Indivíduos portadores do glutamato na posição 27 do gene que codifica para o receptor beta2-adrenérgico têm resposta vasodilatadora muscular aumentada durante manobras fisiológicas. No entanto, o impacto do consumo agudo de gordura nessa resposta não é conhecido. Neste estudo, testou-se a hipótese de que o consumo gordura afetaria a resposta vasodilatadora aumentada destes indivíduos durante manobras fisiológicas. Vinte e cinco indivíduos saudáveis foram subdivididos em dois grupos: 11 homozigotos para o glutamato (Glu27Glu, 40+-3 anos; 65+-3kg) e 14 homozigotos para a glutamina (Gln27Gln, 40+-2 anos; 64+-2kg). O fluxo sangüíneo muscular foi medido por pletismografia de oclusão venosa. A resposta vasodilatadora muscular foi avaliada durante 3 minutos de exercício e estresse mental em jejum e 3 horas após consumo de 62 g de gordura. A condutância basal foi semelhante entre grupos (Glu27Glu=2,3+-0,1; Gln27Gln=2,2+-0,1; P=0,21). O aumento da condutância vascular durante exercício e durante o estresse mental foi maior no grupo Glu27Glu (0,73+-0,2 vs 0,22+-0,1; P=0,008 e 1,8?0,3 vs 1,2+-0,2; P=0,04, respectivamente). O consumo agudo de uma preparação rica em gordura eliminou esta diferença. A resposta de pressão arterial e freqüência cardíaca foi semelhante antes e após a ingestão de gordura. Os níveis de triglicérides, glicose e insulina foram semelhantes ao longo de todo período de estudo. O consumo agudo de gordura elimina a resposta aumentada do fluxo sangüíneo muscular durante manobras fisiológicas dos indivíduos portadores do genótipo Glu27Glu no receptorbeta2- adrenérgico. / Subjects who have glutamic acid at position 27 in gene encoding to beta2-adrenoceptor have increased muscle vasodilatory response during physiological maneuvers. However, the impact of a high-fat meal in this response is unknown. We tested the hypothesis that a high-fat meal would modify the increased muscle vascular reactivity during handgrip and mental stress in these subjects. Twenty-five healthy subjects were subdivided in two groups: 11 were homozygous to glutamic acid (Glu27Glu, 40?3 years; 65+-3kg) and 14 were homozygous to glutamine (Gln27Gln, 40+-2 years; 64+-2kg). Forearm blood flow was measured by venous occlusion pletysmography. Forearm blood flow was recorded for 3 minutes of handgrip and mental stress during fasting and three hours after 62g of fat consumption. Baseline forearm vascular conductance was similar between groups (Glu27Glu=2.3+-0.1; Gln27Gln=2.2+-0.1; P=0.21). Forearm vascular conductance during handgrip and mental stress was greater in the genotype Glu27Glu (0.73+-0.2 vs 0.22+-0.1; P=0.008 and 1.8+-0.3 vs 1.2+-0.2; P=0.04, respectively). Acute fat consumption eliminated the difference of vasodilatory response previously achieved. Blood pressure and heart rate response were similar before and after fat intake. Triglycerides, glucose and insulin levels were also similar between groups. We concluded that high-fat ingestion abolishes the augmented muscle blood flow responses during physiological maneuvers in individuals who are homozygous for the Glu27 allele of the beta2-adrenoceptor gene.

In unserem Institut wurde in vorangegangenen Studien der Nachweis für die Präsenz des Natrium-Glukose-Kotransporters (SGLT-1) im Pansenepithel von Schafen sowohl auf molekularbiologischer als auch auf funktioneller Ebene erbracht. Im Dünndarm von Monogastriern und Wiederkäuern hängt die Aktivität des SGLT-1 im Wesentlichen vom phylogenetischen Ernährungstyp (Wiederkäuer: Konzentrat-selektierer oder Rauhfutterkonsumenten), vom aktuellen Substratangebot und der Beeinflussung durch stoffwechselaktive Hormone ab. Es ist bekannt, dass bei vermehrter Aufnahme leicht verdaulicher Kohlenhydrate die Präsenz des intestinalen SGLT-1 erhöht ist. Eine Stimulation des SGLT-1 durch den intrazellulären Messenger cAMP ist beschrieben. Im Rattendünndarm wurde eine cAMP-vermittelte Erhöhung der Glukoseaufnahme durch pankreatisches Glukagon 29, enterisches Glukagon 37 und Adrenalin nachgewiesen. Die vorliegenden In-vitro- Studien am Pansenepithel waren der Frage gewidmet, inwiefern sich die bisher am Dünndarm untersuchten Regulationswege auf den ruminalen SGLT-1 übertragen lassen. Dies betraf im Einzelnen: - Phylogenetische Unterschiede bei der ruminalen SGLT-1-Aktivität zwischen verschiedenen Ernährungstypen (Schaf, Damwild) - Regulation des SGLT-1 durch kurzfristige Änderungen des Substratangebots - Steigerung der Glukoseaufnahme durch Hormone (Katecholamine u. Glukagon), Die Studien wurden mittels einer modifizierten Ussing-Kammer-Technik durchgeführt. Von Schafen und Damwild wurde die Tunica mucosa der Pansenepithelien präpariert und in Ussing-Kammern inkubiert. Nach einer einminütigen mukosalen Inkubation mit D-Glukose (einschließlich 14C-markierter Glukose) erfolgte die Bestimmung der Glukosekonzentration im Epithelzelllysat mittels Szintillationszählung der radioaktiv markierten Glukose. Der Einfluss verschiedener Substanzen auf die Glukoseaufnahme wurde durch entsprechende Vorinkubation untersucht. Es konnten folgende Befunde erhoben werden: - Die ruminale Glukoseaufnahme bei einer mukosalen Glukosekonzentration von 200 µM erfolgt bei Schafen zu ca. 60 % über den SGLT-1, bei Damwild betrug dieser Anteil nur etwa 25 %. - Eine mehrstündige Erhöhung des mukosalen Glukoseangebotes auf 10 mM resultierte in einer ca. 50%igen Steigerung der SGLT-1-vermittelten Glukoseaufnahme beim Schaf. - Die Hormone Glukagon 37 und Adrenalin bewirkten am Schafspansen eine deutliche Steigerung der SGLT-1-vermittelten Glukoseaufnahme. Glukagon 29 hatte einen geringen Effekt, während Noradrenalin die Glukoseaufnahme nicht nachweisbar beeinflusste. - Im Rahmen einer detaillierteren Charakterisierung des Adrenalineffektes konnte eine Stimulation des SGLT-1 auch durch den -Rezeptoragonist Isoproterenol und den 2-spezifischen Agonisten Terbutalin ausgelöst werden. Andere rezeptorspezifische Agonisten (alpha-1, alpha-2, beta-1 und beta-3) hatten keinen Einfluss. Schlussfolgernd lässt sich postulieren, dass der SGLT-1 bei Schafen der bedeutendste Transportmechanismus für die ruminale Glukoseaufnahme ist und bei Damwild im Gegensatz zu den vom Dünndarm bekannten Verhältnissen eine untergeordnete Rolle spielt. Eine Steuerung ist ähnlich wie im Dünndarm sowohl durch erhöhtes Glukoseangebot als auch durch Erhöhung der intrazellulären cAMP-Konzentration möglich. Die Hormone Adrenalin und Glukagon 37 stimulieren die Glukoseaufnahme in den getesteten Konzentrationen signifikant. Der Signalweg der Adrenalin-vermittelten Stimulation verläuft über Aktivierung der Adenylatzyklase und Proteinkinase A. Dafür scheint ein beta-2-Rezeptor im Pansenepithel verantwortlich zu sein. Die praktische Relevanz der untersuchten Stimulierbarkeit des SGLT-1 könnte in vivo in der schnellen Entfernung überschüssiger Glukosemengen aus dem Vormagen liegen, um eventuell einer bakteriellen Dysfermentation im Pansen entgegenzusteuern. / Recent studies evidenced the presence of the sodium / glucose cotransporter, SGLT-1, in ruminal epithelia of sheep and showed its ability to absorb glucose. Concerning the possibility of regulation of SGLT-1, the influence of substrate concentration has been reported in other tissues. There are phylogenetic adaptions according to the feeding habits of different ruminants (grass and roughage consumer, intermediate mixed feeder and concentrate