Ephrin-A5 induces rounding, blebbing and deadhesion of EphA3-expressing 293T and melanoma cells by CrkII and Rho-mediated signalling

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions, membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.

Recent advances on the role of CD40 and dendritic cells in immunity and tolerance

CD40 is a key signaling pathway for the function of B cells, monocytes, and dendritic cells in the immune system, and plays an important role in inflammatory pathways of nonhemopoietic cells. The NFkappaB family of transcription factors is a critical mediator in inflammation. NFkappaB is involved both in the regulation of CD40 expression and in cell signaling after CD40 ligation. This positive feedback loop linking NFkappaB and CD40 plays an important role in the control of the adaptive immune response, with fundamental implications for immunity and tolerance in vivo.

Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Biological activity of well defined hydantoin derivatives on efflux pump systems of bacteria and cancer cells

A multi-resist��ncia a antibi��ticos e medicamentos usados em quimioterapia �� um dos grandes problemas com os quais as institui����es de sa��de se debatem hoje em dia. A ac����o provocada por bombas de efluxo �� uma das suas causas. Estas bombas t��m uma import��ncia fundamental, uma vez que, ao expelirem todo o tipo de t��xicos para o exterior das c��lulas, tamb��m expelem medicamentos, fazendo com que estes n��o tenham o efeito desejado dentro delas. As bombas de efluxo s��o transportadores que se encontram nas membranas de todo o tipo de c��lulas. Existem dois grandes tipos de bombas de efluxo: as prim��rias e as secund��rias. As primeiras conferem multi-resist��ncia principalmente em c��lulas eucariotas, como as c��lulas do cancro em humanos, tendo como fun����o a media����o da repulsa de subst��ncias t��xicas por interm��dio da hidr��lise de ATP. A primeira a ser descoberta e mais estudada destas bombas foi a ABCB1 que �� o gene que codifica a glicoprote��na-P (P de permeabilidade). Enquanto as secund��rias, que s��o a maior fonte de multi-resist��ncia em bact��rias, promovem a extrus��o de subst��ncias t��xicas atrav��s da for��a motriz de prot��es. Neste tipo de bombas s��o conhecidas quatro fam��lias principais, das quais uma das mais importantes �� a superfam��lia RND, uma vez que inclui a bomba AcrAB-TolC, que �� muito importante no metabolismo xenobi��tico de bact��rias Gramnegativas, nomeadamente a E.coli. Com o objectivo de reverter a multi-resist��ncia, tanto em c��lulas eucariotas como procariotas, t��m-se desenvolvido estrat��gias de combate que envolvem a descoberta de subst��ncias que inibam as bombas de efluxo. Assim sendo, ao longo dos tempos t��m sido descobertas variadas subst��ncias que cumprem este objectivo. �� o caso, por exemplo, dos derivados de fluoroquinolonas usados como inibidores de bombas de efluxo em bact��rias ou do Tamoxifen, utilizado na terapia de pacientes com cancro da mama. Um dos grupos de subst��ncias estudados para o desenvolvimento de poss��veis compostos que actuem como reversores de multi-resist��ncia s��o os compostos derivados de hidanto��nas. Estes, s��o conhecidos por possu��rem uma grande variedade de propriedades bioqu��micas e farmacol��gicas, sendo portanto usados para tratarem algumas doen��as em humanos, como a epilepsia. Nestes, est��o englobados compostos com actividade anti-convuls��o que constitui a sua grande mais-valia e, dependente da substitui����o no anel que os constitui, uma grande variedade de outras propriedades farmacol��gicas como a anti-fungica, a anti-arritmica, a anti-viral, a anti-diab��tica ou por exemplo a antagoniza����o de determinados receptores, como os da serotonina. Apesar de pouco usados em estudos experimentais para desenvolver subst��ncias anti-carcinog��nicas, existem alguns estudos com este efeito. Objectivos: O presente projecto envolve o estudo de bombas de efluxo prim��rias e secund��rias, em c��lulas eucariotas e procariotas, respectivamente. Em