Adipose tissue is an organ that plays an important role in energy storage, nutritional balance and thermogenesis. White and brown adipose tissues have distinct cell morphology and metabolic functions. White adipose tissue (WAT) with unilocular lipid droplets serves as a major site of energy storage, while brown adipose tissue (BAT) with multilocular lipid droplets plays an important role in thermogenesis via a mitochondrial protein, uncoupling protein 1 (UCP1). These cells are derived from mesenchymal stem cells (MSCs). Newly discovered beige adipocytes are derived from the same MSC precursors as WAT but resemble BAT due to expression of UCP1. Due to side effects of drugs for treating obesity, activation of UCP1 positive beige adipocytes in WAT has become a new therapeutic target. The interaction of extracellular matrix (ECM) with integrin was found to regulate cell specification of mesenchymal stem cells (MSCs) via intracellular signaling. However, the role of individual ECM proteins in beige adipogenesis in WAT remains unknown. Therefore, we established a system for culturing stromal vascular fraction (SVF) cells from inguinal WAT on ECM protein coated plates and differentiating the cells into either white or beige adipocytes. We found that cells cultured on type I collagen had more round cell morphology and higher mRNA expression of thermogenic genes, UCP1 and type II iodothyronine deiodinase (DIO2),which was further enhanced in myocardin-related transcription factor A (MRTFA) knockout SVF cells. MRTFA has been reported to regulate beige adipogenesis in BMP-ROCK signaling pathway. Based on our data, we found that type I collagen-integrin signaling regulates beige adipogenesis by controlling the activity of MRTFA in MSCs. Our study has provided an insight into developing therapeutic drugs to enhance beige adipocytes formation in WAT for reducing obesity in the future.

abstract: Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Introdução: O saco endolinfático tem sido apontado como o alvo das reações imuno-alérgicas da orelha interna. A prevalência de alergia em pacientes com Doença de Ménière foi estabelecida em torno de 41,6% para inalantes e 26,6% para alimentos, por Derebery em 2000, dados aumentados em relação à prevalência de alergia na população em geral, que, no Brasil, varia de 9% a 30% para inalantes e de 1% a 3% para alimentos. Objetivos: Avaliar a prevalência de reações de hipersensibilidade tipo I a inalantes e alimentos na população do setor de Otoneurologia do Hospital das Clínicas da Faculdade de Medicina de São Paulo e descrever os sintomas vestibulares dos pacientes. Casuística e método: Setenta e cinco pacientes com distúrbios do equilíbrio de origem periférica foram submetidos a questionário de caracterização clínica de sintomas cócleo-vestibulares e teste cutâneo (prick test) para 13 inalantes e 5 alimentos. Resultados: Vinte e cinco (33,3%) pacientes apresentaram prick test positivo a pelo menos um alérgeno inalante e 6 (8%) a pelo menos um alérgeno alimentar. Quatro pacientes apresentaram prick test positivo na ausência de sintomas alérgicos. Prevaleceu a queixa de tontura de caráter rotatório em proporções semelhantes entre os pacientes com prick test positivo e negativo. Conclusão: A prevalência de reações de hipersensibilidade tipo I a inalantes e a alimentos na população avaliada foi maior do que na população em geral. Os sintomas vestibulares não diferiram entre os pacientes da amostra, com prick test positivo ou negativo. No entanto, deve-se obter maior número de amostra para que os dados sejam confiáveis. / Introduction: The endolymphatic sac has been pointed out as the target of immuno-allergic reactions in the inner ear. The prevalence of allergy in patients with Ménières disease was established as approximately 41,6% for inhalants and 26,6% for food by Derebery in 2000, an increase in the data in relation to that of the prevalence of allergy in the general population, which in Brazil varies from 9% to 30% for inhalants and from 1% to 3% for food. Objectives: To evaluate the prevalence of reactions to type I hypersensitivity to