Nutrient and growth factor receptors internalize through dathrin coated pits. The signal sequences which mediate the association between receptors and the coated pit reside in receptor cytoplasmic tail domains. These signal sequences have been extensively investigated in nutrient receptors, and a minimal functional sequence has been identified consisting of a tyrosine residue in an exposed b turn. Protein-protein contacts between internalization signal sequences and components of the coated pit machinery have been proposed to mediate rapid internalization. In vitro evidence suggests the AP-2 adaptor may be that protein component. The signal sequences of growth factor receptors are less well understood. However, a growth factor- and temperature- dependent binding between the epideimal growth factor receptor and the AP-2 adaptor has been observed. We identified Hsc70 as a cytosolic ligand for the cytoplasmic tail of the transferrin receptor. The binding was mapped to the internalization signal sequence of the receptor tail. Mutations within the signal sequence which inhibit internalization result in alteration of signal sequence secondary structure and reduction in stimulation of the Hsc70 ATPase. Co-immunoprecipitation analysis showed a population of transferrin receptors which are bound to Hsc70, suggesting an association in vivo. We also showed binding of Hsc70 to the epidermal growth factor receptor by co-immunoprecipitation analysis. This binding was increased by treatment with EGF. The binding was transient, and occured prior to the binding of the receptor to AP-2 adaptors. Other agents which induce EGF receptor clustering and internalization also stimulate the transient increase in Hsc70 binding and the later AP-2 binding, suggesting a role in early endocytosis. These data support the hypothesis that Hsc70 is associated with the receptors for transferrin and epidermal growth factor in vitro and in vivo. We propose a role for the 70 kD heat shock protein in the assembly/disassembly of protein complexes involved in receptor signalling and/or internalization.

Orientador: Carlos Alberto de Souza Costa / Resumo: A fixação do tecido conjuntivo à superfície dos abutments de implantes dentários impede a migração apical do epitélio juncional e previne a reabsorção da crista óssea normalmente ocasionada pela inflamação peri-implantar, o qual promove comprometimento estético e o sucesso do procedimento reabilitador. O uso de terapias para favorecer a adesão celular, a fim de induzir um selamento biológico efetivo, pode melhorar as condições estéticas e funcionais nas reabilitações protéticas. O objetivo desta pesquisa foi avaliar o efeito da laserterapia de baixa potência (LBP) e do recobrimento de superfícies de titânio (Ti) e zircônia (ZrO2) com fator de crescimento epidérmico (EGF), sobre a adesão e metabolismo de células da mucosa oral humana expostas ao estímulo inflamatório com o fator de necrose tumoral α (TNF-α). Fibroblastos de gengiva e células epiteliais foram isolados e cultivados sobre discos de Ti e ZrO2, simulando o selamento biológico in vitro. Nos grupos com EGF, este fator de crescimento foi aplicado sobre as superfícies de Ti e ZrO2 previamente ao cultivo celular. Após a semeadura das células, estas foram irradiadas 3 vezes com LBP (LaserTABLE, InGaAsP, 780±3nm), em intervalos de 24 h, nas doses de 0,5 J/cm2; 1,5 J/cm2 e 3,0 J/cm2, de acordo os grupos estabelecidos e então, o TNF-α foi aplicado sobre as mesmas por 24 h. Foram realizadas as análises de rugosidade da superfície dos discos por Microscopia Confocal (n=8) e de liberação do EGF dos substratos recobertos, além ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The attachment of the connective tissue to abutment surface of dental implants prevents the apical migration of the junctional epithelium and the bone crest reabsorption, normally caused by the peri-implant inflammation, which compromises the aesthetics and the success of the rehabilitation procedure. The use of therapies to promote cell adhesion in order to induce effective biological sealing can improve aesthetic and functional conditions in prosthetic rehabilitation. The aim of this study was to evaluate the effects of low-level laser therapy (LLLT) and surfaces coating of titanium (Ti) and zirconia (ZrO2) with epidermal growth factor (EGF) on adhesion and metabolism of oral mucosa cells exposed or not to tumor necrosis alpha (TNF-α) inflammatory stimuli. Gingival fibroblasts and epithelial cells were isolated and seeded on surface Ti or ZrO2 surfaces, simulating the in vitro biological sealing. In EGF-coated groups, this growth factor was applied to Ti and ZrO2 surfaces prior to cell culture. After seeding cells, they were irradiated 3 times with LLLT (LaserTABLE, InGaAsP, 780 ± 3 nm) at 24 h intervals at doses of 0.5 J/cm2, 1.5 J/cm2 and 3.0 J/cm2, according to the established groups and then, TNF-α was applied to them for 24 h. Discs surface roughness analyzes were performed by confocal microscopy (n=8), EGF release of the coated substrates and cell adhesion by direct fluorescence. Cell viability (AlamarBlue, n=8), IL-6, IL-8 and VEGF (qPCR, n=5) gene expression, IL-6, ... (Complete abstract click electronic access below) / Doutor

No description available.

