No description available.

In vielen humanen Neoplasien findet sich eine erhöhte Aktivität des Raf-Mek-Erk-Signaltransduktionsweges. Zunächst wurde davon ausgegangen, dass diese erhöhte Aktivität hauptsächlich durch die Ras-Onkogene hervorgerufen wird. Doch mittlerweile konnten auch Mutationen der Raf Gene in humanen Neoplasien nachgewiesen werden. Gegen Raf und Mek konnten eine Anzahl von Enzyminhibitoren entwickelt werden. Der Nachteil vieler dieser Inhibitoren ist, dass sie nicht zwischen den einzelnen Kinaseisoformen unterscheiden können. In dieser Arbeit ist es nun das erste Mal gelungen, jede Komponente des Raf-Mek-Erk-Signaltransduktionsweges einzeln mittels RNA Interferenz effizient zu inhibieren. Dabei konnte die Rolle der verschiedenen Isoformen in der Proliferation, Morphologie und Genex-pression von transformierten Zellen definiert werden. In den NIH3T3-pEJ Zellen konnte A-Raf erstmals eine antiapoptotische Rolle zugewiesen werden. Diese Hemmung der Apoptose läuft möglicherweise über einen Mek2-abhängigen Weg und ist an die Mitochondrien gekoppelt. Für die beiden Mek Kinasen konnten unter-schiedliche Funktionen in der Signalweiterleitung gezeigt werden. Mek2 spielt die Hauptrolle in der Aktivierung der beiden Substratkinasen Erk1 und Erk2. Der Verlust der Mek1 Isoform wird dagegen möglicherweise durch eine erhöhte Expression von Mek2 kompensiert und wirkt sich nicht so stark auf die Phosphorylierung von Erk1/2 aus. Durch die Verwendung von Erk1 und Erk2 spezifischen siRNAs konnte eine Trennung zwischen der Proliferationsre-gulation und der Kontrolle der morphologischen Transformation herausgearbeitet werden. Durch die Verwendung von Mikroarrays ist es gelungen, beiden Phänotypen ein Genexpres-sionsprofil zuzuordnen. Neben Unterschieden zwischen den verschiedenen Kinaseisoformen konnten neue, potentielle Feedbacks beschrieben werden. / In many human neoplasia an increased activity of the RAF/MEK/ERK- signaling pathway is found. First it was assumed that this raised activity is caused primarily by the RAS onco-genes. However, meanwhile mutations in the RAF genes could be also proved in human neo-plasia. A number of enzyme inhibitors have been developed against the RAF and MEK pro-teins. The disadvantage of many of these inhibitors is that they cannot distinguish between the different kinase isoforms. In this work it has succeeded the first time to inhibit every compo-nent of the RAF/MEK/ERK- signaling pathway individually by means of interference RNA. Beside this, the role of the different isoforms in the proliferation, morphology and genetic profile of transformed cells could be defined. For the first time A-Raf could be assigned an anti-apoptotic role in NIH3T3-pEJ cells. This inhibition of the apoptosis possibly runs through a Mek2-dependent way and is coupled to the mitochondria. For both Mek kinases different functions could be shown in the downstream signaling. Mek2 plays the leading role in the activation of both downstream kinases Erk1 and Erk2. The loss of the Mek1 isoform expression is possibly compensated through an increased expression of Mek2 and does not affect the phosphorylation of Erk1 / 2 so strongly. A discri-mination between the regulation of proliferation and the control of the morphological trans-formation could be worked out by the use of Erk1 and Erk2 specific siRNAs. By the use of micorarray an expression profile of both phenotypes has assigned. Beside differences between the different kinases new, potential feedback pathways could be described.