inhalants and food in the population of the Otoneurological Section of the Clinics Hospital of the University of São Paulo Medicine School and to describe the vestibular symptoms of the patients. Method: Seventy-five patients with peripheral equilibrium disturbances who had answered a questionnaire of clinical characterization regarding cochlear-vestibular symptoms and undergone prick test for 13 inhalants and 5 types of food. Results: Twenty-five (33,3%) of the patients were positive for the prick test and for at least one allergen inhalant and 6 (8%) for at least one food allergen. Four patients were positive for the prick test in the absence of allergy symptoms. There was a prevalence of the complaint of rotatory dizziness in similar proportions among the patients with positive and negative prick test. Conclusion: The presence of type I hypersensitivity reactions to inhalants and food in the population evaluated was greater than in the general population. The vestibular symptoms did not differ among the patients in the sample, neither with positive or negative prick test results. However, a sample of greater number should be obtained for a higher confidence level of data results.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os bufodienolÃdeos sÃo esterÃides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da famÃlia Bufonidae utilizado na medicina chinesa. Os bufodienolÃdeos possuem grande variedade de atividades biolÃgicas, incluindo atividades antineoplÃsicas. Em relaÃÃo à atividade antitumoral, os bufodienolÃdeos tem demonstrado inibir o crescimento de vÃrias linhagens de cÃlulas cancerÃgenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotÃxico e genetÃxico de seis bufodienolÃdeos em seis linhagens tumorais humanos, trÃs linhagens murinas normais e cÃlulas mononucleadas do sangue perifÃrico (CMSP) humano. Todos os seis bufodienolÃdeos foram citotÃxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 ÂM. Os bufodienolÃdeos testados nÃo apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de aÃÃo envolvido. Uma sequÃncia de experimentos in vitro foram realizados utilizando-se a linhagem leucÃmica HL-60. As cÃlulas foram tratadas em diferentes concentraÃÃes da amostra hellebrigenina (0,03, 0,06 e 0,12 ÂM) por 24 horas. A viabilidade das cÃlulas (nÃmero de cÃlulas viÃveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o nÃmero de cÃlulas reduziu a partir da menor concentraÃÃo (0,03 ÂM) testada e a porcentagem de cÃlulas com membrana integra reduziu a partir da concentraÃÃo 0,06 ÂM. A anÃlise morfolÃgica por citometria de fluxo revelou aumento de cÃlulas com padrÃo apoptÃtico a partir da concentraÃÃo de 0,06 ÂM. Jà a anÃlise do conteÃdo nuclear, nos mostrou aumento de fragmentaÃÃo de DNA sub-G1 indicativo de apoptose e acÃmulo de cÃlulas na fase G2/M a partir das concentraÃÃes de 0,03 e 0,06 ÂM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalizaÃÃo da fosfatidilserina, despolarizaÃÃo mitocondrial, ativaÃÃo da caspase iniciadora 8 e consequente ativaÃÃo das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotÃxico por induÃÃo de mais de uma via apoptÃtica. Hellebrigenina nÃo foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberraÃÃes cromossÃmicas em CMSP. Por meio dos estudos de docking molecular foi possÃvel predizer a ligaÃÃo entre hellebrigenina e topoisomeraseIIα humana, resultado compatÃvel com a possÃvel inibiÃÃo dessa enzima. De forma geral, os resultados apontam o potencial citotÃxico do bufodienolÃdeo hellebrigenina / Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frogâs skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolÃdeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 ÂM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 ÂM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 ÂM) tested and the percentage of cells with reduced membrane integrity from 0.06 ÂM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 ÂM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 ÂM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseIIα, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin

No description available.

