The world is continuously moving, and so are the life on it. As our society is constantly evolving and the width of human needs are rising, do organization need to provide new solutions that can satisfy our needs. Consequently, every designer is going to meet new challenges whenever the situation calls for it. This is the situation that CEVT’s engineers have found themselves in and the reason as to why this engineering project has become relevant for their industrial development and innovation. The seatbelt designers at CEVT’s Restraints department have encountered a situation where it forces them to change their product as it can no longer be installed in the cars’ B-pillars. The company must investigate alternative positionings with regard to the car's new design criteria in order to recreate or improve the functionalities in both safety and comfort of their seatbelt system. My project objective is to investigate alternative positionings and components that are part of the classic three-point seatbelt system with an aim to ensure good user experience in the area of comfort. By the end of this thesis I ought to have answered the following Mission Statement: “Determine the most optimal positioning and components of the seatbelt system to reduce inertia for a fontal Belt-in-Seat, where the system’s performance should be comparable to the users’ experience from an installation of a seatbelt in a B-pillar.” To establish the best component combination out of the parts delivered from Autoliv AB and secure an optimal placement for the involved parts, have I followed the three-stage process described by IDEO (2015). The Inspiration phase has included a Literature study, benchmarking, analytical assessments of user needs as well as prepared and performed of a test on the seatbelt system. The Ideation phase was focusing on establishing a placement for the system's components through a brainstorming so that it could be mounted in a seat prototype prepared for the user experience tests performed. The last phase, Implementation, consisted of an analysis that was focusing on the feedback received from users of different anthropometry. But also concentrated on summarizing all data collected throughout the project to select the final concept for this assignment. / Världen och allt liv på jorden är i ständig rörelse. När samhället strävar efter utveckling uppkommer samtidigt nya behov hos människan och dess omgivning, vilket gör att olika organisationer och företag behöver leverera nya lösningar och designa artefakter för att tillfredsställa människans behov. Varje designer kommer att möta nya utmaningar när en situation tvingar dem att ändra sina produkter. Detta är en situation som CEVT:s bilbältesdesigners står inför och anledningen till att detta arbete blivit aktivt för företagets industriella utveckling inom innovation. Situationen som tvingar konstruktörerna att ändra bältessystemets design är att systemet inte längre kan monteras i passagerarbilens B-stolpar. Därför måste företaget undersöka alternativa positioneringar som överensstämmer med bilens designkriterier för att återskapa bältets funktionalitet som berör både säkerhet och komfort av deras bältessystem. Arbetets syfte är att undersöka alternativa positioneringar och de inkluderade komponenterna som utgör ett klassiskt tre-punkts bälte med målet att säkerställa en god användarupplevels inom området komfort. I slutet av projektet skall jag ha uppfyllt följande Mission Statement: “Bestäm den mest optimala positioneringen och komponenter av ett säkerhetsbältes system för att reducera krafterna från ett bältes-integrerat framsäte, en ’Belt-in-Seat’, där systemets prestandard skall vara jämförbart med användarens upplevelse av en installation av bältet i bilens B-stolpe.”   Jag har följt IDEOs (2015) tre-stegs process för att säkerställa den bästa kombinationen av komponenter som har tillhandahållits av Autoliv AB samt fastställa en optimal placering av varje komponent i systemet. Inspirationsfasen har inkluderat en litteraturstudie, benchmarking, analytiska bedömningar av användarens behov samt förberett och genomfört ett test på bältesystemets prestandard. Ideationsfasen fokuserade på att etablera en placering för systemets komponenter genom en brainstorming så att dessa kunde monteras i en sätesprototyp som förberetts inför testerna för att evaluera användarens komfortupplevelse. Den sista fasen, Implementation, bestod av en analys som fokuserade på feedbacken från testpersonerna som erhöll olika antropometri. Fasen kretsade kring att sammanfatta all data som samlats in genom hela projektet för att välja det slutliga konceptet för uppgiften.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21972号 / 農博第2362号 / 新制||農||1071(附属図書館) / 学位論文||R1||N5223(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 木岡 紀幸, 教授 三芳 秀人, 教授 植田 充美 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Previously it was demonstrated that biliary excretion of a single dose of [14C]dieldrin or [3H]7, 12-dimethylbenz/alanthracene (DMBA) was stimulated up to 700% and 300%, respectively, in rainbow trout fed 0.3-0.4 mg dieldrin/kg/d for 9-12 wk. This was not explained by increased activities of hepatic microsomal xenobiotic-metabolizing enzymes or increased amounts of any of six cytochrome P-450 isozymes quantitated by Western blots. It was hypothesized that stimulated excretion was explained by induction of (1) cytosolic binding proteins that facilitated intracellular trafficking of DMBA to sites of metabolism, or (2) ATP-dependent proteins that transport xenobiotic metabolites from liver to bile. Binding of 15 and 60 nmol [3H]DMBA/mg protein increased about 200% in hepatic cytosol from dieldrin-fed fish. A 50-fold molar excess of unlabeled DMBA reduced binding of 15 nmol [3H]DMBA/mg protein (nonspecific binding) by the same amount in cytosol from control and dieldrin-fed fish, indicating that dieldrin induced specific binding. Liver sections from control and dieldrin-fed fish were treated with multidrug resistance (MDR) protein monoclonal antibodies C494, C219, and JSB-1, and polyclonal antibody MDR Ab-1. There were no marked differences in optical densities of immunohistochemical staining near bile canaliculi of control and dieldrin-fed fish. Induction of xenobiotic binding capacity in cytosol of dieldrin-fed rainbow trout at least partially explained altered DMBA disposition in fish pretreated with this cyclodiene insecticide.