bact��rias, foram usados quatro modelos experimentais: Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, E. coli AG 100 e Salmonella Enteritidis NCTC 13349. Em c��lulas de cancro foram usadas, c��lulas T de linfoma de rato parentais e c��lulas T de linfoma de rato transfectadas com o gene humano MDR-1. O principal objectivo deste estudo foi a pesquisa de novos moduladores de bombas de efluxo presentes em bact��rias e c��lulas do cancro, tentando assim contribuir para o desenvolvimento de novos agentes farmacol��gicos que consigam reverter a multi-resist��ncia a medicamentos. Assim sendo foram testados trinta compostos derivados de hidanto��nas: SZ-2, SZ-7, LL-9, BS-1, JH-63, MN-3, TD-7k, GG-5k, P3, P7, P10, P11, RW-15b, AD-26, RW-13, AD-29, KF-2, PDPH-3, Mor-1, KK-XV, Thioam-1, JHF-1, JHC-2, JHP-1, Fur-2, GL-1, GL-7, GL-14, GL-16, GL-18. Como forma de atingir estes objectivos, a actividade biol��gica dos trinta compostos derivados de hidanto��nas foi avaliada nas quatro estirpes de bact��rias da seguinte forma: foram determinadas as concentra����es m��nimas inibit��rias dos trinta compostos como forma de definir as concentra����es em que os compostos seriam utilizados. Os compostos foram posteriormente testadas com um m��todo fluorom��trico de acumula����o de brometo de et��deo, que �� um substrato comum em bombas de efluxo bacterianas, desenvolvido por Viveiros et al. A actividade biol��gica dos compostos derivados de hidanto��nas nas c��lulas de cancro foi demonstrada por diferentes m��todos. O efeito anti-proliferativo e citot��xico dos trinta compostos foi avaliado nas c��lulas T de linfoma de rato transfectadas com o gene humano MDR-1 pelo m��todo de thiazolyl de tetraz��lio (MTT). Como o brometo de et��deo tamb��m �� expelido pelos transportadores ABC, estes compostos foram posteriormente testados com um m��todo fluorom��trico de acumula����o de brometo de et��deo desenvolvido por Spengler et al nos dois diferentes tipos de c��lulas eucariotas. Resultados: A maioria dos compostos derivados de hidanto��nas foi eficaz na modula����o de bombas de efluxo, nas duas estirpes de bact��rias Gram-negativas e nos dois diferentes tipos de c��lulas T de linfoma. Em contraste com estes resultados, nas duas estirpes de c��lulas Gram-positivas, a maioria dos compostos tiveram pouco efeito na inibi����o de bombas de efluxo ou at�� nenhum, em muitos dos casos. De uma maneira geral os melhores compostos nas diferentes estirpes de bact��rias foram: Thioam-1, SZ-2, P3, Rw-15b, AD-26, AD-29, GL-18, GL-7, KF-2, SZ-7, MN-3, GL-16 e GL- 14. Foram portanto estes os compostos que provocaram maior acumula����o de brometo de et��deo, inibindo assim com maior efic��cia as bombas de efluxo. No presente estudo, a maioria dos compostos conseguiu inibir a resist��ncia provocada pela bomba de efluxo ABCB1, tanto nas c��lulas parentais bem como nas c��lulas que sobre-expressam esta bomba, causando a acumula����o de brometo de et��deo dentro das c��lulas. As c��lulas que sobreexpressam a bomba ABCB1 foram posteriormente testadas com citometria de fluxo que �� a t��cnica padr��o para pesquisa de inibidores de bombas de efluxo. Os compostos que foram mais efectivos na inibi����o da bomba ABCB1, causando assim maior acumula����o de brometo de et��deo nas c��lulas que sobre-expressam esta bomba foram: PDPH-3, GL-7, KK-XV, AD-29, Thioam-1, SZ-7, KF-2, MN-3, RW-13, LL-9, P3, AD-26, JH-63 e RW- 15b. Este facto n��o corroborou totalmente os resultados da citometria de fluxo uma vez que os moduladores que provocaram maior inibi����o da bomba ABCB1 foram o MN-3, JH-63 e o BS-1, sendo que o ��ltimo n��o foi seleccionado como um bom composto usando o m��todo fluorom��trico de acumula����o de brometo de et��deo. Conclus��o: Os compostos derivados de hidanto��nas testados tiveram maior efeito nas estirpes de bact��rias Gram-negativas do que nas Gram-positivas. Relativamente ��s c��lulas eucariotas, as estruturas mais activas apresentam substituintes arom��ticos bem como alguns fragmentos aminicos terci��rios.