Cell death is an imperative mechanism for the development, homeostasis and survival of an organism. Various forms of cell death have been documented and recent reports indicate that the mode of cell death elicited can have a profound influence on the development and perpetuation of inflammation. Apoptosis is the predominant, programmed pathway of cell death, which ensures physiological elimination of unwanted cells. On the other hand, another cell death pathway described as programmed necrosis (necroptosis), has recently been revealed. The induction of necroptosis and its impact in host biology is not clear. Herein I have evaluated the mechanisms of necroptosis in macrophages, an important cell type of the immune system. My experiments indicate that type I interferon (IFN-I) signaling through transcription factors STAT1, STAT2 and IRF9, collectively described as the ISGF3 complex, is indispensable for necroptosis of macrophages. Furthermore, my results indicate that IFN-I signaling promotes the sustained phosphorylation of receptor interacting protein kinase 3 (Rip3), a key protein required for the execution of necroptosis. My findings also reveal that dynamin-dependent endocytosis following IFNβ stimulation and caspase inhibition is necessary for the induction of necroptosis. The results presented in this thesis provide new insights into the molecular mechanisms of necroptosis and therefore contribute to a deeper understanding of multiple inflammatory pathologies.

Recognition of non-self, pathogen-associated molecular patterns is a central component of host immune response to pathogens like viruses. Intracellular detection of viral nucleic acids leads to the production of type I interferons (IFN-I) and subsequent establishment of an antiviral state in infected and neighboring cells. Viruses have evolved multiple mechanisms to counteract IFN-I responses in infected cells, however, viral nucleic acids released from dying cells can stimulate IFN-I production in surrounding or distal uninfected cells. This thesis examines the mechanisms by which cells recognize extracellular viral nucleic acids and the subsequent downstream antiviral signaling. Class A scavenger receptors (SR-As) internalize extracellular viral double-stranded RNA (dsRNA) to mediate IFN-I responses, but little is known about extracellular viral DNA. We observed that extracellular DNA is recognized and internalized by SR-As in a manner like extracellular dsRNA. Furthermore, we established that SR-A1 is sufficient in mediating extracellular dsRNA-induced cellular responses and other nucleic acid receptors like SR-J1 and DEC-205 are dispensable. Finally, a direct interaction of RNA and DNA species was demonstrated with the coiled-coil collagenous domain of SR-A1, but not the scavenger receptor cysteine rich domain of SR-A6.We elaborated the role of SR-A1 by identifying the cellular processes activated through SR-A1 following uptake of extracellular dsRNA. Cytosolic sensors are essential in mediating an antiviral response to the endocytosed dsRNA, but the mechanism of endoplasmic release and cytoplasmic entry of dsRNA remains an enigma. We demonstrated that the lack of a dsRNA-channel, SIDT2, impaired the ability of the cells to mediate an antiviral response to extracellular dsRNA. Understanding host responses to extracellular viral nucleic acids will enable the development of novel vaccines and antiviral therapeutics against RNA and DNA viruses that efficiently counteract these responses in infected cells. / Thesis / Doctor of Philosophy (PhD) / Viral infections remain a threat to global health as new diseases continue to emerge. To develop effective vaccines and antivirals to combat viruses and alleviate human disease require a deeper understanding of virus-host interactions. Host cells identify virus-associated molecules to detect viruses and eliminate them whereas, viruses employ tactics to prevent the activation of the immune system. However, virus-induced cell lysis releases viral molecules that can stimulate immune responses in neighbouring uninfected cells. This thesis examines the mechanism by which cells respond to extracellular viral nucleic acids. We showed that a protein present at the cell surface called ‘class A scavenger receptor 1’ is sufficient to internalize extracellular viral nucleic acids, leading to immune responses. The response is impaired when a channel protein, SIDT2, is absent in the cells. Further work is necessary to understand how this knowledge can be harnessed to develop vaccines and antiviral therapeutics.