Flax (Linum usitatissimum L.) is a self-pollinated crop widely cultivated for fiber and oil production. Flaxseed is renowned for its health attributes but the presence of compounds, such as the heavy metal cadmium (Cd), is undesirable. Genomic studies in flax have produced large amounts of data in the last 15 years, providing useful resources to improve the genetic of this crop using genomics-based technologies and strategies. The goal of this thesis is therefore to capitalize on these advances to address the Cd problem and to propose solutions to improve breeding efficiencies. To find genomic-based solutions to Cd content, to the currently low breeding efficiency and to abiotic stress resistance in flax, this study utilized four major strategies: (1) genomic cross prediction, (2) gene family identification, (3) genome-wide association study (GWAS) and (4) genomic selection (GS). Characterization of the ATP-binding cassette (ABC) transporter and heavy metal associated (HMA) gene families was performed using the flax genome sequence. A total of 198 ABC transporter and 12 HMA genes were identified in the flax genome, of which nine were orthologous to Cd-associated genes in Arabidopsis, rice and maize. A transcriptomic analysis of eight tissues provided some support towards the functional annotation of these genes and confirmed the expression of these ABC transporter and HMA genes in flax seeds and other tissues. A diversity panel of 168 flax accessions was grown in the field at multiple locations and years and the seed content of 24 heavy metals (HMs) was measured. The panel was also sequenced and a single nucleotide polymorphism (SNP) dataset of nearly 43,000 SNPs was defined. A GWAS was conducted using these genotypic and phenotypic data and a total of 355 non-redundant quantitative trait nucleotides (QTNs) were identified for ten of the 24 metal contents. Overall, a total of 24 major and 331 minor effect QTNs were detected, including 11 that were pleiotropic. After allelic tests, 108 non-redundant QTNs were retained for eight of the ten metals and ranging from one for copper (Cu) to 70 for strontium (Sr). A total of 20 candidate genes for HM accumulation were identified at 12 of the 24 major QTN loci, of which five belonged to the ABC transporter family. Many of the metal contents, including Cd, appeared to be controlled by many genes of small effects; hence, GS is better suited than marker-assisted selection for application in breeding. To test this, predictive ability using ten GS statistical models was evaluated using trait-specific QTN and the random genome-wide 43K SNP datasets. Significantly higher predictive abilities were observed from the GS models built with the dataset made of QTNs associated with metal contents (70-80%) compared to that of the 43K dataset (10-25%). This study showed the feasibility of using GS to improve the predictive ability of polygenic traits such as metal content in seeds. GS can be applied in early generation selection to accelerate the improvement of abiotic stress resistance and either select low-Cd lines or discard high-Cd lines. These findings validate the use of a QTL-based strategy as a highly effective method for improving the efficiency of predictive ability of GS for highly complex traits such as resistance or tolerance to HM accumulation. Identification of both large and minor effect QTNs and/or pleiotropic effects hold potential for flax breeding improvement. Candidate gene functional validation can be performed using methods such as genome editing or targeting induced local lesions in genomes (TILLING).

No / Hair disorders cause psychological distress but are generally poorly controlled; more effective treatments are required. Despite the long-standing use of minoxidil for balding, its mechanism is unclear; suggestions include action on vasculature or follicle cells. Similar drugs also stimulate hair, implicating ATP-sensitive potassium (K(ATP)) channels. To investigate whether K(ATP) channels are present in human follicles, we used organ culture, molecular biological, and immunohistological approaches. Minoxidil and tolbutamide, a K(ATP) channel blocker, opposed each other's effects on the growing phase (anagen) of scalp follicles cultured in media with and without insulin. Reverse transcriptase-polymerase chain reaction identified K(ATP) channel component gene expression including regulatory sulfonylurea receptors (SUR) SUR1 and SUR2B but not SUR2A and pore-forming subunits (Kir) Kir6.1 and Kir6.2. When hair bulb tissues were examined separately, epithelial matrix expressed SUR1 and Kir6.2, whereas both dermal papilla and sheath exhibited SUR2B and Kir6.1. Immunohistochemistry demonstrated similar protein distributions. Thus, human follicles respond biologically to K(ATP) channel regulators in culture and express genes and proteins for two K(ATP) channels, Kir6.2/SUR1 and Kir6.1/SUR2B; minoxidil only stimulates SUR2 channels. These findings indicate that human follicular dermal papillae contain K(ATP) channels that can respond to minoxidil and that tolbutamide may suppress hair growth clinically; novel drugs designed specifically for these channels could treat hair disorders.

No description available.

