Dissertations / Theses on the topic 'CAMP assay'

Pancreatic cancer is relatively uncommon. Despite its relative scarcity, it is the fourth-ranked cancer killer in the Western world with less than a 5% 5-year survival rate. The high mortality rate is due to the asymptomatic nature of the disease and the advanced stage at which it is usually diagnosed. S100P is a calcium-binding protein that has been shown to be highly expressed in the early stages of pancreatic cancer and has been proposed as a potential therapeutic target via the blocking of its interaction with its receptor RAGE, the receptor for advanced glycation end-products. In this thesis, computational techniques were employed on the NMR ensemble of S100P (PDB Accession code 1OZO) to identify potential inhibitors of the S100P-RAGE interaction in the hope of identifying a series of novel leads that could be developed into clinical candidates for the treatment of pancreatic cancer. In silico studies identified putative binding sites at the S100P dimeric interface capable of accommodating cromolyn, an anti-allergy drug shown to bind to the protein both in vitro and in vivo. Virtual screening of >1 million lead-like compounds using 3D pharmacophore models derived from the predicted binding interactions between S100P and cromolyn, identified 9,408 'hits'. These were hierarchically clustered according to similarities between chemical structures into 299 clusters and 77 singletons. Biological screening of 17 of the 'hits' identified from virtual screening stuidies, 4 of which were synthesised in-house, against pancreatic cancer cell lines identified five compounds that demonstrated an equal or greater capacity to reduce BxPC-3 S100P-expressing pancreatic cells' metastatic potential in vitro relative to cromolyn. Compound 24 in particular, showed significant (p<0.05) inhibition of invasion of these cells at a concentration of 100 μM that was comparable to cromolyn at the same concentration. This compound, structurally distinct from cromolyn, was successfully synthesised, purified and characterised in-house alongside 39 of its analogues. Biological screening of compound 24 and four of its analogues for anti-proliferative activity against BxPC-3 and Panc-1 pancreatic cancer cell lines showed all five compounds significantly (p < 0.0001) inhibiting proliferation in both cell lines at a concentration of 1 μM relative to the non-treated control. Hence, structurally distinct compounds that show promising inhibitory activity on the metastasis and proliferation of pancreatic cancer cells have been identified using a structure-based drug design methodology. These compounds, with further optimisation, could provide good starting points as therapeutic lead candidates for the treatment of pancreatic cancer.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
The Paracoccidioides genus comprises a complex of pathogenic fungi that are the etiologic agents of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The infection begins after inhalation of fungal propagules, which reach the epithelium of the alveoli where the transition from the mycelial to the pathogenic yeast form. Host elevated temperature triggers the morphological switch, which is necessary for fungal pathogenicity. The cAMP/protein kinase A (PKA) signaling pathway has been shown to be important in controlling morphological changes and the pathogenicity of several pathogenic fungi. Evidence also highlights the importance of the cAMP/PKA pathway in the morphological transition of Paracoccidioides. PKA is the major effector of this signaling pathway. The protein is an inactive tetramer composed of regulatory subunit, encoded by the BCY1 gene; and catalytic subunit, encoded by the TPK2 gene. Upon binding of cAMP to the regulatory subunits, the catalytic subunits dissociate and become active. Activated PKA subsequently phosphorylates protein kinases, transcription factors, and other substrates to control several biological processes. In this study, we evaluated the expression and interactions of Tpk2 protein Paracoccididioides spp. The Tpk2 is present in mycelium decreased during the initial stages of transition phases, and increases again at the end of differentiation, with maximal levels in yeast. We analyzed the interactions of recombinant Tpk2p with Paracoccidioides proteins using pull-down assays followed by MS analysis. Two interacting proteins were identified: the heat shock protein 90 (Hsp90) and a conserved hypothetical protein with a MFS domain. Hsp90 is involved in the regulation of morphogenesis, development and virulence in several thermal dimorphic fungi. These data are important for understanding the mechanisms that trigger the transition phases in Paracoccidioides.
