21 |
Identifizierung und Charakterisierung von BitterrezeptorenBufe, Bernd January 2003 (has links)
Menschen nehmen Tausende von Stoffen als bitter wahr. Die chemische Struktur der verschiedenen Bitterstoffe ist sehr vielfältig: Sie reicht von kleinen Molekülen wie Kaliumchlorid oder Harnstoff, bis zu sehr komplexen organischen Verbindungen. Die Größe der einzigen bekannten menschlichen Familie von Bitterrezeptoren (TAS2Rs) wurde auf nur ca. 80-120 Mitglieder geschätzt. In Anbetracht der hohen Zahl und Komplexität der Bitterstoffe erscheint die Zahl von Rezeptoren als sehr gering. Dies führt natürlich zu einer Reihe von Fragen: Wie viele Mitglieder hat die menschliche TAS2R-Genfamilie? Wie viele verschiedene Substanzen können denselben Rezeptor aktivieren? Scheint die Zahl der TAS2R-Rezeptoren ausreichend, alle Bitterstoffe wahrnehmen zu können oder muss es noch andere Bitterrezeptorfamilien geben? Diese Fragen zu beantworten, ist das Ziel der vorliegenden Arbeit. <br />
<br />
Hier durchgeführte Analysen des menschlichen Genomprojektes zeigen, dass Menschen ca. 25 TAS2R-Rezeptoren besitzt, die eine sehr divergente Aminosäurestruktur aufweisen. Diese Rezeptoren wurden in eine neu entwickelte Expressionskassette kloniert, die den Transport des Rezeptors an die Zelloberfläche ermöglicht. Um Liganden für die menschliche TAS2R-Rezeptoren zu identifizieren, wurden die Rezeptoren in HEK293 Zellen exprimiert und mit verschiedenen Bitterstoffen stimuliert. Der Nachweis der Rezeptoraktivierung erfolgte durch Calcium-Imaging. Es konnte gezeigt werden, dass hTAS2R16 der menschliche Rezeptor zur Wahrnehmung von Salicin und verwandten bitteren Pyranosiden ist. So wird hTAS2R16 in HEK293 Zellen durch Salicin und chemisch verwandte Substanzen aktiviert. Ein Vergleich der in diesem Messsystem erhaltenen Daten mit psychophysikalisch ermittelten Geschmackswahrnehmungen beim Menschen, ergab eine hohe Übereinstimmung. Die Ergebnisse deuten auch darauf hin, dass die Desensitiverung einzelner Rezeptoren die Ursache für die Adaption des Bittergeschmacks ist. Der Nachweis der Expression des Rezeptors in menschlichen Geschmackspapillen, sowie die festgestellte Assoziation des G/A Polymorpphismus an Position 665 des hTAS2R16 Gens mit einer reduzierten Salicinwahrnehmung, sind weitere unabhängige Beweise für diese These. Ein anderer menschlicher Rezeptor, hTAS2R10, wird durch die Bitterstoffe Strychnin, Brucin und Denatonium aktiviert. Dies sowie die Tatsache, dass die zur Aktivierung benutzten Konzentrationen eine sinnvolle Korrelation zu dem menschlichen Geschmacksschwellwert von Strychnin zeigen, sind starke Hinweise, dass hTAS2R10 der menschliche Rezeptor zur Wahrnehmung von Strychnin und verwandten Substanzen ist. <br />
<br />
Die vorliegenden Daten zeigen eindeutig, dass die TAS2R-Rezeptoren auch beim Menschen Bitterrezeptoren darstellen. Sowohl hTAS2R16, als auch hTAS2R10 werden durch ein Spektrum strukturell sehr unterschiedlicher Bitterstoffe aktiviert. Falls die anderen Mitglieder der TAS2R-Familie ebenfalls dieses Verhalten zeigen, wäre es möglich, dass die nur ca. 25 Mitglieder umfassende TAS2R-Rezeptorfamilie des Menschen tatsächlich zur Wahrnehmung aller Bitterstoffe ausreicht. / Bitter taste generally is aversive and protects vertebrates from ingestion of toxic substances, but bitter tastants also contribute to the enjoyment and palatability of food influencing human nutritional habits. Although bitter taste has been extensively studied, receptor mechanisms are still poorly understood. Anatomic, functional and genetic evidence from rodents suggested, however, that the T2R genes encode a family of bitter receptors while definite proof is missing in humans 6. We here report that the hT2R16 receptor is present in vallate papilla taste buds of the human tongue and activated by bitter b-glucopyranosides. These phytonutrients did not activate any other human T2R and showed similar concentration-response relations and cross-desensitization in hT2R16 expressing cells and psychobiological experiments. hT2R16 is broadly tuned. It binds bitter compounds that share only a b-glycosidic bond and a glucose residue. The broad tuning of T2Rs could explain how a limited number of receptors permits the perception and coding of numerous and different bitter substances. Together our data identify the hT2R16 as a broadly tuned, genuine human bitter receptor for b-glucopyranosides.
|
22 |
Differentielle pharmakologische Sensitivität von humanen 5-HT3-Rezeptor-SubtypenBrünker, Sandra 12 November 2010 (has links) (PDF)
Ziel dieser Arbeit war die elektrophysiologische Charakterisierung des vor kurzem erstmals von NIESLER et al. (2003) klonierten humanen 5-HT3A+E-Rezeptors. Da dieser Rezeptor-Subtyp ausschließlich im Gastrointestinaltrakt exprimiert wird, ist ein Einfluss auf Nausea und Emesis sehr wahrscheinlich. Es stellt sich demnach die Frage, ob funktionelle Unterschiede zum homomeren 5-HT3A-Rezeptor und zum heteromeren 5-HT3A+B-Rezeptor bestehen, und ob auf molekularer Ebene unterschiedliche Wirkungen emetogener bzw. antiemetischer Pharmaka festzustellen sind.