Introdução: A quantidade e o tipo de gordura alimentar exercem importante influência no desenvolvimento de doença cardiovascular (DCV) e podem contribuir para o desenvolvimento de esteatose hepática. Os ácidos graxos saturados e trans são consensualmente apontados como aterogênicos; já os poli-insaturados parecem exercer ação antiaterogênica. Com relação a esteatose hepática, sabe-se que os ácidos graxos saturados estão associados com o seu desenvolvimento; porém, a ação dos ácidos graxos trans na gênese e no desenvolvimento de esteatose hepática não está totalmente elucidada. Neste estudo, avaliou-se o efeito do consumo de dietas enriquecidas com ácidos graxos saturados (SAT), poli-insaturados (POLI) ou trans (TRANS) sobre componentes envolvidos na indução e na progressão da placa aterosclerótica, bem como sobre o desenvolvimento da doença hepática gordurosa não alcoólica. Métodos: Camundongos com ablação gênica para o receptor de LDL (LDLr-KO) foram alimentados com dietas hiperlipídicas (40% do valor calórico total sob a forma de gordura), enriquecidas com ácidos graxos SAT, POLI ou TRANS por 16 semanas e ao final submetidos a: 1) análises plasmáticas: colesterol total (CT), triglicérides (TG), insulina, glicose, aspartato aminotransferase (AST) e alanina aminotransferase (ALT), interleucina-6 (IL-6), fator de necrose tumoral-? (TNF-alfa) e perfil de lipoproteínas; 2) determinação da lesão aterosclerótica: área de lesão (Oil Red-O), conteúdo de ATP-binding cassette transporter A1 (ABCA1) e infiltrado de macrófagos (imuno-histoquímica), colocalização de ABCA1 e macrófagos (microscopia confocal) e conteúdo de colágeno (Picrosirius-Red) na raiz aórtica; 3) conteúdo de CT, colesterol éster (CE) e colesterol livre (CL) na aorta total; 4) macrófagos peritoneais foram tratados com lipopolissacarídeo (LPS), e IL-6, TNF-alfa e interleucina-10 (IL-10) medidas no meio de cultura; 5) no fígado: grau da doença hepática gordurosa não alcoólica, concentração de CT e TG e expressão de RNA mensageiro (mRNA) de PPAR-gama, PPAR-gama, SREBP-1c, MTP, CPT-1 e ABCA1 por RT-qPCR; 6) determinação do conteúdo de tecido adiposo visceral e subcutâneo na carcaça. Resultados: O consumo de dieta não diferiu entre os grupos; comparado à dieta POLI, TRANS induziu menor ganho de peso refletido por menor conteúdo de tecido adiposo. TRANS induziu hepatomegalia, desenvolvimento de esteato-hepatite não alcoólica (NASH) e piora da sensibilidade insulínica (evidenciada pelo índice HOMAIR). As concentrações de AST e ALT não diferiram entre os grupos. A dieta TRANS elevou a expressão de mRNA de genes relacionados à lipogênese hepática (PPAR-gama e SREBP-1c) comparada à SAT e POLI e reduziu a expressão de MTP comparada à dieta POLI. Não houve diferença entre os grupos com relação à expressão de genes envolvidos na oxidação hepática de lípides (PPAR-gama e CPT-1). As concentrações plasmáticas de CT e TG foram maiores no grupo TRANS comparado a SAT e POLI. POLI apresentou menor área de lesão, infiltrado de macrófagos e conteúdo de ABCA1 comparados a SAT e TRANS. Macrófagos e ABCA1 não se colocalizaram na área de lesão. O conteúdo de CT na parede arterial foi menor no grupo POLI comparado a TRANS; CL foi menor no grupo POLI comparado a SAT e TRANS; CE não diferiu entre os grupos. Comparado a POLI, SAT e TRANS apresentaram maior conteúdo de colágeno e núcleos necróticos na placa aterosclerótica. A concentração plasmática de IL-6 não diferiu entre os grupos; já a concentração de TNF-alfa foi maior nos grupos POLI e TRANS em comparação a SAT. Em relação à resposta inflamatória de macrófagos ao LPS, POLI e TRANS apresentaram maiores concentrações de IL-6 e TNF-alfa comparadas a SAT. POLI apresentou menores concentrações de IL-10 em comparação aos demais grupos. A expressão hepática de ABCA1 não diferiu entre os grupos. Conclusão: O consumo de dieta TRANS induziu perfil lipídico proaterogênico, hipercolesterolemia, hipertrigliceridemia, hiperglicemia e severo desenvolvimento de aterosclerose, além de hepatomegalia, maior acúmulo hepático de lípides e desenvolvimento de NASH. Por outro lado, POLI preveniu o desenvolvimento de aterosclerose, independentemente de sua ação inflamatória. / Introduction: The amount and type of dietary fat play important roles on the development of cardiovascular disease (CVD) and on the development of hepatic steatosis. Saturated (SAT) and trans (TRANS) fatty acids are known as pro-atherogenic, while the polyunsaturated (POLY) fats seem to exert an antiatherogenic action. Regarding hepatic steatosis, it is known that SAT are associated with its development, however, the role of TRANS in the genesis and development of hepatic steatosis is not fully undestood. This study evaluated the effect of the intake of diets enriched with SAT, POLY or TRANS on the parameters involved in the progression of