Thesis (Ph.D.). / Written for the Dept. of Medicine, Division of Experimental Medicine. Title from title page of PDF (viewed 2009/06/09). Includes bibliographical references.

Abscisic acid is an important plant hormone in the responses to biotic and abiotic stresses, which also regulates various growth and developmental processes in plants. Three major components-receptors (PYRs), the PP2C type phosphatases and the SnRK2 subtype kinases form a double negative regulatory system: PYR/PYL/RCARs inhibit the activity of PP2Cs while PP2Cs inhibit that of SnRK2s in ABA signaling pathway. The results of my studies showed that ABA would directly affect the interaction between SnRK2.2 and ABI1 in absence of PYRs. Furthermore, ABA can inhibit the catalytic activity of the SnRK2.2 kinase. These findings indicated that ABA may directly interact with SnRK2.2. Posttranslational modifications play a key role in signal transduction. Phosphorylation is the most important posttranscriptional modification in ABA signal transduction. To dissect new components in ABA signaling network in plants, proteomics studies were carried out in Arabidopsis for identifying ABA- responsive phosphoproteins. Ten phosphoproteins, ATPB, ATPC1, FBA1, CTIMC, GGAT1, GAPC1, GAPC2, GAPA1 and ALDH11A3, were identified by 2-DE proteomic approach and LC-MS/MS analysis. These proteins are likely to be the potential phosphorylated targets of SnRK2s in ABA signaling network. Lysine acetylation (LysAc) also emerges as one of the important posttranslational modifications for protein regulation in plants. Eleven lysine acetylated proteins with altered acetylation in response to ABA were identified in Arabidopsis using proteomic approach. The increased LysAc of Rubisco and the decreased Rubisco activity by ABA treatment indicates the acetylation of Rubisco caused by ABA resulted in the inhibition of Rubisco activity. ABA has also been shown to exist and function in both lower animals and mammalians. The medical application of ABA has become a new area of investigation. To explore the role of protein phosphorylation in ABA-mediated function in mammalians, phosphoproteomic study was conducted in mouse 3T3-L1 cells. Ten phosphoproteins with significant changes in serine/threonine phosphorylation in response to ABA were identified. These results suggest these phosphoproteins are involved in ABA signaling network in mouse cells. The significance of the function of SFRS1, ANXA1 and Galectin-3 on human diseases indicated that ABA could be a potential treatment for some human diseases, such as cancer.

Vitamin D plays an important role in preserving healthy bones, and has additional roles in the body, including modulation of cell growth, differentiation, neuromuscular and immune function, and anti-inflammatory function. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily and regulates transcription of vitamin D-dependent target genes, such as those for key proteins involved in calcium and phosphorus absorption and bone development. Histone acetylation weakens the association of histones with DNA, and increases the accessibility of transcriptional regulatory proteins to chromatin templates, thereby increasing transcriptional activity of gene expression. Histone deacetylases remove the acetyl groups and condense chromatin structure, thereby preventing transcription. TSA is a potent histone deacetylase inhibitor and can significantly enhance gene expression. Bioactive food component, sulforaphane (SFN) is found in cruciferous vegetables and is known to be a histone deacetylase inhibitor, leading to transcriptional activation of gene expression. The objective of this study is to demonstrate that the bioactive food components modulate vitamin D action in adipocytes. To investigate the effects of TSA and SFN on vitamin D response, 3T3L1 mouse preadipocytes were treated with the combination of various concentrations of 1,25(OH)2 vitamin D, TSA, and SFN. Upon harvesting cells, the amounts of 24-hydroxylase mRNA, marker of vitamin D response, were measured by semiquantitative reverse transcriptase-PCR analysis. The results showed that the cells treated with 1μM TSA increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 3.5-fold (p < 0.05) at 1nM 1,25(OH)2 vitamin D and nearly 2.5-fold (p < 0.05) in 10 nM 1,25(OH)2 vitamin D, and the cells treated with 5μM SFN increased 1,25(OH)2 vitamin D-induced CYP24 mRNA level nearly 1.4-fold at 1nM 1,25(OH)2 vitamin D and nearly 1.2-fold at 10 nM 1,25(OH)2 vitamin D.

No description available.