Um die Wirkmechanismen und die Interaktionen eines Pharmakons mit den 5-HT3-Rezeptor-Subtypen beurteilen zu können, erfordert dies genaue Kenntnisse über das biophysikalische Verhalten und die pharmakologische Sensitivität der 5-HT3-Rezeptor-Untereinheiten. Die Experimente erfolgten in-vitro an heterolog in HEK293-Zellen exprimierten Rezeptoren, wobei alleinig die 5-HT3A-Untereinheit in der Lage ist, funktionelle homopentamere Rezeptoren auszubilden. Die 5-HT3E- und 5-HT3B-Untereinheiten können nur zusammen mit der 5-HT3A-Untereinheit an die Zelloberfläche exprimiert werden und funktionelle heteropentamere Rezeptoren bilden. Im Verlauf der Untersuchungen hat sich herausgestellt, dass bei der Transfektion die 5-HT3E- und die 5-HT3B-Untereinheiten im Verhältnis zur 5-HT3A-Untereinheit signifikant schwächer exprimiert werden. Mittels der experimentellen Methode der Patch-Clamp Technik im „excised-patch“ („outside-out“)- und im Ganzzell-Modus war es möglich, die biophysikalischen und pharmakologischen Eigenschaften des heteromeren 5-HT3A+E-Rezeptors im Vergleich mit dem homomeren 5-HT3A-Rezeptor und dem heteromeren 5-HT3A+B-Rezeptor zu analysieren.
Bei den Experimenten zur Grundcharakterisierung des humanen 5-HT3A+E-Rezeptor-Subtyps zeigte die Agonisten-Konzentrations-Wirkungskurve mit einem Hill-Koeffizienten von 1,0 einen deutlichen flacheren Verlauf als die Kurve des 5-HT3A-Rezeptor-Subtyps, die einen Hill-Koeffizienten von 1,5 aufwies. Dies spricht für eine geringe Agonisten-Bindungskooperativität des 5-HT3A+E-Rezeptors. Kein Unterschied zeigte sich allerdings in der Affinität zu 5-HT, da die EC50-Werte von beiden Rezeptor-Subtypen im Bereich von ca. 7 µM lagen. Aus dem biphasischen Verlauf der Kurve konnte der Rückschluss gezogen werden, dass bei der Transfektion des heteromeren 5-HT3A+E-Rezeptors der homomere 5-HT3A-Rezeptor parallel exprimiert wird. Dasselbe Verhalten wurde auch schon für den heteromeren 5-HT3A+B-Rezeptor beschrieben (WALSTAB et al. 2008). Bei der Charakterisierung eines heteromeren Rezeptor-Subtyps ergibt sich dadurch die Schwierigkeit, dessen Eigenschaften nicht eindeutig von denen des homomeren Rezeptors unterscheiden zu können. Des Weiteren konnte im Vergleich zum homomeren 5-HT3A-Rezeptor eine schnellere Desensibilisierungszeitkonstante des heteromeren 5-HT3A+E-Rezeptors nachgewiesen werden. Insgesamt deuten die beschriebenen Ergebnisse auf eine erhöhte Sensitivität des Rezeptors für Serotonin hin. Da der 5-HT3A+E-Rezeptor ausschließlich im Gastrointestinaltrakt exprimiert wird, könnte dies ein Hinweis auf eine Beteiligung dieses Rezeptors bei der Vermittlung von Emesis sein.
Bei der pharmakologischen Charakterisierung wurden der partielle 5-HT3-Rezeptoragonist Tryptamin, der volle 5-HT3-Rezeptorantagonisten Tropisetron sowie die partiellen 5-HT3-Rezeptorantagonisten Metoclopramid, Tubocurarin, Mirtazapin und der Cannabinoid-Rezeptoragonist Anandamid, welcher eine emetogene Wirkung aufweist, untersucht. Auffällig war ein deutlich flacherer Verlauf der Konzentrations-Wirkungskurve von Metoclopramid (5-HT3A+E-Rezeptor: Hill-Koeffizient = -0,8; 5-HT3A-Rezeptor: Hill-Koeffizient = -1,2) und von Mirtazapin (5-HT3A+E-Rezeptor: Hill-Koeffizient = -0,9; 5-HT3A-Rezeptor: Hill-Koeffizient = -1,3) am heteromeren 5-HT3A+E-Rezeptor. Des Weiteren konnte für Mirtazapin am 5-HT3A+E-Rezeptor ein IC50-Wert von 8,4 nM im Vergleich zu 25,4 nM am 5-HT3A-Rezeptor festgestellt werden. Diese deutlich höhere Potenz von Mirtazapin am untersuchten heteromeren Rezeptor-Subtyp sowie die geringere Bindungskooperativität von Mirtazapin und Metoclopramid am 5-HT3A+E-Rezeptor, stellen einen interessanten Ansatz für eine effektive Pharmakotherapie gastrointestinaler Erkrankungen dar.
