Thesis (Ph. D.)--Virginia Commonwealth University, 2010. / Prepared for: Dept. of Pathology. Title from resource description page. Includes bibliographical references . Unavailable until 5/13/2105.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 180-194.

Yung, Lai Hang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 93-115). / Abstract also in Chinese. / Declaration --- p.i / Acknowledgements --- p.ii / Abbreviations --- p.iii / Abstract in English --- p.v / Abstract in Chinese --- p.ix / Contents --- p.xi / Chapter Chapter I - --- Introduction / Chapter 1.1) --- Endothelial cells function --- p.1 / Chapter 1.2) --- Oxidative stress in the vascular wall --- p.2 / Chapter 1.2.1) --- Sources of ROS --- p.3 / Chapter 1.2.2) --- Actions of ROS --- p.3 / Chapter 1.2.2.1) --- Impaired endothelium-dependent vasodilatation --- p.3 / Chapter 1.2.2.2) --- VSMC migration --- p.4 / Chapter 1.2.2.3) --- Programmed cell death (cell apoptosis) --- p.4 / Chapter 1.3) --- Endothelial cell apoptosis --- p.7 / Chapter 1.3.1) --- Apoptosis and cardiovascular diseases --- p.7 / Chapter 1.3.2) --- Mechanisms of endothelial cells apoptosis --- p.7 / Chapter 1.3.2.1) --- What are caspases? --- p.8 / Chapter 1.3.2.2) --- Death receptor-mediated apoptosis --- p.9 / Chapter 1.3.2.3) --- Mitochondria-dependent pathway --- p.9 / Chapter 1.3.3) --- Regulations of endothelial cells apoptosis --- p.10 / Chapter 1.3.3.1) --- Oxidative stress --- p.10 / Chapter 1.3.3.2) --- Shear Stress --- p.11 / Chapter 1.3.3.3) --- Growth factors --- p.12 / Chapter 1.3.3.4) --- NO --- p.12 / Chapter 1.3.3.5) --- Inflammatory mediators --- p.13 / Chapter 1.4) --- Mitogen activated kinases signaling in apoptosis --- p.15 / Chapter 1.5) --- Bone morphogenic proteins (BMPs) --- p.17 / Chapter 1.5.1) --- BMPs functions and cardiovascular system --- p.17 / Chapter 1.5.2) --- BMPs signaling pathways --- p.18 / Chapter 1.5.2.1) --- Smad-dependent pathway --- p.18 / Chapter 1.5.2.2) --- MAPKs and SAPKs pathways --- p.19 / Chapter 1.5.2.3) --- Antagonists of BMPs signaling --- p.20 / Chapter 1.5.3) --- BMP4 and cardiovascular diseases --- p.20 / Chapter 1.6) --- "Justification, long-term significance and objectives of the present project" --- p.23 / Chapter Chapter II - --- Methods and Materials / Chapter 2.1) --- Animal handling --- p.24 / Chapter 2.2) --- Endothelial cell isolation and culture --- p.24 / Chapter 2.2.1) --- Primary culture of rat endothelial cells --- p.24 / Chapter 2.2.2) --- Culture of human umbilical cord vein endothelial cells… --- p.25 / Chapter 2.3) --- Apoptosis assessment --- p.25 / Chapter 2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.25 / Chapter 2.3.2) --- Cell death detection ELISA kit --- p.26 / Chapter 2.3.3) --- Flow cytometry --- p.27 / Chapter 2.4) --- Western blot analysis --- p.28 / Chapter 2.4.1) --- Sample preparation --- p.28 / Chapter 2.4.2) --- SDS-PAGE and transfer --- p.28 / Chapter 2.5) --- DHE fluorescence --- p.29 / Chapter 2.6) --- "Drugs, chemicals and other reagents" --- p.30 / Chapter 2.6.1) --- Drugs and chemicals used in the present experiments --- p.30 / Chapter 2.6.2) --- Reagents for Western blot analysis --- p.30 / Chapter 2.6.3) --- Primary antibodies --- p.33 / Chapter 2.7) --- Small interfering RNA experiment --- p.34 / Chapter 2.8) --- Statistical analysis --- p.34 / Chapter Chapter III - --- BMP4 induces endothelial cell apoptosis in ROS related p38 MAPK and JNK mediated caspase-3 dependent pathway / Chapter 