selectors) and the possibility to short-term adaption in response to the availability of easely fermentable carbohydrates. Furthermore, some hormones are known to stimulate glucose uptake. The important role of cyclic AMP (cAMP) as an intracellular mediator and stimulator of glucose uptake is well described. There is evidence for the influence of hormones stimulating the SGLT-1 by rising the cAMP level. Stimulation of intestinal glucose uptake occured after preincubation with pancreatic glucagon (glucagon 29), enteric glucagon (glucagon 37) and epinephrine in the rat small intestine. To proove whether the regulatory priciples of the intestinal SGLT-1 apply also to the SGLT-1 in the ruminant forestomach, the heredescribed studies addressed the following topics: -Quantification of SGLT-1-mediated glucose uptake in the rumen of ruminants with different feeding habits (sheep vs. fallow deer) -Short-term regulation of glucose uptake by substrate availability -Modulation of glucose uptake by hormones with stimulatory effects on cAMP level Glucose uptake studies were performed in vitro using a modified Ussing chamber technique. Ruminal epithelia were incubated on the mucosal side with glucose (including 50 kBq 14C-glucose) for 1 min. Glucose content of the lysed epithelia was measured by scintillation counting. The influence of certain substances on glucose uptake was investigated by preincubation with these substances either on the serosal, mucosal or both sides of the epithelia. RESULTS - Ruminal glucose absorption in sheep is mediated to approximately 60 % by SGLT-1, whereas the SGLT-1 of fallow deer transports only 25 % of the glucose (at a mucosal glucose concentration of 200 µM). - After mucosal preincubation with 10 mM glucose for several hours, the SGLT-1-mediated glucose uptake of sheep rumen raised up to 150 %. - The hormones glucagon 37 and epinephrine increased SGLT-1-mediated glucose transport in the sheep rumen. A minor stimulation was also observed after addition of glucagon 29, whereas norepinephrine had no effect on glucose uptake. - The effect of epinephrine was characterized in detail. It could be simulated by using the beta-receptoragonist isoproterenol as well as the beta-2-receptor agonist terbutaline. No effect was observed with alpha-1-, alpha-2-, beta-1- and beta-3-receptor agonists. These results lead to the following conclusions: SGLT-1 is the most important transport mechanism for ruminal glucose uptake in sheep. In contrast to the high SGLT-1-activity in the intestine of fallow deer, there is only a minor relevance in the rumen of this species. A fast significant stimulation of the ruminal SGLT-1 of sheep in vitro is possible by rising glucose supply and serosal addition of epinephrine and glucagon 37 in the tested concentrations. Glucagon 29 and norepinephrine had no effect on SGLT-1 under the used experimental conditions. The intracellular signalling of the epinephrine action involves the pathway via adenylate cyclase and protein kinase A. The investigated effect of epinephrine is mediated by beta-2-adrenoceptors. The priciples of regulation of the ruminal SGLT-1 are similar to the intestine. Perhaps, in vivo, up-regulation of glucose absorption by substrates and hormones is important for a fast removal of excess glucose from rumen to prevent dysfermentation.

<p>Uvod: Pu&scaron;enje predstavlja jedan od najznačajnijih uzroka lo&scaron;e kontrole astme, zbog iritativnog dejstva duvanskog dima na disajne puteve i razvoja rezistencije na inhalatorne kortikosteroide. Stoga je pu&scaron;ače sa astmom često potrebno lečiti kombinovanom antiinflamatornom terapijom, iako je efikasnost ovakvog tretmana jo&scaron; uvek nedovoljno ispitana. Cilj: utvrditi efikasnost kombinovane antiinflamatorne terapije: inhalatorni kortikosteroidi (ICS) u kombinaciji sa dugodelujućim beta2-adrenergičkim agonistima (DDBA) u odnosu na ICS u kombinaciji sa antagonistima leukotrijenskih receptora (ALTR) u postizanju dobre kontrole astme, pobolj&scaron;anju kvaliteta života i plućne funkcije kod pu&scaron;ača u odnosu na nepu&scaron;ače sa astmom. Metod: Pacijenti starosti od 18-50 godina sa astmom (&ge;6meseci), FEV1 većim od 60%, podeljeni su u grupu nepu&scaron;ača &ndash;NP (N=60) i aktivnih pu&scaron;ača &ndash;PU (&le;2 &ge;15 p/g i &ge;10&le;40 cigareta na dan; N=60). Obe grupe su randomizovane u jednu od dve, otvorene, terapijske grupe (ICS uz dodatak DDBA ili ALTR) u trajanju od 24 nedelje. Rezultati: u svakoj od 4 randomizovane grupe (NP-DDBA, NP-ALTR, PU-DDBA, PU-ALTR) je bilo po 30 pacijenata. Tokom 24 nedelje, PU su imali lo&scaron;ije kontrolisanu astmu od NP (p=0,02), bez ralizke između DDBA vs ALTR (0,677 vs 0,634). Konstantno dobru kontrolu astme (ACQ&lt;0,75) tokom 24 nedelje je postiglo 48% NP i 32% PU (p=0,094), bez značajne razlike u odnosu na terapiju (DDBA vs ALTR; p=1,000). NP su imali bolji kvalitet života od PU, ali razlika nije dostigla statističku značajnost (p=0,056)- Kod NP i kod PU u oba modaliteta lečenja (LABA, ALTR) je do&scaron;lo do statistički značajne promene srednjeg skora AQLQ (p&lt;0,001). Povećanje FEV1(%) je bilo statistički značajno i u grupi NP i u grupi PU (p=0,001 vs. p=0,002). Kod pacijenata lečenih DDBA povećenje FEV(%) je bilo na nivou p=0,001, dok je u grupu ALTR bilo na nivou p=0,005. Multivarijantnom analizom je utvrđeno da su nezavisni faktori postizanja dobre kontrole astme BMI&ge;24, nepu&scaron;ač, FEV1&ge;90%, ACQ&le;2,2 i AQLQ&ge;4,2 Zaključak: Kombinovana antiinflamatorna terapija je efikasnija kod NP u odnosu na PU, dok su u populaciji aktivnih pu&scaron;ača, oba dodatna leka (DDBA, ALTR) bila podjednako efikasna u pobolj&scaron;anju kontrole astme, kvaliteta života i plućne funkcije.</p> / <p>Introduction: Smoking is one of the major causes of a bad asthma control, due to negative effects of the tobacco smoke on the airways and consequent resistance to inhalant corticosteroids. Smoking asthmatics should therefore often be treated with combined anti-inflammatory therapy, although the efficacy of this treatment regimen has not been completely examined yet. Objective: To examine the efficacy of the combined anti-inflammatory therapy (ICS combined to LABA vs.LTRA) in achieving a good asthma control, better quality of life and improved lung function in smoking vs. nonsmoking asthmatics. Method: The patients at 18-50 years of age with asthma (&ge;6 months), FEV1 &gt; 60%, were subclassified into the group of nonsmokers &ndash;NS (N=60), and the group of active smokers - SM (&le;2 &ge;15 p/g and &ge;10&le;40 cigarettes a day; N=60). Both groups were randomized into one of the two open therapy groups (ICS combined to DDBA or ALTR), receiving the selected treatment for 24 weeks. Results: Any of the four randomized groups (NS-LABA, NS-LTRA, SM-LABA, SM-LTRA) consisted of 30 patients. During the 24-week period, SM had a worse control of their asthma than NS (p=0.02), but no difference was registered between DDBA vs. ALTR therapy subgroups (0.677 vs. 0.634). Over the 24-week period, a constantly good asthma control (ACQ&le;0,75) was achieved by 48% of NS and 32% of SM (p=0.094), and no significant difference related to the applied therapy regimen (LABA vs. LTRA; p=1.000). NS had a better life quality than SM, but this difference remained statistically insignificant (p=0.056). Both the NS and the SM group in either treatment modality (LABA, ALTR) had a statistically significant change of the AQLQ score (p&lt;0.001). FEV1 (%) improvement was statistically significant t in both the NS and the SM group (p=0.001 vs. p=0.002). The LABA and LTRA treated patients had their FEV (%) improvement at the level of p=0.001, and p=0.005 respectively. The multivariate analysis has established the following independent factors of a good asthma control: BMI&ge;24, nonsmoker, FEV1&ge;90%, ACQ&le;2.2, and AQLQ&ge;4.2. Conclusion: The combined anti-inflammatory therapy is more efficient in NS than in SM asthmatics, while in the population of active smokers, both additional drugs (LABA, LTRA) were equally efficient in improving asthma control, life quality, and lung function.</p>