Die Ergebnisse dieser Arbeit zeigen erstmalig auf molekularer Ebene, die elektrophysiologischen Eigenschaften der humanen 5-HT3A+E-Rezeptoren sowie deren Beeinflussung durch emetogenen und antiemetische Pharmaka. Aufgrund der schwachen Expression der 5-HT3E-Untereinheit gilt es in Zukunft durch einen alternativen Weg der Transfektion, die Effizienz der Ausbeute von 5-HT3A+E-Rezeptoren zu erhöhen. / The aim of this doctor thesis was the electrophysiological characterization of the human 5-HT3A+E receptor which was recently cloned for the first time by NIESLER et al. (2003). Since the expression of this receptor subtype takes place exclusively in gastrointestinal tract, an influence on nausea and emesis is very likely. The question is if functional differences exist between homomeric 5-HT3A receptors and heteromeric 5-HT3A+B receptors, and whether different effects from emetic and antiemetic drugs can be detected at the molecular level.
To assess the mechanisms and the interactions of a drug with the 5-HT3 receptor subtypes, knowledge of the biophysical characteristics and the pharmacological sensitivity of the 5-HT3 receptor subunit is required. The experiments were developed in-vitro on heterologous expressed receptors in HEK293-cells, whereat only the 5-HT3A subunit is able to form functional homopentameric receptors. The 5-HT3E and the 5-HT3B subunit can only be expressed on the cell surface and build functional heteropentameric receptors in combination with the 5-HT3A subunit. In the course of the investigations it became obvious that during transfection the 5-HT3E subunit and the 5-HT3B subunit are significantly lesser expressed than the 5-HT3A subunit. Using the patch-clamp technique in the excised-patch (outside-out) and whole-cell configuration it was possible to analyse the pharmacological and biophysical properties of the heteromeric 5-HT3A+E receptor compared with the homomeric 5-HT3A-receptor and the heteromeric 5-HT3A+B receptor.
During the characterisation of the human 5-HT3A+E receptor subtype, the agonist concentration-response curve with the hillslope of 1,0 showed a significant flatter course than the graph of the 5-HT3A receptor subtype with a hillslope of 1,5. This indicates a diminished agonist binding-cooperativeness of the 5-HT3A+E receptor. No difference could be detected in the affinity to 5-HT, since the EC50 values of both receptor-subtypes were at the range of 7 µM. The biphasic course of the graph showed that by transfection of the heteromeric 5-HT3A+E receptor the homomeric 5-HT3A-receptor is expressed parallel. The same properties were described also for the 5-HT3A+B receptor (WALSTAB et al. 2008). Therefore it is difficult to distinguish the properties of a homomeric receptor by characterisation of a heteromeric receptor subtype. Furthermore, a faster desensitization of the heteromeric 5-HT3A+E-receptor could be demonstrated in comparison to homomeric 5-HT3A-receptor. Overall, the results described above indicate an increased sensitivity to the receptor for serotonin. As the 5-HT3A+E receptor is expressed exclusively in the gastro-intestinal tract, this could be an indication of involvement of this receptor in the mediation of emesis.
During the pharmacological characterisation the partial 5-HT3 receptor agonist tryptamine, the full 5-HT3 receptor antagonist tropisetron as well as the partial 5-HT3 receptor antagonists metoclopramide, tubocurarin, mirtazapin and the cannabinoid receptor agonist anandamide, which has an emetic effect, were examined. The agonist concentration-response curve of metoclopramide (5-HT3A+E receptor: hillslope = -0,8; 5-HT3A receptor: hillslope = -1,2) and of mirtazapin (5-HT3A+E receptor: hillslope = -0,9; 5-HT3A receptor: hillslope = -1,3) showed a significant flatter course at the 5-HT3A+E receptor. Mirtazapin has an IC50 value of 8,4 nM at the 5-HT3A+E receptor in comparison to 25,4 nM at the 5-HT3A receptor. This significant higher potency of mirtazapin at the heteromeric 5-HT3 receptor subtype and the decreased binding-cooperativeness of mirtazapin and meteclopramide at the 5-HT3A+E receptor represent interesting approaches for an effective pharmacotherapy for gastrointestinal diseases.
For the first time the results of this thesis showed the electrophysiological properties of the human 5-HT3A+E receptors and their interference by emetic and antiemetic drugs on the molecular level. Due to the decreased expression of 5-HT3E subunit, the goal for the future is to find an alternative way of transfection which increases the rate of yield for the 5-HT3A+E receptors.
|
23 |
Transportní studie in vitro na 2D a 3D buněčné úrovni / Transport studies in vitro on 2D and 3D cellular levelUrbanová, Johana January 2017 (has links)
in Hradec Králové Student: Johana Urbanová Supervisor: PharmDr. Jana Mandíková, Ph.D. = 38.02 μM), lowest indometacin μM
|
24 |
Développement d'un biosenseur BRET permettant le criblage de drogues qui causent l'activation de canaux Kir3 via les récepteurs couplés aux protéines GRichard-Lalonde, Mélissa 08 1900 (has links)
Les récepteurs couplés aux protéines G forment des complexes multimériques comprenant protéines G et effecteurs. Nous cherchons à caractériser de tels complexes comprenant les récepteurs opioïdes delta (DOR) et les canaux Kir3, qui nous sont d’intérêt vu leur implication dans l’analgésie des opioïdes.
