cJun NH2-terminal kinase (JNK) is a member of the MAPK (mitogen- activated protein kinase) signaling family that responds to various extracellular stimuli, such as stress, growth factors, cytokines, or UV radiation. JNK activation can lead to cellular responses including gene expression, growth, survival, and apoptosis. JNK has been implicated in normal developmental processes, including tissue morphogenesis, as well as pathological processes, such as cellular transformation and cancer. JNK exists in three isoforms, and knockout mice have been generated for each isoform; the ubiquitously expressed Jnk1 and Jnk2 have been studied independently, however, the two isoforms are partially functionally redundant. Jnk1-/- Jnk2-/-mice are nonviable, therefore studies of compound JNK-deficiency have been limited to mouse embryonic fibroblasts (MEF). Understanding the role of JNK in epithelial cells is now possible with the creation of conditional JNK knockout animals. I sought to elucidate the role of JNK in cellular transformation, cancer, and normal development. I employed both in vitro and in vivo approaches. First, I evaluated the role of JNK in cellular transformation using p53-/- Jnk1-/- Jnk2-/- MEF transduced with oncogenic Ras. To extend this study, I examined JNK-deficiency in a Kras-induced model of lung tumorigenesis. Second, I investigated JNK1- and JNK2-deficiency in a p53-mediated model of mammary tumorigenesis. Finally, I examined the role of JNK in mouse mammary gland development by establishing JNK-deficient primary mouse mammary epithelial cells and evaluating JNK-deficient mammary gland transplants. Taken together, this work provides evidence of context-dependent roles for JNK in both normal and pathological cell biology.

The central nervous system (CNS) is composed of two types of cells: neurons that send electrical signals to transmit information throughout the animal and glial cells. Glial cells were long thought to be merely support cells for the neurons; however, recent work has identified many critical roles for these cells during development and in the mature animal. In the CNS, glial cells act as the resident immune cell and they are responsible for the clearance of dead or dying material. After neuronal injury or death, glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. This rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery, but molecular pathways mediating these engulfment events remain poorly defined. Drosophila melanogaster is a genetically tractable model system in which to study glial biology. It has been shown that Drosophila glia rapidly respond to axonal injury both morphologically and molecularly and that they ultimately phagocytose the degenerating axonal debris. This glial response to axonal debris requires the engulfment receptor Draper and downstream signaling molecules dCed-6, Shark, and Rac1. However, much remains unknown about the molecular details of this response. In this thesis I show that Drosophila c-Jun kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, glial dJNK signals through a cascade involving the upstream MAPKKKs Slipper and TAK1, the MAPKK MKK4, and ultimately the Drosophila AP-1 transcriptional complex composed of JRA and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced up-regulation of Draper levels in glia and glial-specific over-expression of Draper was sufficient to rescue phenotypes associated with loss of dJNK signaling. I have identified the dJNK pathway as a novel mediator of glial engulfment activity and show that a primary role for the glial Slipper/Tak1→MKK4→dJNK→dAP-1 signaling cascade is activation of draper expression after axon injury.

The c-Jun NH2-terminal kinase (JNK) group and the p38 group of mitogen-activated protein kinases (MAPK) are stress-activated protein kinases that regulate cell proliferation, differentiation, development, and apoptosis. These protein kinases are involved in a signal transduction cascade that includes a MAP kinase (MAPK), a MAP kinase kinase (MAP2K), and a MAP kinase kinase kinase (MAP3K). MAPK are phosphorylated and activated by the MAP2K, which are phosphorylated and activated by various MAP3K. The work presented in this dissertation focuses on understanding the regulation and function of the JNK and p38 MAPK pathways. Two different strategies were utilized. First, I used molecular and biochemical techniques to examine how MAP2K and MAP3K mediate signaling specificity and to define their role in the MAPK pathway. Second, I used gene targeted disruption studies to determine the in vivo role ofMAP2K and MAP3K in MAPK activation. I specifically used these approaches to examine: (1) docking interactions between p38 MAPK and MAP2K [MKK3 and MKK6 (Chapter II)]; (2) the differential activation of p38 MAPK by MAP2K [MKK3, MKK4, and MKK6 (Chapter III)]; and (3) the selective involvement of the mixed lineage kinase (MLK) group of MAP3K in JNK and p38 MAPK activation (Chapter IV and Appendix). In addition, I analyzed the role of the MKK3 and MKK6 MAP2K in cell proliferation and the role of the MLK MAP3K in adipocyte differentiation (Chapter III and Chapter IV). Together, these data provide insight into the regulation and function of the stress-activated MAPK signal transduction pathways.