Studying molecular mechanisms underlying adipocyte differentiation is imperative to understanding adipocyte function and its role in obesity. However, the majority of research exploring adipogenesis is conducted with cell lines cultured directly on tissue culture plastic. Culturing cells on plastic may result in altered proliferation and differentiation, and subsequent change in pharmacological response. The extracellular matrix (ECM) plays a critical role in adipocyte development and survival. It is suggested that cells in vitro express high levels of ECM proteins to compensate for lack of an ECM. Differentiating preadipocytes on a substrate representative of the mature adipocyte extracellular environment may provide a more physiological response to drugs and environmental chemicals. The purpose of this study was to investigate the impact of Matrigel on 3T3-L1 cell growth, differentiation, lipid accumulation and responsiveness to Rosiglitazone. Matrigel decreased 3T3-L1 cell proliferation, enhanced lipid accumulation, and increased expression of adipogenic and lipogenic markers, including PPARγ, C/EBPα, SREBP1c, FAS, LPL, FABP4 and PLIN1. This was accompanied by a decrease in gene expression of ECM proteins, including fibronectin, collagen 1, collagen 3, collagen 4, laminin and collagen 6 in 3T3-L1 cells on Matrigel. Finally, Matrigel enhanced the response of 3T3-L1 cells to Rosiglitazone, which is a known PPARγ agonist and significantly increases lipid accumulation in 3T3-L1 cells. Our results suggest that enhanced lipid accumulation in 3T3-L1 cells on Matrigel is associated with decreased expression of ECM genes. Future studies require investigation of the cell-to-ECM interaction to confirm these findings. This study proposes that the nature of the ECM for cultured adipocytes alters temporal lipid accumulation patterns and response to various drugs as compared to 3T3-L1 cells grown on tissue culture plastic. / Thesis / Master of Science (MSc)

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No. of bitstreams: 1 DanieleDeSouzaMarinhoDoNascimento_DISSERT.pdf: 2192295 bytes, checksum: 73b11671b4d2ac4a205f4fba79f5180a (MD5) Previous issue date: 2017-06-22 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico (CNPq) / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES) / A obesidade ? considerada um importante problema de sa?de p?blica, sendo um fator de risco para v?rias doen?as cr?nicas como doen?as cardiovasculares, hipertens?o, diabetes e outras. A Baccharis trimera (Less.) DC, conhecida popularmente como carqueja, ? uma planta medicinal utilizada na medicina tradicional em v?rias partes do Brasil. Para tal, infus?es, decoc??es e tinturas de suas folhas s?o produzidas e utilizadas para o tratamento da obesidade, diabetes, assim como diur?ticos, agentes digestivos, antiinflamat?rios, dentre outros. Neste trabalho, com intuito de respaldar o potencial medicinal da carqueja, extratos de folhas de Baccharis trimera foram obtidos, caracterizados qu?mica e fitoquimicamente e avaliados com rela??o ? sua atividade antioxidante e antiadipog?nica. Foram obtidos tr?s extratos: aquoso (AE), decoco (AE-D) e metan?lico (ME); a partir destes, foram realizados seis diferentes ensaios antioxidantes in vitro: ensaio do radical super?xido e hidroxila, poder redutor, capacidade antioxidante total e quela??o dos ?ons ferro e cobre. Bem como, foi avaliada sua poss?vel atividade antiadipog?nica com os testes de oil red O, glicerol livre e mensura??o de fatores de transcri??o adipog?nicos C/EBP?, C/EBP? e PPAR?. Na caracteriza??o fitoqu?mica, revelou-se a presen?a de flavonoides (?cido clorog?nico e apigenina) e compostos fen?licos nos extratos AE e AE-D. Quanto a atividade antioxidante, verificou-se uma atividade dose-dependente. Em rela??o ? atividade antiadipog?nica, a Baccharis trimera inibiu significantemente tanto a diferencia??o e ac?mulo de gordura nos adip?citos pelo MDA, quanto a express?o dos fatores de transcri??o C/EBP?, C/EBP? e PPAR?, durante a adipog?nese todas em rela??o dose-dependente. Este trabalho sugere que Baccharis trimera possui ?tima atividade antioxidante, prevenindo o estresse oxidativo, podendo contribuir para a diminui??o da adipog?nese, al?m de possuir ?tima atividade antiadipog?nica. Este foi o primeiro trabalho que demonstrou o potencial efeito de extratos de Baccharis trimera na diferencia??o de adip?citos 3T3-L1 em adip?citos. / Obesity is considered an important public health problem, being a risk factor for several chronic diseases such as cardiovascular diseases, hypertension, diabetes and other. Baccharis trimera (Less.) DC (carqueja) is a medicinal plant used in traditional medicine in various parts of Brazil. Infusions, decoctions and tinctures of its leaves are produced and used for the treatment of obesity and diabetes, and also as diuretics, digestive agents, anti-inflammatory drugs, among others. In this work, in order to support the medicinal potential of carqueja, extracts of Baccharis trimera leaves were obtained, characterized chemically and phytochemically and evaluated in relation to their antioxidant and antiadipogenic activities. Three extracts were obtained: aqueous (AE), decoco (AE-D) and methanolic (ME); From these, six different in vitro antioxidant assays were performed: superoxide and hydroxyl radical assay, reducing power, total antioxidant capacity and chelation of iron and copper ions. As well, its possible antiadipogenic activity was evaluated with oil red O, free glycerol and measurement of adipogenic transcription factors C / EBP?, C / EBP? and PPAR?. Phytochemical characterization revealed the presence of flavonoids (chlorogenic acid and apigenin) and phenolic compounds in extracts AE and AE-D. As for the antioxidant activity, a dose-dependent activity was observed. Regarding antiadipogenic activity, Baccharis trimera significantly inhibited the differentiation and accumulation of fat in adipocytes by MDA in a dose-dependent relationship and significantly reduced the expression of the transcription factors C / EBP?, C / EBP? and PPAR?, during adipogenesis in a relation dose-dependent too. This work suggests that Baccharis trimera has an excellent antioxidant activity, preventing oxidative stress, and may contribute to the decrease of adipogenesis, besides having an excellent antiadipogenic activity. This was the first work that demonstrated the potential effect of Baccharis trimera extracts on the differentiation of 3T3-L1 adipocytes into adipocytes.