the atherosclerotic plaque and also on the development of the nonalcoholic fatty liver disease (NAFLD). Methods: LDL receptor knock-out (LDLR-KO) male mice were fed for 16 weeks a high fat diet (40% of calories as fat) enriched with SAT, POLY or TRANS, for 16 weeks. The following parameters were mesured: 1) plasma: total cholesterol (TC), triglyceride (TG), insulin, glucose, aspartate aminotransferase (AST) and alanine amino transferase (ALT), interleukin-6 (IL-6), tumor necrosis factor- ? (TNF-?) and lipoprotein profile; 2) atherosclerotic lesion - lesion area (Oil Red-O), ATP-binding cassette transporter A1 (ABCA1) content and macrophage infiltration (immunohistochemistry), co-localization of ABCA1 and macrophages (confocal microscopy) and collagen content (Picrosirius-Red); 3) TC, cholesteryl ester (CE) and free cholesterol (FC) content of the total aorta; 4) interleukin-6 and 10 (IL-6 and IL-10) and TNF-alfa in the culture medium of peritoneal macrophages after treatment with lipopolysaccharide (LPS); 5) liver: degree of fat liver disease, concentration of TC and TG and mRNA expression (RT-qPCR) of PPAR-gama, PPAR-gama, SREBP-1c, MTP, ABCA1 and CPT-1; 6) visceral and subcutaneous adipose tissue contents in the carcass of the animals. Results: Food intake did not differ amongst the groups, however, compared to POLY, TRANS induced less weight gain, due to lower adipose tissue content. TRANS induced hepatomegaly, nonalcoholic steatohepatitis (NASH) and worsening of insulin sensitivity, as evidenced by the index HOMAIR. The concentrations of ALT and AST did not differ among groups. TRANS increased the mRNA expression of the hepatic lipogenic genes (PPAR-gama and SREBP-1c) compared to the SAT and POLY and reduced the mRNA expression of MTP compared to POLI. There was no difference among the groups regarding the mRNA expression of genes involved in hepatic lipid oxidation (PPAR-gama and CPT-1). Plasma concentrations of TC and TG were higher in TRANS compared to SAT and POLY. POLY showed lower arterial lesion area, macrophage infiltration and ABCA1 content compared to SAT and TRANS. ABCA1 and macrophages did not colocalize in the lesion area. The TC content in the arterial wall was lower on POLY compared to TRANS; FC was lower on POLY compared to SAT and TRANS; CE did not differ among groups. Compared to POLY, SAT and TRANS showed higher collagen content and necrotic core in atherosclerotic plaques. The plasma concentration of IL-6 did not differ among groups, however, TNF-alfa plasma concentration was higher in POLY and TRANS compared to SAT. Regarding the macrophage inflammatory response to LPS, POLY and TRANS showed higher concentrations of IL-6 and TNF-alfa compared to SAT. Moreover, POLY had the lowest concentration of the anti-inflammatory cytokine IL-10. The hepatic expression of ABCA1 did not differ amongst the groups. Conclusion: TRANS induced pro-atherogenic lipid profile, hypercholesterolemia, hypertriglyceridemia, hyperglycemia, and severe atherosclerosis, and in addition, elicted hepatomegaly, increased hepatic lipid accumulation and NASH. On the other hand, POLY prevented the development of atherosclerosis, independently of their pro-inflammatory activity.

Produtos de glicação avançada, carbamilação e estresse oxidativo contribuem como fatores de risco não tradicionais para a aterosclerose na doença renal crônica (DRC), em parte, por prejudicarem o metabolismo lipídico e por representarem um mecanismo de injúria memorizado ao longo do desenvolvimento da doença renal. A albumina sérica, isolada de animais com DRC, reduz a remoção de colesterol mediado por apoA-I e subfrações de HDL, prejudicando o fluxo de colesterol de macrófagos arteriais ao fígado por meio do transporte reverso de colesterol. Objetivo: Avaliou-se a influência do tratamento com N-acetilcisteína (NAC) em ratos com DRC sobre a concentração plasmática de produtos de oxidação e glicação avançada e o reflexo sobre os efeitos da albumina sérica sobre o efluxo de colesterol e o estresse de retículo endoplasmático em macrófagos. Métodos: Ratos Wistar com 2 meses de idade, pesando aproximadamente 200-250g foram submetidos à nefrectomia 5/6 e mantidos por 60 dias (grupo DRC) com ou sem tratamento com N-acetilcisteína na água (600mg/L), após o 7° dia de indução da DRC (grupo DRC + NAC). Animais controles foram falso-operados (grupo C) e um subgrupo submetido ao tratamento com NAC (C + NAC). No início e no final do estudo foram determinadas as concentrações plasmáticas de glicose, colesterol (CT), triglicérides (TG), ureia, creatinina e na urina, excreção urinária de proteína de 24 h. AGE totais, pentosidina, TBARS (marcador de peroxidação lipídica) e pressão arterial sistólica (PAS) foram determinados no final do estudo. A albumina sérica foi isolada por cromatografia rápida para separação de proteínas e