related to some premalignant lesions and to some types of mouth cancer. In order to evaluate the relation between HPVs and one type of precancerous lesion that affects the lips called chronic actinic cheilitis (CAC), 29 CAC patients were assessed and considered eligible for the study. The polymerase chain reaction (PCR) was used to detect the presence of HPV in fresh tissue samples of affected lips. All results were negative. All CAC patients were Caucasian, 19 males and 10 women, mean age of 56.14 years. The clinical and histological aspects of CAC were also assessed - there were 14 cases of discreet (48.27%), 10 cases of moderate (34.49%) and 5 cases of severe epithelial atypia (17.24%). By statistical analysis we concluded that, clinically, the presence of leukoplastic areas and progression of the lesion for over five years are directly related to more severe epithelial atypia. Smoking and drinking habits seem to contribute to the condition but achieved no statistical significance regarding onset of CAC.