Made available in DSpace on 2014-07-29T15:13:52Z (GMT). No. of bitstreams: 1 pre textuais.pdf: 419949 bytes, checksum: 16c33c78cc5fa05473d5d08f666f2827 (MD5) Previous issue date: 2009-08-21 / Bovine estrous cycle activates the pathways of MAPKs (mitogen activated protein) via oscillation of hypothalamus-pituitary-gonad axis hormones. The objective of this work was to evaluate in vivo the frequency of expression of active (phosphorylated) form, of p-MEK 1/2 and p-ERK1/2 in all types of ovarian follicles and corpora lutea in different phases of the estrous cycle of cattle. Bovine ovaries were harvested in a local slaughterhouse and the phase of the estrous cycle was determined, considering seven stages, defined as experimental groups: days 1-3 (n = 2), 4-6 (n = 5), 7-9 (n = 5), 10-12 (n = 3), 13-15 (n = 5), 16-18 (n = 5) and 19-21 (n = 5). After processing, the histological sections were subjected to immunohistochemistry using antibodies that specifically recognize the phosphorylated form of MEK 1/2 and ERK 1/2. Histological sections containing corpora lutea were also subjected to immunohistochemistry with antibodies that recognize p-BCL2 (B cell phospho lymphoma 2) and COX-2 (COX-2). The frequency of marked follicles was evaluated for the presence or absence of markup for the two cellular enzymes. The fragments of the corpus luteum were evaluated according to the scores of intensity for positive staining of the four antibodies. The frequencies of the markers studied in the follicles were compared using the chi-square. In preantral follicles, both MEK and ERK 1/2 showed high levels between 16 to 18 days. In the case of p-MEK, the greater activation of primordial follicles and primary held from 7 to 9. In secondary follicles, the less marked for p-MEK occurred in group 4 to 6. Levels of p-ERK began to rise from 13 to 15 days and subsequently decreased. Comparing the frequencies of MEK and ERK, there was statistic difference (p <0.0001) in antral follicles compared to other types of follicles. It was concluded that the intensity of activation of MAPKs MEK / ERK varies with the phases of the estrous cycle and follicular class studied. Since the corpora lutea were evaluated by analysis of variance after transforming the data, it was observed that the proteins studied showed variation in expression profile, in relation to the development, survival and death of the corpus luteum. Since COX-2, a precursor of prostaglandin, is capable of activating the pathways of MEK / ERK 1/2, it reduces the frequency of expression of cell survival factor BCL-2, leading to the corpus luteum at luteolysis. The in vivo evaluation of the activation profile of MAPKs without the use of hormonal protocols demonstrated the importance of this enzyme during the physiological reproductive process, raising new inquiries and providing opportunities to expand knowledge about the functioning of the hormone and its association with the cellular mechanisms activated by hormones / A ocorrência do ciclo estral bovino, a partir de variações dos hormônios do eixohipotálamo- hipófise-gônadas, ativa as vias das MAPKs (proteína ativada por mitógenos) O objetivo desta tese foi avaliar in vivo a freqüência de expressão da forma ativa (fosforilada), de todos os tipos foliculares ovarianos e corpo lúteo para p- MEK 1/2 e p-ERK1/2, em diferentes fases do ciclo estral de bovinos. Para tal, foram coletados em abatedouros locais ovários de bovinos e determinada a fase do ciclo estral, caracterizando sete fases, definidas como grupos experimentais: dias 1-3 (n=2), 4-6 (n=5), 7-9 (n=5), 10-12 (n=3), 13-15 (n=5), 16-18 (n=5) e 19-21 (n=5). Após o processamento histológico os cortes foram submetidos à imunoistoquímica, utilizando anticorpos que reconhecem especificamente a forma fosforilada da MEK 1/2 e ERK 1/2. Cortes histológico que apresentavam corpos lúteos foram submetidos ainda a imunoistoquímica com anticorpos que reconhecem p-BCL2 (fosfo células B de linfoma 2) e COX-2 (ciclooxigenase 2). As freqüências de folículos marcados foram avaliadas em relação à presença ou não de marcação celular para as duas enzimas. Já os fragmentos de corpo lúteo foram avaliados de acordo com escores de intensidade de marcação para os quatro anticorpos. As freqüências das marcações nos folículos estudados foram comparadas por meio do teste qui-quadrado. Em folículos pré-antrais, tanto a MEK quanto a ERK 1/2 apresentaram níveis elevados entre os dias 16 a 18. No caso da p-MEK, a maior ativação dos folículos primordiais e primários ocorreu entre os dias 7 a 9. Em folículos secundários, a menor presença de folículos marcados para p-MEK ocorreu no grupo 4 a 6. Os níveis de p-ERK começaram a se elevar a partir de 13 a 15 dias, e posteriormente diminuíram. Quando comparadas as freqüências de MEK e ERK, foram registradas diferenças (p<0,0001) nos folículos antrais em comparação aos demais tipos foliculares. Concluiu-se que a intensidade de ativação das MAPKs MEK/ERK varia de acordo com as fases do ciclo estral e com a classe folicular estudada. Já os corpos lúteos foram avaliados por meio de análise de variância, após transformação dos dados, na qual foi observada que as proteínas estudadas apresentam variação no perfil de expressão, estando relacionada com o desenvolvimento, sobrevivência e morte do corpo lúteo. Sendo que o COX-2, um precursor da prostaglandina, tem capacidade de ativar as vias das MEK/ERK 1/2, diminuindo a freqüência de expressão do fator de sobrevivência celular BCL-2, levando o corpo lúteo ao momento da luteólise. Assim a avaliação do perfil de ativação das MAPKs in vivo, sem a utilização de protocolos hormonais, demonstrou a importância desta via enzimática durante o processo fisiológico reprodutivo, abrindo espaço para futuras indagações e possibilidades de ampliação de conhecimento a cerca do funcionamento hormonal e sua associação com os mecanismos celulares ativados pelos hormônios