Des expériences d’immunopurification, de BRET et de liaison GTPgS ont été réalisées à l’intérieur de cellules HEK293 transfectées. Les canaux Kir3 ont été co-immunopurifiés avec les DOR, suggérant une interaction spontanée entre récepteur et effecteur. Des essais BRET ont corroboré que l’interaction était présente dans des cellules vivantes et nous ont permis d’identifier une interaction spontanée et spécifique entre DOR/Gg et Gg/Kir3, indiquant leur coexistence en un même complexe. Puisque l’activation du récepteur implique la présence de changements conformationnels à l’intérieur de celui-ci, nous étions intéressés à vérifier si l’information conformationnelle circule à partir du récepteur lié au ligand jusqu’à l’effecteur en aval. Ainsi, nous avons déterminé l’effet de différents ligands sur le signal BRET généré par les paires suivantes : DOR/Gbg, DOR/Kir3 et Kir3/Gbg. Nous avons constaté une modulation de l’interaction DOR/Gbg et Gbg/Kir3 suivant l’ordre d’efficacité des ligands à stimuler la protéine G, ce que nous n’avons pas observé entre DOR et Kir3.
Donc, nous concluons que l’information conformationnelle circule du récepteur au canal Kir3 via la protéine Gbg. Ces résultats nous ont permis de développer un biosenseur BRET (EYFP-Gg2/Kir3.1-Rluc) qui pourrait être utilisé dans le criblage à haut débit afin de détecter de nouvelles molécules ayant une grande efficacité à activer les canaux Kir3. / G protein-coupled receptors form multimeric complexes comprising G protein and effectors. We want to characterize such complexes comprising delta opioid receptors (DOR) and Kir3 channels, which interest us due to their involvement in opioid analgesia.
Immunopurification, BRET and GTPgS binding experiments were done in transfected HEK293 cells. Kir3 channels were co-immunopurified with DOR, implying a spontaneous interaction between the receptor and effector. BRET assays corroborated the presence of this interaction in living cells and allowed us to identify a spontaneous and specific interaction between DOR/Gg and Gg/Kir3, indicating their co-existence within the same complex. Since the activation of the receptor implies it undergoes conformational changes, we were interested in evaluating if the conformational information flows from the ligand-bound receptor until the downstream effector. Hence, we determined the effect of different ligands on the BRET signal that was generated by the following pairs: DOR/Gbg, DOR/Kir3 and Kir3/Gbg. We noticed a modulation of the DOR/Gbg and Gbg/Kir3 interactions that followed the order of efficacy of the ligands to activate the G protein, which we did not observe between DOR and Kir3.
Therefore, we concluded that the conformational information flows from the receptor to the Kir3 channel via the Gbg protein. These results allowed us to develop a BRET biosensor (EYFP-Gg2/Kir3.1-Rluc), which could be used in high throughput screening to detect new molecules that activate Kir3 channels with high efficacy.
|
25 |
Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual / Generation of HEK293 cell clones overexpressing recombinant isoforms of human VEGF-A (Vascular Endothelial Growth Factor A) with the aim of producing biopharmaceuticals for molecular therapy and tissue engineeringBelchior, Gustavo Gross 25 April 2014 (has links)
Os primeiros vasos sanguíneos do embrião de vertebrados, formados de novo a partir de células originadas da mesoderme, originam os vasos linfáticos em um processo denominado vasculogênese. Já no adulto, novos vasos são formados principalmente através da angiogênese (ou linfoangiogênese) a partir da vasculatura pré-existente. Em indivíduos saudáveis, a arquitetura vascular é relativamente estática, sendo que o excesso ou a insuficiência de vasos são comumente relacionados à angiogênese patológica, vinculada a diversas doenças, como câncer, degeneração macular relacionada à idade, isquemia de membros e muitas outras. Dessa forma, o controle local da densidade de vasos sanguíneos torna-se interessante para o tratamento de condições patológicas visando melhoria de prognóstico e cura. Dentre os diversos fatores de crescimento conhecidos, o fator de crescimento endotelial vascular, VEGF, destaca-se como o principal regulador do processo de angiogênese através de isoformas pró-angiogênicas (VEGFxxx) e antiangiogênicas (VEGFxxxb) do gene VEGF-A. Consequentemente, as proteínas codificadas por esse gene constituem um alvo com alto potencial terapêutico. No presente estudo, propusemos a produção das isoformas proteicas recombinantes rhVEGF165, rhVEGF165b e rhVEGF121, oriundas do gene VEGF-A humano, visando à geração de biofármacos que podem ser utilizados para terapia molecular e engenharia tecidual. As sequências codificadoras das isoformas rhVEGF165 e rhVEGF121 foram amplificadas a partir do cDNA total sintetizado de amostras de RNA total de pulmão humano, enquanto que a da isoforma rhVEGF165b foi gerada através da mutação sítio-dirigida da sequência de rhVEGF165. As sequências foram clonadas no vetor de clonagem pGEM®-T Easy, sendo em seguida subclonadas no vetor pLV-eGFP, um vetor plasmidial de transferência lentiviral, que