A variety of soybean oils (SOs) were developed with improved functional properties. Some of the modified SOs contain altered fatty acid (FA) composition by selective breeding methods. Currently, low α- linolenic acid soybean oil (LLSO) and low α- linolenic acid and mid oleic acid soybean oil (LLMOSO) are available FA modified SOs in the market. The consumption of FA modified SOs has been increased because the United States Food and Drug Administration required listing trans fat content in food products sold in U.S. as an effort to reduce possible health risks caused by trans fat beginning 2006. However, the effects of these FA modified SOs on human chronic diseases including breast cancer (BC) have not been studied. BC has become the most frequently diagnosed cancer and is the second leading cause of cancer death among American women. The type of dietary fat, FA composition, and n-6/n-3 ratio are known to influence BC development. Therefore, it is possible that the changed FA composition and n-6/n-3 ratio in the FA modified SOs may affect BC progression, and its critical health concern needs to be investigated. Increased human epithelial growth factor receptor 2 (Her-2/neu) and fatty acid synthase (FAS) are associated with BC progression. In fact, FAS activity and expression are affected by dietary FA composition and FA metabolism. Hypothesis of this research is that LLSO and LLMOSO may affect Her-2/neu and FAS expressing human BC (SK-Br3) cell growth in vitro and in vivo. To test our hypothesis, we investigated the potential adverse or beneficial effects of LLSO and LLMOSO in comparison with conventional SO and lard on human BC cells and then examined the possible mechanisms of action by evaluating the expression level of genes markers involved in growth factor mediated signal transduction pathway, specifically Her-2/neu PI 3-kinase (phophoinositide 3- kinase)-FAS signal transduction pathway. In vitro study demonstrated that all the tested oils at 0-2 μl/ml level have cytotoxic effects. LLMOSO had less cytotoxic effects on the growth of SK-Br3 cells compared to SO. However, there was no difference in SK-Br3 cell growth between LLSO and SO. The apoptotic protein markers (mutant p53 and caspase-3) analysis revealed that the cell growth inhibition by oil treatments was cytotoxic by triggering apoptosis. Western blot analysis demonstrated that LLSO- and LLMOSO- induced changes on cell growth involve Her-2/neu and FAS signaling transduction pathway and sterol regulatory element binding protein-1 (SREBP-1), mitogen activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI 3-kinase) are possible down-stream effectors of Her-2/neu signaling pathway. We also evaluated the dietary effects of LLSO (20% fat of total calorie), SO (20%), and lard (20%) on the growth of SK-Br3 tumors implanted in athymic mice. Changes in tumor surface area, body weight, and food intake were monitored during the 6 months feeding study. After termination, tumor net weight, Her-2/neu and FAS mRNA expression in tumors, FAS protein expression in liver, lipid composition in diets, abdominal fat, and serum, as well as plasma total cholesterol and triglyceride levels were analyzed. In vivo study showed that there were no statistical differences in tumor size and tumor net weight among SO, LLSO, and lard groups. No differences in FAS mRNA and protein expression levels between the LLSO and SO groups were observed. Tumors from the lard group expressed higher Her-2/neu and FAS mRNA than those from the LLSO and SO group. The lipid analysis demonstrated that LLSO was not significantly distinct from SO in trans fat concentration after metabolism. Serum cholesterol and triglyceride levels were unchanged in LLSO fed compared to SO fed mice. In summary, LLSO which contained modification in αLA concentration showed similar effects on SK-Br3 as SO in both in vitro and in vivo. However, LLMOSO which contained more drastic modifications on FA composition exhibited less cytotoxicity compared to SO in vitro. / Master of Science

No description available.