3.1) --- Introduction --- p.35 / Chapter 3.2) --- Methods and materials --- p.39 / Chapter 3.2.1) --- Isolation and culture of endothelial cells --- p.39 / Chapter 3.2.2) --- Drugs treatment --- p.39 / Chapter 3.2.3) --- Assay for cell apoptosis --- p.40 / Chapter 3.2.3.1) --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay --- p.40 / Chapter 3.2.3.2) --- Cell death detection ELISA kit --- p.41 / Chapter 3.2.3.3) --- Flow cytometric analysis --- p.41 / Chapter 3.2.4) --- Western blot analysis --- p.41 / Chapter 3.2.5) --- Dihydroethidium (DHE) staining --- p.42 / Chapter 3.2.6) --- Statistical analysis --- p.42 / Chapter 3.3) --- Results --- p.43 / Chapter 3.3.1) --- Dose- and time-dependent effect of BMP4 --- p.43 / Chapter 3.3.2) --- Role of caspases in apoptosis of RAECs and HUVECs --- p.43 / Chapter 3.3.3) --- Roles of BMP4 and ROS in endothelial cell apoptosis --- p.44 / Chapter 3.3.3.1) --- Noggin antagonism of BMP4-induced effect --- p.44 / Chapter 3.3.3.2) --- NAD(P)H oxidase-mediated ROS production --- p.44 / Chapter 3.3.3.3) --- Inhibition of endothelial cell apoptosis by ROS scavengers --- p.45 / Chapter 3.3.4) --- Roles of MAPKs/SAPKs in BMP4-induced endothelial cell apoptosis --- p.45 / Chapter 3.3.5) --- Relationship between ROS and MAPKs/SAPKs --- p.46 / Chapter 3.3.6) --- Relationship between p38 MAPK and JNK --- p.46 / Chapter 3.4) --- Discussion --- p.82 / Chapter 3.4.1) --- Caspase-dependent pathways --- p.82 / Chapter 3.4.2) --- Oxidative stress --- p.85 / Chapter 3.4.3) --- Role of MAPKs activation in BMP4-induced endothelial cell apoptosis --- p.87 / Chapter 3.4.4) --- ROS mediates BMP4-induced activation of MAPKs --- p.88 / Chapter 3.4.5) --- Role of p38 MAPK in the activation of JNK 1 --- p.89 / Chapter 3.5) --- Concluding remarks --- p.91 / References --- p.93 / Publications and Awards --- p.116

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2007. / Typescript. Non-Latin script record Includes bibliographical references (leaves 112-128). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

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.

The cytokine tumor necrosis factor α (TNFα) stimulates the NF-кB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting Rip1 and Traf2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that Rip1 links the TNFR1 to the IкB kinase (IKK) complex, whereas Traf2 couples the TNFR1 to the SAPK/JNK cascade. We found TNFα-induced p38 MAP kinase activation and interleukin-6 (IL-6) production is impaired in rip1-/- murine embryonic fibroblasts (MEF) but unaffected in traj2-/- MEF, demonstrating that Rip1 is also a specific mediator of the p38 MAP kinase response to TNFα. Moreover, we demonstrate that endogenous Rip1 associates with the MAP3K, Mekk3 in response to TNFα and that TNFα-induced p38 MAP kinase activation is impaired in mekk3-/- cells, indicating that Rip1 may mediate the p38 MAP kinase response to TNFα by recruiting Mekk3. We also demonstrate that Rip1 is phosphorylated and ubiquitinated in response to Tnfα and that Rip1 phosphorylation is not required for ubiquitination of Rip1. Furthermore, TNFα-induced ubiquitination of Rip1 is impaired in Traf2-/- cells, suggesting that Traf2 is the E3 ubiquitin ligase responsible for the TNFα-dependent ubiquitination of Rip1. Finally, recruitment of the ubiquitinated Tak1 complex is dependent on the presence of Rip1, suggesting that Rip1 ubiquitination rather than its phosphorylation is critical in TNFR1 signaling.

Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease is a fatal neurodegenerative disease. ALS is typically adult onset and is characterized by rapidly progressive loss of both upper and lower motor neurons that leads to death usually within 3-5 years. About 90% of all the cases are sporadic with no family history while the remaining 10% are familial cases with mutations in several genes including SOD1, FUS/TLS, TDP43 and C9ORF72. FUS/TLS (Fused in Sarcoma/Translocated in Liposarcoma or FUS) is an RNA/DNA binding protein that is involved in multiple cellular functions including DNA damage repair, transcription, mRNA splicing, RNA transport and stress response. More than 40 mutations have now been identified in FUS that account for about 5% of all the familial cases of ALS. However, the exact mechanism by which FUS causes ALS is unknown. While significant progress has been made in understanding the disease mechanism and identifying therapeutic strategies, several questions still remain largely unknown. The work presented here aims at understanding the normal functions of FUS as well as the pathogenic mechanisms by which it leads to disease. Several studies showed the association of mutant-FUS with structures made up of RNA and proteins, called stress granules that form under various stress conditions. However, little is known about the role of endogenous FUS under stress conditions. I have shown that under hyperosmolar conditions, the predominantly nuclear FUS translocates into the cytoplasm and incorporates into stress granules. The response is specific to hyperosmolar stress because FUS remains nuclear under other stress conditions tested, such as oxidative stress, ER stress and heat shock. The response of FUS is rapid, and cells with reduced FUS levels are susceptible to the hyperosmolar stress, indicating a pro-survival role for FUS. In addition to investigating the functions of endogenous wild-type (WT) FUS, the work presented also focuses on identifying the pathogenic mechanism(s) of FUS variants. Using various biochemical techniques, I have shown that ALS-causing FUS variants are misfolded compared to the WT protein. Furthermore, in a squid axoplasm based vesicle motility assay, the FUS variants inhibit fast axonal transport (FAT) in a p38 MAPK dependent manner, indicating a role for the kinase in mutant-FUS mediated disease pathogenesis. Analysis of human ALS patient samples indicates higher levels of total and phospho p38, supporting the notion that aberrant regulation of p38 MAPK is involved in ALS. The results presented in this dissertation 1) support a novel prosurvival role for FUS under hyperosmolar stress conditions and, 2) demonstrate that protein misfolding and aberrant kinase activation contribute to ALS pathogenesis by FUS variants.

Introdução: Pearlman e colaboradores relacionou a presença de mutações ativadoras no gene MAP3K1 com o desenvolvimento testicular anormal em pacientes com disgenesia gonadal 46,XY familial, embora os estudos em camundongos tenham demonstrado que o gene Map3k1 não é essencial para a determinação testicular. No desenvolvimento gonadal masculino, a ligação do MAP3K1 à proteína RHOA promove uma fosforilação normal de p38 e ERK1/2, o que determina um bloqueio da via da beta-catenina pela MAP3K4. Já no desenvolvimento feminino, ocorre uma hiper fosforilação de p38 e ERK1/2, o que determina a ativação da via da beta-catenina e o bloqueio da via de retroalimentação positiva do SOX9 e o desenvolvimento testicular. Objetivos: Pesquisar a presença de variantes alélicas do gene MAP3K1 em pacientes portadores de distúrbios do desenvolvimento sexual (1) 46,XY por anormalidades do desenvolvimento gonadal e avaliar a repercussão funcional das variantes identificadas. Casuística e Métodos: Quarenta e sete pacientes com disgenesia gonadal 46,XY (17 com a forma completa e 29 com a forma parcial) e uma paciente com DDS 46,XY de causa etiológica não conhecida foram estudados. As regiões codificadoras do gene MAP3K1 foram amplificadas e sequenciadas pelo método de Sanger ou painel customizado de genes-alvo associados ao DDS. Estudo in vitro utilizando o método de detecção colorimétrica In-Cell ELISA com anticorpos específicos para detecção de ERK1/2 e AKT, fosforilado e não fosforilado foi realizado em fibroblastos obtidos por biópsia de pele e mantidos em cultura celular de 3 indivíduos portadores de variantes no MAP3K1. A quantificação da fosforilação de p38 e ERK por ensaio de citometria