permite a expressão de transgenes em células de mamíferos e, também, da proteína repórter eGFP. Células humanas HEK293 em cultura aderente foram independentemente cotransfectadas com cada uma das construções geradas (pLV-rhVEGF165, pLV-rhVEGF165b e pLV-rhVEGFl21) juntamente com o vetor pTK-Hyg na proporção de 40:1 (m/m), viabilizando a seleção dos transfectantes com o antibiótico higromicina B, além da detecção de eGFP. Os clones celulares superprodutores das proteínas de interesse foram avaliados quanto à cinética de expressão em meio carente de soro e adaptados para a cultura em suspensão estática na presença de meio na ausência de componentes derivados de animais, demostrando a capacidade de expressão das isoformas rhVEGFs nestas condições de cultivo. Para o nosso conhecimento, este é o primeiro trabalho a descrever a expressão de isoformas de VEGF-A em células HEK293 mantidas em suspensão. As isoformas rhVEGF165 e rhVEGF165b foram purificadas por cromatografia de afinidade a heparina, a partir do meio condicionado pelos clones superprodutores gerados. Ensaios in vitro, utilizando o AngioPhaseTM Kit, e in vivo, através do ensaio da membrana corioalantoide em embriões de galinha (CAM Assay), ambos próprios para avaliação da atividade pró- e antiangiogênica de diferentes compostos, demonstraram que a isoforma rhVEGF165 possui atividade biológica, enquanto a isoforma rhVEGF165b não apresentou a atividade esperada (inibição da angiogênese). Estas isoformas foram testadas em modelo murino de engenharia tecidual do intestino curto, com indícios de que poderiam contribuir para o uso terapêutico neste contexto. A purificação da isoforma rhVEGF121, bem como as análises estruturais das proteínas produzidas, estão em processo de otimização. / The first blood vessels of the vertebrate embryo are formed de novo from mesoderm-derived cells and give rise to lymph vessels in a process termed vasculogenesis. In the adult, new blood vessels are formed mainly through angiogenesis (or lymphangiogenesis) from the pre-existing vasculature. In healthy individuals, the vascular architecture is fairly static, and both the excess and the insufficiency of vessels comprise a pathological angiogenic state, to which is credited the onset and/or progression of several diseases such as cancer, age-related macular degeneration, limb ischemia, and many others. Therefore, locally controlling the blood vessel density becomes interesting for the treatment of pathological conditions aiming at prognosis improvement and cure. Among the various known growth factors, the vascular endothelial growth factor, VEGF, stands out as the major regulator of the angiogenic process. This process is mediated through the action of pro- (VEGFxxx) and antiangiogenic (VEGFxxxb) isoforms, which are derived from the VEGF-A gene. Consequently, the proteins encoded by this gene are potential therapeutic targets. In this work, we set out to produce the recombinant protein isoforms rhVEGF165, rhVEGF165b, and rhVEGF121, which originate from the human VEGF-A gene, with the aim of generating biopharmaceuticals to be used for molecular therapy and tissue engineering. The rhVEGF165 and rhVEGF121 coding sequences were amplified from total cDNA sythesized from human lung total RNA. Conversely, the rhVEGF165b coding sequence was generated by site-directed mutagenesis of the rhVEGF165 sequence. The sequences were cloned into the pGEM®-T Easy cloning vector. These cDNAs were then subcloned into pLV-eGFP, a plasmid lentiviral transfer vector that allows for expression of transgenes and the eGFP reporter protein in mammalian cells. Human HEK293 cells cultivated under adherent conditions were independently co-transfected with each of the obtained constructs (pLV-rhVEGF165, pLV-rhVEGF165b, and pLV-rhVEGF121) and the pTK-Hyg vector at a proportion of 40:1 (m/m), enabling for the selection of transfectants with hygromycin B, apart from the detection of eGFP. The cell clones overexpressing the proteins of interest were evaluated for expression kinetics in serum-deprived conditioned media and adapted to static suspension culture in medium free of animal-derived components, demonstrating that expression of the protein isoforms was possible in these culture conditions. To the best of our knowledge, this is the first work that describes the expression of VEGF-A isoforms in HEK293 cells in suspension culture. The rhVEGF165 and rhVEGF165b isoforms were purified by affinity chromatography from media previously conditioned by the overexpressing cell clones. The biological activity of rhVEGF165 was demonstrated in vitro, by the AngioPhaseTM Kit assay, and in vivo with the CAM (chorioallantoic membrane) assay, both of which are suitable for evaluating the pro- and antiangiogenic activity of different compounds. The rhVEGF165b did not show the expected antiangiogenic activity. These isoforms were tested in a model of murine tissue-engineered small intestine, indicating a possible contribution to therapeutic use in this context. The purification of rhVEGF121, as well as the structural analysis of all three proteins, are in the process of being optimized.
