Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^

Cellular Uptake of Neutrohpil Elastase Links Inflammation to Adaptive Immunity

Many tumors arise from sites of inflammation providing evidence that innate immunity is a critical component in the development and progression of cancer. Neutrophils are primary mediators of the innate immune response. Upon activation, an important function of neutrophils is release of an assortment of proteins from their granules including the serine protease neutrophil elastase (NE). The effect of NE on cancer has been attributed primarily to its ability to degrade the extracellular matrix thereby promoting invasion and metastasis. Recently, it was shown that NE could be taken up by lung cancer cells leading to degradation of insulin receptor substrate-1 thereby promoting hyperactivity of the phosphatidylinositol-3 kinase (PI3K) pathway and tumor cell proliferation. To our knowledge, nobody has investigated uptake of NE by other tumor types. In addition, NE has broad substrate specificity suggesting that uptake of NE by tumor cells could impact processes regulating tumorigenensis other than activation of the PI3K pathway. Neutrophil elastase has been identified in breast cancer specimens where high levels of NE have prognostic significance. These studies have assessed NE levels in whole tumor lysates. Because the major source of NE is from activated neutrophils, we hypothesized that breast cancer cells do not have endogenous NE but may take up NE released by tumor associated neutrophils in the tumor microenvironment and that this could provide a link between the innate immune response to tumors and specific adaptive immune responses. In this thesis, we show that breast cancer cells lack endogenous NE expression and that they are able to take up NE resulting in increased generation of low molecular weight cyclin E (CCNE) and enhanced susceptibility to lysis by CCNE-specific cytotoxic T lymphocytes. We also show that after taking up NE and proteinase 3 (PR3), a second primary granule protease with significant homology to NE, breast cancer cells cross-present the NE- and PR3-derived peptide PR1 rendering them susceptible to PR1-targeted therapies. Taken together, our data support a role for NE uptake in modulating adaptive immune responses against breast cancer.

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

Assessing high-priority mixtures: A review of methanol as influenced by ethanol

The intent of this research was to identify the level of risk methanol posed to a fetus during an ethanol co-exposure. This investigation was prompted by the known competitive inhibition properties of ethanol and the developmental toxicity of methanol. Integrated into this research was the practicality necessitated by regulatory processes, namely: does the risk justify the expense of additional research. To this end, the scope and nature of exposures were summarized to illustrate the ubiquity of these chemicals and the potential for dual exposure. Similarly, severity of outcome was evaluated by systematically reviewing the LOAELs, NOAELs, and statistical significance contained in methanol-induced developmental studies. Results. Blood methanol levels corresponding to developmental effects in laboratory studies were found to be substantially higher than the blood methanol levels predicted in high-risk methanol-ethanol exposure scenarios. This indicates that ethanol would not likely exacerbate methanol toxicity to the point of teratogenicity; however, it is important to note that the developmental toxicity of ethanol—an established human teratogen—was not included in the evaluation. Ethanol's contribution as a developmental toxicant rather than merely as an attenuator of methanol toxicity undermines the severity of effects possible from this chemical combination. Therefore further evaluation is needed to assess the developmental toxicities following dual exposures before rendering methanol and ethanol a high-priority mixture.^

In vitro propagation of Glandularia peruviana (L.) Small, an ornamental native plant from South America

The flower market is characterized by being both eager for novelties and highly competitive. The exploration of native species with ornamental potential represents a remarkable area of research, since it entails the introduction and development of novel promising ornamental crops. The genus Glandularia, widely distributed in Argentina, holds an enormous ornamental potential, due to the variety of colors of its inflorescences (red, violet, white, rose and lily), and extended flowering period. There is little information on tissue culture of Glandularia, thus highlighting the relevance of this research. In this work, the conditions for in vitro multiplication of G. peruviana were optimized. It was concluded that WPM supplemented with TDZ, in concentrations ranging from 1.1 to 9.0 μM, was the most adequate treatment, rendering a multiplication rate of approximately 10 de novo shoots per explant. This paper presents a protocol for the in vitro propagation of this species and introduces interesting prospects in the application of biotechnological tools to breed Glandularia.