O gênero Paracoccidioides compreende um complexo de fungos patogênicos, que são os agentes etiológicos da paracoccidioidomicose (PCM), a micose sistêmica mais prevalente na América Latina. A infecção inicia-se com a inalação de propágulos do fungo, que atingem o epitélio dos alvéolos pulmonares, onde ocorre à transição da forma de micélio para a forma patogênica, de levedura. Há evidências de que a temperatura seja o principal fator responsável pela diferenciação celular desses fungos, e sua patogenicidade é frequentemente associada com a transição dimórfica. A via de sinalização cAMP/ proteína quinase A (PKA) controla alterações morfológicas e de virulência/patogenicidade em várias espécies de fungos patogênicos humanos. Evidências apontam também para a importância da via cAMP/PKA em Paracoccidioides spp. A PKA é o principal efetor desta via de sinalização. A proteína na forma inativa é um tetrâmero composto de subunidade regulatória, codificada pelo gene BCY1; e subunidade catalítica, codificada pelo gene TPK2. Após a ligação de cAMP às subunidades regulatórias, as subunidades catalíticas dissociam-se e tornam-se ativas. Ativada a PKA fosforila proteína-quinases, fatores de transcrição, e outros substratos para controlar diversos processos biológicos. Neste estudo, avaliamos a expressão e as interações da proteína Tpk2 de Paracoccididioides spp. A Tpk2 está presente em micélio, diminui nos estágios iniciais da transição de fases e volta a aumentar no final da diferenciação, apresentando níveis máximos na levedura. Foram analisadas as interações de Tpk2p recombinante com proteínas de Paracoccidioides utilizando ensaios de pull-down, seguido por análise de MS. Foram identificadas duas proteínas que interagem: a proteína de choque térmico 90 (Hsp90) e uma proteína hipotética conservada com um domínio MFS. Hsp90 está envolvido na regulação da morfogênese, desenvolvimento e virulência em vários fungos dimórficos térmicos. Estes dados são importantes para entendimento dos mecanismos que disparam a transição de fases em Paracoccidioides spp.

Initiation der Translation ist ein komplexer und stark regulierter Prozess, bei dem Ribosomen die mRNA binden. Die überwiegende Mehrheit eukaryotischer mRNAs wird durch einen 5‘-Cap-abhängigen Mechanismus translatiert. Dazu bindet der eIF4F-Proteinkomplex die mRNA an der 5'-Cap-Struktur, um weitere eIFs und die kleine ribosomale Untereinheit zu rekrutieren, welche dann die 5'UTR von 5'- in 3'-Richtung bis zu einem Startcodon scannt. Anschließend trifft die große ribosomale Untereinheit dazu und die Proteinsynthese beginnt. Darüber hinaus kann die Translation durch IRES, interne ribosomale Eintrittsstellen, vermittelt werden, welche das Ribosom unabhängig von Cap und 5‘-Ende zum Startcodon rekrutieren. Die zelluläre IRES-vermittelte Translation gilt als ineffizient unter physiologischen Bedingungen, wird aber durch Stress aktiviert. Da die Regulation dieses Mechanismus weitaus unbekannt ist, haben wir die zelluläre, Cap-unabhängige Translationsinitiation untersucht. Dafür haben wir eine embryonale Stammzelllinie generiert, welche eine dominant-negative Mutante von 4E-BP1 exprimiert. 4E-BP1 bindet das 5‘-Cap-bindende Protein, sodass eIF4F nicht am 5'-Cap andocken kann. Wir haben das Proteom während der Überexpression von 4E-BP1 und der neuronalen Differenzierung bestimmt, um Translationsdynamiken systemisch zu erfassen. Gene mit verminderter Sensitivität für die Cap-abhängige Translation wurden so identifiziert und in bicistronischen Reporter-Assays getestet. Nach strenger Validierung wurde eine Cap-unabhängig translatierte mRNA, Pqbp1, entdeckt. Der zweite Teil dieser Studie untersuchte die Cap-unabhängige Translation einer circRNA, welche keine freien Enden hat und daher per IRES translatiert werden muss. Wir konnten bestätigen, dass circMbl in vitro translatiert wird und konnten so innerhalb eines Kooperationsprojekts zu der Erkenntnis beitragen, dass circRNAs im Fliegengehirn translatiert werden.