|
26 |
Expressão e caracterização do hormônio folículo estimulante (FSH) de pirarucu (Arapaima gigas) / Expression and characterization of follicle stimulating hormone (FSH) of pirarucu (Arapaima gigas)Sevilhano, Thais Cristina dos Anjos 17 July 2019 (has links)
O Arapaima gigas, conhecido popularmente no Brasil como pirarucu, pertence ao grupo mais primitivo dos teleósteos, sendo um dos maiores peixes de água doce da América do Sul. Desde os anos 40 informações divergentes têm sido dadas em relação ao seu tamanho e peso máximo, porém todas concordam com o fato de que se trata de uma espécie de grande porte, com exemplares pesando de 125 a 200 quilos e um comprimento de 2 a 3 metros. Sua carne é desprovida de espinhas e apresenta baixo teor de gordura. Os níveis de rendimento da carcaça são elevados e o crescimento juvenil da espécie é consideravelmente rápido. Por isso, o pirarucu, apresenta um atraente valor de mercado e grande importância para o comércio de pescado amazônico. A pesca predatória tem reduzido significativamente a população dessa espécie nas regiões amazônicas ao longo dos anos. Sendo assim, desde os anos 2000, o pirarucu foi incluído na lista de animais sobre explorados ou em perigo de extinção do IBAMA. A ausência de um empreendimento de aquicultura capaz de produzir o pirarucu a preços competitivos, de forma previsível e em larga escala deve-se principalmente ao fato de que essa espécie, de um modo geral, apresenta dificuldades no processo reprodutivo em cativeiro, apresentando uma ovulação, espermiação e/ou maturação final gonadal insatisfatória. O hormônio folículo estimulante (FSH) é um hormônio gonadotrófico que exerce um papel importante na maturação folicular inicial das gônodas. Assim como os mamíferos, os peixes também apresentam hormônios gonadotróficos (GTHs) heterodiméricos, onde a subunidade α é comum entre os hormônios pertencentes a essa família e a subunidade β determina a atividade hormonal específica. As subunidades α e β do hormônio folículo estimulante de Arapaima gigas (ag-FSH) foram previamente isoladas pelo nosso grupo de pesquisa ao longo dos anos e possibilitaram um estudo de modelagem tridimensional baseada nas sequências de aminoácidos obtidas. Paralelamente, foi realizada a expressão do ag-FSH recombinante com níveis de rendimento satisfatórios (28 mg/L) através da expressão em células de embrião de rim humano (HEK 293), purificando-o e caracterizando-o a partir de técnicas cromatográficas padronizadas pelo nosso grupo de pesquisa. / Arapaima gigas, popularly known in Brazil as pirarucu, belongs to the most primitive group of teleosts, being one of the largest freshwater fish in South America. Since 1940, divergent information has been given regarding its size and maximum weight, but all agree that it is a large species, with specimens weighing from 125 to 200 kilograms and with a 2 to 3 meters of length. Its meat is devoid of fish bones and has low fat content. Carcass yield levels are high and juvenile growth of the species is considerably fast. Therefore, the pirarucu, presents an attractive market value and great importance for the Amazonian fish trade. Predatory fishing has significantly reduced the population of this species in Amazonian regions over the years. Thus, since 2000, pirarucu has been included in IBAMA\'s list of exploited or endangered animals. The absence of an aquaculture enterprise able to produce pirarucu at competitive prices in a predictable and large scale is mainly because this species, in general, presents difficulties in the reproductive process in captivity, presenting an unsatisfactory ovulation, spermiation and / or late gonadal maturation. The follicle-stimulating hormone (FSH) is a gonadotrophic hormone that plays an important role in the initial follicular maturation of the gonads. Like mammals, fish also have heterodimeric gonadotrophic hormones (GTHs), where the α subunit is common between the hormones belonging to this family and the β subunit determines the specific hormonal activity. The α and β subunits of the follicle stimulating hormone of Arapaima gigas (ag-FSH) were previously isolated by our research group over the years and provided a three-dimensional modeling study based on the obtained amino acid sequences. At the same time, the recombinant ag-FSH was expressed with satisfactory yields (28 mg/L) in human kidney embryonic cells (HEK 293), purified and characterized from chromatographic techniques, standardized by our group.
|
27 |
Développement d'un biosenseur BRET permettant le criblage de drogues qui causent l'activation de canaux Kir3 via les récepteurs couplés aux protéines GRichard-Lalonde, Mélissa 08 1900 (has links)
Les récepteurs couplés aux protéines G forment des complexes multimériques comprenant protéines G et effecteurs. Nous cherchons à caractériser de tels complexes comprenant les récepteurs opioïdes delta (DOR) et les canaux Kir3, qui nous sont d’intérêt vu leur implication dans l’analgésie des opioïdes.
Des expériences d’immunopurification, de BRET et de liaison GTPgS ont été réalisées à l’intérieur de cellules HEK293 transfectées. Les canaux Kir3 ont été co-immunopurifiés avec les DOR, suggérant une interaction spontanée entre récepteur et effecteur. Des essais BRET ont corroboré que l’interaction était présente dans des cellules vivantes et nous ont permis d’identifier une interaction spontanée et spécifique entre DOR/Gg et Gg/Kir3, indiquant leur coexistence en un même complexe. Puisque l’activation du récepteur implique la présence de changements conformationnels à l’intérieur de celui-ci, nous étions intéressés à vérifier si l’information conformationnelle circule à partir du récepteur lié au ligand jusqu’à l’effecteur en aval. Ainsi, nous avons déterminé l’effet de différents ligands sur le signal BRET généré par les paires suivantes : DOR/Gbg, DOR/Kir3 et Kir3/Gbg. Nous avons constaté une modulation de l’interaction DOR/Gbg et Gbg/Kir3 suivant l’ordre d’efficacité des ligands à stimuler la protéine G, ce que nous n’avons pas observé entre DOR et Kir3.