Angiogênese é a formação de novos capilares a partir de vasos pré-existentes, mediada por eventos de sinalização bioquímica que determinam proliferação, migração, diferenciação e morte celular e controlam crescimento e remodelação tecidual. A beta2-glicoproteína I (beta2GPI) é uma proteína plasmática com ação sobre a função vascular e a aterogênese. Monomêros de beta2GPI apresentam efeito anti-inflamatório e anticoagulante; a clivagem enzimática favorece sua dimerização e induz o aparecimento de efeitos opostos. Resultados anteriores mostraram que monômeros e dímeros de beta2GPI têm efeitos diferentes sobre a proliferação e a diferenciação de células endoteliais em culturas bidimensionais utilizadas como modelo de angiogênese. Os monômeros e dímeros de beta2GPI foram obtidos por purificação fracionada e caracterizada por SDS-PAGE e ELISA, como descrito. Neste trabalho, foram utilizadas culturas tridimensionais de células humanas vasculares de cordão umbilical (HUVEC) sobre Matrigel foram utilizadas para identificar efeitos de monômeros e dímeros da beta2GPI sobre a proliferação, migração e formação de estruturas de interação celular in vitro. O monômero de ?2GPI atuou como um fator de diferenciação endotelial dependente da densidade de plaqueamento, induzindo nas culturas tridimensionais de HUVECs a formação de fenótipos alongados, prolongamentos e estruturas de interação célula-célula. A fração dimérica modulou negativamente a proliferação de HUVECs. A membrana corioalantóide (CAM) de embriões de galinha foi empregada para estudar efeitos da beta2GPI sobre a angiogênese. In ovo, o dímero de beta2GPI impediu a angiogênese e induziu a morte embrionária 48h após a exposição, enquanto o monômero permitiu o desenvolvimento do embrião até o 10º dia, apesar de induzir mudanças precoces no desenvolvimento dos vasos da membrana corioalantóide. As estruturas da microvasculatura foram analisadas através de uma abordagem morfológica quantitativa, baseada na classificação de padrões binários locais (LBP). Alvos moleculares de beta2GPI relatados anteriormente foram considerados como fonte dos efeitos observados in vitro e in ovo. Os resultados obtidos suportam dados anteriores sobre a inibição da via de sinalização de anexina-2/Akt pela beta2GPI. Adicionalmente, sugere-se a via de sinalização Notch como um alvo do efeito antiangiogênico de da beta2GPI / Angiogenesis is the formation of new capillaries from pre-existing vessels, mediated by biochemical signaling events that determine proliferation, migration, differentiation and cell death, and control of tissue growth and remodeling. beta2-glycoprotein I (beta2GPI) is a plasma protein active on vascular function and atherogenesis. ?2GPI monomers present anti-inflammatory and anticoagulant effects. Enzymatic cleavage favors beta2GPI dimerization and induces the appearance of opposing effects. Previous results have shown that beta2GPI monomers and dimers induce different effects upon the proliferation and differentiation of endothelial cells in two-dimensional cultures used as an angiogenesis model. beta2GPI monomers and dimers were obtained by fractioned purification and characterized by SDS-PAGE and ELISA, as described. In this work, three-dimensional Human Umbilical Vein Endothelial Cells (HUVEC) cultures on Matrigel were used to investigate the effects of beta2GPI monomers and dimers upon proliferation, migration and in vitro formation of cellular interaction structures. The beta2GPI monomer performed as a density-dependent endothelial differentiation factor, inducing the formation of elongated phenotypes, membrane extensions and cell-cell interaction structures in three-dimensional HUVEC cultures; the dimeric fraction negatively modulated the proliferation and differentiation of HUVECs. The chorioallantoic membrane (CAM) of chicken embryos was employed to study the effects of beta2GPI upon angiogenesis. In ovo, the beta2GPI dimer prevented angiogenesis and induced embryonic death after 48h exposure, while the monomer allowed embryo development up to the 10th day, despite it induced early changes in the development of chorioallantoic membrane vessels. Microvasculature structures were evaluated through a quantitative morphology approach, based on local binary pattern classification. Previously reported molecular beta2GPI targets were then considered as the source of the observed effects in vitro and in ovo. The obtained results support previous data on the inhibition of the annexin-2/Akt signaling pathway by beta2GPI. Additionally, the Notch signaling pathway is suggested as a target of the antiangiogenic effect of beta2GPI

A nefropatia diabética (ND) é uma das principais causas de doença renal terminal. Além da lesão glomerular, o compartimento tubulointersticial é afetado no desenvolvimento da ND, não somente pela proteinúria como pelos efeitos pró-inflamatórios, profibróticos, pró-oxidantes e angiogênicos da hiperglicemia e dos produtos finais de glicação avançada (AGEs). Sabese que as concentrações de marcadores do estresse oxidativo estão aumentadas em pacientes com diabetes mellitus (DM). O sistema tiorredoxina (TXN) é um dos principais sistemas antioxidantes endógenos. A TXN é capaz de interagir com um grande número de fatores de transcrição e proteínas, tal como a Thioredoxin interacting protein (TXNIP) que tem sido reconhecida na patogênese do DM e de suas complicações. Além da TXNIP, a deficiência de tiamina, ou vitamina B1, já foi relatada em modelos experimentais de DM, concomitantemente a um aumento de seu clearance renal. Os transportadores de tiamina 1 (THTR1) e 2 (THTR2) (codificados pelos genes SLC19A2 e SLC19A3, respectivamente) são os responsáveis pela reabsorção de tiamina no túbulo proximal após sua filtração pelo glomérulo. Estudos já demonstraram que a excreção aumentada de tiamina pode ser um fator de risco para o declínio precoce da função renal em pacientes DM. Uma outra proteína de interesse é a beta 2 microglobulina (B2M), expressa em situações que cursam com inflamação, um fenômeno bem caracterizado na tubulopatia diabética. O estudo da ativação intrarenal de vias potencialmente associadas à evolução da ND em humanos é dificultada pela necessidade de biópsia renal. Recentemente o sedimento urinário tem sido utilizado na avaliação das doenças renais, tanto na tentativa de identificar biomarcadores que possam predizer o declínio da função renal, como para o melhor entendimento da patogênese dessa complicação. O objetivo deste trabalho foi estudar a participação dos genesalvo TXNIP, TXN, SLC19A2, SLC19A3 e B2M na patogênese da ND em portadores de DM1. Estudamos o RNA mensageiro (mRNA) do