em células linfoblastóides mutadas foram realizados em amostras de 4 indivíduos portadores de variantes no MAP3K1 em estudo realizado em colaboração. Imunohistoquímica com anticorpos anti Caspase-3 foram realizadas em tecidos gonadais parafinados das pacientes portadoras de variantes alélicas nos genes MAP3K1 e FGFR2. Resultados: Vinte e uma variantes alélicas, sete das quais ainda não descritas na literatura, foram identificadas no gene MAP3K1. Quatro novas variantes alélicas exônicas e não sinônimas (p.Leu639Pro, p.Leu447Trp, p.Thr657Arg e p.Cys691Arg) foram identificadas em heterozigose; todas foram classificadas como deletérias para a proteína nos estudos de predição \"in silico\", não foram identificadas em indivíduos controles brasileiros estudados e não estão descritas nos bancos de dados populacionais. A variante p.Leu639Pro foi identificada em duas irmãs com disgenesia gonadal 46,XY portadoras da variante p.Ser453Leu no gene FGFR2 identificada previamente. A variante intrônica c.834+1G >T identificada em heterozigose foi classificada como deletéria à proteína na análise no site de predição para alteração de \"splicing\". Os ensaios colorimétricos para detecção de ERK1/2 e AKT, fosforilado e não fosforilado foram inconclusivos. Os estudos in vitro de avaliação dos níveis de fosforilação de p38 e ERK evidenciaram uma maior fosforilação nas culturas celulares mutantes para o MAP3K1 quando comparado com a linhagem celular selvagem, resultado estatisticamente significativo ( p < 0,001) e que corrobora com os dados publicados previamente. A imunohistoquímica com anticorpos anti Caspase-3 mostrou uma maior marcação em células germinativas nos tecidos gonadais das pacientes portadoras das variantes no MAP3K1 e FGFR2 do que no tecido testicular normal, porém marcações foram identificadas também em células germinativas de tecidos testiculares de indivíduos com DDS 46,XY de outras etiologias. Conclusões: Os achados sugerem fortemente a participação das mutações identificadas no MAP3K1 na etiologia dos distúrbios do desenvolvimento sexual dos pacientes estudados. Porém, uma melhor compreensão dos mecanismos de participação da via MAPK nas redes gênicas de regulação do processo de determinação testicular humano ainda é necessário / Introduction: Pearlman et al. associated the presence of activating mutations in MAP3K1 gene with abnormal testicular development in patients with familial 46,XY gonadal dysgenesis, although studies in mice have shown that the Map3k1 gene is not essential for testicular determination. In male gonadal development, the binding of MAP3K1 to the RHOA protein promotes a normal phosphorylation of p38 and ERK1/2, and a blockade of the beta- catenin pathway is determined by MAP3K4. In the female development, hyperphosphorylation of p38 and ERK1/2 occurs. p38 and ERK1/2 hyperphosphorylated determine the activation of the beta-catenin pathway, the blockade of the positive feedback pathway of SOX9 and the testicular development. Objectives: To investigate the presence of allelic variants of the MAP3K1 gene in patients with 46,XY disorders of sex development (DSD) due to abnormalities of gonadal development and to evaluate the functional repercussion of the identified variants. Patients and Methods: Forty-seven patients with 46,XY gonadal dysgenesis (17 patients with complete form and 29 with partial form) and one patient with 46,XY DSD of unknown cause were studied. The MAP3K1 coding regions were amplified and sequenced by Sanger method or by custom panel of target genes associated with DSD. In-Cell ELISA assay with specific antibodies for the detection of phosphorylated and non-phosphorylated ERK1/2 and AKT was performed on fibroblasts obtained by skin biopsy and kept in cell culture of 3 individuals with MAP3K1 variants. Quantification of p38 and ERK phosphorylation by cytometric assay on mutated lymphoblastoid cells were performed on samples from 4 subjects with MAP3K1 variants in a collaborative study. Immunohistochemistry with anti-Caspase-3 antibodies were performed on paraffinembedded gonadal tissues of patients with MAP3K1 and FGFR2 allelic variants. Results: Twenty-one allelic variants, seven of them have not yet been described in the literature, were identified in the MAP3K1. Four novel exonic and non-synonymous allelic variants (p.Leu639Pro, p.Leu447Trp, p.Thr657Arg and p.Cys691Arg) were identified in heterozygous state; all of them were classified as deleterious in silico prediction sites; they