The prevalence of type 2 diabetes mellitus is increasing worldwide and is considered one of the top health concerns globally. The occurrence of type 2 diabetes is linked to the rapidly increasing trend of obesity in both adults and children, which is proposed to be a contributing factor in the development of insulin resistance and type 2 diabetes. White adipose tissue, an insulin target tissue, is an important endocrine organ involved in the control of energy homeostasis through its direct influence on metabolism, insulin sensitivity and food intake. To better understand these functions, we studied adipocyte differentiation in 3T3-Ll cells, a white adipose tissue cell line. Many mitochondrial proteins exhibit an increase in expression levels during adipogenesis as identified by mass spectrometry. Moreover, increased mitochondrial mass and altered morphology was observed by light microscopy. Qualitative changes in mitochondrial gene expression were also observed during adipogenesis as revealed by Affymetrix GeneChip analysis. Additionally, striking changes in mitochondrial protein expression and morphology were identified following treatment with the insulin sensitizing agent, rosiglitazone. These results suggest that mitochondrial biogenesis and remodeling is inherent to white adipocyte differentiation. To investigate the physiological relevance of these findings, mRNA and protein expression profiles and mitochondrial morphology were studied during the development of insulin resistance and obesity and following treatment with rosiglitazone in ob/ob mice. These studies reveal a marked decrease in transcript levels for over 50% of mitochondrial genes with the onset of obesity in ob/ob mice. Rosiglitazone treatment stimulates enhanced expression in approximately half of these genes, as well as changes in mitochondrial mass and remodeling. Furthermore, these studies reveal that depressed oxygen consumption and fatty acid oxidation occur with obesity development and these alterations can be reversed with rosiglitazone treatment. This work identifies the previously underscored plasticity of mitochondria in white fat and suggests that mitochondrial biogenesis and remodeling in white adipose tissue may lead to systemic changes in insulin sensitivity and energy homeostasis. Lastly, these studies suggest that mitochondria may be an important therapeutic target for antidiabetic drugs.