sedimento urinário de pacientes DM1 (n=55), portadores de glomeruloesclerose segmentar e focal (GESF, um modelo de nefropatia não diabética, n=12) e de indivíduos controles (n=11) para avaliação da expressão dos genes-alvo, e sua associação com as manifestações da ND. Também foi extraído mRNA de células linfomononucleares de pacientes DM1 (n=162) e indivíduos controles (n=26) para comparação com os resultados obtidos no sedimento urinário e foram dosadas as concentrações plasmáticas de tiamina em um subgrupos de pacientes DM1 e de indivíduos controles. Além disso, uma linhagem de células renais humanas foi tratada com altas concentrações de glicose e albumina glicada ou não glicada in vitro para avaliar se os AGEs estão implicados na alteração de expressão dos genes-alvos. Como resultado observamos (1) um aumento na expressão de TXNIP e TXN no sedimento urinário de pacientes DM1 com doença renal e associação da expressão de TXNIP com a magnitude do declínio da taxa de filtração glomerular; (2) um aumento na expressão de TXNIP e TXN nas células linfomononucleares dos pacientes DM1; (3) um aumento na expressão de SLC19A2 no sedimento urinário de pacientes DM1 com doença renal; (4) uma diminuição nas concentrações plasmáticas de tiamina nos pacientes DM1 em comparação aos controles; (5) um aumento na expressão de B2M no sedimento urinário de pacientes DM1 com doença renal e (6) um aumento na expressão de TXNIP e B2M nas células renais humanas tratadas concomitantemente com altas concentrações de glicose e albumina glicada. Os resultados do presente estudo sugerem fortemente a participação do sistema TXN e da B2M na etiopatogênese da ND / Diabetic nephropathy (DN) is a major cause of end stage renal disease. Glomerular and tubulointerstitial compartments are affected not only by proteinuria but also by the pro-inflammatory, profibrotic, pro-oxidants and pro-angiogenic effects exerted by hyperglycemia and advanced glycation end products (AGEs) in the development of DN. It is well known that concentrations of oxidative stress markers are increased in patients with diabetes mellitus (DM). The thioredoxin system (TXN) is one of the majors endogenous antioxidant systems; TXN molecule is able to interact with a large number of transcription factors and proteins such as Thioredoxin interacting protein (TXNIP), which has been recognized in the pathogenesis of DM and its complications. Deficiency of thiamine, or B1 vitamin, has been reported in experimental models of DM concomitantly with an increase in its renal clearance. Thiamine transporter 1 (THTR1), encoded by the SLC19A2 gene and Thiamine transporter 2 (THTR2), encoded by the SLC19A3 gene are responsible for thiamine reabsorption in the proximal tubule after glomerular filtration. Studies have shown that increased urinary excretion of thiamine may be a risk predictor for an early decline in kidney function in diabetic patients. Another protein of interest is beta-2-microglobulin (B2M), expressed in situations of inflammation, a well-characterized phenomenon in diabetic tubulopathy. The study of activation or inactivation of intrarenal pathways potentially associated with progression of DN in humans is complicated by the need for renal biopsy. Recently urinary sediment has been used in the evaluation of kidney diseases in an attempt to identify biomarkers that can predict kidney function decline and as a tool for a better understanding of the pathogenesis of this complication. The objective of this work was to study the participation of the target genes TXNIP, TXN, SLC19A2, SLC19A3 and B2M in the pathogenesis of DN in type 1 DM (T1D) patients. We studied the urinary sediment messenger RNA (mRNA) from patients with T1D (n=55); with focal and segmental glomerulosclerosis (FSGS, a non diabetic nephropathy model, n=12) and from control subjects (n=11) to assess the expression of the target genes and their association with the severity of DN. We also studied the mRNA expression of peripheral lymphomononuclear (PLMN) cells from T1D patients (n=162) and control subjects (n=26) in order to compare with the results obtained in the urinary sediment. Plasmatic concentrations of thiamine were also measured in a subgroup of T1D patients and control subjects. In addition, a lineage of human kidney cells was exposed to high glucose concentrations and to glycated and non-glycated albumin to evaluate if AGEs are implicated in the modulation of expression of the target genes. As a result we found that (1) TXNIP and TXN are upregulated in the urinary sediment of T1D patients with kidney disease and TXNIP is associated with the magnitude of glomerular filtration rate decline; (2) TXNIP and TXN are upregulated in PLMN cells from T1D patients; (3) SLC19A2 is upregulated in the urinary sediment of T1D patients with kidney disease; (4) T1D patients present decreased plasmatic thiamine concentrations compared to control subjects; (5) B2M is upregulated in the urinary sediment of T1D patients with kidney disease and (6) TXNIP and B2M are upregulated in human kidney cells exposed concomitantly to high glucose concentrations and glycated albumin. The results of the present study strongly suggest the participation of the TXN system and of B2M in the pathogenesis of DN. Descriptors: diabetes mellitus, diabetic nephropathies, urine, glycosylation end products, advanced, thiamine, thioredoxins, beta 2-microglobulin Diabetic nephropathy (DN) is a major cause of end stage renal disease. Glomerular and tubulointerstitial compartments are affected not only by proteinuria but also by the pro-inflammatory, profibrotic, pro-oxidants and pro-angiogenic effects exerted by hyperglycemia and advanced glycation end products (AGEs) in the development of DN. It is well known that concentrations of oxidative stress markers are increased in patients with diabetes mellitus (DM). The thioredoxin system (TXN) is one of the majors endogenous antioxidant systems; TXN molecule is able to interact with a large number of transcription factors and proteins such as Thioredoxin interacting protein (TXNIP), which has been recognized in the pathogenesis of DM and its complications. Deficiency of thiamine, or B1 vitamin, has been reported in experimental models of DM concomitantly with an increase in its renal clearance. Thiamine transporter 1 (THTR1), encoded by the SLC19A2 gene and Thiamine transporter 2 (THTR2), encoded by the SLC19A3 gene are responsible for thiamine reabsorption in the proximal tubule after glomerular filtration. Studies have shown that increased urinary excretion of thiamine may be a risk predictor for an early decline in kidney function in diabetic patients. Another protein of interest is beta-2-microglobulin (B2M), expressed in situations of inflammation, a well-characterized phenomenon in diabetic tubulopathy. The study of activation or inactivation of intrarenal pathways potentially associated with progression of DN in humans is complicated by the need for renal biopsy. Recently urinary sediment has been used in the evaluation of kidney diseases in an attempt to identify biomarkers that can predict kidney function decline and as a tool for a better understanding of the pathogenesis of this complication. The objective of this work was to study the participation of the target genes TXNIP, TXN, SLC19A2, SLC19A3 and B2M in the pathogenesis of DN in type 1 DM (T1D) patients. We