Arterial occlusive diseases are major causes of morbidity and mortality in industrialized countries and represent a huge economic burden. The extent of the native collateral circulation is an important determinant of blood perfusion restoration and therefore the severity of tissue damage and functional impairment that ensues following arterial occlusion. Understanding the mechanisms responsible for collateral artery development may provide avenues for therapeutic intervention. Here, we identify a critical requirement for mixed lineage kinase (MLK) – cJun-NH2-terminal kinase (JNK) signaling in vascular morphogenesis and native collateral artery development. We demonstrate that Mlk2-/-Mlk3-/- mice or mice with compound JNK-deficiency in the vascular endothelium display abnormal collateral arteries, which are unable to restore blood perfusion following arterial occlusion, leading to severe tissue necrosis in animal models of femoral and coronary artery occlusion. Employing constitutive and inducible conditional deletion strategies, we demonstrate that endothelial JNK acts during the embryonic development of collateral arteries to ensure proper patterning and maturation, but is dispensable for angiogenic and arteriogenic responses in adult mice. During developmental vascular morphogenesis, MLK – JNK signaling is required for suppression of excessive sprouting angiogenesis likely via JNK-dependent regulation of Dll4 expression and Notch signaling. This function of JNK may underlie its critical requirement for native collateral artery formation. Thus, this study introduces MLK – JNK signaling as a major regulator of vascular development. In contrast, we find that JNK in hematopoietic cells, which are thought to share a common mesodermally-derived precursor with endothelial cells, is cellautonomously dispensable for normal hematopoietic development and hematopoietic stem cell self-renewal, illustrating the highly context dependent function of JNK.

No / Zinc oxide (ZnO) nanoparticles are the mostly used engineered metal oxide nanoparticles in consumer products. This has increased the likelihood of human exposure to this engineered nanoparticle (ENPs) through different routes. At present, the majority of the studies concerning ZnO ENPs toxicity have been conducted using in vitro and in vivo systems. In this study, for the first time we assessed the effect of ZnO ENPs on the major cellular pathways in the lymphocytes of healthy individuals as well as in susceptible patients suffering from lung cancer, chronic obstructive pulmonary disease (COPD) and asthma. Using the differential expression analysis, we observed a significant (P < 0.05) dose-dependent (10, 20 and 40 microg/ml for 6h) increase in the expression of tumour suppressor protein p53 (40, 60 and 110%); Ras p21 (30, 52 and 80%); c-Jun N-terminal kinases; JNKs) (28, 47 and 78%) in lung cancer patient samples treated with ZnO ENPs compared to healthy controls. A similar trend was also seen in COPD patient samples where a significant (P < 0.05) dose-dependent increase in the expression of tumour suppressor protein p53 (26, 45 and 84%), Ras p21 (21, 40 and 77%), JNKs (17, 32 and 69%) was observed after 6h of ZnO ENPs treatment at the aforesaid concentrations. However, the increase in the expression profile of tested protein was not significant in the asthma patients as compared to controls. Our results reiterate the concern about the safety of ZnO ENPs in consumer products and suggest the need for a complete risk assessment of any new ENPs before its use.

O câncer de pele é um dos mais frequentes entre humanos, sendo o melanoma o tipo menos comum, mas com grande importância devido à agressividade que ele apresenta. Um dos principais agentes etiológicos deste tipo de tumor é a radiação ultravioleta proveniente da luz solar. A fração de radiação ultravioleta B (UVB) gera dano no DNA e induz alterações nas células da pele após a exposição prolongada e sem proteção. A resposta à luz UVB em melanócitos e melanomas é diferente, mostrando a importância do perfil celular. O efeito genotóxico da luz UVB pode alterar a expressão de moléculas como galectina-3 e MAPKs, desencadeando respostas UVB-dependentes. Galectina-3 é uma lectina que reconhece beta-galactosídeos e está envolvida na regulação de diversos processos celulares que modificam a viabilidade celular e a proliferação. Esta molécula é ubiquamente expressa apresentando um comportamento específico dependendo da sua localização subcelular. No presente trabalho mostramos que a distribuição de galectina-3 em melanoma e melanócitos é ampla, encontrando-se tanto no núcleo como no citoplasma, podendo ser modificada após irradiação UVB ou ainda secretada para o meio extracelular. Além disso, observamos que a luz UVB ativa a via de MAPKs, proteínas quinases ativadas por mitógenos envolvidas no crescimento, sobrevivência, diferenciação e resposta a estresse, em melanócitos e em melanomas poucos minutos após a exposição à UVB. Uma maior atividade de p38 e de ERK é evidenciada em melanomas, enquanto que em melanócitos