Glioblastoma multiforme (GBM) is an aggressive cancer that affects millions of patients per year. Conventional therapies combining chemotherapeutic agents with radiation can only extend survival by a few months; therefore, there is a dire need for an effective means of treating this deadly disease. Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), currently in the early stages of FDA pre-IND drug trials, has proven to be an effective cancer specific cytokine, able to trigger the onset of mitochondrial dysfunction and/or autophagy. GBM’s have mutations that often result in the activation of cytoprotective cell signaling pathways, preventing cancer therapeutics and even MDA-7/IL-24 treatments from being effective. Since the discovery of MDA-7/IL-24 a number of groups have shown toxic effects in a variety of tumor cells. However, the lethality of MDA-7/IL-24 is not enough to eradicate the tumor. We hypothesized two xxiii rationales for this minimalistic effect. First, the MDA-7/IL-24 gene delivery mechanisms are not efficient or second, active pro-survival pathways are playing a role in protection. Here we have shown that the inhibition of cytoprotective cell-signaling pathways using small molecule inhibitors of mitogen-activated extracellular regulated kinase (MEK)1/2 and phosphatidyl inositol 3-kinase (PI3K) or AKT; mammalian target of rapamycin (mTOR) and MEK1/2; HSP90 inhibitor 17AAG; and the autophagy-inducing drug OSU-03012 (AR-12), enhances the toxicity of MDA-7/IL-24. In addition, the use of a modified recombinant adenovirus comprised of the tail and shaft domains of a serotype 5 virus and the knob domain of a serotype 3 virus expressing MDA-7/IL-24, Ad.5/3-mda-7, proved to be a more effective, CAR-independent means of infecting and killing GBM cells in vitro and in vivo when compared to Ad.5-mda-7. Collectively, our data demonstrate that the induction of autophagy and mitochondrial dysfunction by a combinatorial treatment approach represents a potentially viable strategy to kill primary human GBM cells.

Ovarian cancer is the most lethal gynecological malignancy among women. The current first-line treatments for ovarian cancer are cisplatin, carboplatin and paclitaxel. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Therefore, the resistance to cisplatin therapy has been a critical hurdle in the management of recurrent ovarian cancer. The mechanisms responsible for cisplatin resistance are not completely understood. In the search for new therapies to overcome/bypass cisplatin resistance, melanoma differentiation gene-7 (MDA-7) IL-24, which is a new cytokine, has anti-cancer efficacy by suppressing cell growth and inducing apoptosis in a broad range of tumor cells and does not induce any toxicity in normal cells, thus, making it a potentially effective therapeutic gene for ovarian cancer. The purpose of this study was to evaluate the potential therapeutic efficacy of MDA-7 to treat ovarian carcinoma. Since adenoviral-mediated MDA-7 gene therapy has been shown to be well tolerated and showed biological activity in clinical studies in the context of other carcinomas we assessed the anticancer effects of Ad.mda-7 and in combination with cis-platinum on ovarian cancer cells. Our results show that the purified recombinant MDA-7 protein, GST-MDA-7, and Ad.mda7 virus (5) induced growth arresst and apoptosis in ovarian cancer cells. However, the apoptosis induction was low and directly correlated with infectivity of Ad.mda-7 virus (5). The use of a modified Ad.mda-7 virus type5, Ad.mda-7 virus type(5/3), inhanced infectivity and significantly enhanced ovarian cancer cell killing in human ovarian cancer cell lines in vitro compared to unmodified Ad.mda-7 virus, Ad.mda-7 virus type5. Also Ad-mda7 synergizes with cis-platinum in vitro and enhances ovarian cancer cell death. Taken together, these findings demonstrate that MDA-7 is capable of promoting growth suppression and inducing cell death in ovarian cancer cells, at least OVCAR cells and support the pharmacological interest of the combination of MDA-7 and cis-platinum.