Anomalous persistent photoconductivity in Cu2ZnSnS4 thin films and solar cells

A persistent photoconductivity effect (PPC) has been investigated in Cu2ZnSnS4 thin films and solar cells as a function of temperature. An anomalous increase of the PPC decay time with temperature was observed in all samples. The PPC decay time activation energy was found to increase when temperature rises above a crossover value, and also to grow with the increase of the sulfurization temperature and pressure. Both the anomalous behavior of the PPC decay time and the existence of two different activation energies are explained in terms of local potential fluctuations in the band edges of CZTS.

Cytokines and T-Lymphocute count in patients in the acute and chronic phases of Bartonella bacilliformis infection in an endemic area in peru: a pilot study

Human Bartonellosis has an acute phase characterized by fever and hemolytic anemia, and a chronic phase with bacillary angiomatosis-like lesions. This cross-sectional pilot study evaluated the immunology patterns using pre- and post-treatment samples in patients with Human Bartonellosis. Patients between five and 60 years of age, from endemic areas in Peru, in the acute or chronic phases were included. In patients in the acute phase of Bartonellosis a state of immune peripheral tolerance should be established for persistence of the infection. Our findings were that elevation of the anti-inflammatory cytokine IL-10 and numeric abnormalities of CD4+ and CD8+ T-Lymphocyte counts correlated significantly with an unfavorable immune state. During the chronic phase, the elevated levels of IFN-γ and IL-4 observed in our series correlated with previous findings of endothelial invasion of B. henselae in animal models.

Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment

This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-na��ve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21). Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma) and 3.2 log10 copies/ml (semen). At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments.

Interaction of an opportunistic fungus Purpureocillium lilacinum with human macrophages and dendritic cells

IntroductionPurpureocillium lilacinum is emerging as a causal agent of hyalohyphomycosis that is refractory to antifungal drugs; however, the pathogenic mechanisms underlying P. lilacinum infection are not understood. In this study, we investigated the interaction of P. lilacinum conidia with human macrophages and dendritic cells in vitro.MethodsSpores of a P. lilacinum clinical isolate were obtained by chill-heat shock. Mononuclear cells were isolated from eight healthy individuals. Monocytes were separated by cold aggregation and differentiated into macrophages by incubation for 7 to 10 days at 37��C or into dendritic cells by the addition of the cytokines human granulocyte-macrophage colony stimulating factor and interleukin-4. Conidial suspension was added to the human cells at 1:1, 2:1, and 5:1 (conidia:cells) ratios for 1h, 6h, and 24h, and the infection was evaluated by Giemsa staining and light microscopy.ResultsAfter 1h interaction, P. lilacinum conidia were internalized by human cells and after 6h contact, some conidia became inflated. After 24h interaction, the conidia produced germ tubes and hyphae, leading to the disruption of macrophage and dendritic cell membranes. The infection rate analyzed after 6h incubation of P. lilacinumconidia with cells at 2:1 and 1:1 ratios was 76.5% and 25.5%, respectively, for macrophages and 54.3% and 19.5%, respectively, for cultured dendritic cells.ConclusionsP. lilacinum conidia are capable of infecting and destroying both macrophages and dendritic cells, clearly demonstrating the ability of this pathogenic fungus to invade human phagocytic cells.