studied the urinary sediment messenger RNA (mRNA) from patients with T1D (n=55); with focal and segmental glomerulosclerosis (FSGS, a non diabetic nephropathy model, n=12) and from control subjects (n=11) to assess the expression of the target genes and their association with the severity of DN. We also studied the mRNA expression of peripheral lymphomononuclear (PLMN) cells from T1D patients (n=162) and control subjects (n=26) in order to compare with the results obtained in the urinary sediment. Plasmatic concentrations of thiamine were also measured in a subgroup of T1D patients and control subjects. In addition, a lineage of human kidney cells was exposed to high glucose concentrations and to glycated and non-glycated albumin to evaluate if AGEs are implicated in the modulation of expression of the target genes. As a result we found that (1) TXNIP and TXN are upregulated in the urinary sediment of T1D patients with kidney disease and TXNIP is associated with the magnitude of glomerular filtration rate decline; (2) TXNIP and TXN are upregulated in PLMN cells from T1D patients; (3) SLC19A2 is upregulated in the urinary sediment of T1D patients with kidney disease; (4) T1D patients present decreased plasmatic thiamine concentrations compared to control subjects; (5) B2M is upregulated in the urinary sediment of T1D patients with kidney disease and (6) TXNIP and B2M are upregulated in human kidney cells exposed concomitantly to high glucose concentrations and glycated albumin. The results of the present study strongly suggest the participation of the TXN system and of B2M in the pathogenesis of DN

Angiogênese é a formação de novos capilares a partir de vasos pré-existentes, mediada por eventos de sinalização bioquímica que determinam proliferação, migração, diferenciação e morte celular e controlam crescimento e remodelação tecidual. A beta2-glicoproteína I (beta2GPI) é uma proteína plasmática com ação sobre a função vascular e a aterogênese. Monomêros de beta2GPI apresentam efeito anti-inflamatório e anticoagulante; a clivagem enzimática favorece sua dimerização e induz o aparecimento de efeitos opostos. Resultados anteriores mostraram que monômeros e dímeros de beta2GPI têm efeitos diferentes sobre a proliferação e a diferenciação de células endoteliais em culturas bidimensionais utilizadas como modelo de angiogênese. Os monômeros e dímeros de beta2GPI foram obtidos por purificação fracionada e caracterizada por SDS-PAGE e ELISA, como descrito. Neste trabalho, foram utilizadas culturas tridimensionais de células humanas vasculares de cordão umbilical (HUVEC) sobre Matrigel foram utilizadas para identificar efeitos de monômeros e dímeros da beta2GPI sobre a proliferação, migração e formação de estruturas de interação celular in vitro. O monômero de ?2GPI atuou como um fator de diferenciação endotelial dependente da densidade de plaqueamento, induzindo nas culturas tridimensionais de HUVECs a formação de fenótipos alongados, prolongamentos e estruturas de interação célula-célula. A fração dimérica modulou negativamente a proliferação de HUVECs. A membrana corioalantóide (CAM) de embriões de galinha foi empregada para estudar efeitos da beta2GPI sobre a angiogênese. In ovo, o dímero de beta2GPI impediu a angiogênese e induziu a morte embrionária 48h após a exposição, enquanto o monômero permitiu o desenvolvimento do embrião até o 10º dia, apesar de induzir mudanças precoces no desenvolvimento dos vasos da membrana corioalantóide. As estruturas da microvasculatura foram analisadas através de uma abordagem morfológica quantitativa, baseada na classificação de padrões binários locais (LBP). Alvos moleculares de beta2GPI relatados anteriormente foram considerados como fonte dos efeitos observados in vitro e in ovo. Os resultados obtidos suportam dados anteriores sobre a inibição da via de sinalização de anexina-2/Akt pela beta2GPI. Adicionalmente, sugere-se a via de sinalização Notch como um alvo do efeito antiangiogênico de da beta2GPI / Angiogenesis is the formation of new capillaries from pre-existing vessels, mediated by biochemical signaling events that determine proliferation, migration, differentiation and cell death, and control of tissue growth and remodeling. beta2-glycoprotein I (beta2GPI) is a plasma protein active on vascular function and atherogenesis. ?2GPI monomers present anti-inflammatory and anticoagulant effects. Enzymatic cleavage favors beta2GPI dimerization and induces the appearance of opposing effects. Previous results have shown that beta2GPI monomers and dimers induce different effects upon the proliferation and differentiation of endothelial cells in two-dimensional cultures used as an angiogenesis model. beta2GPI monomers and dimers were obtained by fractioned purification and characterized by SDS-PAGE and ELISA, as described. In this work, three-dimensional Human Umbilical Vein Endothelial Cells (HUVEC) cultures on Matrigel were used to investigate the effects of beta2GPI monomers and dimers upon proliferation, migration and in vitro formation of cellular interaction structures. The beta2GPI monomer performed as a density-dependent endothelial differentiation factor, inducing the formation of elongated phenotypes, membrane extensions and cell-cell interaction structures in three-dimensional HUVEC cultures; the dimeric fraction negatively modulated the proliferation and differentiation of HUVECs. The chorioallantoic membrane (CAM) of chicken embryos was employed to study the effects of beta2GPI upon angiogenesis. In ovo, the beta2GPI dimer prevented angiogenesis and induced embryonic death after 48h exposure, while the monomer allowed embryo development up to the 10th day, despite it induced early changes in the development of chorioallantoic membrane vessels. Microvasculature structures were evaluated through a quantitative morphology approach, based on local binary pattern classification. Previously reported molecular beta2GPI targets were then considered as the source of the observed effects in vitro and in ovo. The obtained results support previous data on the inhibition of the annexin-2/Akt signaling pathway by beta2GPI. Additionally, the Notch signaling pathway is suggested as a target of the antiangiogenic effect of beta2GPI

A nefropatia diabética (ND) é uma das principais causas de doença renal terminal. Além da lesão glomerular, o compartimento tubulointersticial é afetado no desenvolvimento da ND, não somente pela proteinúria como pelos efeitos pró-inflamatórios, profibróticos, pró-oxidantes e angiogênicos da hiperglicemia e dos produtos finais de glicação avançada (AGEs). Sabese que as concentrações de marcadores do estresse oxidativo estão aumentadas em pacientes com diabetes mellitus (DM). O sistema tiorredoxina (TXN) é um dos principais sistemas antioxidantes endógenos. A TXN é capaz de interagir com um grande número de fatores de transcrição e proteínas, tal como a Thioredoxin interacting protein (TXNIP) que tem sido reconhecida na patogênese do DM e de suas complicações. Além da TXNIP, a deficiência de tiamina, ou vitamina B1, já foi relatada em modelos experimentais de DM, concomitantemente a um aumento de seu clearance renal. Os transportadores de tiamina 1 (THTR1) e 2 (THTR2) (codificados pelos genes SLC19A2 e SLC19A3, respectivamente) são os responsáveis pela reabsorção de tiamina no túbulo proximal após sua filtração pelo glomérulo. Estudos