The present study analyzes a selection of topics in the grammar of Nalca (Mek language; Papua), with a focus on verbs and nominals. No published grammar or dictionary is available for Nalca, but a translation of the New Testament was used as a parallel text. The results showed that Nalca is split-ergative, strongly suffixing and agglutinating, with subject-object-verb (SOV) as the dominant word order. Verbs consist of a stem and a series of suffixes expressing tense/aspect/mood, negation, number and person. The case alignment is ergative-absolutive for nouns, for which syntactic function is indicated by a series of postpositions. These postpositions agree with nouns in gender. Ergativity was not observed for pronouns; while the results were inconclusive, they appeared to show a nominative-accusative case alignment. The numeral system is an extended body-part system with the base 27. Many of the features found in Nalca are comparable with other Mek languages, with the gender system and split-ergativity being two major exceptions. Finally, the use of the New Testament as a parallel text was a success, with a basic description of the grammar of Nalca having been made, although further investigation is needed. / Denna studie analyserar ett urval av områden i nalcas (mekspråk; Papua) grammatik, med fokus på verb och nominaler. Det finns ingen publicerad grammatik eller ordlista tillgänglig för nalca, men en översättning av Nya Testamentet användes som parallelltext. Resutltaten visade att nalca är split-ergativt, starkt suffigerande och agglutinerande, med subjekt-objekt-verb (SOV) som dominerande ordföljd. Verb består av en stam och en serie suffix som uttrycker tempus/aspekt/modus, negation, numerus och person. Argumentstrukturen är ergativ-absolutiv för substantiv, för vilka syntaktisk funktion indikeras av en serie postpositioner. Dessa postpositioner kongruerar med substantiven efter genus. Ergativitet observerades inte för pronomen; trots att resultaten inte var slutgiltiga, tycktes dessa istället uppvisa ett nominativ-ackusativt system. Det numeriska systemet är ett utökat kroppsdelssystem med basen 27. Många av karaktärsdragen i nalca hade motsvarigheter i de andra mekspråken, med genussystemet och split-ergativiteten som de största undantagen. Användandet av Nya Testamentet som parallelltext visade sig vara lyckat, eftersom en grundläggande beskrivning av nalcas grammatik åstadkoms, även om ytterligare forskning krävs.