Melanoma differentiation associated gene-7 (mda-7) was discovered in the Fisher laboratory by subtraction hybridization of temporally spaced subtracted cDNA libraries prepared from terminally differentiated human melanoma cells treated with human fibroblast interferon (IFN-β) and the protein kinase C activator mezerein (MEZ), an approach called ‘differentiation induction subtraction hybridization’ (DISH). mda-7 is located in human chromosome 1q32–33 and based on sequence homology, chromosomal localization, and its functional properties, the mda-7 gene is now classified as a member of the IL-10 family of cytokines and named IL-24. The mda-7/IL-24 cDNA encodes a protein of 206-amino acids with a predicted size of ~24-kDa, which contains an interleukin (IL)-10 signature motif at amino acids 101–121 (SDAESCYLVHTLLEFYLKTVF) shared by other members of the IL-10 family of cytokines. Sequence analysis revealed the presence of a 49-amino acid signal peptide suggesting that the molecule could be cleaved and secreted. Expression of MDA-7/IL-24 protein was detected in cells of the immune system (mainly by expression in tissues associated with the immune system, such as spleen, thymus and PBMC) and normal human melanocytes. Of interest, a progressive loss of MDA-7/IL-24 expression during melanoma progression suggests an inverse relationship between MDA-7/IL-24 expression and the evolution of melanocytes to various stages of melanoma. mda-7/IL-24 induces growth suppression in human melanoma and other cancer cells, without affecting normal cells. Subsequent studies provided consistent evidence that ectopic expression of mda-7/IL-24 employing a replication incompetent adenovirus (Ad.mda-7) resulted in apoptosis induction and cell death in a wide variety of solid tumors including melanoma, malignant glioma, carcinomas of the breast, kidney, cervix, colorectum , liver, lung, ovary and prostate sparing normal cellular counterparts, i.e., such as normal melanocytes, astrocytes, fibroblasts, and mesothelial and epithelial cells. The in vitro antitumor activity of mda-7/IL-24 readily translated into the in vivo situation in animal models containing human breast, prostate, lung and colorectal carcinomas and in malignant glioma xenografts. Moreover, the ability of mda-7/IL-24 to induce a potent “bystander cancer-specific killing effect” provides an unprecedented opportunity to use this molecule to target for destruction not only primary tumors, but also metastases. Based on its profound cancer-selective tropism, substantiated by in vivo human xenograft studies in nude mice, mda-7/IL-24 (administered as Ad.mda-7) was evaluated in a Phase I clinical trial in patients with melanomas and solid cancers. These studies document that mda-7/IL-24 is well tolerated and demonstrates evidence of significant (44%) clinical activity. This review focuses on the recent enhancements in our understanding of the mode of action of mda-7/IL-24 and its potential applications as a unique and promising effective cytokine-based gene therapy for human cancers. The first chapter explored the efficacy of a tropism-modified Ad-based cancer gene therapy approach for eradicating low CAR colorectal cancer cells. We show that in low CAR human colorectal cancer cells (RKO), a recombinant Ad.5/3 virus delivering mda-7/IL-24 (Ad.5/3-mda-7) is more efficient than Ad.5 delivering mda-7 (Ad.5-mda-7) in expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis and inhibiting in vivo tumor growth in a nude mouse xenograft model. Additionally, our in vitro and in vivo data confirms that BI-97C1 (Sabutoclax) profoundly sensitizes mda-7/IL-24 mediated toxicity in colorectal cancer. Thus, Ad.5/3-mda-7, alone and/or in combination with BI-97C1 (Sabutoclax), might represent an improved and more effective therapeutic approach for colorectal and other cancers. In view of the essential roles of anti-apoptotic Bcl-2 family proteins in tumorigenesis and chemoresistance, efforts are focused on developing small molecule inhibitors of Bcl-2 family proteins as potential therapeutics for cancer. Unfortunately, due to the unique structure of Mcl-1 as compared with Bcl-2 and Bcl-xL, currently employed inhibitors, such as ABT-737 or its clinical counterpart, ABT-263, display limited affinity for Mcl-1. Using nuclear magnetic resonance (NMR) binding assays and computational docking studies, we have recently identified a series of new Apogossypol derivatives, compound 3 (BI-79D10) and compound 11 (BI-97C1), with pan-Bcl-2- inhibitory potency. BI-79D10 binds to Bcl- xL, Bcl-2, and Mcl-1 with IC50 values of 190, 360, and 520 nmol/L, respectively. BI-97C1 (Sabutoclax) is an optically pure individual Apogossypol derivative that retains all the properties of BI-79D10 along with superior in vitro and in vivo efficacy. Because Mcl-1 is over-expressed in the majority of PCs, we hypothesized that suppressing Mcl-1 by treating human PC cells with BI-97C1 (Sabutoclax) would sensitize them to mda-7/IL-24-mediated cytotoxicity. The second chapter study highlights the noteworthy potential of a combinatorial approach involving mda-7/IL-24, a broad-acting anticancer gene, and BI-97C1 (Sabutoclax), which targets Mcl-1, to sensitize PC to mda-7/IL-24-mediated cytotoxicity, thereby enhancing therapeutic efficacy. Our data suggests that treatment with the combination regimen of mda-7/IL-24 and BI-97C1 (Sabutoclax) induces autophagy that facilitates apoptosis in association with up regulation of NOXA, accumulation of Bim, and activation of Bax and Bak. Treatment with mda-7/IL-24 and BI-97C1 (Sabutoclax) inhibited the growth of PC xenografts and suppressed PC development in an immunocompetent transgenic mouse model of PC. The third chapter study explored the efficacy of a tropism-modified CRCA cancer gene therapy approach for eradicating low CAR prostate cancer cells. We showed that in low CAR PC3 cells Ad.5/3-CTV is more efficient than Ad.5-CTV in delivering transgene (mda-7/IL-24), infecting tumor cells, expressing MDA-7/IL-24 protein, inducing cancer-specific apoptosis, inhibiting in vivo tumor growth and exerting an antitumor ‘bystander’ effect in a nude mouse human prostate cancer xenograft and suppressed PC development in an immunocompetent transgenic mouse model of PC model.