Sol-gel entrapped biosystems: enzyme(s) and whole cells

In this work two different procedures to utilize the sol-gel technology were applied to immobilize/encapsulate enzymes and living cells. CO2 has reached levels in the atmosphere that make it a pollutant. New methods to utilize this gas to obtain products of added value can be very important, both from an environmentally point of view and from an economic standpoint. The first goal of this work was to study the first reaction of a sequential, three-step, enzymatic process that carries out the conversion of CO2 to methanol. Of the three oxidoreductases involved, our focus was on formate dehydrogenase (FateDH) that converts CO2 to formate. This reaction requires the presence of the cofactor ��-nicotinamide adenine dinucleotide in reduced form (NADH). The cofactor is expensive and unstable. Our experiments were directed towards generating NADH from its oxidized form (NAD+), using glutamate dehydrogenase (GDH). The formation of NADH from NAD+ in aqueous medium was studied with both free and sol-gel entrapped GDH. This reaction was then followed by the conversion of CO2 to formate, catalysed by free or sol-gel entrapped FateDH. The quantification of NADH/NAD+ was made using UV/Vis spectroscopy. Our results showed that it was possible to couple the GDH-catalyzed generation of the cofactor NADH with the FateDH-catalyzed conversion of CO2, as confirmed by the detection of formate in the medium, using High Performance Liquid Chromatography (HPLC). The immobilization of living cells can be advantageous from the standpoint of ease of recovery, reutilization and physical separation from the medium. Also dead cells may not always exhibit enzymatic activities found with living cells. In this work cell encapsulation was performed using Escherichia coli bacteria. To reduce toxicity for living organisms, the sol-gel method was different than for enzymes, and involved the use of aqueous-based precursors. Initial encapsulation experiments and viability tests were carried out with E. coli K12. Our results showed that sol-gel entrapment of the cells was achieved, and that cell viability could be increased with additives, namely betaine that led to greater viability improvement and was selected for further studies. For an approach to ���in-cell��� Nuclear Magnetic Resonance (NMR) experiments, the expression of the protein ctCBM11 was performed in E. coli BL21. It was possible to obtain an NMR signal from the entrapped cells, a considerable proportion of which remained alive after the NMR experiments. However, it was not possible to obtain a distinctive NMR signal from the target protein to distinguish it from the other proteins in the cell.

Perylene Diimide acceptors: Fabrication and characterization of electron-only, hole-only devices and solar cells

The thrust towards energy conservation and reduced environmental footprint has fueled intensive research for alternative low cost sources of renewable energy. Organic photovoltaic cells (OPVs), with their low fabrication costs, easy processing and flexibility, represent a possible viable alternative. Perylene diimides (PDIs) are promising electron-acceptor candidates for bulk heterojunction (BHJ) OPVs, as they combine higher absorption and stability with tunable material properties, such as solubility and position of the lowest unoccupied molecular orbital (LUMO) level. A prerequisite for trap free electron transport is for the LUMO to be located at a level deeper than 3.7 eV since electron trapping in organic semiconductors is universal and dominated by a trap level located at 3.6 eV. Although the mostly used fullerene acceptors in polymer:fullerene solar cells feature trap-free electron transport, low optical absorption of fullerene derivatives limits maximum attainable efficiency. In this thesis, we try to get a better understanding of the electronic properties of PDIs, with a focus on charge carrier transport characteristics and the effect of different processing conditions such as annealing temperature and top contact (cathode) material. We report on a commercially available PDI and three PDI derivatives as acceptor materials, and its blends with MEH-PPV (Poly[2-methoxy 5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) and P3HT (Poly(3-hexylthiophene-2,5-diyl)) donor materials in single carrier devices (electron-only and hole-only) and in solar cells. Space-charge limited current measurements and modelling of temperature dependent J-V characteristics confirmed that the electron transport is essentially trap-free in such materials. Different blend ratios of P3HT:PDI-1 (1:1) and (1:3) show increase in the device performance with increasing PDI-1 ratio. Furthermore, thermal annealing of the devices have a significant effect in the solar cells that decreases open-circuit voltage (Voc) and fill factor FF, but increases short-circuit current (Jsc) and overall device performance. Morphological studies show that over-aggregation in traditional donor:PDI blend systems is still a big problem, which hinders charge carrier transport and performance in solar cells.