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Type I collagen, which consists of two alpha1 chains [α1(I), COL1A1 ] and one alpha2 chain [α2(I), COL1A2 ], is the most abundant member of the collagen family. Interferon-gamma (IFN-γ), which is both an important pro-inflammatory and a anti fibrotic cytokine, coordinately suppresses both α1(I) and α2(I) production primarily at the transcriptional level. Previous work identified a regulatory factor for X-box (RFX) binding site in the first exon of the collagen α2(I) gene. RFX5 complex, consisting of three proteins (RFX5, RFXB, and RFXAP), as well as class II transactivator (CIITA), activates major histocompatibility complex class II (MHC II) transcription during IFN-γ stimulation. This thesis demonstrates that these factors mediate repression of collagen gene transcription in response to IFN-γ. All three RFX5 complex proteins were required for maximum repression of COL1A2 promoter activity. Mutations in regions of RFX5 proteins important for complex formation either reversed repression of collagen transcription or activated collagen transcription, presumably due to dominant negative effects. CIITA, recruited to the collagen transcription start site by RFX5, repressed collagen gene transcription mainly through its acidic and proline-serine-threonine rich domains. Repression of the collagen promoter by CIITA was enhanced in the presence of the RFX5 complex. Inhibition of IFN-γ induced expression of CIITA by RNA interference (RNAi) led to partial alleviation of collagen repression and MHC II activation. IFN-γ decreased collagen transcription at the same time that it increased the expression of RFX5/CIITA complex. In addition, IFN-γ increased the expression of RFXB, but decreased the expression of an RFXB splice variant, RFXBSV. RFXBSV reversed collagen repression by IFN-γ. Both RFX5 and CIITA were present in a large repressor/co-repressor complex containing histone deacetylase (HDAC) and Sin3 proteins. IFN-γ promoted the recruitment of RFX5/CIITA complex as well as HDAC2/Sin3B to the collagen transcription start site, which resulted in the deacetylation of histories surrounding this site. IFN-γ also blocked the occupancy of RNA polymerase II (Pol II) on the collagen transcription start site in conjunction with the increase in CIITA binding. Taken together, these data identify the RFX5/CIITA complex as a repressor of collagen gene transcription. / 2031-01-01

The Poisson distribution is commonly used in analyzing count data, and many insurance companies are interested in studying the related risk models and ruin probability theory. Over the past century, many different bivariate models have been developed in the literature. The bivariate Poisson distribution was first introduced by Campbell (1934) for modelling bivariate accident data. However, in some situations, a given dataset may possess over-dispersion compared to Poisson distribution which moti- vated researchers to develop alternative models to handle such situations. In this regard, Minkova and Balakrishnan (2014a) developed the Type I bivariate Polya- Aeppli distribution by using compounding with Geometric random variables and the trivariate reduction method. Inference for this Type I bivariate Polya-Aeppli distribution is the topic of this thesis. The parameters in a model are used to describe and summarize a given sample within a specific distribution. So, their estimation becomes important and the goal of estimation theory is to seek a method to find estimators for the parameters of interest that have some good properties. There exist many methods of finding estimators such as Method of Moments, Bayesian estimators, Least Squares, and Maximum Likelihood Estimators (MLEs). Each method of estimation has its own strength and weakness (Casella and Berger (2008)). Minkova and Balakrishnan (2014a) discussed the moment estimation of the parameters of the Type I bivariate Polya-Aeppli dis- tribution. In this thesis, we develop the likelihood inference for this model. A simulation study is carried out with various parameter settings. The obtained results show that the MLEs require more computational time compared to Moment estimation. However, Method of Moments (MoM) did not result in good estimates for all the simulation settings. In terms of mean squared error and bias, we observed that MLEs performed, in most of the settings, better than MoM. Finally, we apply the Type I bivariate Polya-Aeppli model to a real dataset containing the frequencies of railway accidents in two subsequent six year periods. We also carry out some hypothesis tests using the Wald test statistic. From these results, we conclude that the two variables belong to the same univariate Polya-Aeppli distribution but are correlated. / Thesis / Master of Science (MSc)

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24329号 / 生博第488号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 杉田 昌彦, 教授 垣塚 彰 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM