published_or_final_version / abstract / toc / Anatomy / Master / Master of Philosophy

Aging is a universal process that is accompanied by the loss of proliferative potential of cells. However, factors governing this age-dependent decline in proliferation remain largely undefined. The pancreatic β-cells of the islets of Langerhans serve as a unique model to explore the effect of cellular age on proliferation and function within the same organ. During early juvenile stage, the zebrafish islet is rapidly expanding and newly differentiated β-cells are added to the pool of older β-cells that were formed during embryogenesis. In this thesis, using accurate reporters for cell-cycle stages and intra-cellular calcium sensors, it was shown that younger β-cells are more proliferative but less functional compared to older β-cells. Furthermore, as the animal ages, the overall rate of β-cell proliferation declines. Transcriptomic analysis of β-cells from young adult and older adult islets revealed that older cells display an inflammatory signature. Transgenic reporter line for inflammatory NF-kB activity showed that β-cells of younger islets display varying levels of NF-kB activity, which becomes homogenous in older β-cells. Furthermore, the cells with higher NF-kB-activity proliferate less compared to their neighbors with lower activity. Specifically, younger NF-kBhigh cells upregulate socs2, a negative regulator of proliferation that is also enriched in older β-cells. Interestingly, activated macrophages were observed infiltrating the islet during late juvenile stages, thus pointing to an important role of the microenvironment in activation of inflammatory signature in the islet. Overall, this study shows that cells of different ages co-exist within the same micro-organ. This age-related cellular heterogeneity governs the rate of proliferation of the tissue. The loss of cellular heterogeneity with age reduces the proliferative pool of the tissue. Finally, the expression of inflammatory NF-kB activity acts as a marker of this loss of proliferative heterogeneity.

肺癌是一個世界性的健康難題。大量研究證據顯示，煙草及其致癌物NNK對環氧酶（COX）-2及其下游產物具有促進效應。血栓素（TxA）2是COX-2的關鍵性下游產物之一，該論文闡述了TxA2在NNK導致的肺癌增長中的可能作用。 / 我們發現相对于非吸烟者，吸煙者肺癌組織表达更高水平的TxA2合酶（TxAS）。NNK可以刺激培養的肺癌細胞TxA2合成。用TxAS抑制劑和TxA2受體（TP）拮抗劑分別阻抑TxA2的合成與功能可以引起細胞凋亡，從而有效抑制NNK導致的細胞增殖效應。在TxA2合成受抑制的情況下，TP激動劑U46619幾乎可以重建NNK效應，說明TP在NNK效應中的重要作用。研究還顯示，激活的TP可以通過PI3K/Akt和ERK通路進一步激活CREB，從而參與NNK對肺癌細胞的促生長效應。 / 緊接著，我們的研究顯示TP 可以調節NNK對COX-2 和TxA2的誘導，而且發現NNK刺激的TxA2合成主要依賴於COX-2活性。COX-2和TxA2功能抑製劑對NNK的促細胞生長作用具有相似的抑制效用。考慮到TP是TxA2的功能受體，該資料說明TP在NNK處理的肺癌細胞中傳遞了上游因子COX-2的促腫瘤作用。在使用COX-2小干擾RNA（siRNA）抑制NNK作用的情況下，TP激動劑U46619幾乎可以恢復NNK的效應證實了TP的傳遞者角色。研究還發現 TPα而不是TPβ在培養的肺癌細胞系中廣泛表達，並且過表達TPα具有促進腫瘤生長的作用。在用NNK處理細胞的條件下，TPα還具有促COX-2表達和TxA2生成的作用。 / 我們的研究進一步發現，在吸煙者肺癌組織中TPα表達增高，這與TxAS的表達相似。与此结果相一致，在經NNK處理的A/J小鼠肺癌組織中，TxAS和TP表達水準也是明顯上升的。在細胞培養實驗中，NNK能夠提高TxAS蛋白和信使RNA（mRNA）的表達水準。但是，在TP的兩個亞型TPα和TPβ中， NNK僅能促進TPα的蛋白表達，對它們的mRNA均無影響。NNK對TxAS的促表達作用是核轉錄因數(NF)-κB依賴性的。其他的幾個關鍵轉錄因數，諸如特異性蛋白(SP)-1，CREB和活化受體 （PPAR）γ均未參與NNK對TxAS和TPα的表達促進作用。進一步的，轉錄後機理被證實參與了NNK對TPα的作用。TPα而不是TPβ經鑒別在NNK的促NF-κB 激活 和 促TxAS 表達效應中起正向調節作用。 / 總之， 我們的研究說明TxA2相關通路在NNK的促肺癌細胞生長效應中起正向調節作用。我們的研究揭示了TPα的自我激活環路。通過該環路，TxA2，或者說TxAS和TPα參與了NNK的肺癌促生長效應。因此，我們的研究為肺癌的防治了提供了一個新的方向，即靶向TxAS和TPα是一種可能有效的策略。 / Lung cancer concerns a world-wide health problem. There is considerable evidence of that tobacco smoke and its carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) have the potential effects on the production of cyclooxygenase (COX)-2 and its downstream products in tumor cells. This thesis is constructed to describe the study focused on the role of thromboxane A2 (TxA2), one of the key downstream products of COX-2, in NNK-induced lung tumor growth. / We found that as compared to non-smokers, lung cancer tissues obtained from smokers tended to express more TxA2 synthase (TxAS). Moreover, NNK could stimulate TxA2 synthesis in lung cancer cells. Blockade of TxA2 synthesis and action by TxAS inhibitor and TxA2 receptor (TP) antagonist completely blocked NNK-promoted cell proliferation via inducing apoptosis. Moreover, TP agonist U46619 reconstituted a near full proliferative response to NNK when TxAS was inhibited, affirming the role of TP in NNK-induced cell growth. Furthermore, we revealed that the activated TP may then activate CREB through PI3K/Akt and ERK pathways, thereby contributing to the NNK-induced lung cancer cell growth. / We subsequently showed that TP could modulate the induction of COX-2 and TxA2 by NNK. The synthesis of TxA2 stimulated by NNK was found to be mainly dependent on COX-2 activity. Intriguingly, there are similar inhibitory effects on NNK-induced cell growth between pharmacological inhibition of COX-2 and the blockade of TxA2 synthesis and action. Because TP is the natural receptor of TxA2, these results suggest that TP may function as a mediator for the tumor-promoting effects of COX-2 upon NNK treatment, which was confirmed by the data showing that U46619 almost restored NNK effects in the presence of COX-2-siRNA. Importantly, TPα, but not TPβ was found to be widely expressed in lung cancer cells and be able to promote tumor growth, COX-2 expression and TxA2 synthesis upon NNK treatment. / We further demonstrated that in lung tumor tissues obtained from smoker, TPα protein was increased, which was similar to the change in TxAS protein. The increased levels of TxAS and TP proteins were also found in lung cancer tissues of A/J mice treated with NNK. In cell culture experiments, NNK could increase TxAS at both protein and mRNA levels. However, TPα rather than TPβ was increased by NNK at protein but not mRNA level. NNK-stimulated TxAS expression was dependent on nuclear factor (NF)-κB signaling. Other key transcriptional factors, such as specificity protein(SP)-1, CREB and peroxisome proliferator-activated receptor-gamma (PPARγ), were not involved in NNK-induced TxAS and TPα expression. Further experiments revealed that post-transcriptional mechanisms were responsible for NNK-induced TPα expression. TPα rather than TPβ was finally identified to have a positive role in NNK-induced NF-κB activation and TxAS expression. / Taken together, our study suggests that TxA2 pathway has a positive role in NNK-induced lung cancer cell growth. An auto-positive feedback loop of TPα activation to facilitate lung tumor growth in the presence of NNK is delineated by these results. Therefore, targeting TxAS or/and TPα may represent a promising strategy for prevention and treatment of lung cancer. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Runyue. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 119-146). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.I / 摘要 / Publications / Acknowledgement / Abbreviations / Table of contents / Chapter Chapter 1 --- General introduction--Tobacco smoking, COX-2 pathway and cancer / Chapter 1.1 --- Abstract --- p.1 / Chapter 1.2 --- Introduction --- p.2 / Chapter 1.3 --- Cyclooxygenase and prostanoids --- p.5 / Chapter 1.4 --- The effects of tobacco smoking on COX-2 pathway, and the related pathologies --- p.8 / Chapter 1.4.1 --- Smoking, PGE2, inflammation and immunosupression --- p.8 / Chapter 1.4.2 --- Smoking, TxA2, platelet activation, cell contraction and angiogenesis --- p.11 / Chapter 1.4.3 --- Smoking and PGI2 --- p.16 / Chapter 1.5 --- The role of cyclooxygenase-2 pathway in the progression of tobacco smoke-related cancers --- p.19 / Chapter 1.5.1 --- Lung cancer --- p.19 / Chapter 1.5.2 --- Gastrointestinal cancer --- p.23 / Chapter 1.5.3 --- Bladder cancer --- p.24 / Chapter 1.5.4 --- Head and neck squamous cell carcinoma --- p.25 / Chapter 1.5.5 --- The signaling mechanisms underlying the induction of COX-2 by smoking in tumors --- p.26 / Chapter 1.6 --- Summary, future directions and key questions --- p.28 / Chapter Chapter 2 --- NNK induces lung cancer cell growth by stimulating TxA2 and its receptor / Chapter 2.1 --- Abstract --- p.32 / Chapter 2.2 --- Introduction --- p.33 / Chapter 2.3 --- Materials and Methods --- p.35 / Chapter 2.3.1 --- Cell lines and cell culture --- p.35 / Chapter 2.3.2 --- Chemicals and drug treatment --- p.35 / Chapter 2.3.3 --- Thromboxane B2 EIA assay --- p.36 / Chapter 2.3.4 --- MTT assay --- p.36 / Chapter 2.3.5 --- BrdU cell proliferation assay --- p.37 / Chapter 2.3.6 --- Flow cytometry for analysis of apoptosis --- p.37 / Chapter 2.3.7 --- Transfection of cells with CREB siRNA --- p.38 / Chapter 2.3.8 --- Western blot analysis and antibodies --- p.38 / Chapter 2.3.9 --- Statistical analysis --- p.39 / Chapter 2.4 --- Results --- p.41 / Chapter 2.4.1 --- High expression of TxAS in lung cancer tissues of smoker --- p.41 / Chapter 2.4.2 --- NNK stimulated TxA2 synthesis in lung cancer cells --- p.43 / Chapter 2.4.3 --- Blockade of TxA2 synthesis and action prevented NNK-induced cell growth --- p.44 / Chapter 2.4.4 --- TxA2 mimetic U46619 reconstituted NNK-enhanced cell proliferation under TxA2-inhibited condition --- p.47 / Chapter 2.4.5 --- Blockade of TxA2 synthesis or action induced the apoptosis of the NNK-exposed cells --- p.47 / Chapter 2.4.6 --- CREB is accountable for the key role of TxA2 in NNK-enhanced cell proliferation --- p.49 / Chapter 2.4.7 --- PI3K/Akt and ERK rather than JNK and p38 pathways were mediated by TxA2 in the NNK-exposed cells --- p.52 / Chapter 2.4.8 --- CREB is located downstream of the PI3K/Akt and ERK pathways in NNK-treated cells --- p.53 / Chapter 2.5 --- Discussion --- p.55 / Chapter Chapter 3 --- The positive role of TPα in the induction of COX-2, TxA2 and cell growth by NNK in human lung cancer cells / Chapter 3.1 --- Abstract --- p.62 / Chapter 3.2 --- Introduction --- p.63 / Chapter 3.3 --- Materials and methods --- p.65 / Chapter 3.3.1 --- Cell culture and chemicals --- p.65 / Chapter 3.3.2 --- Transient transfections --- p.66 / Chapter 3.3.3 --- TxB2 measurement --- p.66 / Chapter 3.3.4 --- Cell growth detection --- p.67 / Chapter 3.3.5 --- Analysis of apoptosis --- p.67 / Chapter 3.3.6 --- Western blot analysis and antibodies --- p.67 / Chapter 3.3.7 --- Statistical analysis --- p.68 / Chapter 3.4 --- Results --- p.70 / Chapter 3.4.1 --- Examination of TP as the modulator for induction of COX-2 and TxA2 by NNK --- p.70 / Chapter 3.4.2 --- The TxA2 generated in cells treated with NNK is mainly dependent on COX-2 activity --- p.72 / Chapter 3.4.3 --- Examination of TP as the key mediator for the tumor-promoting effect of COX-2 --- p.72 / Chapter 3.4.4 --- The expression and action of α and β isoforms of TP in human lung cancer cells --- p.77 / Chapter 3.4.5 --- the identification of positive role of TPα in NNK-induced COX-2, TxA2 and cell growth in lung cancer cells --- p.79 / Chapter 3.5 --- Discussion --- p.81 / Chapter Chapter 4 --- TP-α facilitates lung tumor growth through an autoregulatory feedback mechanism / Chapter 4.1 --- Abstract --- p.88 / Chapter 4.2 --- Introduction --- p.89 / Chapter 4.3 --- Materials and methods --- p.91 / Chapter 4.3.1 --- Human lung tissue and immunohistochemical analysis --- p.91 / Chapter 4.3.2 --- Animal treatment --- p.91 / Chapter 4.3.3 --- Cell