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Charakterisierung muriner und humaner Fibroblasten mit knorpelerosivem Potential / Characterization of murine and humane fibroblasts with cartilage-erosive potentialHoffmann, Matthias 31 January 2014 (has links) (PDF)
Die rheumatoide Arthritis (RA) ist eine chronisch-entzündliche Bindegewebserkrankung mit bevorzugtem Befall der Gelenke. Es bestimmen Knorpel- und Knochendestruktionen das Krankheitsbild. Eine Schlüsselrolle in der Pathogenese nehmen proliferierende, synoviale Fibroblasten (RASF) durch Auflösung der extrazellulären Matrix (EZM), durch Interaktion mit immunkompetenten Zellen und durch Produktion pro-inflammatorischer Zytokine ein. Die vorliegende Arbeit zeigt die Migrationseigenschaften von RASF und zwei verschiedenen murinen (LS48) und humanen (TK188) Fibroblastenzelllinien in einem In-vitro-Migrationsassay. Es wird der Einfluss von Antikörpern sowie verschiedener EZM-Komponenten auf die Migration der Zelllinien untersucht. Die nachfolgende Analyse phänotypischer Charakteristika stellt dabei die besondere Rolle der genannten Fibroblastenzelllinien heraus, welche eine Reihe von Gemeinsamkeiten untereinander und mit RASF besitzen. Sie zeigen ebenso erhöhte Migrationsaktivität unter dem Einfluss eines Chemoattraktants und besitzen ähnliche Destruktionsmuster von Kollagenmatrizen. Beide Zellreihen exprimieren mehr RASF-typische Proteasen, Adhäsionsmoleküle und immunologisch agierende Proteine als nicht pathologisch transformierte Fibroblasten. Ebenso weisen sie eine gesteigerte Stoffwechselaktivität und Proliferationstätigkeit auf. Diese in vitro erbrachten Hinweise auf mögliche Knorpeldestruktionen sollten Anlass für weitere In-vivo-Studien zu den genannten Zelllinien geben.
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Atividades biol?gicas de xilana de sabugo de milhoSilveira, Raniere Fagundes de Melo 25 February 2010 (has links)
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Previous issue date: 2010-02-25 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study
aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that
combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in
the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (?H and ??C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical
and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase
cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 ?g of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods,
pharmaceuticals and chemical industries. / O sabugo de milho ? um subproduto agr?cola ainda pouco utilizado, isto se deve em parte ao baixo conhecimento do potencial biotecnol?gico de suas biomol?culas. Xilana de sabugo de milho (XSM) ? um polissacar?deo presente em maior quantidade na estrutura do vegetal e seu potencial biotecnol?gico ? pouco conhecido. Este trabalho teve como objetivo a extra??o, caracteriza??o qu?mica e avalia??o de atividades biol?gicas de XSM. Sabugos de milho foram limpos, cortados, desidratados e triturados, dando origem a uma farinha. Esta foi submetida a uma metodologia que combina o uso de meio alcalino com ondas de ultra-som. Ap?s precipita??o metan?lica, centrifuga??o e secagem obteve-se um rendimento de 40% (g/g de farinha). An?lises qu?micas indicaram um alto percentual de polissacar?deos na amostra (60%) e baixa contamina??o por prote?nas (0.4%) e compostos fen?licos (>0.01%). An?lises da composi??o monossacar?dica por cromatografia em papel e por cromatografia l?quida de alta performance (CLAE) indicaram a presen?a de xilose:glicose:arabinose:galactose:manose:?cido glucur?nico em uma propor??o molar de 50:20:15:10:2,5:2,5. A presen?a de xilana na amostra foi confirmada por resson?ncia magn?tica nuclear (13C e 1H) e por espectroscopia de infravermelho (IR). Testes foram realizados para avalia??o do potencial antioxidante de XSM. Esta mostrou uma capacidade antioxidante total de 48.45 EAA/g de amostra. Contudo, a mesma n?o mostrou atividade sequestradora de super?xido, radical hidroxila bem como poder redutor. Em contra partida,
apresentou 70% de atividade quelante de ?ons de ferro na concentra??o de 2 mg/mL. A capacidade de XSM em influenciar a prolifera??o celular em cultura tamb?m foi avaliada. Este polissacar?deo n?o influenciou a prolifera??o de c?lulas fibrobl?sticas normais (3T3), entretanto, diminuiu a taxa de prolifera??o de c?lulas tumorais (HeLa) de maneira dose-dependente, chegando a uma inibi??o de aproximadamente 50% com concentra??o em torno de 2 mg/mL.