purificada por extração alcoólica. Macrófagos J774 foram incubados por 18 h com as albuminas dos diferentes grupos experimentais para determinação do conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) e de marcadores de estresse de retículo endoplasmático (chaperonas Grp 78, Grp94 e proteína dissulfeto isomerase, PDI) por imunolbot e efluxo de colesterol, mediado por apo A-I e HDL2. Para isto, as células foram previamente enriquecidas com LDL-acetilada e 14C-colesterol. Macrófagos foram também incubados isoladamente com concentrações crescentes de NAC para avaliação do conteúdo dos receptores de HDL. Resultados: Ao final do estudo, o peso corporal foi 10% menor no grupo DRC em comparação ao C (p=0,006). Esta alteração foi prevenida pelo tratamento com NAC. A PAS (mmHg) foi maior no grupo DRC (130 ± 3) em comparação ao grupo DRC+NAC (109±3; p=0,0004). Ureia, creatinina, CT, TG (mg/dL), proteinúria (mg/24 h), AGE total, pentosidina (unidades arbitrárias de fluorescência) e TBARS (nmol/mL) foram maiores nos grupos DRC em comparação ao grupo C (122 ± 8 vs. 41 ± 0,9 ; 0,9 ± 0,07 vs. 0,4 ± 0,03; 151 ± 6 vs. 76 ± 2,7; 83 ± 4 vs. 51,5 ± 3; 46 ± 2,5 vs. 14 ± 0,9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6,6 ± 0,5 vs. 2 ± 0,2, respectivamente) (p < 0,0001) e nos grupos DRC+NAC em comparação ao grupo C+NAC (91,4 ± 5 vs. 40 ± 0,9 ; 0,6 ± 0,02 vs. 0,3 ± 0,02; 126 ± 7,5 vs. 76 ± 2,6; 73 ± 6 vs. 68 ± 4; 51 ± 3,5 vs. 18,4 ± 1,5; 24720 ± 1114 vs. 20040 ± 700; 10080 ± 748 vs. 5050 ± 267; 4,5 ± 0,5 vs. 1,8 ± 0,2, respectivamente) (p < 0,0001). No grupo DRC + NAC, PAS, CT, ureia, creatinina, AGE total, pentosidina e TBARS foram, respectivamente, 17% (p=0,0004), 17% (p=0,02), 25% (p=0,02), 33% (p=0,06), 24% (p < 0,0001), 40% (p=0,0008), 28% (p=0,009) menores do que no grupo DRC. A glicemia foi maior nos grupos C + NAC (107+-4,6) e DRC + NAC (107+-2,6) em comparação ao C (96+-1,8) e DRC (98+-1,6), respectivamente. Macrófagos tratados com albumina-DRC apresentaram maior conteúdo de PDI (5 vezes; p=0,02 e 7 vezes p=0,02) e Grp94 (66 %; p =0,02 e 20 %; p=0,02) quando comparados aos tratados com albumina-C ou albumina-DRC + NAC, respectivamente. O conteúdo do receptor ABCA-1 foi menor 87% e 70% (p < 0,01) nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação com albumina-C. O conteúdo de ABCG-1 foi, respectivamente, 4 e 7,5 vezes maior nos macrófagos tratados com albumina-C+NAC e albumina-DRC+NAC em comparação as respectivas situações sem tratamento. O efluxo de colesterol mediado por apo A-I foi 59 % e 70 % (p < 0,0001) menor nos macrófagos tratados com albumina-C+NAC e albumina-DRC, respectivamente em comparação a albumina-C. O efluxo de colesterol mediado pela HDL2 foi 52 % maior nos macrófagos tratados com albumina-C+NAC em comparação as células tratadas com albumina-C. Não houve diferença no conteúdo do receptor ABCA-1 nos macrófagos tratados com concentrações crescentes NAC por 8 h. No entanto, após 18 h, o ABCA-1 diminuiu 50 %, 69 % e 72 % nos macrófagos tratados respectivamente com 10 mM, 20 mM e 30 mM de NAC isoladamente em comparação aos macrófagos controles. O conteúdo de ABCG-1 nos macrófagos tratados com NAC, em 8 h e 18 h não sofreu alteração. Conclusão: A N-acetilcisteína reduz produtos de oxidação e glicação avançada no plasma de animais com DRC e previne o estresse de RE em macrófagos, induzido pela albumina isolada destes animais. Apesar de diminuir o conteúdo de ABCA-1 e o efluxo de colesterol mediado por apo A-I, a NAC aumenta o conteúdo de ABCG-1. Desta forma, a NAC pode contribuir para atenuar os efeitos deletérios da albumina modificada na DRC sobre o acúmulo lipídico em macrófagos, contribuindo para a prevenção da aterosclerose / Advanced glycation, carbamylation and oxidative stress c contribute to atherosclerosis in chronic kidney disease (CKD) as nontraditional risk factors. They impair lipid metabolism and promote a long last injury during the development of CKD. Serum albumin isolated from CKD-animals reduces cholesterol efflux mediated by apoa A-I and HDl subfractions, impairing the cholesterol flux from arterial wall macrophage to the the liver by the reverse cholesterol transport (RCT).Objective: In the present study it was analyzed the influence of N-acetylcysteine treatment in CKD-rats in plasma concentration of lipid peroxides and advanced glycation end products and the effect of serum albumin in macrophage cholesterol efflux and endoplasmic reticulum stress development. Methods: Two months male Wistar weighting 200-250g were submitted to a 5/6 nephrectomized maintained for 60 days (CKD group) treated or not with N-acetylcysteine in water (600 mg/L), after the seventh day of CKD induction (CKD+NAC group). Sham animals were false-operated (SHAM group) and a subgroup was treated with NAC (SHAM+NAC group). In the basal and final periods it was