culture and chemicals --- p.92 / Chapter 4.3.4 --- Transient transfection --- p.93 / Chapter 4.3.5 --- Real-time PCR --- p.93 / Chapter 4.3.6 --- Western blot analysis and antibodies --- p.94 / Chapter 4.3.7 --- Statistical analysis --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- The effects of smoking on the expression of TP in human lung cancer tissue --- p.96 / Chapter 4.4.2 --- The effects of NNK on the expression of TxAS and TP in lung tissues of A/J mice --- p.98 / Chapter 4.4.3 --- The effects of NNK on the expression of TxAS and TPα in lung cancer cells --- p.99 / Chapter 4.4.4 --- Identification of the roles of NF-κB, CREB and SP1 in NNK-induced TxAS and TPα expression --- p.101 / Chapter 4.4.5 --- The negative role of PPARγ in NNK-induced TxAS and TPα expression --- p.104 / Chapter 4.4.6 --- NNK-induced TPα expression via post-transcriptional mechanism --- p.105 / Chapter 4.4.7 --- Examination of TPα auto-activation mechanism in lung cancer cells stimulated with NNK --- p.106 / Chapter 4.5 --- Discussion --- p.109 / Chapter Chapter 5 --- Conclusion and future works / Chapter 5.1 --- Conclusion --- p.114 / Chapter 5.2 --- Future works --- p.115 / Chapter 5.2.1 --- The possible role of miR-34c in the auto-regulatory loop of TxAS expression or TPα activation --- p.116 / Chapter 5.2.2 --- The possible role of FOXO3a in the auto-regulatory loop of TxAS expression or TPα activation --- p.116 / References --- p.119

Hyperthermia damages both developing and adult brains, especially when it occurs after ischemia or stroke. Work presented in this dissertation used in vitro models of these stresses to investigate mechanisms underlying damage to immature neurons and neural precursors cultured from embryonic rat brain. Studies described in Chapter 2 investigated the effects of a brief, intense hyperthermic stress (30-45 min at 43ºC). This stress produced a selective depletion of nestin-immunoreactive neural precursor cells, and reduced proliferation, as evidenced by reduced BrdU incorporation into young Tuj1-immunoreactive neurons. The stress activated caspase 3, and produced multiple signs of nuclear damage as well as early and persisting mitochondrial depolarization. Cycloheximide, an inhibitor of protein synthesis, reduced cell death. All these findings suggest an apoptotic death process. Studies described in Chapter 3 used a combination of oxygen-glucose deprivation (OGD, 2 h) followed by mild 41ºC hyperthermia for 90 min (T). The combined OGDT stress reduced both survival in monolayer cultures and colony-forming ability in neurospheres. Cell death occurred gradually over 2 days, and was accompanied by caspase activation that began within 6 h post-stress. Post-stress application of cycloheximide or a general caspase inhibitor (especially qVD-OPH) reduced cell death, but specific inhibitors of caspases 2, 3, 8 or 9 were ineffective. OGDT led to upregulation of the pro-apoptotic protein Bim as well as redistribution of Bax from cytoplasm to mitochondria within 6 h. Persisting mitochondrial depolarization began within 3 h following the combined OGDT stress, but not following individual OGD or T stresses alone. These findings suggest that OGD sensitizes neural precursor cells to hyperthermia-induced damage, and that the combined OGDT stress kills neural precursors via apoptotic mechanisms that include activation of mitochondrial death pathways. Results of these studies suggest that immature neurons and neural precursors are especially vulnerable to hyperthermia-induced damage via apoptotic mechanisms. Pan-caspase inhibitors may be a promising therapeutic strategy to preserve viability of these cells following stroke with hyperthermia.