The most common malignant glioma, glioblastoma multiforme (GBM), remains an intractable tumor despite advances in therapy. Its proclivity to infiltrate surrounding brain tissue contributes greatly to its treatment failure and the grim prognosis of patients. Radiation is a staple in modern therapeutic regimens, though cells surviving radiation become more aggressive and invasive. Consequently, it is imperative to define further the cellular mechanisms that control GBM invasion and identify promising novel therapeutic targets. Melanoma differentiation associated gene-9 (MDA-9/Syntenin) is a highly conserved PDZ domain-containing scaffolding protein that promotes invasion and metastasis in human melanoma models. We show that MDA-9/Syntenin is robustly expressed in GBM cell lines and patient samples, and expression increases by tumor grade. These findings are confirmed through database analysis, which revealed MDA-9/Syntenin expression correlates with shorter survival times and patient tumors high in MDA-9/Syntenin have a worse prognosis when undergoing radiotherapy. Modulating MDA-9/Syntenin levels produced changes in invasion, angiogenesis, and signaling, indicating MDA-9/Syntenin enhances glioma pathogenesis. Overexpression of MDA-9/Syntenin enhances invasion, while knockdown inhibits invasion, migration, and anchorage-independent growth in soft agar. MDA-9/Syntenin increases activation of c-Src, P38MAPK, and NF-kB, leading to elevated MMP2 expression and IL-8 secretion. Through an orthotopic tumor model, we show that shmda-9 tumor cells formed smaller tumors and had a less invasive phenotype in vivo. Knockdown of MDA-9/Syntenin radiosensitizes GBM cells and significantly reduces post-radiation invasion gains through abrogation of radiation-induced Src and EphA2 activity. In efforts to pharmacologically inhibit MDA-9/Syntenin, we describe the effects of a novel small molecule, PDZ1i, which targets the PDZ1 domain of MDA-9/Syntenin and successfully reduces invasion gains in GBM cells following radiation. While it does not effect astrocyte radiosensitivity, PDZ1i radiosensitizes GBM cells. PDZ1i inhibits crucial GBM signaling including FAK and mutant EGFR, EGFRvIII, and can negate gains in secreted proteases, such as MMP2 and MMP9, following radiation. In a model of glioma, PDZ1i treatment combined with radiation results in less invasive tumors and extends survival. Our findings indicate that MDA-9/Syntenin is a novel and important mediator of GBM pathogenesis, and further identify it as a targetable protein that enhances radiotherapy for treatment in glioma.