Είναι ευρέως αποδεκτό και καλά τεκμηριωμένο ότι ο υποδοχέας του επιδερμικού αυξητικού παράγοντα (epidermal growth factor receptor, EGFR) ελέγχει σημαντικές λειτουργίες των καρκινικών κυττάρων, όπως τον πολλαπλασιασμό και την απόπτωση, αλλά και διαδικασίες όπου συμμετέχουν περισσότεροι του ενός τύποι κυττάρων, όπως τη διήθηση και την αγγειογένεση. Μεταξύ των τύπων καρκίνου, στην ανάπτυξη των οποίων συμμετέχει ο EGFR, είναι και ο μη μικροκυτταρικός καρκίνος του πνεύμονα (ΜΜΚΠ). Πολύ πρόσφατα δεδομένα δείχνουν ότι ένα άλλο μόριο που εμπλέκεται στην ανάπτυξη του καρκίνου του πνεύμονα είναι το Notch. Ο ρόλος του είναι περίπλοκος και διττός: Έχει προταθεί ότι το Notch επάγει την ανάπτυξη του ΜΜΚΠ και αναστέλλει την ανάπτυξη του μικροκυτταρικού καρκίνου του πνεύμονα (ΜΚΠ). Επιπλέον, έχει βρεθεί ότι το μονοπάτι μεταγωγής σήματος του Notch επηρεάζει, αλλά και επηρεάζεται από άλλα μόρια. Στην παρούσα μεταπτυχιακή εργασία διερευνήθηκε ο ρόλος του EGFR και του Notch στην ανάπτυξη κυττάρων ΜΜΚΠ χρησιμοποιώντας τον προσδέτη του EGFR, EGF και τον αναστολέα της γ-σεκρετάσης DAPT. Για τη διεξαγωγή των πειραμάτων χρησιμοποιήθηκαν οι ανθρώπινες καρκινικές κυτταρικές σειρές ΜΜΚΠ Η23, Α549, Η661 και ΗCC827. Οι κυτταρικές σειρές Η23, Α549 και Η661 εκφράζουν τον αγρίου τύπου (wild type, wt) EGFR και η κυτταρική σειρά HCC827 εκφράζει EGFR που φέρει τη μετάλλαξη (mutation) (DE746- A750). Αρχικά με ανάλυση κατά western μελετήθηκε το προφίλ των κυττάρων ως προς τα επίπεδα έκφρασης του ενδοκυττάριου τμήματος του Notch (Notch Intracellular Domain, NICD). Βρέθηκε ότι τα κύτταρα Η23 εκφράζουν τα υψηλότερα επίπεδα Notch ICD, τα κύτταρα Η661 και HCC827 μέτρια επίπεδα και τα κύτταρα Α549 τα χαμηλότερα. Στη συνέχεια με τη μέθοδο του ΜΤΤ έγινε έλεγχος του DAPT στον πολλαπλασιασμό των κυττάρων και βρέθηκε ότι τα κύτταρα Η661 είχαν τη μεγαλύτερη αναστολή, παρόμοια συμπεριφορά έδειξαν και τα Α549. Τα κύτταρα Η23 εμφάνισαν μικρότερη ανταπόκριση σε σχέση με τα Η661 ενώ τα κύτταρα HCC827 εμφανίστηκαν ανθεκτικά στο DAPT. Η ανασταλτική δράση του DAPT στα κύτταρα Η661 συνοδεύτηκε με επαγωγή της απόπτωσης η οποία προσδιορίστηκε με τη μέθοδο αννεξίνης V καθώς και με επαγωγή της αυτοφαγίας η οποία ανιχνεύτηκε κάνοντας ανάλυση κατά western για τα πρωτεϊνικά επίπεδα της beclin-1. Περαιτέρω τα κύτταρα ενεργοποιήθηκαν με EGF και εν συνεχεία προστέθηκε DAPT. Παρατηρήθηκε ότι στα κύτταρα Η23 η προσθήκη του EGF δεν επέτρεψε να δράσει ανασταλτικά το DAPT ενώ στα Η661 εν μέρει ο EGF αντέστρεψε την ανασταλτική δράση του DAPT. Επιλέγοντας τις κυτταρικές σειρές Η23 και Η661, μελετήθηκε η δράση του DAPT και του EGF στα επίπεδα του Notch ICD. Παρατηρήθηκε ότι στα κύτταρα Η23, το DAPT μείωσε με χρονοεξαρτώμενο τρόπο τα πρωτεϊνικά επίπεδα του Notch ICD μέχρι και 6 ώρες μετά την προσθήκη του στα κύτταρα ενώ 24 ώρες μετά το φαινόμενο αντιστράφηκε. Η προσθήκη του EGF δεν επηρέασε τα επίπεδα του Notch ICD σε καμία από τις χρονικές στιγμές που μελετήθηκαν. Στα Η661 κύτταρα το DAPT προκάλεσε χρονοεξαρτώμενη μείωση των επιπέδων