já demonstraram que a excreção aumentada de tiamina pode ser um fator de risco para o declínio precoce da função renal em pacientes DM. Uma outra proteína de interesse é a beta 2 microglobulina (B2M), expressa em situações que cursam com inflamação, um fenômeno bem caracterizado na tubulopatia diabética. O estudo da ativação intrarenal de vias potencialmente associadas à evolução da ND em humanos é dificultada pela necessidade de biópsia renal. Recentemente o sedimento urinário tem sido utilizado na avaliação das doenças renais, tanto na tentativa de identificar biomarcadores que possam predizer o declínio da função renal, como para o melhor entendimento da patogênese dessa complicação. O objetivo deste trabalho foi estudar a participação dos genesalvo TXNIP, TXN, SLC19A2, SLC19A3 e B2M na patogênese da ND em portadores de DM1. Estudamos o RNA mensageiro (mRNA) do sedimento urinário de pacientes DM1 (n=55), portadores de glomeruloesclerose segmentar e focal (GESF, um modelo de nefropatia não diabética, n=12) e de indivíduos controles (n=11) para avaliação da expressão dos genes-alvo, e sua associação com as manifestações da ND. Também foi extraído mRNA de células linfomononucleares de pacientes DM1 (n=162) e indivíduos controles (n=26) para comparação com os resultados obtidos no sedimento urinário e foram dosadas as concentrações plasmáticas de tiamina em um subgrupos de pacientes DM1 e de indivíduos controles. Além disso, uma linhagem de células renais humanas foi tratada com altas concentrações de glicose e albumina glicada ou não glicada in vitro para avaliar se os AGEs estão implicados na alteração de expressão dos genes-alvos. Como resultado observamos (1) um aumento na expressão de TXNIP e TXN no sedimento urinário de pacientes DM1 com doença renal e associação da expressão de TXNIP com a magnitude do declínio da taxa de filtração glomerular; (2) um aumento na expressão de TXNIP e TXN nas células linfomononucleares dos pacientes DM1; (3) um aumento na expressão de SLC19A2 no sedimento urinário de pacientes DM1 com doença renal; (4) uma diminuição nas concentrações plasmáticas de tiamina nos pacientes DM1 em comparação aos controles; (5) um aumento na expressão de B2M no sedimento urinário de pacientes DM1 com doença renal e (6) um aumento na expressão de TXNIP e B2M nas células renais humanas tratadas concomitantemente com altas concentrações de glicose e albumina glicada. Os resultados do presente estudo sugerem fortemente a participação do sistema TXN e da B2M na etiopatogênese da ND / Diabetic nephropathy (DN) is a major cause of end stage renal disease. Glomerular and tubulointerstitial compartments are affected not only by proteinuria but also by the pro-inflammatory, profibrotic, pro-oxidants and pro-angiogenic effects exerted by hyperglycemia and advanced glycation end products (AGEs) in the development of DN. It is well known that concentrations of oxidative stress markers are increased in patients with diabetes mellitus (DM). The thioredoxin system (TXN) is one of the majors endogenous antioxidant systems; TXN molecule is able to interact with a large number of transcription factors and proteins such as Thioredoxin interacting protein (TXNIP), which has been recognized in the pathogenesis of DM and its complications. Deficiency of thiamine, or B1 vitamin, has been reported in experimental models of DM concomitantly with an increase in its renal clearance. Thiamine transporter 1 (THTR1), encoded by the SLC19A2 gene and Thiamine transporter 2 (THTR2), encoded by the SLC19A3 gene are responsible for thiamine reabsorption in the proximal tubule after glomerular filtration. Studies have shown that increased urinary excretion of thiamine may be a risk predictor for an early decline in kidney function in diabetic patients. Another protein of interest is beta-2-microglobulin (B2M), expressed in situations of inflammation, a well-characterized phenomenon in diabetic tubulopathy. The study of activation or inactivation of intrarenal pathways potentially associated with progression of DN in humans is complicated by the need for renal biopsy. Recently urinary sediment has been used in the evaluation of kidney diseases in an attempt to identify biomarkers that can predict kidney function decline and as a tool for a better understanding of the pathogenesis of this complication. The objective of this work was to study the participation of the target genes TXNIP, TXN, SLC19A2, SLC19A3 and B2M in the pathogenesis of DN in type 1 DM (T1D) patients. We studied the urinary sediment messenger RNA (mRNA) from patients with T1D (n=55); with focal and segmental glomerulosclerosis (FSGS, a non diabetic nephropathy model, n=12) and from control subjects (n=11) to assess the expression of the target genes and their association with the severity of DN. We also studied the mRNA expression of peripheral lymphomononuclear (PLMN) cells from T1D patients (n=162) and control subjects (n=26) in order to compare with the results obtained in the urinary sediment. Plasmatic concentrations of thiamine were also measured in a subgroup of T1D patients and control subjects. In addition, a lineage of human kidney cells was exposed to high glucose concentrations and to glycated and non-glycated albumin to evaluate if AGEs are implicated in the modulation of expression of the target genes. As a result we found that (1) TXNIP and TXN are upregulated in the urinary sediment of T1D patients with kidney disease and TXNIP is associated with the magnitude of glomerular filtration rate decline; (2) TXNIP and TXN are upregulated in PLMN cells from T1D patients; (3) SLC19A2 is upregulated in the urinary sediment of T1D patients with kidney disease; (4) T1D patients present decreased plasmatic thiamine concentrations compared to control subjects; (5) B2M is upregulated in the urinary sediment of T1D patients with kidney disease and (6) TXNIP and B2M are upregulated in human kidney cells exposed concomitantly to high glucose concentrations and glycated albumin. The results of the present study strongly suggest the participation of the TXN system and of B2M in the pathogenesis of DN. Descriptors: diabetes mellitus, diabetic nephropathies, urine, glycosylation end products, advanced, thiamine, thioredoxins, beta 2-microglobulin Diabetic nephropathy (DN) is a major cause of end stage renal disease. Glomerular and tubulointerstitial compartments are affected not only by proteinuria but also by the pro-inflammatory, profibrotic, pro-oxidants and pro-angiogenic effects exerted by hyperglycemia and advanced glycation end products (AGEs) in the development of DN. It is well known that concentrations of oxidative stress markers are increased in patients with diabetes mellitus (DM). The thioredoxin system (TXN) is one of the majors endogenous antioxidant systems; TXN molecule is able to interact with a large number of transcription factors and proteins such as Thioredoxin interacting protein (TXNIP), which has been recognized in the pathogenesis of DM and its complications. Deficiency of thiamine, or B1 vitamin, has been reported in experimental models of DM concomitantly with an increase in its renal clearance. Thiamine transporter 1 (THTR1), encoded by the SLC19A2 gene and Thiamine transporter 2 (THTR2), encoded by the SLC19A3 gene are responsible for thiamine reabsorption in the proximal tubule after glomerular