Lung cancer arises from epithelial cells that line the air passages of the lungs. It is the second most common malignancy in the United States; trends suggest that over 228,000 new patients will be diagnosed with lung cancer in 2013. Due to the fact that lung cancer is highly aggressive, it has proven difficult to control. The 5-year survival rate has been shown to be only 15.9%, despite the advances made in terms of diagnosis and treatment. Therefore, we are faced with the problem of finding more effective methods that allow for an earlier diagnosis and the improved treatment of lung cancer. This study attempts to address these issues by investigating Tarsh, a novel molecule that is involved in the regulation of cellular senescence. Previous studies have shown that Tarsh is expressed in normal lung cells, but is significantly downregulated in lung tumors. These studies also determined that Tarsh is likely dependent upon the expression of p53, a tumor suppressor gene. The current study investigated these results, in addition to the biological effects of ectopically increasing Tarsh and/or knocking down p53 expression in two lung cancer cell lines: A549 and H1299 cell lines. It was determined that increasing the expression of Tarsh decreased the rate of proliferation in both cell lines. Additionally, it was shown that the knockdown of p53 increased proliferation in A549 cells. In regards to the migration rate of these cell lines, the overexpression of Tarsh decreased migration in A549 cells, but had no effect on H1299 cells. However, the role of p53 in migration is still unclear. The results of this study suggest that the knockdown of p53 decreases cell migration in A549 cells. This contradicts the fact that H1299 cells do not express p53, yet was found to have the highest migration rate. It is evident that a further investigation is needed to make more concrete conclusions. Nevertheless, the suppressive features of Tarsh on cell proliferation, and possibly migration, make it a promising target of research for lung cancer therapy.

This work has focused on improving our knowledge of the thyroid specification process. Thyroid cells are derived from mouse embryonic stem cells (mESCs) by directed differentiation through multiple intermediate developmental stages, including anterior foregut endoderm (AFE), prior to NKX2-1+ thyroid progenitor specification. To investigate if transient Nkx2-1 expression can increase the efficiency of thyroid specification, we utilized a mESC line double knock-in GFP-T/hCD4-Foxa2 with a doxycycline inducible (Tet-On) Nkx2-1 transgene. Transient activation of the Nkx2-1 transgene at the AFE stage leads to stable induction of high levels of endogenous Nkx2-1 as well as early and mature thyroid-specific markers including Pax8, Foxe1, Tg, Nis, and Tshr. Lung and neuronal NKX2-1+ lineages were not derived in this system. The thyroid progeny mature and organize into follicle-like structures in 3D culture. These follicles express adherens and tight junction proteins indicative of an epithelial character and produce the thyroid hormone thyroxine (T4) in the presence of iodide. Critical determinants of thyroid lineage specification have been revealed by variations in developmental stage timing, signaling pathways, and sorting of AFE-stage subpopulations. To provide further insights into the mechanisms of thyroid specification, RNA-Seq data acquired from relevant stages has identified potential genes involved in early thyroid development. The results demonstrate that Nkx2-1 can act as a stage-specific inductive signal during directed differentiation of mESCs to thyroid follicular cells. We have also developed a mouse model to recapitulate these results in an in vivo context. This work has provided novel insights into thyroid specification and provides an efficient system for deriving and studying thyroid cells, which can be used for in vitro modeling of development and disease.

The CCAAT/enhancer-binding protein delta (C/EBPδ) is a highly conserved transcription factor capable of regulating numerous cell fate processes, such as cell growth, differentiation, proliferation and apoptosis. C/EBPδ is inducible during cellular stress responses, including inflammation and responses to growth factor deprivation or thermal stress. C/EBPδ is stress-inducible in a diversity of fishes, including the zebrafish Danio rerio; however, little is known about its role in fish development. Here I show that overexpression of C/EBPδ leads to severe developmental defects, including reduced body length, edema, liver malformation and retinal abnormalities. The proportion of individuals that display developmental abnormalities is significantly greater in C/EBPδ-overexpressing embryos compared to control embryos and overexpression significantly reduces survival of larvae over time. TUNEL analysis suggests C/EBPδ-overexpressing embryos exhibit a pattern of apoptotic cell death which is spatially distinct from control embryos. These data support a critical role for C/EBPδ in numerous developmental processes, including promoting programmed cell death during development. Mutations in C/EBPδ have been implicated in the progression of human tumors, including those of myeloid, hepatocellular and breast cancers. Therefore, the C/EBPδ-overexpressing zebrafish will serve as a valuable model for examining the role of this gene during development, as a part of the cellular response to stress and in pathological states such as tumor progression.

Membrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.

ABSTRACT TRANSCRIPTION PATTERN COMPARISON OF TWO UBIQUITIN SPECIFIC PROTEASES (USP6 AND USP32) Akhavan Tabasi, Shiva M.Sc., Department of Biology Supervisor: Assist. Prof. Dr. A. Elif Erson August 2007, 93 pages Breast cancer is the most common type of cancer among women worldwide. The incidence of breast cancer is 1 in 8 among women. Usually loss of tumor suppressor genes and overexpression of proto-oncogenes are known to be involved during mammary tumorigenesis. USP32 (Ubiquitin Specific Protease 32) gene is located on chromosomal band 17q23, a region of amplification in breast cancer. Gene amplification is known to be a common mechanism in breast cancer cells, through which proto-oncogenes are overexpressed and contribute to tumor progression. Presence of multiple oncogene candidates on 17q23 requires individual characterization of these genes. USPs (Ubiquitin Specific Protease), have various roles in protein degradation pathways (e.g / by editing the ubiquitin chains, recycling of ubiquitin, v deubiquitinating the target proteins and inhibiting their degradation by the proteasome). Deregulated expression of USPs is likely to interfere with the degradation of many key regulatory proteins in the cell. Therefore, USP32 becomes an interesting oncogene candidate that may have roles in protein degradation pathways based on the fact that it is located on an amplicon region and that it is overexpressed in breast tumors. On the other hand, USP6 (Ubiquitin Specific Protease 6), a known oncogene on 17p13, is also a deubiquitinating enzyme, with conserved histidine and cysteine domains, which are also shared by USP32. Interestingly there is a 97% sequence similarity between bases 3,197 to 7,831 of USP6 and 2,390 to 7,024 of USP32 gene. In this study, we aimed to investigate the expression patterns of USP32 and USP6 (including alternative transcripts) in breast tissue to avoid any possibility of overlapping functions of two enzymes due to their high sequence similarity. In addition, we sub-cloned USP32 gene into TOPO-TA vector, so that further functional studies (e.g / localization and overexpression) can be performed. Further characterizations of Ubiquitin Specific Protease 32, may help us understand its importance in the protein degradation pathway during breast tumorigenesis.

No description available.

The monotopic membrane protein alMGS, a glycosyltransferase catalyzing glucolipid synthesis in Acholeplasma laidlawii, was overexpressed in Escherichia coli. Optimization of basic growth parameters was performed, and a novel method for detergent and buffer screening using a small size-exclusion chromatography was developed. This resulted in a tremendous increase in protein yields, as well as the unexpected discovery that the protein induces intracellular vesicle formation in E. coli. This was confirmed by sucrose density separation and Cryo-TEM of membranes, and the properties of the vesicles were analyzed using SDS-PAGE, western blot and lipid composition analysis. It is concluded that both alMGS and alDGS, the next enzyme in glucolipid pathway, have the ability to make the membrane bend and eventually form vesicles. This is likely due to structural and electrostatic properties, such as the way the proteins penetrate the membrane interface and thereby expand one monolayer. The highly positively charged binding surfaces of the glycosyltransferases may bind negatively charged lipids, such as Phosphatidylglycerol (PG), in the membrane and withdraw it from the general pool of lipids. This would increase the overall lipid synthesis, since PG is a pace-keeper, and the local concentration of nonbilayer prone lipids, such as Phosphatidylethanolamine, can increase and also induce bending of the membrane. The formation of surplus membrane inside the E. coli cell was used to develop a generic method for overexpression of membrane proteins. A proof-of-principle experiment with a test set of twenty membrane proteins from E. coli resulted in elevated expression levels for about half of the set. Thus, we believe that this method will be a useful tool for overexpression of many membrane proteins. By engineering E. coli mutants with different lipid compositions, fine-tuning membrane properties for different proteins is also possible. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.

Made available in DSpace on 2014-12-17T14:10:25Z (GMT). No. of bitstreams: 1 ValeskaDSS_DISSERT.pdf: 2384968 bytes, checksum: cf4765fd0b42fd627fcb0ce0d604be1e (MD5) Previous issue date: 2011-10-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The flowering is a physiological process that it is vital for plants. This physiological process has been well studied in the plant model Arabidopsis, but in sugarcane this process is not well known. The transition of the shoot apical meristem from vegetative to flowering is a critical factor for plant development. At Brazil northeastern region, the transition to flowering in sugarcane has an important effect as it may reduce up to 60% its production. This is a consequence of the sugar translocation from stalks to the shoot apical meristem which is necessary during the flowering process. Therefore, the aim of this work was to explore and analyze cDNAs previously identified using subtractive cDNA libraries. The results showed that these cDNAs showed differential expression profile in varieties of sugarcane (early x late flowering). The in silico analysis suggested that these cDNAs had homology to calmodulin, NAC transcription factor and phosphatidylinositol, a SEC14, which were described in the literature as having a role in the process of floral development. To better understand the role of the cDNA homologous to calmodulin, tobacco plants were transformed with overexpression cassettes in sense and antissense orientation. Plants overexpressing the cassette in sense orientation did not flowered, while plants overexpressing the cassette in the antissense orientation produced flowers. The data obtained in this study suggested the possible role from CAM sequence, SEC14 and NAC in the induction/floral development pathway in sugarcane, this is the first study in order to analyze these genes in the sugarcane flowering process. / A flora??o ? um processo fisiol?gico que demanda um alto gasto energ?tico, e ? vital para a planta, pois ? dela que depende sua propaga??o gen?tica. Este processo fisiol?gico tem sido bem estudado no modelo vegetal Arabidopsis, mas em cana-dea??car n?o ? muito conhecido. A transi??o do meristema apical no processo de flora??o ? um fator cr?tico no ciclo de desenvolvimento da planta. No nordeste brasileiro, a transi??o para a flora??o na cana-de-a??car tem um efeito dram?tico, pois pode reduzir em at? 60% a produ??o de a??car ou etanol. Isso porque, o florescimento da cana resulta na isoporiza??o do colmo que ? caracterizado pela transloca??o do a??car presente no colmo para os ?pices meristem?ticos com a finalidade de suprir a necessidade energ?tica durante o processo de flora??o. Portanto, o objetivo desse trabalho foi de prospectar e analisar cDNAs de cana-dea??car que possam estar associados ? flora??o utilizando ferramentas de bioinform?tica e de transgenia. Os resultados mostraram que os cDNAs em estudo apontaram um perfil de express?o diferencial em variedades de cana-de-a??car. As an?lises in silico sugeriram que os cDNAs estudados apresentam similaridade para calmodulina, fator de transcri??o NAC e fosfatidilinositol, uma SEC14, as quais foram descritas na literatura como tendo um papel no processo de desenvolvimento floral. Para entender melhor o papel do cDNA hom?logo ? calmodulina, plantas de tabaco foram transformadas com cassetes de superexpress?o nas orienta??es sense e antisense contendo o promotor 35S. Plantas superexpressando o cassete na orienta??o senso n?o floresceram, enquanto que plantas superexpressando o cassete na orienta??o antissense apresentaram o desenvolvimento reprodutivo. Os dados obtidos nesse trabalho apontam para o poss?vel papel de CAM, NAC e SEC14 na via de indu??o/desenvolvimento floral em cana-de-a??car, sendo um dos primeiros trabalhos a se estudar esses genes no processo de flora??o nesse organismo

The aim of this work was to characterise the influence of adenosine on imaginal disc cell line Cl8+ of Drosophila. I prepared stable cell lines with the overexpression or RNA interference of genes coding adenosine receptor AdoR (CG9753) and adenosine transporter DmENT2 (CG11045) in D. melanogaster. These cell lines were subsequently used to test their response to extracellular adenosine signal by the measurement of cell viability and level of second messengers cAMP and Ca2+ in Cl8+ cells.

Orientadores: Selma Giorgio, Fernanda Ramos Gadelha / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:07:59Z (GMT). No. of bitstreams: 1 Codonho_BarbaraSantoni_M.pdf: 2501497 bytes, checksum: d7ceeef1653b2df4a7d52b28ebd16543 (MD5) Previous issue date: 2014 / Resumo: As leishmanioses são um conjunto de doenças causadas pelo protozoário do genêro Leishmania, que atingem milhões de pessoas por ano. O tratamento é realizado primeiramente com antimoniais pentavalentes e, em casos de resistência, são indicadas a pentamidina ou anfotericina B. Todos estes fármacos são tóxicos e induzem efeitos colaterais nos pacientes. Devido a dificuldades no tratamento, o estudo de moléculas presentes no parasita se torna importante. Dentre essas, as heat shock proteins 70 (HSP70) são proteínas essenciais para o ciclo de vida da Leishmania. Durante a passagem do vetor para o hospedeiro vertebrado, o parasita encontra vários tipos de estresses que induzem a uma maior expressão da HSP70. Nesse projeto avaliou-se os efeitos da superexpressão da HSP70 em Leishmania (Leishmania) amazonensis, comparando-se parasitas que superexpressam a proteína HSP70 (pTEX-HSP70) com parasitas contendo somente o vetor (pTEX). Os resultados mostraram que os promastigotas transfectados pTEX e pTEX-HSP70 apresentaram vários aspectos ultraestruturais semelhantes aos não transfectados (WT), porém mostraram ser maiores e com o tamanho da área nuclear maior. A superexpressão da proteína HSP70 conferiu aos parasitas uma fase estacionária de proliferação mais estendida do que a observada em parasitas pTEX. Uma maior resistência e capacidade proliferativa foram observadas nos parasitas pTEX-HSP70 quando submetidos a diferentes condições de estresses (tratamentos com H2O2, choque térmico e ambiente hiperbárico), em relação a parasitas pTEX. Os resultados também mostraram que parasitas pTEX e pTEX-HSP70 infectam culturas de macrófagos peritoneais e macrófagos humanos derivados de sangue periférico, em taxas (% de infecção e número de amastigotas/macrófago) semelhantes a de parasitas WT. O processo de infecção em camundongos BALB/c mostrou que o tamanho da lesão induzida pelos parasitas pTEX e pTEX-HSP70 na pata foi diferente nas primeiras semanas, mas semelhante no curso final da infecção. Adicionalmente, as cargas parasitárias nas lesões dos camundongos BALB/c infectados com os parasitas pTEX e pTEX-HSP70 foram semelhantes, mas maiores que as cargas parasitárias nas lesões induzidas por WT. Além disso, os baços dos camundongos infectados com os parasitas pTEX e pTEX-HSP70 apresentaram visceralização. Ensaios da bioenergética destes promastigotas mostraram que parasitas pTEX-HSP70 apresentam maiores taxas de consumo de O2 do que parasitas pTEX, apesar de apresentarem produção de ATP semelhante. A produção de superóxido nos parasitas pTEX-HSP70 e pTEX foram similares, apesar da liberação de H2O2 ser bem inferior nos de parasitas pTEX-HSP70. Os resultados obtidos indicam que a superexpressão da proteína HSP70 protege a L.(L.) amazonensis de situações de estresse imediato, mas não interfere com a sua capacidade infectiva / Abstract: Leishmaniasis are a group of diseases caused by the protozoan genus Leishmania, which affect millions of people each year. The treatment is performed primarily with pentavalent antimony and resistance cases are indicated pentamidine or amphotericin B. All these drugs are toxic and induce side effects in patients. Due to difficulties in treatment, the study of molecules present in the parasite becomes important. Among these, the heat shock protein 70 (HSP70) proteins are essential for the life cycle of Leishmania. During the transition from vector to vertebrate host, the parasite finds various types of stresses that induce a higher expression of HSP70. In this project was evaluated the effects of overexpression of HSP70 in Leishmania (Leishmania) amazonensis, comparing parasites that overexpressing HSP70 (pTEX-HSP70) protein with parasites containing the empty vector (pTEX). The results showed that transfected promastigotes pTEX and pTEX-HSP70 showed several similar ultrastructural aspects similar to promastigotes of L.(L.) amazonensis untransfected (WT), but proved to be larger and the size of the largest nuclear area. Overexpression of HSP70 protein gave the parasites a stationary phase of proliferation more extended than that observed in parasites pTEX. Higher strength and better proliferative capacity were observed in parasites pTEX-HSP70 when submitted to different stress conditions (hydrogen peroxide, heat shock treatments and hyperbaric environment), in relation to parasites pTEX. The results also showed that pTEX and pTEX-HSP70 parasites infect cultures of peritoneal macrophages and human peripheral blood-derived macrophages in rate (% infection and the number of amastigotes / macrophage) similar to WT parasites. The process of infection in BALB/c mice showed that the size of the induced parasitic pTEX and pTEX-HSP70 foot injury was different in the first few weeks but similar in the final course of infection. Additionally, parasitic loads on the lesions of BALB/c mice infected with pTEX and pTEX-HSP70 parasites were similar, but larger than the parasitic loads in lesions induced by WT. Moreover, spleens from infected with pTEX and pTEX-HSP70 parasites mice showed visceralization. Assays of bioenergetics promastigotes showed that these pTEX-HSP70 parasites consume more O2 than pTEX parasites, despite showing similar ATP production. Superoxide production in parasites pTEX and pTEX-HSP70 were similar, despite the release of hydrogen peroxide is considerably lower than pTEX-HSP70 parasites. The results indicate that overexpression of HSP70 protein protect L.(L.) amazonensis in immediate situations of stress, but does not interfere with its infective capacity / Mestrado / Imunologia / Mestra em Genética e Biologia Molecular