Notch ICD η οποία διήρκησε μέχρι και 24 ώρες μετά τη προσθήκη του DAPT. Ο EGF όπως και προηγουμένως δεν επηρέασε τα επίπεδα του Notch ICD. Παρατηρώντας ότι στα Η661 το DAPT ασκεί δράση με μεγαλύτερη διάρκεια σε σχέση με τα κύτταρα Η23, τα κύτταρα Η661 ενεργοποιήθηκαν με EGF και στη συνέχεια προστέθηκε το DAPT προκειμένου να δούμε τη δράση του συνδυασμού στα επίπεδα του Notch ICD. Βρέθηκε ότι ο EGF αντέστρεψε την μείωση των Notch ICD επιπέδων που προκαλεί μόνο του το DAPT. Τα αποτελέσματα αναδεικνύουν ότι τα μονοπάτια του EGFR και του Notch, συνηγορούν προς την ίδια κατεύθυνση για τη μείωση του όγκου και αυτό υποδηλώνει έναν ελκυστικό δρόμο συνδυαστικών προσεγγίσεων για τη θεραπεία του ΜΜΚΠ, που μπορεί να ενισχύσει τη δράση των ανασταλτικών παραγόντων του EGFR σε όγκους. Συμπερασματικά, θα μπορούσαμε να υποθέσουμε ότι στο ΜΜΚΠ: α) τα δύο μονοπάτια EGFR και Notch συνεπικουρούν για την ανάπτυξη του όγκου, β) η αναστολή του Notch είναι πιο αποτελεσματική σε κύτταρα με ενδιάμεσα επίπεδα ενεργού Notch 1, προκαλώντας τόσο απόπτωση όσο και αυτοφαγία, και γ) η μετάλλαξη του EGFR προσφέρει αντίσταση στη δράση αναστολέα της γ-σεκρετάσης. / It is widely accepted and well established that the epidermal growth factor receptor (EGFR) controls important processes of tumor cells, such as proliferation and apoptosis, but also processes involving more than one type of cells such as invasion and angiogenesis. It has been found that the EGFR has an important role in the development of several types of cancer including non-small cell lung cancer (NSCLC). Very recent data indicate that another molecule, which is involved in the development of lung cancer, is Notch. Its role is complicated and is under investigation. It is suspected that Notch has a growth promoting function in NSCLC, whereas exerts an inhibitory effect in small cell lung cancer (SCLC). Furthermore it has been found that the signaling pathway of Notch can affect/ can be affected by other molecules. This thesis investigated the role of EGFR and Notch in cell growth of NSCLC cells using the ligand of EGFR, EGF and gamma-secretase inhibitor, DAPT. To conduct the experiments the human NSCLC cell lines H23, A549, H661 and HCC827 were used. The cell lines H23, A549 and H661 express the wild type (wt) EGFR and the cell line HCC827 expresses EGFR bearing the mutation (mt) DE746-A750. Initially, we studied the profile of NSCLC cells regarding the protein levels of Notch intracellular domain (Notch ICD) using western blot analysis. It was found that H23 cells express the higher levels Notch ICD, H661 and HCC827 cells express intermediate levels and A549 cells express the lowest levels of Notch ICD. The next step was the evaluation of DAPT effect in cell proliferation using the MTT assay. We found that DAPT caused the greatest inhibition to H661 and A549 cells. DAPT was less effective to H23 cells while had no effect to HCC827 cells. The inhibitory effect of DAPT in H661 cells was in line with the induction of apoptosis and