Translation initiation is a complex and highly regulated process which involves the assembly of an elongation competent ribosome on the mRNA. The vast majority of eukaryotic mRNAs is translated by a canonical cap-dependent mechanism. This requires the eIF4F protein complex to bind the mRNA at the 5’-cap to recruit further eIFs and the small ribosomal subunit which then scans the 5’UTR in 5’ to 3’ direction until a start codon is encountered. Afterwards the large ribosomal subunit joins and protein synthesis begins. Besides that, translation of mRNAs can be mediated by IRESs, internal ribosome entry sites, which recruit the ribosome in a cap and 5’-end-independent manner to the start codon. Such cellular IRES-mediated translation is thought to be inefficient under physiological conditions but activated during stress. As the regulation of this mechanism is not well understood, we aimed to elucidate cellular cap-independent translation events. Therefore, we generated a mouse embryonic stem cell line with inducible overexpression of a dominant negative mutant of 4E-BP1. 4E-BP1 sequesters the cap-binding protein eIF4E so that the eIF4F protein complex fails to assemble at the 5’-cap. We performed shotgun proteomics during 4E‑BP1 overexpression and neuronal differentiation to globally monitor translation dynamics. Genes with reduced sensitivity for cap-dependent translation were identified and tested for internal translation initiation in bicistronic reporter assays. After stringent validation one cap-independently translated mRNA, Pqbp1, was discovered. The second part of this study investigated cap-independent translation initiation on a circRNA, which by nature lacks free ends and thus requires IRES-mediated translation. We could show that circMbl is translated in vitro and thus contributed to the scientific evidence for the translation of circRNAs in fly brain, which was studied in a collaboration project.

Os avanços tecnológicos vêm contribuindo cada vez mais nas áreas biológicas e científicas oferecendo ferramentas computacionais e sistemas específicos que em análise de dados in silico fornecem resultados rápidos e confiáveis. O presente projeto propõe o desenvolvimento de um portal na Internet para instalação e utilização de um banco de dados laboratoriais das principais serpentes brasileiras com seus respectivos venenos e antivenenos naturais, e a análise dos dados obtidos em ensaios farmacológicos, e bioquímicos. Utilizando a via de comunicação e interação mais simples atualmente, a Internet permite o compartilhamento de dados entre comunidades de pesquisadores, viabilizando recursos e tempo, além de permitir uma significante interação entre pesquisadores de todo o mundo, principalmente brasileira, na troca de informações e compartilhamento de dados. Dados elementares relacionados às serpentes foram armazenados no banco de dados, bem como as atividades tóxicas, farmacológicas e enzimáticas dos componentes dos venenos, e ainda, as aplicações biotecnológicas dos produtos que podem ser obtidos destes venenos, abrangendo ainda dados clínicos e valores estatísticos dos acidentes ofídicos. Aspectos bioquímicos dos ensaios realizados em laboratório permitiram a construção de uma ferramenta para análise comparativa de estruturas primárias de PLA2s, depositadas em bancos de dados internacionais. Além da interatividade entre pesquisadores, em Fóruns de Discussões, o sistema conta com listas dos principais artigos publicados em periódicos indexados, e devidamente atualizados periodicamente, com revisões bibliográficas.
Technological advances have been contributing, more and more, with biological and scientific areas, offering computational tools and specific systems which in silico data analysis supply reliable and fast results. The present project considers the development of an Internet portal for installation and use of laboratory data base for the main Brazilian serpents, with its respective venom and natural anti-venom, and the analysis of obtained data in pharmacological assays and biochemists. Using the easiest way of communication and interaction, the Internet allows sharing of data and information between communities of researchers around the world, especially for Brazilian researchers, making resources and time possible. Elementary data about serpents have been stored in the data base, as well as toxic, pharmacological and enzymatic activities of venom components, besides biotechnological applications of the products that can be obtained from these venom, enclosing clinical data and statistical values of ophidian accidents. Biochemists aspects of the assays carried through in laboratory allowed the construction of a comparative analysis tool for primary structures of PLA2s, deposited in international data bases. Beyond interactivity between researchers, in discussion forums, the system counts with lists of main articles published in indexed periodic, duly and constantly updated with bibliographical revisions.

La question moyenne-orientale est dans l'actualité depuis 1948. C'est en cette année que se crée l'Etat d'Israël sur les décombres du mandat britannique en Palestine. Dès le commencement, la guerre va commencer à fixer les frontières entre Israël et ses voisins arabes. Cependant, après 1967, une nouvelle question va apparaître, celle des relations avec les Territoires occupés. En conséquence, l'Etat d'Israël aura deux questions frontalières à gérer : la question interétatique classique et la question interne avec les Palestiniens. Cette recherche tente de démontrer les voies employées par les différents acteurs régionaux et internationaux pour trouver une solution à cette question juridique qui cause l'instabilité régionale. On s'appuiera sur le droit, l'Histoire, la science politique (en particulier, l'étude des idéologies sioniste et arabiste) et les relations internationales pour trouver une cohérence aux réussites et aux échecs qui ont émaillé l'histoire du Moyen-Orient depuis 1948 et le fait qu'Israël n'ait encore que deux frontières internationalement reconnues, une avec l'Egypte et l'autre avec le royaume de Jordanie.