No description available.

As transformações de modelos são um ponto vital para os enfoques de desenvolvimento dirigido por modelos, incluindo a MDA. No presente trabalho apresenta-se alguns dos conceitos da MDA relacionados a transformações de modelos. São vistos os conceitos de PIM, PSM, transformações, mapeamentos e marcas. Estes conceitos são analisados no contexto de três ferramentas MDA, a OptimalJ, a AndroMDA e a ArcStyler, para verificar se as ferramentas os implementam de acordo com a MDA. Também é analisado como e quais transformações e mapeamentos de modelos estão sendo implementados pelas ferramentas, através da comparação qualitativa de definições de conceitos e de exemplos do uso destes. Além de verificar os conceitos relacionados às transformações, faz-se um estudo de caso qualitativo para analisar as transformações e mapeamentos providos pelas ferramentas e como as ferramentas trabalham com eles. Mostram-se as dificuldades de se criar um modelo PIM do sistema do estudo de caso e adaptá-lo para cada uma das ferramentas. / Model transformations are a vital point for model driven development, including MDA. In the present essay we introduce some of the MDA concepts related with model transformations. We present the concept of PIM, PSM, transformations, mappings and marks. These concepts are analyzed in the context of three MDA tools, OptimalJ, AndroMDA and ArcStyler, to verify if the tools implement the concepts according to MDA. We also analyze how and which model transformations and model mappings are being implemented by the tools, through a qualitative comparison of the definition of these concepts and using some examples of their intended use. In addition to verifying the concepts related to transformations, we create a qualitative case study to analyze the model transformations and model mappings provided by the tools, and how the tools work with them. We show a few of the problems faced when creating a PIM model of the case study\'s system and the adaptations it required for each tool.

As transformações de modelos são um ponto vital para os enfoques de desenvolvimento dirigido por modelos, incluindo a MDA. No presente trabalho apresenta-se alguns dos conceitos da MDA relacionados a transformações de modelos. São vistos os conceitos de PIM, PSM, transformações, mapeamentos e marcas. Estes conceitos são analisados no contexto de três ferramentas MDA, a OptimalJ, a AndroMDA e a ArcStyler, para verificar se as ferramentas os implementam de acordo com a MDA. Também é analisado como e quais transformações e mapeamentos de modelos estão sendo implementados pelas ferramentas, através da comparação qualitativa de definições de conceitos e de exemplos do uso destes. Além de verificar os conceitos relacionados às transformações, faz-se um estudo de caso qualitativo para analisar as transformações e mapeamentos providos pelas ferramentas e como as ferramentas trabalham com eles. Mostram-se as dificuldades de se criar um modelo PIM do sistema do estudo de caso e adaptá-lo para cada uma das ferramentas. / Model transformations are a vital point for model driven development, including MDA. In the present essay we introduce some of the MDA concepts related with model transformations. We present the concept of PIM, PSM, transformations, mappings and marks. These concepts are analyzed in the context of three MDA tools, OptimalJ, AndroMDA and ArcStyler, to verify if the tools implement the concepts according to MDA. We also analyze how and which model transformations and model mappings are being implemented by the tools, through a qualitative comparison of the definition of these concepts and using some examples of their intended use. In addition to verifying the concepts related to transformations, we create a qualitative case study to analyze the model transformations and model mappings provided by the tools, and how the tools work with them. We show a few of the problems faced when creating a PIM model of the case study\'s system and the adaptations it required for each tool.

Thesis (M.A. (English) -- University of Limpopo, 2010 / This dissertation examines the way in which two South African novelists, Zakes Mda and J.M. Coetzee, portray the sexual abuse of women during the apartheid and post-apartheid eras. The two selected novels used in this research are The Madonna of Excelsior (2002) by Mda and Disgrace (2000) by Coetzee. The research, furthermore, analyses the attitudes of the sexual abusers and their victims in both eras as well as examines the effects of socio-economic imbalances that might have prompted the men to sexually abuse women whom they should be offering protection against any form of violence and abuse. The study furthermore identifies and analyses the factors that hinder victims from reporting their sexual abuse to the police. Finally, this dissertation also presents a comparative study of the differences and the similarities between the way the two novelists portray sexual abuse.