autophagy, as was detected using annexin V assay and western blot analysis for beclin-1, respectively. Furthermore, cells were stimulated with EGF and subsequently DAPT was added. We found that the stimulatory effect of EGF was not reversed by DAPT in H23 cells. However a partial reverse of EGF stimulation was observed in H661 cells. The next step was to study the effect of DAPT and EGF at Notch ICD protein levels, in H23 and H661 cells. We found that DAPT reduced the protein levels of Notch ICD in H23 cells, with a time-dependent manner, up to 6 hours after DAPT addition and this effect reversed 24 hour later. The addition of EGF did not affect the levels of Notch ICD at any time point tested. In H661 cells, DAPT caused a time-dependent reduction of Notch ICD protein levels up to 24 hours after DAPT addition to cells. EGF as previously, did not affect the levels of Notch ICD in these cells. Since DAPT was more effective to H661 cells, these cells stimulated with EGF and then DAPT was added in order to study the effect of the combination at the levels of Notch ICD. We found that EGF reversed the decrease of Notch ICD protein levels caused by DAPT alone. These results indicate that the pathways of EGFR and Notch might act with a synergistic fashion and this could be an attractive approach for the treatment of NSCLC. Summarizing our results, we might assume that in NSCLC: a) both pathways of EGFR and Notch exert a significant role in tumor growth, b) the inhibition of Notch is more effective in cells with intermediate levels of activated Notch 1, causing both apoptosis and autophagy, and c) the EGFR mutation confers resistance to the effect of γ- secretase inhibitor.

Štěpána Boukalová Mechanisms of activation and modulation of vanilloid TRP channels TRPV1 and TRPV3 are thermosensitive ion channels from the vanilloid subfamily of TRP receptors. TRPV1, which is primarily expressed in nociceptive sensory neurons, is an important transducer of painful stimuli and is also involved in the detection of noxious heat. TRPV3 is expressed mainly in the skin where it regulates proliferation and differentiation of keratinocytes. Similarly to voltage-dependent potassium (Kv) channels, TRP receptors are comprised of four subunits, each with six transmembrane segments (S1-S6). Using mutational approach, we tried to elucidate the role of S1 in TRPV1 functioning. Our results indicate that the extracellular portion of S1 plays a crucial role in TRPV1 gating. TRPV1 channels with a conservative mutation of positively charged residue in this region (R455K substitution) were overactive. However, they were neither activated nor potentiated by low pH; on the contrary, protons stabilized the closed conformation of this mutant channel. Very similar phenotypic properties were found in other TRPV1 mutants with substitution in S4/S5-S5 region and in the pore helix. In Kv channels, extracelular portion of S1 forms a small contact surface with the pore helix, which allows efficient transmission of...