Fosforilaci��n de l��pidos se��ales disparada por ABA, NaCl y manitol en cole��ptilos, ra��ces y c��lulas de aleurona de cebada (<i>Hordeum vulgare</i>). Lipid signaling phosphorylation triggered by ABA, NaCl and mannitol in coleoptile, roots and aleurone cells of barley (<i>Hordeum vulgare</i>).

Es bien conocido que los fosfol��pidos son un conjunto de mol��culas capaces de funcionar como reguladores en diversos procesos celulares. Al respecto, este proyecto tiene como objetivo dilucidar la participaci��n de los mismos, en particular ��cido fosfat��dico (PA) y diacilglicerol pirofosfato (DGPP) durante el efecto antag��nico de ABA en la germinaci��n y como reguladores de la respuesta al estr��s salino en la pl��ntula. Se sabe que las plantas responden de forma r��pida y adecuada a una situaci��n de estr��s modificando el patr��n de fosfol��pidos de sus membranas, lo cual lleva a un cambio global en las actividad de l��pido quinasas, fosfatasas y a la expresi��n/represi��n de genes particulares. El desarrollo de la propuesta permitir��a responder dos cuestiones b��sicas: conocer la relaci��n entre fosfol��pidos y ABA e indagar su participaci��n durante la se��al de estr��s. La relevancia de la propuesta radica en la necesidad de ampliar el conocimiento sobre una de las causas mas importantes "estr��s salino" que afecta la germinaci��n de la semilla y luego el crecimiento y desarrollo de la pl��ntula. En principio se evaluara a nivel morfol��gico, bioqu��mico y molecular el efecto de ABA y de fosfol��pidos. Se pretende indagar sobre cambios a nivel de vacuolizaci��n en protoplastos aislados, actividad de enzimas relacionadas, pH intracelular, nivel de fosfol��pidos y enzimas implicadas en su metabolismo y tambi��n efectos sobre la expresi��n g��nica. Por otro lado, se analizara los niveles de fosfol��pidos y enzimas relacionadas con su metabolismo en ra��ces y coleoptilos de semillas que germinaron bajo condiciones de estr��s. Asimismo, se identificaran los cambios morfol��gicos provocados por el estr��s en la longitud de cole��ptilos y ra��ces. Por ultimo como indicador de una respuesta al estr��s se evaluara los cambios en los niveles de prolina. La importancia del proyecto es determinar el papel que desempe��an PA y DGPP en la germinaci��n y durante la respuesta al estr��s.

Caracterizaci��n funcional de zeb1 en c��lulas en diferenciaci��n y metast��sicas. Functional characterization of zeb1 in differentiating and metastatic cells