碩士
國立臺灣海洋大學
食品科學系
97
Abstract High concentration of zinc was found in the digestive tract tissue of common carp which was not seen in other fish species. A zinc-binding protein with a molecular weight of 43kDa located on the plasma membrane of the connective tissue cells was responsible for the high zinc in common carp. Using the “carp zinc-binding protein” as antigen, polyclonal antibody of the protein was prepared. An immunochemical assay method using the antibody against the “carp zinc-binding protein” to quantitatively determine the amount of the protein in fish tissue was developed to study the existence of the protein in fish tissue. The immunochemical assay method developed could specifically detect a amount of “carp zinc-binding protein” between 2-40 ng. Based on this immunochemical assay, it was found that common carp had high amount of “carp zinc-binding protein” in its digestive tract tissue, kidney, head kidney, and blood with values of about 2, 1, 12, 7, and 1 mg/(g fresh tissue), respectively; but lower amount in its heaptopancreas and muscles (<0.20mg/ �� g fresh tissue ��). In the other fishes, crucian carp had high amount of “carp zinc-binding protein” in its kidney, head kidney and spleen, but grass carp, silver carp and tilapia have little or no “carp zinc-binding protein” (<0.04 mg/ �� g fresh tissue ��) in their tissues. The zinc and “carp zinc-binding protein” in the tissues of common carp fed basal diet was compared with those fed high zinc diet. It was found that the amount of zinc and “carp zinc-binding protein” in the digestive tract tissue and head kidney increased in the common carp fed high zinc diet. The zinc concentration in the blood of the common carp didn’t increase after feeding high zinc diet, but the amount of “carp zinc-binding protein” increased. This immunochemical assay method might be used to study the existence and distribution of “carp zinc-binding protein” in other organism. Besides, using immunochemical assay to determine the change of the “carp zinc-binding protein” in common carp under different circumstances or conditions might be very useful in understanding the function of “carp zinc-binding protein” and its physiological meaning.

by Michael Yiu-kwong Leung.
Thesis (Ph.D.)--Chinese University of Hong Kong, 1998.
Includes bibliographical references (p. 158-168).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

The chorioallantoic membrane (CAM) of chicken embryos belongs to the in vivo model systems frequently used for the study of angiogenesis and cell invasiveness. Using CAM assay we have tested selected chicken sarcoma cell lines characterized by different angiogenic properties and different ability to form metastasis. In addition to CAM assay, several other methods have been used to characterize the phenotype of these cell lines. We have selected a few proteins which could significantly influence the angiogenic and metastatic properties of investigated cell lines. We have established cell lines stably overexpressing these genes and compared their phenotypes with parental cell lines. We have shown that genes encoding ISL1, ARNT2, PROM1, HOXA11 proteins participate, in our experimental model, in activation of programes controlling angiogenesis and cell invasion.