Adipositas und ihre Folgeerkrankungen stellen eine wachsende medizinische Herausforde- rung globalen Ausmaßes dar. Im Rahmen der Adipositasforschung wurde das Fettgewebe als endokrines Organ identifiziert. Von ihm sezernierte Proteine, die sogenannten Adipo- kine, beeinflussen maßgeblich Insulinresistenz und Gefäßverletzbarkeit. Adipositas geht im Fettgewebe mit einer subklinischen chronischen Entzündung einher, die zu einer erhöhten Sekretion von proinflammatorischen Adipokinen führt. Die verstärkte Anwesenheit dieser Proteine ist mit den Komplikationen der Adipositas assoziiert. Die vorliegende Arbeit be- fasst sich mit dem Einfluss von Interleukin (IL)-1β, einem wichtigen Entzündungsmediator des Organismus, auf die Sekretion der proinflammatorischen Adipokine tissue inhibitor of metalloproteinase (TIMP)-1, serum amyloid A (SAA)-3, Lipocalin-2 und Chemerin. Die zugrundeliegenden Untersuchungen wurden mit 3T3-L1- und braunen Adipozyten durch- geführt. Es erfolgte der Nachweis auf mRNA- sowie auf Proteinebene. Der Einsatz von spe- zifischen Inhibitoren erlaubte den Rückschluss auf grundlegende Signalwege. Für alle vier untersuchten Adipokine konnte eine signifikante dosis- und zeitabhängige Steige- rung der mRNA- und Proteinexpression durch IL-1β nachgewiesen werden. Die Transduktion des IL-1β-Signals erfolgte im Falle von Lipocalin-2 und SAA-3 über nuclear factor (NF)-κB und janus kinase (Jak)-2, bei TIMP-1 lediglich über Jak-2 und in Bezug auf Chemerin über NFκB, Jak-2, p44/42 mitogen-activated protein kinase und Phosphatidylinositol-3-Kinase. Die in dieser Arbeit nachgewiesenen Expressions- und Sekretionssteigerungen von TIMP-1, SAA-3, Lipocalin-2 und Chemerin in braunen und weißen Adipozyten festigen IL-1β als einen entscheidenden Mediator proinflammatorischer Prozesse im Fettgewebe. Eine umfassende Bewertung der Funktion von IL-1β im Fettgewebe, insbesondere im Zustand der Adipositas, muss jedoch in weitergehenden Studien erfolgen.

Internalization and trafficking of ligand-receptor complex rely on a particular set of proteins, e.g. small GTPase protein Rab5 and its activators called guanine nucleotide exchange factors. Rab5-activating protein 6 (RAP6), a Vps9-containing protein, may participate in Rab5-mediated insulin signaling and receptor trafficking. A dicarbonyl compound methylglyoxal was found to alter insulin signaling in preadipocytes. This dissertation aimed to investigate the association of RAP6 activity on 3T3-L1 preadipocyte differentiation and those driven by methylglyoxal. Overexpression of RAP6 inhibited preadipocyte differentiation, Ser473-phosphorylation of Akt1, and expression of adipogenic marker PPARγ, but not C/EBPα. Methylglyoxal (10 µM) increased preadipocyte differentiation, proliferation and expression of PPARγ, C/EBPα and p-Akt1-Ser473, but appeared to be neutralized by RAP6 overexpression. The findings suggest that RAP6 may be a key modulator in regulating the stimulatory effect of methylglyoxal on preadipocyte differentiation. The associations of predominant methylglyoxal-derived adduct, methylglyoxal hydroimidazolone 1 (MGH1), with selected risk factors of chronic diseases in Black participants with and without type 2 diabetes (n=234 controls and n=254 cases) were also investigated. Only in individuals with diabetes, MGH1 levels were positively associated with fasting plasma glucose (B=0.240, p=0.037), homocysteine (B=0.355, p=0.014) and triglyceride (B=0.190, p=0.049). Being African Americans with type 2 diabetes was associated with lower MGH1 levels as compared to being Haitian American with diabetes (B=-0.334, p=0.016). The findings suggest that methylglyoxal may be linked to hyperglycemia and metabolic changes in type 2 diabetes, and may differently impact the development of diabetes across Black subgroups.

No description available.