filtration. Studies have shown that increased urinary excretion of thiamine may be a risk predictor for an early decline in kidney function in diabetic patients. Another protein of interest is beta-2-microglobulin (B2M), expressed in situations of inflammation, a well-characterized phenomenon in diabetic tubulopathy. The study of activation or inactivation of intrarenal pathways potentially associated with progression of DN in humans is complicated by the need for renal biopsy. Recently urinary sediment has been used in the evaluation of kidney diseases in an attempt to identify biomarkers that can predict kidney function decline and as a tool for a better understanding of the pathogenesis of this complication. The objective of this work was to study the participation of the target genes TXNIP, TXN, SLC19A2, SLC19A3 and B2M in the pathogenesis of DN in type 1 DM (T1D) patients. We studied the urinary sediment messenger RNA (mRNA) from patients with T1D (n=55); with focal and segmental glomerulosclerosis (FSGS, a non diabetic nephropathy model, n=12) and from control subjects (n=11) to assess the expression of the target genes and their association with the severity of DN. We also studied the mRNA expression of peripheral lymphomononuclear (PLMN) cells from T1D patients (n=162) and control subjects (n=26) in order to compare with the results obtained in the urinary sediment. Plasmatic concentrations of thiamine were also measured in a subgroup of T1D patients and control subjects. In addition, a lineage of human kidney cells was exposed to high glucose concentrations and to glycated and non-glycated albumin to evaluate if AGEs are implicated in the modulation of expression of the target genes. As a result we found that (1) TXNIP and TXN are upregulated in the urinary sediment of T1D patients with kidney disease and TXNIP is associated with the magnitude of glomerular filtration rate decline; (2) TXNIP and TXN are upregulated in PLMN cells from T1D patients; (3) SLC19A2 is upregulated in the urinary sediment of T1D patients with kidney disease; (4) T1D patients present decreased plasmatic thiamine concentrations compared to control subjects; (5) B2M is upregulated in the urinary sediment of T1D patients with kidney disease and (6) TXNIP and B2M are upregulated in human kidney cells exposed concomitantly to high glucose concentrations and glycated albumin. The results of the present study strongly suggest the participation of the TXN system and of B2M in the pathogenesis of DN

Orientador: Alexandre Leite Rodrigues de Oliveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T03:01:25Z (GMT). No. of bitstreams: 1 SabhaJunior_MarioJoseJorge_D.pdf: 14971588 bytes, checksum: 25740c7d964d2a5c4eb70c84cb462b08 (MD5) Previous issue date: 2007 / Resumo: o estabelecimento das redes nervosas que compõem o Sistema Nervoso Central (SNC) é um processo imensamente complexo dependente, não somente da formação de novos pontos de comunicação, as sinapses, mas também da eliminação de sinapses supranumerárias ou incorretas durante o desenvolvimento. Recentemente, foi descrito um mecanismo demonstrando que a ausência da expressão do complexo de histocompatibilidade principal (MHC classe I) no SNC, diminui a remoção de conexões sinápticas extranumerárias durante o desenvolvimento e aumenta a retração sináptica no animal adulto. Interessantemente, a transecção do axônio induz uma extensa retração dos terminais pré-sinápticos da superfície do corpo celular e dendritos dos neurônios axotomizados. No presente trabalho, investigamos as alterações sinaptológicas nos motoneurônios alfa da intumescência lombar em três linhagens de camundongos isogênicos (C57BL/6J, A/J e Balb/cJ), após 1 e 3 semanas da transecção do nervo ciático. Nesse sentido, estudamos a cobertura sináptica dos corpos celulares dos motoneurônios após a lesão. Foram utilizadas medulas espinhais de camundongos machos adultos, as quais foram processadas e analisadas para imunohistoquímica (lH) e microscopia eletrônica de transmissão . (MET). Para IH, foram utilizados anticorpos anti-MHC e anti-sinaptofisina conjugados com anticorpos secundários CY-2 ou CY-3 e analisados em microscópio confocal. Os resultados mostraram aumento significativo da expressão de MHC I na linhagem AIJ, comparativamente à Balb/cJ e C57BL/6J, 1 semana após axotomia. Contudo, mostraram níveis similares de expressão desta molécula 3 semanas após axotomia. Adicionalmente, observamos uma diminuição significativa da expressão de sinaptofisina na linhagem AIJ, após 1 semana da transecção do nervo ciático. Após 3 semanas da lesão nervosa, todas as linhagens, apresentaram níveis similares de expressão de sinaptofisina. Os resultados da MET, após 1 semana da transecção do nervo ciático, mostraram menor cobertura sináptica na linhagem A/J, comparada à Balb/cJ e C57BL/6J. Contudo, 3 semanas após a lesão a linhagem C57BL/6J apresentou menor cobertura sináptica, enquanto AIJ e Balb/cJ recuperam suas aferências. Concluímos que a expressão de MHC I influencia o processo de eliminação sináptica e, possivelmente contribui para o potencial regenerativo dos neurônios axotomizados / Abstract: The wiring of the Central Nervous System (CNS) is an immensely complex process, not only dependent on new communication points, the synapses, but also on the elimination of exuberant or inappropriate synapses during development. Earlier studies have shown that the abscence of class I major histocompatibility complex (class I MHC) in the CNS decreases synaptic elimination during CNS development and increases synaptic retraction in adult. Thus, an axon transection has been shown to induce an extensive detachment of presynaptic terminais from perikarya and dendrites ofaxotomized neurons. In the present work, we investigated synaptological changes in alpha motoneurons from lumbar intumescence in three mice isogenic strains (C57/BL6J, AIJ and Balb/cJ), 1 and 3 weeks after sciatic nerve transection. For this purpose we studied ultrastructurally the synaptic covering of the cell soma of sciatic motoneurons after the lesion. Therefore, spinal cords from adult male mice were processed for transmission electron microscopy (TEM) and imunohistochemistry (IH). For IH, anti-MHC I and anti-synaptofisin antibodies were used, conjugated with CY2 or CY3 secondary antibodies and analyzed with a confocal microscope. The results showed a significant increased expression of MHC I in AIJ strain in comparison to Balb/cJ and C57BL/6J, 1 week after axotomy. Nevertheless, the immunoreactivity levels of this molecule 3 weeks after axotomy did not differ among the studied mice strains. Additionally, a conspicuous decrease of synaptofisin expression in A/J mice was observed 1 week after sciatic transection. Similarly to the MHC I immunolabelirlg, 3 weeks after lesion, ali mice strains showed similar levels of synaptofisin expression. The results from TEM 1 week after lesion showed a lower synaptic covering in AIJ mice in comparison to Balb/cJ and C57BL/6J, although 3 weeks after axotomy C57BL/6J displayed a lower synaptic covering, while AIJ and Balb/cJ strains recovered their afferents, We conclude that the levei of MHC I expression influences the synaptic elimination process and possibly contributes to the regenerative potencial of the axotomized neurons / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

No description available.