IDENTIFICACI��N ZEB1 (Zinc Finger E-box Binding Homeobox) es un factor de transcripci��n funcionalmente asociado con la diferenciaci��n de c��lulas como miocitos, neuronas, c��lulas de sost��n y linfocitos T, adem��s de estar involucrado en la Transici��n Epitelial-Mesenquimatosa (EMT) de los tumores s��lidos epiteliales. A��n no se ha revelado en profundidad la participaci��n de ZEB1 en los procesos de proliferaci��n y diferenciaci��n en los que participa. Estamos interesados en los mecanismos de regulaci��n de ZEB1 y los factores que intervienen en los procesos de diferenciaci��n y transformaci��n celular. HIP��TESIS 1. Las v��as de se��alamiento regulan el estado de fosforilaci��n y la funci��n de ZEB1 en la c��lula normal, el cual se desregular��a en la c��lula neopl��sica llevando a cambios en la funci��n normal de ZEB1 y consecuentemente a met��stasis. 2. IGF-1 es la se��al que, en asociaci��n con el supresor de tumores CCN6, juega un rol causal en la regulaci��n de ZEB1 y esto a su vez en la met��stasis del c��ncer de mama. OBJETIVO GENERAL: establecer el rol funcional de ZEB1, su interrelaci��n con otros factores y su regulaci��n en los procesos de diferenciaci��n y transformaci��n celular. OBJETIVOS ESPECIFICOS (incluye Materiales y M��todos) 1. Estudiar la participaci��n de v��as de se��alizaci��n sobre la funci��n biol��gica de ZEB1 en c��lulas normales y neopl��sicas. Analizaremos la participaci��n de se��ales intracelulares en la fosforilaci��n de ZEB1 por experimentos de ganancia/p��rdida de funci��n de la v��a (por uso de inhibidores farmacologicos, mutantes silenciadoras y siRNAs), lo cual sera evaluado en EMSAs, ChIP, transfecciones, inmunofluoresc, etc. 2. Estudiar el rol de IGF-1 y CCN6 sobre la expresi��n y el estado de fosforilaci��n de ZEB1 en tumores mamarios benignos, no invasivos e invasivos y metastatizantes. A) Se estudiar�� la expresi��n y localizaci��n subcelular de ZEB1 en l��neas celulares de c��ncer mamario y en xenotransplantes de rat��n con variada expresi��n de CCN6. B) Investigar la relevancia de la fosforilaci��n de ZEB1 mediada por IGF-1 en el EMT por experimentos con ganancia/p��rdida de funci��n. RESULTADOS ESPERADOS Esperamos poder delinear la/s v��a/s de se��alizaci��n intracelular que fosforilan ZEB1 y as�� conocer sobre la regulaci��n del mismo. Podremos establecer algunas bases para entender la biolog��a b��sica del c��ncer de mama e identificar blancos terap��uticos. IMPORTANCIA Un amplio conocimiento de los factores de transcripci��n y sus v��as de se��alamiento es necesario para el desarrollo tanto de pruebas diagn��sticas como para la identificaci��n de nuevos blancos terap��uticos para neoplasias. De modo que resulta de gran importancia cl��nica determinar el rol de ZEB1, sus prote��nas y v��as reguladoras en el proceso de oncog��nesis. El desarrollo del proyecto prev�� la formaci��n de dos tesistas. Se continuaran colaboraciones con dos grupos extranjeros y se iniciara una tercera. ZEB1 (Zinc Finger E-box Binding Homeobox) is a transcription factor involved in cell differentiation and Epithelial Mesenchymal Transition (EMT) of epithelial tumors. We are interested in the study of mechanisms of regulation (pre and post transcriptional). S.A.1. To investigate post translational mechanisms of ZEB1 regulation in normal and cancer cells. We will analyze the involvement of intracellular signals in phosphorylation of ZEB1 by gain- and lost-of-function experiments. S.A.2. A) To determine the role of IGF-1 signaling and CCN6 in regulating the expression of hypo- and hyperphosphorylated forms of ZEB1 in benign and malignant breast cell lines and in xenograft mouse models by overexpressing and inhibiting CCN6 in breast cancer cells. B) To investigate the relevance of CCN6-mediated ZEB1 phosphorylation to EMT, breast cancer invasion and metastasis. The role of CCN6 on ZEB1 phosphorylation and regulation of E-cadherin, induction of EMT, invasion and metastasis of breast cells will be investigated using gain- and loss-of-function experiments.

Molecular biological analysis of dynamic interactions between influenza viruses and host cells : host cell proteomes and viral replication dynamics

Magdeburg, Univ., Fak. f��r Verfahrens- und Systemtechnik, Diss., 2011