were not identified in Brazilian control subjects and they were not described in the human genetic variation databases. The p.Leu639Pro variant was identified in two sisters with 46,XY gonadal dysgenesis carrying the previously identified FGFR2 variant (p Ser453Leu). The intronic c.834+1G > T variant identified in heterozygous state was classified as deleterious in the prediction sites. Colorimetric assays for the detection of phosphorylated and nonphosphorylated ERK1/2 and AKT were not significant. In vitro studies to evaluate p38 and ERK phosphorylation levels evidenced increased phosphorylation in the MAP3K1 mutant cells when compared to the wild type cells line; a statistically significant result (p < 0.001) that confirmed previously published data. The immunohistochemistry study with anti-Caspase-3 antibodies showed that the gonadal tissues of patients with MAP3K1 and FGFR2 variants exhibited more apoptotic germ ceIls than normal testicular tissue, but stained germ cells were also identified in the testicular tissues of the 46,XY DSD controls.Conclusions: These findings strongly suggest the participation of MAP3K1 mutations in the etiology of the testicular abnormalities of the 46,XY DSD patients of this study. However, a better understanding of the mechanisms of MAPK pathway in the gene regulatory networks of the human testicular determination process is still necessary

Introdução: Pearlman e colaboradores relacionou a presença de mutações ativadoras no gene MAP3K1 com o desenvolvimento testicular anormal em pacientes com disgenesia gonadal 46,XY familial, embora os estudos em camundongos tenham demonstrado que o gene Map3k1 não é essencial para a determinação testicular. No desenvolvimento gonadal masculino, a ligação do MAP3K1 à proteína RHOA promove uma fosforilação normal de p38 e ERK1/2, o que determina um bloqueio da via da beta-catenina pela MAP3K4. Já no desenvolvimento feminino, ocorre uma hiper fosforilação de p38 e ERK1/2, o que determina a ativação da via da beta-catenina e o bloqueio da via de retroalimentação positiva do SOX9 e o desenvolvimento testicular. Objetivos: Pesquisar a presença de variantes alélicas do gene MAP3K1 em pacientes portadores de distúrbios do desenvolvimento sexual (1) 46,XY por anormalidades do desenvolvimento gonadal e avaliar a repercussão funcional das variantes identificadas. Casuística e Métodos: Quarenta e sete pacientes com disgenesia gonadal 46,XY (17 com a forma completa e 29 com a forma parcial) e uma paciente com DDS 46,XY de causa etiológica não conhecida foram estudados. As regiões codificadoras do gene MAP3K1 foram amplificadas e sequenciadas pelo método de Sanger ou painel customizado de genes-alvo associados ao DDS. Estudo in vitro utilizando o método de detecção colorimétrica In-Cell ELISA com anticorpos específicos para detecção de ERK1/2 e AKT, fosforilado e não fosforilado foi realizado em fibroblastos obtidos por biópsia de pele e mantidos em cultura celular de 3 indivíduos portadores de variantes no MAP3K1. A quantificação da fosforilação de p38 e ERK por ensaio de citometria em células linfoblastóides mutadas foram realizados em amostras de 4 indivíduos portadores de variantes no MAP3K1 em estudo realizado em colaboração. Imunohistoquímica com anticorpos anti Caspase-3 foram realizadas em tecidos gonadais parafinados das pacientes portadoras de variantes alélicas nos genes MAP3K1 e FGFR2. Resultados: Vinte e uma variantes alélicas, sete das quais ainda não descritas na literatura, foram identificadas no gene MAP3K1. Quatro novas variantes alélicas exônicas e não sinônimas (p.Leu639Pro, p.Leu447Trp, p.Thr657Arg e p.Cys691Arg) foram identificadas em heterozigose; todas foram classificadas como deletérias para a proteína nos estudos de predição \"in silico\", não foram identificadas em indivíduos controles brasileiros estudados e não estão descritas nos bancos de dados populacionais. A variante p.Leu639Pro foi identificada em duas irmãs com disgenesia gonadal 46,XY portadoras da variante p.Ser453Leu no gene FGFR2 identificada previamente. A variante intrônica c.834+1G >T identificada em heterozigose foi classificada como deletéria à proteína na análise no site de predição para alteração de \"splicing\". Os ensaios colorimétricos para detecção de ERK1/2 e AKT, fosforilado e não fosforilado foram inconclusivos. Os estudos in vitro de avaliação dos níveis de fosforilação de p38 e ERK evidenciaram uma maior fosforilação nas culturas celulares mutantes