determined plasma concentration of glucose, total cholesterol (TC), triglycerides (TG), urea, creatinine and 24h-urinary protein excretion (UPE). Total AGE, pentosidine, thiobarbituric reactive substances (TBARS) levels and systolic blood pressure (SBP) were measured at the final period only. Serum albumin was isolated by fast protein liquid chromatography and purified by alcoholic extraction. J774 macrophage were incubated for 18 h with albumin isolated from the experimental groups in order to determine the content of HDL receptors and markers of endoplasmic reticulum stress (Grp78, Grp94 and protein dissulfide isomerase, PDI) by immunioblot and cholesterol efflux mediated by apo A-I and HDL2. For this, cells were previously overloaded with acetylated LDL and 14C-cholesterol. Macrophage were also incubated with different concentrations of NAC alone in order to measure HDL-receptors and cholesterole efflux. Results: In the end of the protocol, body weight was 10% lower in CKD group in comparison to SHAM group (p=0.006). This change was preserved by treatment with NAC. SBP (mmHg) was higher in CKD group (130±3) in comparison to CKD+NAC (109±3; p=0.0004). Urea, creatinine, TC, TG (mg/dL), UPE (mg/24 h), total AGE, pentosidine (arbitrary units of fluorescence) and TBARS (nmol/mL) were higher in CKD group in comparison to SHAM (122±8 vs. 41 ± 0.9; 0.9 ± 0.07 vs. 0.4 ± 0.03; 151 ± 6 vs. 76±2.7; 83 ± 4 vs. 51.5 ± 3; 46 ± 2.5 vs. 14 ± 0.9; 32620 ± 673 vs. 21750 ± 960; 16700 ± 1370 vs. 5314 ± 129; 6.6 ± 0.5 vs. 2 ± 0.2, respectively) (p < 0.0001) and in CKD+NAC in comparison to C+NAC (91.4±5 vs. 40±0.9 ; 0.6±0.02 vs. 0.3 ± 0.02; 126±7.5 vs. 76 ± 2.6; 73±6 vs. 68±4; 51 ± 3.5 vs. 18.4±1.5; 24720 ± 1114 vs. 20040±700; 10080±748 vs. 5050 ± 267; 4.5±0.5 vs. 1.8±0.2, respectively) (p < 0.0001). In CKD+NAC group, SBP, TC, urea, creatinine, total AGE, pentosidine and TBARS were, respectively, 17 % (p=0.0004), 17 % (p=0.02), 25 % (p=0.02), 33 % (p=0.06), 24 % (p<0.0001), 40 % (p=0.0008), 28 % (p=0.009) lower than CKD group. Glycemia was higher in SHAM+NAC (107+-4.6) and CKD+NAC (107+-2.6) in comparison to SHAM (96+-1.8) and CKD group (98+-1.6), respectively. Macrophages treat with CKD-albumin presented higher content of PDI (5 times; p=0.02 e 7 times p=0.02) and Grp94 (66 %; p=0.02 e 20 %; p=0.02) when compared to SHAM-albumin and CKD+NAC-albumin- treated cells, respectively. ABCA-1 protein content was 87 % and 70 % (p < 0.01) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively compared with SHAM-albumin. ABCG-1 protein level was respectively 4 and 7.5 times higher in macrophages treated with SHAM+NAC-albumin and CKD+NAC-albumin in comparison to their respective controls without treatment. The cholesterol efflux mediated by apo A-I was 59 % and 70 % (p < 0.0001) lower in macrophages treated with SHAM+NAC-albumin and CKD-albumin, respectively, when compared to SHAM-albumin. The HDL2-mediated cholesterol efflux was 52 % higher in macrophages treated with SHAM+NAC-albumin compared to macrophages treated with SHAM-albumin. No difference was observed in the ABCA-1 protein level in macrophages treated with crescent concentrations of NAC alone for 8 h. Nonetheless, after 18 h, ABCA-1 was 50 %, 69 % and 72 % reduced in macrophages treated, respectively, with 10 mM, 20 mM and 30 mM NAC in comparison to control cells. ABCG-1 content in macrophages treated with NAC for 8 h and 18 h was not changed. Conclusion: NAC reduces plasma lipid peroxidation and AGE in CKD animals and prevents the endoplasmic reticulum stress induced by CKD-albumin in macrophages. Despite diminishing ABCA-1 and apo A-I-mediated cholesterol efflux, NAC increases ABCG-1. Then, NAC may contribute to attenuate the deleterious effects of the in vivo modified albumin on lipid accumulation in macrophages helping to prevent atherosclerosis in CKD

O anticoagulante oral cumarínico varfarina é vastamente utilizado para o tratamento e prevenção de eventos tromboembólicos, que configuram uma das principais causas de mortalidade mundial. Contudo, de acordo com fatores genéticos e ambientais, os cumarínicos apresentam grande variação em sua farmacocinética e farmacodinâmica, implicando em respostas variáveis entre os indivíduos. Para auxílio na tomada de decisão pelo corpo clínico na terapia com a varfarina, algoritmos farmacogenéticos estimadores de dose têm sido extensivamente estudados e desenvolvidos, com o intuito de estabelecer terapias personalizadas. Neste sentido, o presente estudo teve como objetivos investigar a associação de polimorfismos nos genes ABCB1 e CYP4F2 com a variabilidade do requerimento de dose de varfarina, e, primariamente, avaliar o impacto da inclusão desses polimorfismos como covariáveis do algoritmo farmacogenético estimador de dosagem de varfarina previamente desenvolvido para a população brasileira por Santos