Cancer has been a disease, which has generated intense research interest for many years. Misexpression of two oncoproteins, TAL 1 and LMO 1, has been found to help induce a particular type of leukemia, called T-cell acute lymphoblastic leukemia (T-ALL). Presently, it is not completely understood how these proteins induce leukemogenesis or what other cellular proteins they interact with to drive this progression. In this study, a series of experiments were conducted to identify downstream targets of TALI and LMO1. Using retroviral gene transfer, both genes were introduced, either singly or in combination, into a murine T-cell line called AKR-DP-603. Empty vectors were introduced as controls. In order to assay the effects of TALI and LMO I expression on expression of other proteins, a series of Western blots were completed on all populations of engineered cells. It was determined that there were differences in expression of Bcl-2 and p16 as indicated by differences in band intensities on the blots. This is important because it implies an effect on protein levels by TAL 1 and LMO 1. However, there were no differences in protein expression levels for Bax or cyclin D1. This suggests that TAL1 and LMOI do not have any regulatory effects on these proteins. In addition, apoptotic assays were completed on all populations of cells. The results of both a TUNEL assay and ethidium bromide/acridine orange staining protocol showed TAL1- and LMO1expressing cells to have an increase in cell survival under starvation conditions and a lower frequency of apoptosis. Statistical analysis verified significant difference in the apoptosis assays. The data suggests an up-regulation of anti-apoptotic proteins. The finding of this research allow a clearer understanding of the process of leukemogenesis and may lead to a development of better cancer treatments. / Department of Biology

Indiana University-Purdue University Indianapolis (IUPUI) / Targeting uncontrolled cell proliferation and resistance to DNA damaging chemotherapeutics using small molecule inhibitors of proteins involved in these pathways has significant potential in cancer treatment. Several proteins involved in genomic maintenance and stability have been implicated both in the development of cancer and the response to chemotherapeutic treatment. Replication Protein A, RPA, the eukaryotic single-strand DNA binding protein, is essential for genomic maintenance and stability via roles in both DNA replication and repair. Xeroderma Pigmentosum Group A, XPA, is required for nucleotide excision repair, the main pathway cells employ to repair bulky DNA adducts. Both of these proteins have been implicated in tumor progression and chemotherapeutic response. We have identified a novel small molecule that inhibits the in vitro and cellular ssDNA binding activity of RPA, prevents cell cycle progression, induces cytotoxicity and increases the efficacy of chemotherapeutic DNA damaging agents. These results provide new insight into the mechanism of RPA-ssDNA interactions in chromosome maintenance and stability. We have also identified small molecules that prevent the XPA-DNA interaction, which are being investigated for cellular and tumor activity. These results demonstrate the first molecularly targeted eukaryotic DNA binding inhibitors and reveal the utility of targeting a protein-DNA interaction as a therapeutic strategy for cancer treatment.

INTRODUÇÃO: Atualmente as células da microglia têm recebido grande atenção dentro da resposta imune, isto devido ao fato de que sua ativação por citocinas inflamatórias é capaz de promover a infiltração e destruição do sistema nervoso central (SNC) durante algumas doenças, principalmente no caso da esclerose múltipla (EM). Além de seu papel pró-inflamatório, já demonstrou-se que estas também são capazes de expressar moléculas supressoras como a indoleamina-2,3-dioxigenase (IDO), capaz de suprimir a proliferação de células T. Contudo, ainda pouco se sabe sobre seu verdadeiro papel na patogenia da EM. Recentemente tem sido descrita uma população de células T chamadas Th17, capaz de secretar grandes quantidades de IL-17, IL-21 e GM-CSF possuindo uma importância fundamental na patogenia da EM e de seu modelo murino, a EAE. Nesse contexto, a relação entre as Th17 e as células da microglia pode nos fornecer dados importantes acerca dos mecanismos envolvidos nas lesões observadas no SNC. OBJETIVO: Este trabalho teve como objetivo melhor elucidar a relação existente entre a expressão das moléculas imunes por células da microglia e a ação que estas promovem sobre as células T. MÉTODOS: Utilizamos culturas de células da microglia de linhagem, chamadas C8-B4, assim como cultura primária de células da microglia obtidas a partir sistema nervoso de camundongos C57BL/6 adultos. Caracterizamos o perfil imune da microglia, avaliando a transcrição de genes para citocinas através de PCR em tempo real assim como a expressão de suas moléculas ativadoras por citometria de fluxo. A avaliação da IDO se deu através da expressão da mesma por células da microglia ativadas ou não por LPS ou IFN-?. Ja sua capacidade funcional foi medida através da atividade proliferativa de linfócitos T CD4 específicos para MOG 35-55. RESULTADOS: Nossos resultados demonstraram que as células de ambas as culturas possuem a capacidade de expressar diversas moléculas imunes, tanto pró quanto anti-inflamatórios. Dentre estas observamos TLR-4, TLR-2, IL-6, IL-10 e TGF-?. Além disso, confirmamos a expressão da enzima IDO por estas células. O bloqueio de tal enzima impede o controle que a microglia tem sobre