Results of magnetic analysis

Microorganisms are a primary control on the redox-induced cycling of iron in the environment. Despite the ability of bacteria to grow using both Fe(II) and Fe(III) bound in solid-phase iron minerals, it is currently unknown if changing environmental conditions enable the sharing of electrons in mixed-valent iron oxides between bacteria with different metabolisms. We show through magnetic and spectroscopic measurements that the phototrophic Fe(II)-oxidizing bacterium Rhodopseudomonas palustris TIE-1 oxidizes magnetite (Fe3O4) nanoparticles using light energy. This process is reversible in co-cultures by the anaerobic Fe(III)-reducing bacterium Geobacter sulfurreducens. These results demonstrate that Fe ions bound in the highly crystalline mineral magnetite are bioavailable as electron sinks and electron sources under varying environmental conditions, effectively rendering a naturally occurring battery.

Clarice Lispector e o simbolismo da montanha : Retrato de um ser entre dois mundos

Clarice Lispector, en su relato 'Onde estivestes de noite', produce con su divinidad andrógina El-ella y con sus seguidores un movimiento que aborda la sexualidad a partir de nuevos paradigmas y configura una escritura en que el hombre es retratado como un ser irremediablemente escindido entre naturaleza y cultura, entre deseo y razón. Esa dicotomía se extiende a otros personajes clariceanos aquí rescatados, cuya particular caracterización psicológica posibilita un análisis basado en los postulados psicoanalíticos, a partir del texto Cinco lecciones de psicoanálisis de Sigmund Freud.

School counsellor's roles in minimising adolescents' attrition from schools

Counselling ways (rendering financial advice, providing encouragement, reassurances and support), which the school counselllors could adopt to help the school system minimize the increasing incidence of school dropout, were advanced.

La sinrazón de mi vida : Ficción autobiográfica, relato antiestatal y peronismo en La madriguera de Tununa Mercado

La presente investigación se enmarca en el proyecto mayor de nuestro plan de Doctorado en Letras. Luego de realizar una breve presentación de la obra de Tununa Mercado, procedemos a plantear nuestra hipótesis de trabajo: la de que a partir de la publicación de su libro La madriguera (1996) una ficción autobiográfica de infancia (categoría que involucra variables cuya fiabilidad han sido fuertemente sospechadas y cuestionadas por líneas teóricas desde el giro lingüístico en adelante) la autora funda un modo de réplica literaria (Avellaneda, 1983) al gobierno menemista y su ideología, bajo el procedimiento de una operación sustitutoria, desviada, en la que alude al gobierno de Juan Domingo Perón. Narrando y aludiendo a ese gobierno, desestabiliza las ficciones estatales oficiales (en especial el texto de lectura obligatoria La razón de mi vida, 1951) que sirvieron para legitimarlo y producir cohesión política y social, al tiempo que realiza una respuesta cultural de resistencia a la ideología y las prácticas sociales menemistas. Al mismo tiempo, construye un sujeto autobiográfico femenino, lo que involucra operaciones fuertemente innovadoras en orden de imponer una torsión en un género de fuerte tradición patriarcal en Occidente (Klein, 2001). Como toda ficción del origen, La madriguera no escapa a ciertos relatos míticos, fundantes de la identidad, en especial de carácter letrado, e interroga las categorías de la percepción, fundando una nueva sensibilidad y problematizando así el dominio de lo real. Esta es su forma de dar cuenta de los conflictos de fundamento social y no la que opera a partir de una doxa tranquilizadora y deformadora propia de los relatos estatales.

Clarice Lispector e o simbolismo da montanha : Retrato de um ser entre dois mundos

Clarice Lispector, en su relato 'Onde estivestes de noite', produce con su divinidad andrógina El-ella y con sus seguidores un movimiento que aborda la sexualidad a partir de nuevos paradigmas y configura una escritura en que el hombre es retratado como un ser irremediablemente escindido entre naturaleza y cultura, entre deseo y razón. Esa dicotomía se extiende a otros personajes clariceanos aquí rescatados, cuya particular caracterización psicológica posibilita un análisis basado en los postulados psicoanalíticos, a partir del texto Cinco lecciones de psicoanálisis de Sigmund Freud.

School counsellor's roles in minimising adolescents' attrition from schools

Counselling ways (rendering financial advice, providing encouragement, reassurances and support), which the school counselllors could adopt to help the school system minimize the increasing incidence of school dropout, were advanced.