Melanoma differentiation associated gene 7/Interleukin-24 (MDA-7/IL-24) is a secreted cytokine which acts as a tumor suppressor. It is capable of selectively killing cancer cells, regardless of anatomic origin, while sparing normal cells. miRNAs are master regulators of gene expression that can play two roles in cancer: tumor-suppression and oncogenesis. We identified a number of miRNAs that are regulated by MDA-7/IL-24 using a PCR plate array containing probes for miRNAs known to play a role in prostate cancer. We independently validated the array with qRT-PCR to identify three miRNAs which are downregulated by MDA-7/IL-24 treatment in DU145, PC3, and PC3ML prostate cancer lines. These miRNAs were miR-125a, miR-145, and miR-23b. Their gene targets were identified using TargetScan and confirmed to be regulated in our prostate cancer model. NLRC5, KLF4, and KLF15, respectively, were upregulated after treatment with MDA-7/IL-24. We focused on NLRC5 as a novel target of MDA-7/IL-24, which plays a role in immune evasion by cancer cells. NLRC5 is upregulated following inhibition of miR-125a. It is not downregulated by overexpression of miR-125a which suggests that more than one miRNA may be acting to regulate its expression. Finally, we determined that miR-125a is downregulated by MDA-7 through DICER, an important processing enzyme for miRNA biogenesis.

The Unified Modelling Language (UML) is intended to express complex ideas in an intuitive and easily understood way. It is important because it is widely used in software engineering and other disciplines. Although an official definition document exists, there is much debate over the precise meaning of UML models. ¶ In response, the academic community have put forward many different proposals for formalising UML, but it is not at all obvious how to decide between them. Indeed, given that UML practitioners are inclined to reject formalisms as non-intuitive, it is not even obvious that the definition should be “formal” at all. Rather than searching for yet another formalisation of UML, our main aim is to determine what would constitute a good definition of UML. ¶ The first chapter sets the UML definition problem in a broad context, relating it to work in logic and the philosophy of science. More specific conclusions about the nature of model driven development are reached in the beginning of Chapter 2. We then develop criteria for a definition of UML. Applying these criteria to the existing definition, we find that it is lacking in clarity. We then set out to test the precision of the definition. The test is to take an apparently inconsistent model, and determine whether it really is inconsistent according to the definition. ¶ Many people have proposed that UML models are graphs, but few have justified this choice using the official definition of UML. We begin Chapter 3 by arguing from the official definition that UML models are graphs and that instantiation is a graph homomorphism into an interpretation functor. The official definition of UML defines the semantics against its abstract syntax, which is in turn defined by a UML model. Chapters 3 and 4 prepare for our test by resolving this apparent circularity. The result is a semantics for the metamodel fragment of the language. ¶ In Chapter 5, we find, contrary to popular belief, that the official definition does provide sufficient semantics to classify the example model as inconsistent. Moreover, the sustained study of the semantics in Chapters 3 to 5 confirms our initial argument that the semantic domain is graphs. The Actions are the building blocks of UML’s prescriptive dynamics. We see that they can be naturally defined as graph transformation rules. Sequence diagrams are the main example of descriptive dynamics, but we find that their official semantics are broken. The “recorded history” approach should be replaced, we suggest, by a graph-oriented dynamic logic. ¶ Chapter 6 presents our early work on dynamic logic for UML sequence diagrams and further explores the proposed semantic repairs. In Chapter 7, guided by the criteria developed in Chapter 2, we critically survey the UML formalisation literature and conclude that an existing body of graph transformation based work known as “dynamic metamodelling” is very close to what is required. ¶ The final chapter draws together our conclusions. It proposes a category theoretic construction to merge models of the syntax and semantic domain, yielding a type graph for the graph transformation system which defines the dynamic semantics of the language. Finally, it outlines the further work required to realise a satisfactory definition of UML.