a proliferação dos linfócitos T CD4, tanto in vitro quanto in vivo. No modelo in vivo tal efeito repercute em uma encefalomilite mais severa, onde o quadro clínico do animal não regride. CONCLUSÃO: Os resultados aqui obtidos nos dão a certeza da influência das microglias dentro do contexto inflamatório, afirmando sua capacidade de modular a resposta imune. Além disto, fica clara a importância da enzima IDO, cuja ação dentro do controle de uma autoimunidade demonstra ser altamente necessária / INTRODUCTION: Microglia cells has gained great attention recently because its activation by inflammatory cytokines can promote infiltration and destruction of Central Nervous System (CNS) during some disease, mainly in the case of Multiple Sclerosis (MS). On the other hand, these cells may also express suppressor molecules such as the indoleamine-2,3-dioxygenase (IDO), able to suppress T cell proliferation. However, still little is known about its role in MS pathogenesis. Recently it has been described a new population of T cells called Th17, able to secrete high amounts of IL-17, IL-21 and GM-CSF, with a fundamental importance on MS and its murine model, EAE. In this context, the relationship between Th17 and microglia cells can provide us important data about the mechanisms involved in the establishment of CNS lesions. OBJECTIVES: This work had the objective to better elucidate the relationship between the expression of some molecules by microglia and its role T cell activation. METHODS: Through a cellular lineage knowing as C8-B4 and primary cultures of microglia obtained from CNS of adult mice C57BL6 we investigated the transcription of several genes for cytokines and membrane expression of several pattern recognition receptors. The IDO evaluation was performed after activation with LPS or rIFN-?. Its functional capacity was measured trough its action over T cell proliferation. RESULTS: Our results demonstrated that both cells have the capacity of express several immune molecules, both pro and anti-inflammatory. Among this, we observed TLR-4, TLR-2, IL-6, IL-10 and TGF-?. We also confirmed IDO expression by these cells. The blockade of such enzyme prevents the control of microglia above T CD4 lymphocytes proliferation, both in vitro and in vivo. Using the in vivo model, IDO blocker rendered a encephalomyelitis more severe. Conclusion: The results here obtained give us the certainty of microglia influence in inflammatory context, stating its capacity of modulating the immune response

[Truncated abstract] For this thesis, the molecular biology of cancer was approached from two directions. Firstly, an investigation was conducted on the role of growth factor receptor-bound protein 7 (Grb7) in breast cancer. Grb7 is an adapter molecule that binds to a variety of proteins, including the growth factor receptor and proto-oncogene, ErbB2, and mediates signalling to downstream pathways. It has been linked to cell migration and an invasive phenotype, and is of interest as a therapeutic target. To investigate the role of Grb7 in breast cancer, preliminary experiments were performed that, firstly, determined the expression of wild-type Grb7 and a splice variant, Grb7V, in a range of cell lines, and secondly, aided the development of a protocol for treating cells with short interfering RNA (siRNA) against Grb7 and the ErbB ligand, heregulin (HRG), in a cell system appropriate for measuring the functional outcomes. Using this protocol in conjunction with CellTitre (CT) proliferation assays, it was demonstrated that Grb7 does not play a role in the proliferation of either unstimulated or HRG-stimulated SK-BR-3 breast cancer cells. Furthermore, using the protocol in conjunction with Boyden chamber migration assays, it was shown that inhibition of Grb7 expression has a slight stimulatory effect on HRG-stimulated SK-BR-3 cell migration. Thus, Grb7 was found to play only a minor role in the migration of SK-BR-3 cells, suggesting that it is not an ideal anti-cancer target for breast cancers modelled by this cell system. Concurrently, a second investigation was conducted, which similarly sought insight into the molecular biology of cancer, but adopted a more strategic approach. ... These results provide evidence for a biologically significant role for the miR-7-mediated regulation of EGFR expression. A microarray experiment was also performed to identify genes that were down-regulated following treatment with miR-7 compared to NS precursor. Of 248 down-regulated genes, including EGFR, 37 promising new miR-7 target candidates were identified. Functional clustering of down-regulated genes and promising target candidates suggested that miR-7 may have functionally-related targets involved in processes including cell motility and brain-associated functions. This investigation thus yielded a program capable of accurately predicting a miRNA target not predicted by any other target prediction program, verified a previously unknown miRNA:target interaction with functional consequences in cancer cells and provided the first steps towards investigating miR-7-mediated regulation in greater depth. Furthermore, EGFR was, to our knowledge, the first example of a verified miRNA target with target sites that are not conserved across mammals, an observation with important implications for computational target prediction and the evolution of miRNA regulatory systems. In addition, the demonstrated growth inhibitory and cytotoxic effects of miR-7 on lung cancer cells raise the possibility of a miR-7-based therapeutic for the treatment of EGFR-overexpressing tumours.