O Receptor do Fator de Crescimento Epidérmico (do inglês, EGFR) é uma proteína de membrana celular que consiste em um domínio extracelular para o acoplamento do ligante e em um domínio intracelular apresentando sítio catalítico de tirosinoquinase. Em ~40% dos GBMs primários é observada a amplificação de EGFR resultando na sua hiperexpressão, o que raramente ocorre em GBM secundário. Mais da metade dos casos de GBM com amplificação do receptor está associado com rearranjo do gene, uma forma deletada de EGFR (EGFRvIII). Adicionalmente, o receptor de interleucina 13 alfa-2 (IL-13R 2), apresenta-se abundante e especificamente hiperexpresso em gliomas de alto grau, em particular, GBM. O objetivo do presente estudo é analisar a expressão, amplificação, e deleção de EGFR em astrocitomas, bem como a coexpressão entre esse gene e o da IL-13R 2. Foram analisadas 145 astrocitomas (22 astrocitomas pilocíticos (AP); 22 astrocitomas grau II (AGII); 17 astrocitomas anaplásico (AA); e 84 GBM) e 17 tecidos cerebrais não tumorais provenientes de cirurgia de epilepsia. A deleção EGFRvIII foi analisada por RT-PCR, e confirmada por PCR em tempo real (RQ-PCR). A expressão relativa de EGFR e IL-13R 2 foi estudada por RQ-PCR utilizando-se o método SYBR Green, comparado ao tecido não tumoral, e normalizado para os genes de referência endógena, HPRT e Gus- . A amplificação de EGFR foi também determinada por RQ-PCR com relação ao gene da beta-hemoglobina, descrito como um gene de cópia única. Foi realizada imunohistoquímica para analisar a expressão da proteína EGFR. A deleção EGFRvIII foi somente encontrada em GBM (19/84, 23%), demonstrando a exclusividade dessa alteração num grau tumoral de maior malignidade e uma diminuição da sobrevida desses pacientes (p = 0,030). A hiperexpressão de EGFR foi encontrada em 88 casos (61%), correspondendo a 50% de GBM, 88% de AA, e interessantemente em 77% de AGII e 64% de AP. Da mesma maneira, a hiperexpressão de IL-13R 2 foi encontrada em 62 casos (43%), correspondendo a 48% de GBM, 29% de AA, 18% de AGII e, surpreendentemente em 59% de AP. Embora tenha havido um aumento de expressão de ambos os genes em todos os graus de astrocitomas, não houve coexpressão dos mesmos. A amplificação de EGFR foi observada em 29 casos (20%) correspondendo a 31% de GBM e ainda um caso para cada um dos demais graus de astrocitomas, sendo que dos 29 casos amplificados, 21 pacientes eram mais velhos que 45 anos (p < 0,001) e 50% dos casos de GBM com amplificação de EGFR, apresentaram simultaneamente EGFRvIII. Adicionalmente, o acúmulo citoplasmático da proteína foi detectado em 74 casos (51%), correspondendo a 55% de GBM, 47% de AA, 54,5% de AGII e 37% de AP, além de um acúmulo nuclear detectado em 15% dos casos de astrocitomas difusamente infiltrativos. Assim, a alta freqüência de hiperexpressão dos genes estudados em todos os graus de astrocitomas, principalmente a amplificação de EGFR e a presença da deleção EGFRvIII foram observados entre os astrocitomas de alto grau, especialmente em GBM. Os resultados apresentados contribuem para um melhor direcionamento no futuro em métodos terapêuticos específicos, salientando a importância da análise de expressão molecular, protéica e das alterações mutacionais que envolvem genes candidatos, em particular, EGFR e IL-13R 2 / Epidermal Growth Factor Receptor, EGFR is a transmembrane protein consisting of an extracellular EGF-binding domain and an intracellular domain with ligand-activated tyrosine kinase activity. In ~40% of primary GBM is observed amplification leading to overexpression of EGFR, but rarely in secondary GBM. Over the half of primary GBM with EGFR amplification is associated to gene rearrangement, a deleted form of EGFR (EGFRvIII). Additionally, the interleukin-13 alpha 2 receptor (IL13R 2) is abundant and specifically overexpressed in high-grade gliomas, particularly in GBM. The aim of the present study is to analyze the EGFR expression, amplification, and deletion in astrocytomas as well as its coexpression with IL13R 2. We have analyzed 145 surgical astrocytoma samples (22 pilocytic astrocytomas (PA); 22 low-grade astrocytomas (LGA); 17 anaplastic astrocytomas (AA); and 84 GBM) and 17 