a via de p38 é a mais ativa, corroborando a noção de que a resposta celular à luz UVB difere entre melanócitos e melanoma. As moléculas p38 e JNK são proteínas quinases ativada pelo estresse (SAPK). A via de JNK não é tão responsiva em alguns melanomas, mas ativação desta molécula parece estar envolvida com a sobrevivência celular e a translocação mitocondrial após UVB. Em adição, a inibição de JNK leva ao aumento de morte celular em linhagens melanocíticas irradiadas e não irradiadas, e em melanoma induz morte e aumenta autofagia após irradiação. Esta molécula parece interagir com galectina-3 em modelos murinos, mas não em melanomas humanos, enquanto que ERK interage fisicamente com galectina-3 em melanócitos e melanomas humanos, independente de UVB. Através do silenciamento de galectina-3 pela técnica de RNA de interferência, mostramos o aumento da ativação da via de ERK após irradiação e de proteínas downstream de ERK, promovendo a proliferação celular em melanomas nessas condições. Em melanócitos parece existir uma regulação negativa da via de ERK por galectina-3 acompanhada de uma diminuição da viabilidade celular após o silenciamento dessa lectina, independente de UVB. Estes resultados mostram que galectina-3 é uma importante reguladora de eventos associados com sobrevivência e morte celular em melanoma. Por outro lado, em melanomas a ausência de galectina-3 induz aumento da proliferação associada à ativação de ERK, evidenciando a importância do tipo celular na ação de galectina-3 / Skin cancer is the most common cancer among humans, melanoma being the least common type but very important due to its aggressive behavior. A major etiologic agent of this type of tumor is ultraviolet radiation from the sunlight. The ultraviolet B rays (UVB) cause DNA damage and induce alterations over the skin cells after prolonged exposition without protection. The UVB response in melanocytes and melanoma cells is different. This shows the importance of the cellular profile. The genotoxic effect of UVB light can alter the expression of molecules such as galectine-3 and MAPKs and also triggers multiple responses UVB-dependent. Galectin-3 is a lectin that recognizes beta-galactosides. It is involved in the regulation of many cellular processes that modify cellular viability and proliferation and presents specific behavior depending on its subcellular localization. In the present study we showed that galectine-3 distribution in melanoma cells and melanocytes is large, lying both in the nucleus and in the cytoplasm. After UVB irradiation this distribution could be modified or even galactine-3 secreted itself into the extracellular space. Moreover, we observed that UVB light activates the mitogen-activated protein kinase pathway (MAPK) involved in growth, survival, differentiation and stress-response in melanocytes and in melanoma cells just a few minutes after exposure. An increased activity of p38 and ERK was observed in melanomas, while in melanocytes just p38 pathway was highly active, supporting the notion that the cellular response to UVB light differs between melanocytes and melanoma cells. The molecules p38 and JNK are stress-activated protein kinases (SAPK). The JNK pathway is not responsive in some melanoma cells, but the activation of this molecule appears to be involved in cell survival and mitochondrial translocation after being exposed to UVB. Inhibition of JNK leads to increased cell death in irradiated and non-irradiated melanocytic lineage, but in melanoma cells induces cell death and increased autophagy only after irradiation. This molecule seems to interact with galectin-3 in mouse models but not in human melanomas, whereas ERK physically interacts with galectin-3 in human melanocytes and melanoma cells, regardless of UVB exposure. Through the knockdown of galectin-3 by siRNA, we showed increased activation of the ERK and its downstream pathway after irradiation, thus inducing cell proliferation. In melanocytes seems to be a negative regulation of the ERK pathway by galectin-3 accompanied by a decrease in cell viability after its knockdown regardless of UVB exposure. These results show that galectin-3 is an important regulatory molecule of events associated with cell death and survival in melanoma, which has different behavior depending on the cell type