Analisando prote?nas relacionadas ? morte celular, atrav?s de immunoblotting, XSM aumenta a quantidade de Bax, citocromo c e AIF e diminui Bcl-2 e procaspase- 3, indicando a indu??o de morte celular por apoptose dependente e independente de caspase. XSM n?o apresentou atividade anticoagulante pelo
teste de PT. Todavia, no teste de tempo de tromboplastina parcial ativada (aPTT), XSM aumentou o tempo de coagula??o em cerca de 5 vezes utilizando 600 ?g de amostra, quando comparadas com o controle negativo. A presen?a de sulfato ligado a XSM foi descartada por eletroforese em gel de agarose e por IR. Ap?s carboxirredu??o de XSM a atividade anticoagulante diminuiu drasticamente. Os dados deste trabalho demonstram que XSM apresenta potencial como composto antioxidante, antiproliferativo e anticoagulante. Estudos futuros de caracteriza??o dessas atividades do XSM contribuir?o para
aumentar o conhecimento sobre esta mol?cula extra?da de milho e permitir?o a sua utiliza??o em alimentos funcionais, produtos farmac?uticos e ind?strias qu?micas.
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Extratos da planta Baccharis trimera (Less.) DC, carqueja, possui atividades antioxidante e anti-adipog?nica por inibir a express?o de prote?nas envolvidas na diferencia??o adipocit?ria in vitroNascimento, Daniele de Souza Marinho do 22 June 2017 (has links)
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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.
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Analysis of Mitochondrial Remodeling in Adipocytes during Adipogenesis and Obesity Development: a DissertationWilson-Fritch, Leanne 15 April 2004 (has links)
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.
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Expression, purification, and characterization of recombinant basic fibroblast growth factor in pichia pastorisLe, Henry Hieu Minh 01 January 2019 (has links)
Wounds in the mouth, occurring after oral surgery, take time to heal. No ointment can be added to help with the healing process because mouth saliva will constantly wash it away. In order to combat this problem, we propose engineering a normal flora microbe to grow at the site of injury and secrete a recombinant growth factor to promote healing of the damaged tissue. Our goal is to have the yeast Pichia pastoris produce human basic fibroblast growth factor (bFGF), which aids in cellular proliferation. P. pastoris is a good choice for this application because not only is it considered generally recognized as safe (GRAS) by the FDA, but it is a eukaryote that is able to perform posttranslational modifications and secrete large amounts of recombinant protein.
Previous studies have shown that a strain of P. pastoris can be engineered to express bFGF from a methanol-sensitive promoter. The study also showed that the bFGF, which was purified from the yeast’s extracellular medium, was able to promote the growth of NIH/3T3 cells (mice fibroblasts). Because we needed the P. pastoris to express the bFGF in glucose –based tissue culture medium in the presence of mammalian cells, we expressed the bFGF from the constitutive promoter GAP promoter. Along with optimizing and characterizing expression of bFGF, we also investigated the effect of the recombinant protein on mammalian cell growth using both scratch ad MTS assays. In addition, the effects of the yeast being co-cultured with mammalian cells was studied. Our results provide a basis for how a recombinant protein can be clinically used to improve wound healing in the mouth using a yeast strain to produce and secrete a growth factor at the site of injury.