non-neoplastic brain tissue from epilepsy surgery. EGFRvIII deletion was analyzed by RT-PCR, and also confirmed by real time PCR (RQ-PCR). The relative EGFR and IL-13R 2 expression was studied by RQ-PCR using SYBR Green method, compared to non-neoplastic tissue, normalized for HPRT and Gus- genes. The EGFR amplification was also determined by RQ-PCR relative to the hemoglobin beta gene, described as a single copy gene. Immunohistochemistry was performed to analyze the protein expression in tumor samples. The EGFRvIII deletion was found only in GBM cases (19/84, 23%) demonstrating the exclusivity of this alteration in higher tumor grade and survival was decreased in these patients (p = 0.030). EGFR overexpression was found in 88 cases (61%), corresponding to 50% of GBM, 88% of AA, and interestingly in 77% of LGA and 64% of PA. In the same way, the overexpression of IL-13R 2 was found in 62 cases (43%), corresponding to 48% of GBM, 29% of AA, 18% of LGA and, surprising in 59% of PA. Although increased expression of both genes was demonstrated in all astrocytoma grades, it was no coexpression of the genes. The amplification was observed in 29 cases (20%) corresponding to 31% of GBM and only one case each of PA, LGA and AA. Among 29 cases with EGFR amplification, 21 patients were older than 45 years (p < 0.001) and 50% of GBM with EGFR amplification presented simultaneously the EGFRvIII. Moreover, the EGFR cytoplasmic accumulation was detected in 74 cases (51%), corresponding to 55% of GBM, 47% of AA, 54.5% of LGA and 37% of PA, and nuclear accumulation was detected in 15% of diffusely infiltrative astrocytomas. Thus, the high overexpression frequency of the genes studied in all grades of astrocytomas, mainly the EGFR amplification and presence of EGFRvIII deletion were observed among high-grade astrocytomas, mainly in GBM. The present results contribute to better tailoring specific future therapeutical approach in patients with astrocytomas, pointing out the importance of the molecular, protein expressions and mutational analyses of candidates genes, in particular, EGFR and IL-13R 2

A amplificação gênica mais freqüente em câncer de mama é a do oncogene ERBB-2, observada em aproximadamente 30% dos tumores de mama e que está relacionada com menor intervalo livre de doença e sobrevida total das pacientes com câncer de mama. O ERBB-2 ativa importantes vias de sinalização celular, incluindo as vias MAPK e PI3K. Utilizando PCR em tempo real analisou-se o efeito do EGF e da HRG na regulação da expressão dos genes ANP32B, MATR3, ATAD4, NDRG1, ACTN1, SPARC, TPM1 e CENPH, nas células HB4a, C5.2 e SKBr3, que expressam diferentes níveis de ERBB2. Avaliou-se também o perfil de expressão destes transcritos após a supressão do ERBB2 pela técnica de siRNA nas células C5.2. O tratamento com EGF modulou de forma diferente a expressão dos genes estudados nas células HB4a, C5.2 e SKBr3. Nas células HB4a e SKBr3 a HRG também regulou a expressão dos genes acima. Após a transfecção das células C5.2 com siERBB2 houve alteração na expressão dos genes ATAD4, NDRG1, ACTN1, SPARC, MATR3, CENPH e TPM1. / The more frequent genic amplification observed in breast cancer is that of the ERBB2 oncogene, which occurs in approximately 30% of the breast cancers, and is associated with lower disease-free interval and survival of all patients with breast cancer. The ERBB-2 protein activates important cell signaling pathways such as MAPK and PI3K. Using real time PCR, it was investigated the effect of EGF and HRG on ANP32B, MATR3, ATAD4, NDRG1, ACTN1, SPARC, TPM1 and CENPH transcripts regulation in the HB4a, C5.2 and SKBr3 cell, that express different levels of ERBB2. It was also evaluated the expression profile of these transcripts in the C5.2 cell line after the suppression of ERBB2 expression by the siRNA technique. The treatments with EGF modulate differently the expression of the analysed transcripts in HB4a, C5.2 and SKBr3 cells. In HB4a and SKBr3 cells the treatments with HRG also modulate the expression of the transcripts above. The C5.2 cells transfected with siERBB2 showed alteration in the expression of ATAD4, NDRG1, ACTN1, SPARC, MATR3, CENPH and TPM1 transcripts.