et al. (2015). Neste estudo retrospectivo, foram utilizadas amostras de 965 pacientes registrados no Instituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC/FMUSP). As genotipagens dos polimorfismos ABCB1 c.3435C>T e CYP4F2 c.1297G>A foram realizadas por meio da amplificação do DNA genômico através da reação em cadeia da polimerase seguida por análise de curva de dissociação (PCR-HRM) ou ensaio TaqMan®, respectivamente para cada variante. Para as análises estatísticas, utilizamos a abordagem de regressão linear múltipla, considerando a dose estável de varfarina como variável resposta e como covariáveis os polimorfismos de interesse nos genes ABCB1 e CYP4F2, bem como outros fatores genéticos, clínicos e demográficos. Nossos resultados sugerem que carreadores da variante ABCB1 c.3435C>T requerem doses médias de manutenção de varfarina inferiores quando comparados aos indivíduos com genótipo selvagem (redução de 2,5 e 4,3 mg/semana, respectivamente para carreadores dos genótipos CT e TT). Ainda, observamos uma grande variabilidade de dose de varfarina no subgrupo de pacientes autodeclarados não-brancos, de acordo com os genótipos ABCB1 (redução de 5,5 e 10,2 mg/semana, respectivamente para carreadores dos genótipos CT e TT). Além disso, verificamos que ambos os polimorfismos ABCB1 c.3435C>T e CYP4F2 c.1297G>A contribuíram para a predição de dose de varfarina, quando associados a outros fatores genotípicos, demográficos e clínicos relevantes, sendo estatisticamente significativos, aumentando o coeficiente de determinação do algoritmo em 2,6% e explicando um adicional de 3,6% da variabilidade interindividual de dosagem. Em conclusão, demonstramos que as genotipagens das variantes ABCB1 c.3435C>T e CYP4F2 c.1297G>A podem ser relevantes para acurar a terapêutica com varfarina na população brasileira / The coumarin oral anticoagulant warfarin has been widely used for treating and preventing thromboembolic events, which are one of the main causes of mortality worldwide. However, according to genetic and environmental factors, coumarins show high variance in pharmacokinetics and pharmacodynamics, resulting in variable interindividual responses. For supporting warfarin clinical decisions, genetic-guided algorithms have been extensively studied and developed, in order to set personalized therapeutics. In this context, the aims of this master\'s research project were to investigate the association of ABCB1 and CYP4F2 polymorphisms with individual\'s warfarin dose requirements and to assess the impact of the inclusion of these polymorphisms as covariates in the genetic-guided dosing algorithm developed by Santos PC et al. (2015) for the Brazilian population. For this retrospective study, 965 patients enrolled in the Heart Institute (InCor), University of São Paulo Medical School (FMUSP) were involved. Genotyping of ABCB1 c.3435C>T and CYP4F2 c.1297G>A were performed by genomic DNA amplification by polymerase chain reaction (PCR), followed by melting curve analysis (HRM-PCR) and TaqMan® assay, respectively. For statistical analysis, we utilized multiple linear regression approach considering warfarin stable dose as the dependent variable and the ABCB1 and CYP4F2 variants, as well as other genetic, clinical and demographic factors as covariates. Our data suggests that carriers of ABCB1 c.3435C>T genotypes require lower mean warfarin maintenance doses when compared to wild-type individuals (reduction of 2.5 and 4.3 mg/week, respectively for CT and TT genotype carriers). Furthermore, we observed large warfarin dose variability for the subgroup of patients who self-declared themselves as non-white according to ABCB1 genotypes (lowering of 5.5 and 10.2 mg/week, respectively for CT and TT genotype carriers). Finally, we verified that both ABCB1 c.3435C>T and CYP4F2 c.1297G>A polymorphisms were able to contribute to warfarin dose prediction, when associated to other relevant genetic, clinical and demographic data, being statistically significant, improving the algorithm\'s coefficient of determination by 2.6% and explaining an additional of 3.6% of the interindividual warfarin dosage variability. In conclusion, in this study we have demonstrated that the genotyping of ABCB1 c.3435C>T and CYP4F2 c.1297G>A may be relevant for improving the management of warfarin therapeutics in Brazilian patients