No description available.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Lukáš Slatinský Supervisor: Assoc. prof. PharmDr. Martina Čečková, Ph.D. Title of diploma thesis: Interactions of selected anticancer drugs of the MAPK/ERK signaling pathway inhibitors group with the ABC drug transporters ABCB1 (Pgp, P-glycoprotein) and ABCG2 (BCRP, breast cancer resistance protein) are members of a transmembrane efflux ATP dependent transporter family, so called ATP-binding cassettes (ABC). Physiologicaly they are expressed in the cellular membrane and protect body tissues against potentially toxic xenobiotics including drugs. They represent also one of the tumor defense mechanisms when being able to efflux a wide variety of cytotoxic drugs out of the cancer cells leading to treatment failure. BRAF protein plays an important regulatory and signal role in MAPK/ERK pathway affecting cell division, differentiation and secretion. Mutations of BRAF lead to overactivity in MAPK/ERK pathway in many cancer cells and can be therefore targeted by anticancer therapy. Cobimetinib and dabrafenib are relatively new anticancer therapeutics inhibiting the signal pathway mentioned above and they are used in treatment of melanoma carrying the BRAF mutation. The aims of this project were to...

A pesar de que el uso del cannabis y el alcohol como sustancias psicoactivas es muy antiguo y que su uso como drogas recreacionales está muy extendido, los mecanismos a través de los cuales producen sus efectos psicoactivos se han empezado a conocer hace relativamente poco tiempo. En la presente tesis se estudia como afectan distintos tratamientos sistémicos, tanto con etanol como con distintos compuestos cannabinoides, simultáneamente sobre la síntesis de las distintas monoaminas en diferentes regiones cerebrales de rata. Además, se analizó como afectaban estos tratamientos a los distintos componentes de la vía de señalización intracelular de las MAPK en la corteza frontal de rata. Se realizaron distintos tratamientos agudos con estos compuestos así como tratamientos a más largo plazo (crónicos) y además se estudiaron estos parámetros durante el llamado síndrome de abstinencia que aparece una vez que se detiene esta administración crónica.

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. The goal of my dissertation research is to identify the MEK kinase responsible for activation of the MAPK cascade during mitosis and to elucidate the biochemical mechanisms that regulate its activity. In the described herein work I purified and characterized the MEK kinase activity present in M-phase arrested Xenopus egg extracts. I demonstrate that B-Raf is the critical MEK kinase required for activation of the MAPK pathway at mitosis. Consistent with this, I show that B-Raf is activated in an M-phase dependent manner. Further, I provide data linking Cdk1/cyclin B to mitotic activation of B-Raf. Cdk1/cyclin B associates with and phosphorylates B-Raf in M-phase arrested extracts and directly targets Xenopus B-Raf in vitro at a conserved Ser-144 residue. Phosphorylation at Ser-144 is critical for M-phase dependent activation of B-Raf and for B-Raf's ability to trigger activation of the MAPK cascade at mitosis. Finally, I demonstrate that mitotic B-Raf undergoes feedback phosphorylation by MAPK at its conserved C-terminal SPKTP motif. Mutation of both phosphorylation sites within the SPKTP sequence to alanines increases activity of mitotic B-Raf. Further, inhibition or over-activation of MAPK during mitosis enhances or diminishes B-Raf activity, respectively. These results indicate that MAPK-mediated feedback phosphorylation negatively regulates B-Raf activity. Additionally, I show that active mitotic B-Raf exists in large multi-protein complex(s). By utilizing a proteomics approach I identify a set of proteins, which potentially associate with B-Raf at M-phase. Future studies are necessary to elucidate the involvement of these proteins in regulating B-Raf mitotic functions. In summary, my dissertation studies demonstrate that B-Raf activates MAPK signaling at mitosis and undergoes an M-phase dependent regulation. I propose that B-Raf has important functions at mitosis that contributes to its overall role in promoting cell proliferation.

No description available.

No description available.