Donc, nous concluons que l’information conformationnelle circule du récepteur au canal Kir3 via la protéine Gbg. Ces résultats nous ont permis de développer un biosenseur BRET (EYFP-Gg2/Kir3.1-Rluc) qui pourrait être utilisé dans le criblage à haut débit afin de détecter de nouvelles molécules ayant une grande efficacité à activer les canaux Kir3. / G protein-coupled receptors form multimeric complexes comprising G protein and effectors. We want to characterize such complexes comprising delta opioid receptors (DOR) and Kir3 channels, which interest us due to their involvement in opioid analgesia.
Immunopurification, BRET and GTPgS binding experiments were done in transfected HEK293 cells. Kir3 channels were co-immunopurified with DOR, implying a spontaneous interaction between the receptor and effector. BRET assays corroborated the presence of this interaction in living cells and allowed us to identify a spontaneous and specific interaction between DOR/Gg and Gg/Kir3, indicating their co-existence within the same complex. Since the activation of the receptor implies it undergoes conformational changes, we were interested in evaluating if the conformational information flows from the ligand-bound receptor until the downstream effector. Hence, we determined the effect of different ligands on the BRET signal that was generated by the following pairs: DOR/Gbg, DOR/Kir3 and Kir3/Gbg. We noticed a modulation of the DOR/Gbg and Gbg/Kir3 interactions that followed the order of efficacy of the ligands to activate the G protein, which we did not observe between DOR and Kir3.
Therefore, we concluded that the conformational information flows from the receptor to the Kir3 channel via the Gbg protein. These results allowed us to develop a BRET biosensor (EYFP-Gg2/Kir3.1-Rluc), which could be used in high throughput screening to detect new molecules that activate Kir3 channels with high efficacy.
|
28 |
Geração de clones de células HEK293 superprodutores de isoformas recombinantes de VEGF-A (Fator de Crescimento Endotelial Vascular A) humano visando à produção de biofármacos para terapia molecular e engenharia tecidual / Generation of HEK293 cell clones overexpressing recombinant isoforms of human VEGF-A (Vascular Endothelial Growth Factor A) with the aim of producing biopharmaceuticals for molecular therapy and tissue engineeringGustavo Gross Belchior 25 April 2014 (has links)
Os primeiros vasos sanguíneos do embrião de vertebrados, formados de novo a partir de células originadas da mesoderme, originam os vasos linfáticos em um processo denominado vasculogênese. Já no adulto, novos vasos são formados principalmente através da angiogênese (ou linfoangiogênese) a partir da vasculatura pré-existente. Em indivíduos saudáveis, a arquitetura vascular é relativamente estática, sendo que o excesso ou a insuficiência de vasos são comumente relacionados à angiogênese patológica, vinculada a diversas doenças, como câncer, degeneração macular relacionada à idade, isquemia de membros e muitas outras. Dessa forma, o controle local da densidade de vasos sanguíneos torna-se interessante para o tratamento de condições patológicas visando melhoria de prognóstico e cura. Dentre os diversos fatores de crescimento conhecidos, o fator de crescimento endotelial vascular, VEGF, destaca-se como o principal regulador do processo de angiogênese através de isoformas pró-angiogênicas (VEGFxxx) e antiangiogênicas (VEGFxxxb) do gene VEGF-A. Consequentemente, as proteínas codificadas por esse gene constituem um alvo com alto potencial terapêutico. No presente estudo, propusemos a produção das isoformas proteicas recombinantes rhVEGF165, rhVEGF165b e rhVEGF121, oriundas do gene VEGF-A humano, visando à geração de biofármacos que podem ser utilizados para terapia molecular e engenharia tecidual. As sequências codificadoras das isoformas rhVEGF165 e rhVEGF121 foram amplificadas a partir do cDNA total sintetizado de amostras de RNA total de pulmão humano, enquanto que a da isoforma rhVEGF165b foi gerada através da mutação sítio-dirigida da sequência de rhVEGF165. As sequências foram clonadas no vetor de clonagem pGEM®-T Easy, sendo em seguida subclonadas no vetor pLV-eGFP, um vetor plasmidial de transferência lentiviral, que permite a expressão de transgenes em células de mamíferos e, também, da proteína repórter eGFP. Células humanas HEK293 em cultura aderente foram independentemente cotransfectadas com cada uma das construções geradas (pLV-rhVEGF165, pLV-rhVEGF165b e pLV-rhVEGFl21) juntamente com o vetor pTK-Hyg na proporção de 40:1 (m/m), viabilizando a seleção dos transfectantes com o antibiótico higromicina B, além da detecção de eGFP. Os clones celulares superprodutores das proteínas de interesse foram avaliados quanto à cinética de expressão em meio carente de soro e adaptados para a cultura em suspensão estática na presença de meio na ausência de componentes derivados de animais, demostrando a capacidade de expressão das isoformas rhVEGFs nestas condições de cultivo. Para o nosso conhecimento, este é o primeiro trabalho a descrever a expressão de isoformas de VEGF-A em células HEK293 mantidas em suspensão. As isoformas rhVEGF165 e rhVEGF165b foram purificadas por cromatografia de afinidade a heparina, a partir do meio condicionado pelos clones superprodutores gerados. Ensaios in vitro, utilizando o AngioPhaseTM Kit, e in vivo, através do ensaio da membrana corioalantoide em embriões de galinha (CAM Assay), ambos próprios para avaliação da atividade pró- e antiangiogênica de diferentes compostos, demonstraram que a isoforma rhVEGF165 possui atividade biológica, enquanto a isoforma rhVEGF165b não apresentou a atividade