INTRODUÇÃO: Atualmente as células da microglia têm recebido grande atenção dentro da resposta imune, isto devido ao fato de que sua ativação por citocinas inflamatórias é capaz de promover a infiltração e destruição do sistema nervoso central (SNC) durante algumas doenças, principalmente no caso da esclerose múltipla (EM). Além de seu papel pró-inflamatório, já demonstrou-se que estas também são capazes de expressar moléculas supressoras como a indoleamina-2,3-dioxigenase (IDO), capaz de suprimir a proliferação de células T. Contudo, ainda pouco se sabe sobre seu verdadeiro papel na patogenia da EM. Recentemente tem sido descrita uma população de células T chamadas Th17, capaz de secretar grandes quantidades de IL-17, IL-21 e GM-CSF possuindo uma importância fundamental na patogenia da EM e de seu modelo murino, a EAE. Nesse contexto, a relação entre as Th17 e as células da microglia pode nos fornecer dados importantes acerca dos mecanismos envolvidos nas lesões observadas no SNC. OBJETIVO: Este trabalho teve como objetivo melhor elucidar a relação existente entre a expressão das moléculas imunes por células da microglia e a ação que estas promovem sobre as células T. MÉTODOS: Utilizamos culturas de células da microglia de linhagem, chamadas C8-B4, assim como cultura primária de células da microglia obtidas a partir sistema nervoso de camundongos C57BL/6 adultos. Caracterizamos o perfil imune da microglia, avaliando a transcrição de genes para citocinas através de PCR em tempo real assim como a expressão de suas moléculas ativadoras por citometria de fluxo. A avaliação da IDO se deu através da expressão da mesma por células da microglia ativadas ou não por LPS ou IFN-?. Ja sua capacidade funcional foi medida através da atividade proliferativa de linfócitos T CD4 específicos para MOG 35-55. RESULTADOS: Nossos resultados demonstraram que as células de ambas as culturas possuem a capacidade de expressar diversas moléculas imunes, tanto pró quanto anti-inflamatórios. Dentre estas observamos TLR-4, TLR-2, IL-6, IL-10 e TGF-?. Além disso, confirmamos a expressão da enzima IDO por estas células. O bloqueio de tal enzima impede o controle que a microglia tem sobre a proliferação dos linfócitos T CD4, tanto in vitro quanto in vivo. No modelo in vivo tal efeito repercute em uma encefalomilite mais severa, onde o quadro clínico do animal não regride. CONCLUSÃO: Os resultados aqui obtidos nos dão a certeza da influência das microglias dentro do contexto inflamatório, afirmando sua capacidade de modular a resposta imune. Além disto, fica clara a importância da enzima IDO, cuja ação dentro do controle de uma autoimunidade demonstra ser altamente necessária / INTRODUCTION: Microglia cells has gained great attention recently because its activation by inflammatory cytokines can promote infiltration and destruction of Central Nervous System (CNS) during some disease, mainly in the case of Multiple Sclerosis (MS). On the other hand, these cells may also express suppressor molecules such as the indoleamine-2,3-dioxygenase (IDO), able to suppress T cell proliferation. However, still little is known about its role in MS pathogenesis. Recently it has been described a new population of T cells called Th17, able to secrete high amounts of IL-17, IL-21 and GM-CSF, with a fundamental importance on MS and its murine model, EAE. In this context, the relationship between Th17 and microglia cells can provide us important data about the mechanisms involved in the establishment of CNS lesions. OBJECTIVES: This work had the objective to better elucidate the relationship between the expression of some molecules by microglia and its role T cell activation. METHODS: Through a cellular lineage knowing as C8-B4 and primary cultures of microglia obtained from CNS of adult mice C57BL6 we investigated the transcription of several genes for cytokines and membrane expression of several pattern recognition receptors. The IDO evaluation was performed after activation with LPS or rIFN-?. Its functional capacity was measured trough its action over T cell proliferation. RESULTS: Our results demonstrated that both cells have the capacity of express several immune molecules, both pro and anti-inflammatory. Among this, we observed TLR-4, TLR-2, IL-6, IL-10 and TGF-?. We also confirmed IDO expression by these cells. The blockade of such enzyme prevents the control of microglia above T CD4 lymphocytes proliferation, both in vitro and in vivo. Using the in vivo model, IDO blocker rendered a encephalomyelitis more severe. Conclusion: The results here obtained give us the certainty of microglia influence in inflammatory context, stating its capacity of modulating the immune response

Le cancer du sein est le cancer le plus fréquent chez les femmes. Malgré les progrès thérapeutiques, les mécanismes de résistance restent la cause de la morbidité et de la mortalité. L'acide lipoïque (LA) est un cofacteur essentiel du métabolisme oxydatif via sa fonction dans les complexes de pyruvate déshydrogénase et d'α-céto déshydrogénase. Il a été démontré des effets anticancéreux, mais ses mécanismes d'action ne sont pas entièrement compris. Mon projet de thèse vise à évaluer l’effet inhibiteur de LA sur la prolifération de diverses lignées cellulaires du cancer du sein et d’étudier ses mécanismes. Nos résultats ont montré que LA inhibe la prolifération cellulaire en inhibant les voies de signalisation prolifératives PI3K/Akt et MAPK/ERK. Nos résultats ont contribué à une meilleure compréhension des mécanismes d’action conduisant à cet effet anti-prolifératif de LA. En effet, nous avons mis en évidence la réduction de l’expression de la proprotéine convertase, furine, responsable de la maturation d’IGF-1R en réponse à LA aboutissant in fine à l’inhibition de la maturation de ce récepteur. En outre, nous avons démontré que l’effet pro-oxydant de LA aboutit à la réduction de l’expression du facteur de transcription, CREB, qui est impliqué dans l’induction de l’expression de la furine. En conclusion, nous avons élucidé, pour la première fois, le mécanisme d’action suivant : LA induit rapidement et brutalement des ROS par la phosphorylation oxydative qui vont inhiber l’expression du facteur de transcription CREB. Cette inhibition bloque alors l’activation transcriptionnelle de la furine, qui est cruciale pour permettre le passage de la pro-forme d’IGF-1R, qui est immature et non fonctionnelle, à la forme mature, IGF-1R qui est alors recrutée à la membrane plasmique / Breast cancer is the most common cancer in women. Despite therapeutic advances, the mechanisms of resistance remain the underlying cause of morbidity and mortality. Lipoic acid (LA) is and an essential cofactor of oxidative metabolism via its function in pyruvate dehydrogenase and α-keto dehydrogenase complexes. Its potential therapeutic effects have been well documented in the treatment of pathologies associated with oxidative stress, such as diabetes, atherosclerosis, liver diseases and neurodegenerative diseases. In addition, it has been demonstrated its anticancer effects in various cancers, but its mechanisms of action are not fully understood. My PhD project aims to evaluate the inhibitory effect of LA on the proliferation of various breast cancer cell lines and to study the mechanisms of action likely to be involved in this process. Our results showed that LA inhibits cell proliferation by inhibiting PI3K/Akt and MAPK/ERK proliferative signaling. Our results contributed to a better understanding of the mechanisms of action leading to this anti-proliferative effect of LA. Indeed, we have demonstrated the reduction of the expression of the proprotein convertase, furin, responsible for the maturation of IGF-1R in response to LA resulting the inhibition of the maturation of this receptor. In addition, we have demonstrated that the pro-oxidative effect of LA reduces the the expression of the transcription factor, CREB, which is involved in the induction of furin expression. In conclusion, we demonstrated for the first the following mechanism of action: LA rapidly induces ROS by oxidative phosphorylation that inhibit the expression of the CREB transcription factor. This inhibition then blocks the transcriptional activation of furin, an enzyme that is crucial to allow the passage of the IGF-1R pro-form, which is immature and non-functional, to the mature form, IGF-1R, which is then recruited to the plasma membrane