La Résonance Magnétique Nucléaire (RMN) à l’état solide avec rotation à l’angle magique (MAS) est une technique de choix pour l’étude de la structure et de la dynamique de molécules biologiques peu ou non solubles. Un grand nombre d’approches ont été développées pour la reconstitution de structures tridimensionelles à partir de mesures précises de proximités internucléaires, ainsi que pour la détection de mouvements moléculaires avec une résolution atomique sur des échelles de temps couvrant plusieurs ordres de grandeur. Malgré d’impressionnants progrès, les études par RMN MAS sont cependant loin d’être réalisées en routine. Les déterminations structurelles et de dynamique sont souvent démontrées sur des préparations microcristallines modèles, mais sont encore rares pour des systèmes plus complexes tels que les fibrilles amyloïdes non cristallines ou les protéines trans-membranaires insérées dans des bi- couches lipidiques. Mon travail a pour objectif d’étendre les possibilités de la RMN MAS pour l’étude de systèmes biomoléculaires complexes dans différents états d’agrégation. Pour cela, j’ai exploité les possibilités uniques offertes par les hauts champs magnétiques (fréquence de Larmor du 1H 700, 800 et 1000 MHz) combinés avec les sondes MAS de dernières générations capables d’atteindre des vitesses de rotations supérieures à 60 kHz. Ces conditions expérimentales per- mettent d’augmenter la sensibilité de la RMN MAS à l’aide de la détection 1H à haute résolution et d’enrichir la palette de paramètres RMN rapporteurs de la dynamique des protéines. La première partie de cette thèse décrit le développement de nouvelles stratégies pour l’attribution des résonances du squelette de protéines, pour l’élucidation de structures, et pour l’étude de la dynamique du squelette peptidique et des chaînes latérales. Les méthodes présentées réduisent significative- ment les besoins en termes de temps expérimental, de quantités d’échantillon et de marquage isotopique, et permettent d’analyser par RMN des systèmes de plus hauts poids moléculaire. La seconde partie décrit l’application de la RMN MAS avec détection en 1H pour l’évaluation du rôle de la dynamique des protéines dans des processus tels que la formation de fibrilles amyloïdes et le fonctionnement de protéines membranaires. Une première application est l’étude de la tendance de la β-2 microglobuline humaine à former des fibrilles amyloïdes. Une comparaison de la dynamique du squelette peptidique de la protéine sauvage et du mutant D76N dans leur forme cristalline, ainsi que la détermination de propriétés structurales de la forme fibrillaire m’ont permis d’identifier la présence de repliements pathologiques et de formuler des hypothèses sur le mécanisme de formation des fibrilles. Finalement, la dynamique locale et globale de protéines membranaires dans des bicouches lipidiques a été étudiée. En particulier, le mécanisme d’action d’un transporteur d’alkanes, AlkL, de P. putida a été examiné dans un environnement lipidique. La détermination de paramètres pour la dynamique rapide (ps-ns) et lente (μs-ms) du squelette peptidique de la protéine en présence ou en absence de substrat met en évidence des acheminements possibles pour le transfert de molécules vers la membrane et jette les bases pour une meilleure compréhension du processus. / Solid-state NMR with magic angle spinning (MAS) has emerged as a powerful technique for investigating structure and dynamics of insoluble or poorly soluble biomolecules. A number of approaches has been designed for reconstructing molecular structures from the accurate measurement of internuclear proximities, and for probing motions at atomic resolution over timescales spanning several orders of magnitude. Despite this impressive progress, however, MAS NMR studies are still far from routine. Complete determinations, which are often demonstrated on model microcrystalline preparations, are still rare when it comes to more complex systems such as non-crystalline amyloid fibrils or transmembrane proteins in lipid bilayers. My work aimed at extending the possibilities of MAS NMR for applications on complex biomolecular systems in different aggregation states. For this, I exploited the unique possibilities provided by high magnetic fields (700, 800 and 1000 MHz 1H Larmor frequency) in combination with the newest MAS probes capable of spinning rates exceeding 60 kHz. These experimental conditions al- low to boost the sensitivity of MAS NMR through 1H detection at high resolution and to enrich the palette of probes for protein dynamics. The first part of the thesis reports on my contribution to the development of new strategies for backbone resonance assignment, for structure elucidation, and for investigation of backbone and side-chain dynamics. These methodologies significantly reduce the requirements in terms of experimental time, sample quantities and isotopic labeling, and enlarge the molecular size of systems amenable to NMR analysis. The second part describes the application of 1H detected MAS NMR to evaluate the role of protein dynamics in problems such as amyloid fibril formation and membrane protein function. I first addressed the amyloid fibril formation propensity of human beta-2 microglobulin, the light chain of the major histocompatibility complex I. I performed comparative studies of backbone dynamics of the wild type protein as well as a D76N mutant in crystals, and determined some of the structural features of the fibrillar form. This allowed to identify the presence of pathological folding intermediates and to formulate hypotheses on the mechanism of fibrils formation. Finally, I studied the local and global dynamics of membrane proteins in lipid bilayers. In particular, I investigated the mechanism of action of the alkane trans- porter AlkL from P. putida in lipid bilayers. The measurement of parameters for fast (ps-ns) and slow (μs-ms) backbone dynamics of the protein in presence or in absence of a substrate highlights possible routes for molecular uptake and lays the basis for a more detailed mechanistic understanding of the process.