esperada (inibição da angiogênese). Estas isoformas foram testadas em modelo murino de engenharia tecidual do intestino curto, com indícios de que poderiam contribuir para o uso terapêutico neste contexto. A purificação da isoforma rhVEGF121, bem como as análises estruturais das proteínas produzidas, estão em processo de otimização. / The first blood vessels of the vertebrate embryo are formed de novo from mesoderm-derived cells and give rise to lymph vessels in a process termed vasculogenesis. In the adult, new blood vessels are formed mainly through angiogenesis (or lymphangiogenesis) from the pre-existing vasculature. In healthy individuals, the vascular architecture is fairly static, and both the excess and the insufficiency of vessels comprise a pathological angiogenic state, to which is credited the onset and/or progression of several diseases such as cancer, age-related macular degeneration, limb ischemia, and many others. Therefore, locally controlling the blood vessel density becomes interesting for the treatment of pathological conditions aiming at prognosis improvement and cure. Among the various known growth factors, the vascular endothelial growth factor, VEGF, stands out as the major regulator of the angiogenic process. This process is mediated through the action of pro- (VEGFxxx) and antiangiogenic (VEGFxxxb) isoforms, which are derived from the VEGF-A gene. Consequently, the proteins encoded by this gene are potential therapeutic targets. In this work, we set out to produce the recombinant protein isoforms rhVEGF165, rhVEGF165b, and rhVEGF121, which originate from the human VEGF-A gene, with the aim of generating biopharmaceuticals to be used for molecular therapy and tissue engineering. The rhVEGF165 and rhVEGF121 coding sequences were amplified from total cDNA sythesized from human lung total RNA. Conversely, the rhVEGF165b coding sequence was generated by site-directed mutagenesis of the rhVEGF165 sequence. The sequences were cloned into the pGEM®-T Easy cloning vector. These cDNAs were then subcloned into pLV-eGFP, a plasmid lentiviral transfer vector that allows for expression of transgenes and the eGFP reporter protein in mammalian cells. Human HEK293 cells cultivated under adherent conditions were independently co-transfected with each of the obtained constructs (pLV-rhVEGF165, pLV-rhVEGF165b, and pLV-rhVEGF121) and the pTK-Hyg vector at a proportion of 40:1 (m/m), enabling for the selection of transfectants with hygromycin B, apart from the detection of eGFP. The cell clones overexpressing the proteins of interest were evaluated for expression kinetics in serum-deprived conditioned media and adapted to static suspension culture in medium free of animal-derived components, demonstrating that expression of the protein isoforms was possible in these culture conditions. To the best of our knowledge, this is the first work that describes the expression of VEGF-A isoforms in HEK293 cells in suspension culture. The rhVEGF165 and rhVEGF165b isoforms were purified by affinity chromatography from media previously conditioned by the overexpressing cell clones. The biological activity of rhVEGF165 was demonstrated in vitro, by the AngioPhaseTM Kit assay, and in vivo with the CAM (chorioallantoic membrane) assay, both of which are suitable for evaluating the pro- and antiangiogenic activity of different compounds. The rhVEGF165b did not show the expected antiangiogenic activity. These isoforms were tested in a model of murine tissue-engineered small intestine, indicating a possible contribution to therapeutic use in this context. The purification of rhVEGF121, as well as the structural analysis of all three proteins, are in the process of being optimized.
|
29 |
Studium vlastností membránového napěťového senzoru ASAP1 exprimovaného v buněčné linii HEK 293 / Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell lineSanetrníková, Dominika January 2016 (has links)
In the beginning of this thesis is a short introduction into plasmid DNA which is in the form of a vector used in molecular biology. Plasmids can be used in the form of fluorescent probes to measure changes in membrane potential. Into their structure is added a dye called fluorophore. As an important representative of this thesis is a fluorescent probe ASAP1 which contains green fluorescent protein whose response to the membrane potential change is the decrease in the intensity of emitted light. The aim of this thesis was to make chemical transfection of this plasmid into the HEK293 cell line and carry out its characterization. In the work is also described the design of a method for the analysis of the time course of changes in fluorescence depending on the cell membrane depolarisation. In the end of this thesis is also desribed realized experiment including the discussion of aquired results.
|
30 |
Studium vlastností membránového napěťového senzoru ASAP1 exprimovaného v buněčné linii HEK 293 / Study of properties of voltage membrane sensor ASAP1 expressed in HEK293 cell lineJablonská, Dominika January 2017 (has links)
This thesis deals with the problematice of measuring membrane potential and monitoring the propagation of electrical activity of cells. For this purpose, fluorescence membrane voltage sensors have been developed to detect changes in the membrane potential by changing their fluorescence intensity. The practical part is focused on the study of the properties of the ASAP1 fluorescence probe, which was transfected into the HEK293 cell line, which are kidney cells from the human embryo. Cell membrane potential was changed using the patch-clamp technique.
|
Page generated in 0.022 seconds