para o MAP3K1 quando comparado com a linhagem celular selvagem, resultado estatisticamente significativo ( p < 0,001) e que corrobora com os dados publicados previamente. A imunohistoquímica com anticorpos anti Caspase-3 mostrou uma maior marcação em células germinativas nos tecidos gonadais das pacientes portadoras das variantes no MAP3K1 e FGFR2 do que no tecido testicular normal, porém marcações foram identificadas também em células germinativas de tecidos testiculares de indivíduos com DDS 46,XY de outras etiologias. Conclusões: Os achados sugerem fortemente a participação das mutações identificadas no MAP3K1 na etiologia dos distúrbios do desenvolvimento sexual dos pacientes estudados. Porém, uma melhor compreensão dos mecanismos de participação da via MAPK nas redes gênicas de regulação do processo de determinação testicular humano ainda é necessário / Introduction: Pearlman et al. associated the presence of activating mutations in MAP3K1 gene with abnormal testicular development in patients with familial 46,XY gonadal dysgenesis, although studies in mice have shown that the Map3k1 gene is not essential for testicular determination. In male gonadal development, the binding of MAP3K1 to the RHOA protein promotes a normal phosphorylation of p38 and ERK1/2, and a blockade of the beta- catenin pathway is determined by MAP3K4. In the female development, hyperphosphorylation of p38 and ERK1/2 occurs. p38 and ERK1/2 hyperphosphorylated determine the activation of the beta-catenin pathway, the blockade of the positive feedback pathway of SOX9 and the testicular development. Objectives: To investigate the presence of allelic variants of the MAP3K1 gene in patients with 46,XY disorders of sex development (DSD) due to abnormalities of gonadal development and to evaluate the functional repercussion of the identified variants. Patients and Methods: Forty-seven patients with 46,XY gonadal dysgenesis (17 patients with complete form and 29 with partial form) and one patient with 46,XY DSD of unknown cause were studied. The MAP3K1 coding regions were amplified and sequenced by Sanger method or by custom panel of target genes associated with DSD. In-Cell ELISA assay with specific antibodies for the detection of phosphorylated and non-phosphorylated ERK1/2 and AKT was performed on fibroblasts obtained by skin biopsy and kept in cell culture of 3 individuals with MAP3K1 variants. Quantification of p38 and ERK phosphorylation by cytometric assay on mutated lymphoblastoid cells were performed on samples from 4 subjects with MAP3K1 variants in a collaborative study. Immunohistochemistry with anti-Caspase-3 antibodies were performed on paraffinembedded gonadal tissues of patients with MAP3K1 and FGFR2 allelic variants. Results: Twenty-one allelic variants, seven of them have not yet been described in the literature, were identified in the MAP3K1. Four novel exonic and non-synonymous allelic variants (p.Leu639Pro, p.Leu447Trp, p.Thr657Arg and p.Cys691Arg) were identified in heterozygous state; all of them were classified as deleterious in silico prediction sites; they were not identified in Brazilian control subjects and they were not described in the human genetic variation databases. The p.Leu639Pro variant was identified in two sisters with 46,XY gonadal dysgenesis carrying the previously identified FGFR2 variant (p Ser453Leu). The intronic c.834+1G > T variant identified in heterozygous state was classified as deleterious in the prediction sites. Colorimetric assays for the detection of phosphorylated and nonphosphorylated ERK1/2 and AKT were not significant. In vitro studies to evaluate p38 and ERK phosphorylation levels evidenced increased phosphorylation in the MAP3K1 mutant cells when compared to the wild type cells line; a statistically significant result (p < 0.001) that confirmed previously published data. The immunohistochemistry study with anti-Caspase-3 antibodies showed that the gonadal tissues of patients with MAP3K1 and FGFR2 variants exhibited more apoptotic germ ceIls than normal testicular tissue, but stained germ cells were also identified in the testicular tissues of the 46,XY DSD controls.Conclusions: These findings strongly suggest the participation of MAP3K1 mutations in the etiology of the testicular abnormalities of the 46,XY DSD patients of this study. However, a better understanding of the mechanisms of MAPK pathway in the gene regulatory networks of the human testicular determination process is still necessary