O câncer de pele é um dos mais frequentes entre humanos, sendo o melanoma o tipo menos comum, mas com grande importância devido à agressividade que ele apresenta. Um dos principais agentes etiológicos deste tipo de tumor é a radiação ultravioleta proveniente da luz solar. A fração de radiação ultravioleta B (UVB) gera dano no DNA e induz alterações nas células da pele após a exposição prolongada e sem proteção. A resposta à luz UVB em melanócitos e melanomas é diferente, mostrando a importância do perfil celular. O efeito genotóxico da luz UVB pode alterar a expressão de moléculas como galectina-3 e MAPKs, desencadeando respostas UVB-dependentes. Galectina-3 é uma lectina que reconhece beta-galactosídeos e está envolvida na regulação de diversos processos celulares que modificam a viabilidade celular e a proliferação. Esta molécula é ubiquamente expressa apresentando um comportamento específico dependendo da sua localização subcelular. No presente trabalho mostramos que a distribuição de galectina-3 em melanoma e melanócitos é ampla, encontrando-se tanto no núcleo como no citoplasma, podendo ser modificada após irradiação UVB ou ainda secretada para o meio extracelular. Além disso, observamos que a luz UVB ativa a via de MAPKs, proteínas quinases ativadas por mitógenos envolvidas no crescimento, sobrevivência, diferenciação e resposta a estresse, em melanócitos e em melanomas poucos minutos após a exposição à UVB. Uma maior atividade de p38 e de ERK é evidenciada em melanomas, enquanto que em melanócitos a via de p38 é a mais ativa, corroborando a noção de que a resposta celular à luz UVB difere entre melanócitos e melanoma. As moléculas p38 e JNK são proteínas quinases ativada pelo estresse (SAPK). A via de JNK não é tão responsiva em alguns melanomas, mas ativação desta molécula parece estar envolvida com a sobrevivência celular e a translocação mitocondrial após UVB. Em adição, a inibição de JNK leva ao aumento de morte celular em linhagens melanocíticas irradiadas e não irradiadas, e em melanoma induz morte e aumenta autofagia após irradiação. Esta molécula parece interagir com galectina-3 em modelos murinos, mas não em melanomas humanos, enquanto que ERK interage fisicamente com galectina-3 em melanócitos e melanomas humanos, independente de UVB. Através do silenciamento de galectina-3 pela técnica de RNA de interferência, mostramos o aumento da ativação da via de ERK após irradiação e de proteínas downstream de ERK, promovendo a proliferação celular em melanomas nessas condições. Em melanócitos parece existir uma regulação negativa da via de ERK por galectina-3 acompanhada de uma diminuição da viabilidade celular após o silenciamento dessa lectina, independente de UVB. Estes resultados mostram que galectina-3 é uma importante reguladora de eventos associados com sobrevivência e morte celular em melanoma. Por outro lado, em melanomas a ausência de galectina-3 induz aumento da proliferação associada à ativação de ERK, evidenciando a importância do tipo celular na ação de galectina-3 / Skin cancer is the most common cancer among humans, melanoma being the least common type but very important due to its aggressive behavior. A major etiologic agent of this type of tumor is ultraviolet radiation from the sunlight. The ultraviolet B rays (UVB) cause DNA damage and induce alterations over the skin cells after prolonged exposition without protection. The UVB response in melanocytes and melanoma cells is different. This shows the importance of the cellular profile. The genotoxic effect of UVB light can alter the expression of molecules such as galectine-3 and MAPKs and also triggers multiple responses UVB-dependent. Galectin-3 is a lectin that recognizes beta-galactosides. It is involved in the regulation of many cellular processes that modify cellular viability and proliferation and presents specific behavior depending on its subcellular localization. In the present study we showed that galectine-3 distribution in melanoma cells and melanocytes is large, lying both in the nucleus and in the cytoplasm. After UVB irradiation this distribution could be modified or even galactine-3 secreted itself into the extracellular space. Moreover, we observed that UVB light activates the mitogen-activated protein kinase pathway (MAPK) involved in growth, survival, differentiation and stress-response in melanocytes and in melanoma cells just a few minutes after exposure. An increased activity of p38 and ERK was observed in melanomas, while in melanocytes just p38 pathway was highly active, supporting the notion that the cellular response to UVB light differs between melanocytes and melanoma cells. The molecules p38 and JNK are stress-activated protein kinases (SAPK). The JNK pathway is not responsive in some melanoma cells, but the activation of this molecule appears to be involved in cell survival and mitochondrial translocation after being exposed to UVB. Inhibition of JNK leads to increased cell death in irradiated and non-irradiated melanocytic lineage, but in melanoma cells induces cell death and increased autophagy only after irradiation. This molecule seems to interact with galectin-3 in mouse models but not in human melanomas, whereas ERK physically interacts with galectin-3 in human melanocytes and melanoma cells, regardless of UVB exposure. Through the knockdown of galectin-3 by siRNA, we showed increased activation of the ERK and its downstream pathway after irradiation, thus inducing cell proliferation. In melanocytes seems to be a negative regulation of the ERK pathway by galectin-3 accompanied by a decrease in cell viability after its knockdown regardless of UVB exposure. These results show that galectin-3 is an important regulatory molecule of events associated with cell death and survival in melanoma, which has different behavior depending on the cell type