Trimetyloguanozyno (TMG) kap wystepuje na 5’ koncu małych jadrowych RNA (sn RNA) u organizmów wyzszych. Jego główna funkcja jest transport małych jadrowych rybonukleoprotein (snRNP) do jadra komórkowego poprzez oddziaływanie z białkiem adaptorowym – snurportyna1. Struktura TMG kapu zbudowana jest z N2,N2,N7-trimetyloguanozyny połaczonej mostkiem 5’,5’-trifosforanowym z pierwszym nukleozydem łancucha RNA. Głównym celem badan zawartych w niniejszej rozprawie doktorskiej, było opracowanie metod syntezy chemicznej analogów kapu, posiadajacych rózne modyfikacje w obrebie mostka 5’,5’- trifosforanowego oraz zbadanie jak dana modyfikacja wpływa na ich własciwosci biologiczne – oddziaływanie ze snurportyna oraz odpornosc na degradacje enzymatyczna. Wazne jest takze, by wprowadzona modyfikacja nie zaburzała funkcjonowania kapu. Analogi o zwiekszonym badz porównywalnym powinowactwie do snurportyny, odporne na degradacje enzymatyczna (o przedłuzonym czasie zycia in vivo, w systemach biologicznych) stanowia potencjalne narzedzie do wprodzania terapeutyków do jadra komórkowego. Ponadto zarówno analogi modyfikowane w mostku fosforanowym, jak i znakowane fluorescencyjnie, mozna takze wykorzystac do badan nad mechanizmem procesów, w które zaangazowany jest TMG kap: transport dojadrowy makromolekuł, biosynteza TMG kapu, powstawanie agregatów snRNA w chorobie Alzheimera. Niniejsza rozprawa podzielona została na trzy główne czesci: Wstep Literaturowy, Badania Własne i Dyskusje Wyników oraz Czesc Eksperymentalna. We Wstepie Literaturowym czytelnik zostaje szczegółowo wprowadzony w role jaka odgrywa struktura TMG kapu w organizmach zywych: gdzie wystepuje, w jaki sposób powstaje, z jakimi białkami oddziałuje oraz na jakiej drodze zostaje zdegradowana. Omówione w tej czesci zostały takze własciwosci fizykochemiczne struktury TMG kapu. Przedstawiono równiez liczne przykłady syntez zarówno TMG kapu, jak i przykładowe sposoby wprowadzania modyfikacji mostka fosforanowego do struktury dinukleotydów. Koncowe rozdziały wprowadzaja czytelnika w zagadnienia zwiazane z absorpcja oraz fluorescencja struktury TMG kapu oraz białek, co jest bardzo przydatne do zrozumienia metod oraz wyników badan opisanych w dalszych rozdziałach. W czesci Badania Własne i Dyskusja Wyników w pierwszej kolejnosci przedstawiono cele pracy, nastepnie przedstawiona została synteza chemiczna analogów TMG kapu modyfikowanych w mostku 5’,5’-trifosforanowym, oraz znakowanych fluorescencyjnie. Przedstawiona została takze charakterystyka własciwosci spektroskopowych zsyntezowanych analogów. Nastepnie opisane zostały wyniki badan biofizycznych majacych na celu wyznaczenie powinowactwa analogów TMG kapu do snurportyny. W tej czesci zostały takze opisane wyniki badan biologicznych nad stabilnoscia zsyntezowanych analogów TMG kapu w warunkach in vitro, które nie były wykonane przez autorke. Wyniki te ukazuja jednak zasadnosc załozen niniejszej pracy oraz podjetych syntez chemicznych. I jednoczesnie ukazuja potencjał zsyntezowanych zwiazków. Wtrzeciej czesci – Czesci Eksperymentalnej- przedstawiony został szczegółowy opis wykonywanych eksperymentów. Poczawszy od syntez chemicznych oraz charakterystyki otrzymanych zwiazków (MS i NMR), poprzez opis metody biofizycznych, termodynamicznych, a skonczywszy na wyprowadzeniach wzorów, które niezbedne były w otrzymaniu niektórych wyników (wymienic jakich). Syntezy chemiczne oraz wyniki badan biofizycznych i biologicznych zostały opisane w czterech publikacjach naukowych, z czego dwóch z listy filadelfijskiej, a dwóch konferencyjnych. Piata publikacja jest w trakcie przygotowywania. Spis publikacji przedstawiono ponizej. Publikacje naukowe w czasopismach z listy filadelfijskiej: 1) Zytek M., Kowalska J., Lukaszewicz M., Wojtczak B. A., Zuberek A., Ferenc-Mrozek A., Darzynkiewicz E., Niedzwiecka A., Jemielity J., Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5’,5’-triphosphate bridge, Org. Biomol. Chem., 2014, 12, 9184-9199. 2) Honcharenko M., Zytek M., Bestas B., Moreno P., Jemielity J., Darzynkiewicz E., Smith E. C. I., Stromberg R., Synthesis and evaluation of stability of m3 G-CAP analogues in serum-supplemented medium and cytosolic extract, Bioorganic Medicinal Chemistry, 2013, 21, 7921-7928. Publikacje konferencyjne: 1) Zytek M., Kowalska J., Darzynkiewicz E., Jemielity J., The First Examples of Phosphate Modified Trimethylguanosine Cap Analogues, Coll. Symp Ser, 2011, 12, 503-504. 2) Zytek M., Kowalska J., Lukaszewicz M., Darzynkiewicz E., Niedzwiecka A., Jemielity J., Evaluation of biological activity of trimethylguanosine cap analogs modified within the 5’,5’-triphosphate bridge, Coll. Symp Ser, 2014, 14, 416-417.