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Emprego da citotoxicidade basal in vitro na redução do número de animais em ensaios de avaliação da toxicidade oral aguda: a grandisina e seu metabólito majoritário como protótipos / Use of basal cytotoxicity in vitro in reducing the number of animals tests in the evaluation of acute oral toxicity: a grandisin and its major metabolite as prototypesVIEIRA, Marcelo de Sousa 14 April 2009 (has links)
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Previous issue date: 2009-04-14 / The animal replacement in expirements has been very encouraged by government and
other institutions. However, in drugs development the animal replacement is not yet a reality, like
at oral acute systemic tests. The validation of in vitro protocols is necessary for data generation
with reproducibility, repetability and accuracy. In this study was validated at the Laboratório de
Farmacologia e Toxicologia Celular- Faculdade de Farmácia/Universidade Federal de Goiás the
protocol already validated by three laboratories (two in the USA and one at United Kingdom) and
coordinated by ICCVAM: In Vitro Cytotoxicity Methods for Estimating Starting Doses for Acute
Systemic Toxicity Tests, using the neutral red uptake in BAL/c 3T3-A31 cell line. It was used the
grandisine, a lignan, and its major metabolite taken by fungi biodegradation. Our research group
has identified a potential anti-tumoral action of grandisine, data not yet published. After in house
validation we estimated the LD50 of grandisin and 4-O-demethylgrandisin: 617.72 mg/kg and
429.95 mg/kg, respectively. Both were classified under the GSH category 4. / A substituição ou redução do uso de animais em experimentos para a avaliação de
toxicidade tem sido bastante encorajada e tem recebido grandes incentivos, inclusive financeiros,
governamentais e institucionais. No entanto, a substituição completa da maioria dos testes
mandatórios por agências reguladoras do setor ainda não é realidade, a exemplo, o teste de
toxicidade oral aguda sistêmica. Neste contexto, se aplica a validação de testes in vitro para
estimar dados in vivo. No presente trabalho, realizamos a validação in house do protocolo
internacional e multilaboratorial recomendado pelo Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM): Utilização da Citotoxicidade In Vitro na
Estimativa de Doses Iniciais para Testes de Toxicidade Oral Aguda Sistêmica. Para tal,
utilizamos o método de captação do corante vermelho neutro e a linhagem celular fibroblástica de
camundongos BALB/c 3T3-A31. Dentre as substâncias recomendadas pelo ICCVAM para a
validação do método foram investigadas 9 substâncias. A metabolização de produtos pelo
organismo vivo é uma grande limitação dos testes in vitro. Utilizamos a grandisina, um tipo de
ligana com grande potencial antitumoral e seu metabólito majoritário como substâncias modelos.
Com utilização do metabólito conseguimos transpor esta barreira dos testes in vitro. Os
resultados demonstraram que os dados obtidos estão em consonância com os dados da literatura
sendo possível classificar ambas as substâncias na categoria GHS 4: grandisina e 4-Odemetilgrandisina:
617,72 mg/kg and 429,95 mg/kg, respectivamente.
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Einfluss von Interleukin-1 beta auf die Expression und Sekretion der Adipokine TIMP-1, SAA-3, Lipocalin-2 und Chemerin in 3T3-L1 AdipozytenWeise, Sebastian 26 February 2013 (has links) (PDF)
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.
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Bcl-2 related ovarian killer, Bok, is cell cycle regulated and sensitizes to stress-induced apoptosisRodríguez, José M. January 2007 (has links)
Dissertation (Ph.D.)--University of South Florida, 2007. / Title from PDF of title page. Document formatted into pages; contains 82 pages. Includes vita. Includes bibliographical references.
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Regulation of Adipocyte Differentiation and Metabolism: Rab5-Guanine Nucleotide Exchange Factors and MethylglyoxalChantarasinlapin, Praew 31 March 2017 (has links)
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.
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Biocompatibility of Carbon Nanomaterials: Materials Characterization and Cytotoxicity EvaluationZhu, Lin 21 August 2012 (has links)
No description available.
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