La sinrazón de mi vida : Ficción autobiográfica, relato antiestatal y peronismo en La madriguera de Tununa Mercado

La presente investigación se enmarca en el proyecto mayor de nuestro plan de Doctorado en Letras. Luego de realizar una breve presentación de la obra de Tununa Mercado, procedemos a plantear nuestra hipótesis de trabajo: la de que a partir de la publicación de su libro La madriguera (1996) una ficción autobiográfica de infancia (categoría que involucra variables cuya fiabilidad han sido fuertemente sospechadas y cuestionadas por líneas teóricas desde el giro lingüístico en adelante) la autora funda un modo de réplica literaria (Avellaneda, 1983) al gobierno menemista y su ideología, bajo el procedimiento de una operación sustitutoria, desviada, en la que alude al gobierno de Juan Domingo Perón. Narrando y aludiendo a ese gobierno, desestabiliza las ficciones estatales oficiales (en especial el texto de lectura obligatoria La razón de mi vida, 1951) que sirvieron para legitimarlo y producir cohesión política y social, al tiempo que realiza una respuesta cultural de resistencia a la ideología y las prácticas sociales menemistas. Al mismo tiempo, construye un sujeto autobiográfico femenino, lo que involucra operaciones fuertemente innovadoras en orden de imponer una torsión en un género de fuerte tradición patriarcal en Occidente (Klein, 2001). Como toda ficción del origen, La madriguera no escapa a ciertos relatos míticos, fundantes de la identidad, en especial de carácter letrado, e interroga las categorías de la percepción, fundando una nueva sensibilidad y problematizando así el dominio de lo real. Esta es su forma de dar cuenta de los conflictos de fundamento social y no la que opera a partir de una doxa tranquilizadora y deformadora propia de los relatos estatales.

School counsellor's roles in minimising adolescents' attrition from schools

Counselling ways (rendering financial advice, providing encouragement, reassurances and support), which the school counselllors could adopt to help the school system minimize the increasing incidence of school dropout, were advanced.

Clarice Lispector e o simbolismo da montanha : Retrato de um ser entre dois mundos

Clarice Lispector, en su relato 'Onde estivestes de noite', produce con su divinidad andrógina El-ella y con sus seguidores un movimiento que aborda la sexualidad a partir de nuevos paradigmas y configura una escritura en que el hombre es retratado como un ser irremediablemente escindido entre naturaleza y cultura, entre deseo y razón. Esa dicotomía se extiende a otros personajes clariceanos aquí rescatados, cuya particular caracterización psicológica posibilita un análisis basado en los postulados psicoanalíticos, a partir del texto Cinco lecciones de psicoanálisis de Sigmund Freud.

La sinrazón de mi vida : Ficción autobiográfica, relato antiestatal y peronismo en La madriguera de Tununa Mercado

La presente investigación se enmarca en el proyecto mayor de nuestro plan de Doctorado en Letras. Luego de realizar una breve presentación de la obra de Tununa Mercado, procedemos a plantear nuestra hipótesis de trabajo: la de que a partir de la publicación de su libro La madriguera (1996) una ficción autobiográfica de infancia (categoría que involucra variables cuya fiabilidad han sido fuertemente sospechadas y cuestionadas por líneas teóricas desde el giro lingüístico en adelante) la autora funda un modo de réplica literaria (Avellaneda, 1983) al gobierno menemista y su ideología, bajo el procedimiento de una operación sustitutoria, desviada, en la que alude al gobierno de Juan Domingo Perón. Narrando y aludiendo a ese gobierno, desestabiliza las ficciones estatales oficiales (en especial el texto de lectura obligatoria La razón de mi vida, 1951) que sirvieron para legitimarlo y producir cohesión política y social, al tiempo que realiza una respuesta cultural de resistencia a la ideología y las prácticas sociales menemistas. Al mismo tiempo, construye un sujeto autobiográfico femenino, lo que involucra operaciones fuertemente innovadoras en orden de imponer una torsión en un género de fuerte tradición patriarcal en Occidente (Klein, 2001). Como toda ficción del origen, La madriguera no escapa a ciertos relatos míticos, fundantes de la identidad, en especial de carácter letrado, e interroga las categorías de la percepción, fundando una nueva sensibilidad y problematizando así el dominio de lo real. Esta es su forma de dar cuenta de los conflictos de fundamento social y no la que opera a partir de una doxa tranquilizadora y deformadora propia de los relatos estatales.