A leucemia mielóide crônica (LMC) é caracterizada pela translocação (9;22) que dá origem ao gene quimérico BCR-ABL. Este gene codifica uma proteína com atividade tirosina quinase, p210, constitutivamente ativa. O três mecanismos envolvidos na patogênese da LMC são o aumento da proliferação celular, alteração da adesão celular ao estroma e matriz medular e inibição da apoptose. A introdução do mesilato de imatinibe (MI), um inibidor de tirosina quinase, revolucionou o tratamento da LMC levando pacientes em fase crônica a remissões duráveis, porém uma parcela destes não responde ou perde a resposta ao longo do tratamento. Os mecanismos de resistência ao MI podem ser classificados como independentes de BCR-ABL (a1- glicoproteína ácida e genes de resistência a múltiplas drogas) ou dependentes de BCR-ABL (superexpressão de BCR-ABL e mutações do domínio quinase do gene ABL). Objetivo: avaliar a presença de mutações no domínio quinase do gene ABL e a expressão dos genes de resistência a múltiplas drogas MDR1 e BCRP em amostras pré-tratamento com MI, acompanhar estes pacientes mensalmente através da quantificação de transcritos BCR-ABL e quando ocorrer resistência reavaliar a presença de mutações do domínio quinase do ABL e a expressão dos genes de resistência a múltiplas drogas. Material e Métodos: Foram avaliados 61 pacientes com LMC em fase crônica. A pesquisa de mutações do domínio quinase foi realizada pela técnica de seqüenciamento direto e a expressão relativa dos genes de resistência a múltiplas drogas foi avaliada por PCR em tempo real. A quantificação absoluta do número de transcritos BCR-ABL foi realizada pela técnica de PCR em tempo real utilizando-se o sistema Taqman de sondas de hibridização. Resultados: Nas amostras pré-tratamento dos 61 pacientes estudados não foram detectadas mutações. Quando relacionamos o aumento da expressão dos genes MDR1 e BCRP à resposta citogenética completa aos 12 meses de tratamento não houve diferença estatística significativa (p>0,05). Quanto ao número de transcritos BCR-ABL, observamos que os pacientes que apresentaram menos de 1% pela escala internacional aos 3 meses de tratamento atingiram a RMM em período menor (7 meses) do que os que apresentaram mais de 1% (12 meses) com diferença estatística significativa (p = 0,03). Conclusões: As mutações do domínio quinase do gene BCR-ABL nas amostras pré-tratamento não foram detectadas ou pela sensibilidade da técnica de seqüenciamento direto (10%) ou porque tais mutações são mais freqüentes nas fases acelerada e blástica. A expressão dos genes de resistência a múltiplas drogas (MDR1) e BCRP) em pacientes com LMC-FC ao diagnóstico não apresentou correlação com o aparecimento de resistência secundária ao MI. Além disso a quantificação mensal dos transcritos BCR-ABL aos 3 meses pode ser considerada um marcador com valor prognóstico. / Chronic myeloid leukemia is characterized by t(9;22) translocation. The chimeric gene BCR-ABL encodes a p210BCRABL protein with constitutive tyrosine kinase activity which is directly related to CML pathogenesis. The imatinib mesylate, a tyrosine kinase inhibitor, is the first-choice treatment for patients in chronic phase but some patients show primary resistance or relapse after initial response. The mechanisms of resistance to the imatinib mesylate treatment are BCR-ABL dependent (amplification of BCR-ABL and mutation of kinase domain of BCR-ABL) or independent of BCR-ABL (1-acid glycoprotein and expression of multidrug resistance genes). Objective: The objective of this work was to evaluate the mechanisms of resistance (kinase domain mutation and MDR1 and BCRP genes expression) to imatinib mesylate in pretreatment samples, quantify of BCR-ABL transcript on a monthly follow up plan, and to re-evaluate the mechanisms of resistance in the absence or loss of treatment response. Patients and Methods: We have evaluated 61 pretreatment samples derived from chronic phase CML patients. The number of BCR-ABL transcripts was quantified by RTQ-PCR with taqman probes and MDR1 and BCRP expression were evaluated by RTQ-PCR with Syber Green. Mutations within the BCR-ABL kinase domain were screened by direct sequencing and we also have screened the T315I mutation in pretreatment samples by allele-specific PCR. Results:We detected no mutations in the 61 pretreatment samples. The correlation analysis between the expression of MDR1/BCRP genes and the cytogenetic response at 12 months of treatment revealed no significant statistical difference (p = > 0.05). The results of BCR-ABL quantification in the follow up of our cohort indicated that patients who had transcripts <1% by the international scale at 3 months of therapy are more likely to achieve rapid MMR (median of 7 months) than those who had >1% (median of 12 months) (p = 0,03). Conclusions: As expected, the kinase domain mutations of BCR-ABL in pretreatment samples of CML chronic phase patients are not detectable by direct sequencing because of the sensitivity of the assay (10%) and also because these mutations are more common in accelerated phase and blast crisis. About the expression of multidrug resistance genes MDR1 and BCRP, they showed no correlation with secondary resistance to imatinib mesylate. And finally the number of BCR-ABL transcripts at 3 months of treatment can be considered a marker with prognostic value.