Danos ao sistema nervoso central (SCN) resultam em perda de conexões axonais, das funções motoras e sensoriais. Uma das estratégias para seu reparo é o transplante de células-tronco mesenquimais (CTMs). Porém essa alternativa requer uma adequada via de aplicação. Nesse sentido, o uso de matrizes alinhadas pode ser usado para apoiar o crescimento e diferenciação das CTMs e, quando incorporadas com fatores de crescimento, otimizam o processo de regeneração tecidual. O objetivo desse trabalho foi avaliar a diferenciação neural das CTMs cultivadas sobre matrizes de nanofibras orientadas com o fator de crescimento epidermal (EGF) incorporado. Os scaffolds com fibras alinhadas foram produzidos por electrospinning de emulsão e avaliados conforme a sua morfologia, o diâmetro das nanofibras, a degradabilidade e a liberação do EGF. As CTMs utilizadas foram provenientes da polpa de dentes decíduos esfoliados humanos. Essas células foram cultivadas nos scaffolds e avaliadas conforme os testes biológicos: adesão, viabilidade, proliferação, citotoxicidade e diferenciação neural. Os scaffolds com fibras alinhadas controle (AC) e contendo o EGF (AE) apresentaram morfologia, diâmetro das nanofibras e tempo de degradação semelhantes. Com base no total de EGF presente na matriz AE, 90,14% foi liberado após 28 dias. O citoesqueleto e o núcleo das CTMs cultivadas nos scaffolds AC e AE estavam mais alongados e alinhados quando comparado com as CTMs cultivadas no poço de cultura (controle). As CTMs aderiram mais nas matrizes AE em relação às matrizes AC, porém a proliferação e viabilidade celular foram similares, exceto no tempo de 72 horas, o qual a viabilidade no grupo controle foi maior, em comparação aos demais grupos. Os scaffolds AC e AE não foram tóxicos para as CTMs. Em relação aos resultados da neuro-diferenciação, a expressão de nestina e neurofilamentos consideravelmente maior em todos os grupos analisados quando comparado ao grupo controle. A expressão de βIII-tubulina e GFAP foi maior em todos os grupos diferenciados quando comparada ao grupo controle. A maioria das CTMs cultivadas nas matrizes AC e AE, induzidas ou não à diferenciação neural, apresentaram correntes dependente de voltagem para sódio. O valor de condutância máxima foi maior para todos os grupos analisados quando comparado ao grupo controle onde as células não foram diferenciadas. Portanto, as matrizes com nanofibras orientadas induzem à diferenciação neural das CTMs em neurônios funcionais tanto na ausência como na presença de EGF incorporado. As matrizes AE ainda mostraram ser capazes de melhorar a adesão celular. Dessa forma, conclui-se que as matrizes de nanofibras estudadas são uma possível estratégia para otimização da regeneração de lesões neurológicas. / Damage to the central nervous system (CNS) results in loss of axonal connections and motor and sensory functions. One of the strategies for its repair is the transplantation of mesenchymal stem cells (MSCs). However, this requires a suitable application route. Accordingly, the use of scaffolds support the growth of MSCs and, when incorporated with growth factors, optimize the regeneration process. The purpose of this study was to evaluate the neural differentiation of MSCs cultured on nanofiber matrices oriented with epidermal growth factor (EGF) incorporated. Aligned scaffolds were produced by electrospinning emulsion and evaluated according to their degradation, the morphology and diameter of the nanofibers, and release of EGF from the nanofibers. MSCs used were from human exfoliated deciduous teeth (SHED). These cells were cultured on the scaffolds and evaluated according to biological tests: adhesion, viability, proliferation, cytotoxicity and neural differentiation. The aligned control scaffolds (AC) containing EGF (AE) presented similar morphology, diameter of nanofibers and degradation time. Based on the total EGF present in the scaffold AE, 90.14% was released after 28 days. The cytoskeleton and the core of the MSCs cultured on scaffolds AC and AE were more aligned and elongated when compared to the MSCs grown on plate wells (control). MSCs adhered more to matrices AE when compared to matrices AC, although proliferation and cell viability were similar, except after 72 hours. In this period, the viability of the control group was higher when compared to the rest of the groups. Scaffolds AC and AE were not toxic to MSCs. In regard to the results of neuro-differentiation, the expression of nestin and neurofilament was much higher in all groups than the control group. The expression of βIII tublin and GFAP was higher in all differentiated groups than the control group. Most of the MSCs grown in matrices AC and AE, induced or not to neural differentiation, showed voltage-dependent sodium currents. The maximum value of conductance of these groups was higher for the cells in all groupscompared to the control group, where the cells were not differentiated. Therefore, oriented nanofiber matrices induce neural differentiation of MSCs into functional neurons both in the absence and in the presence of incorporated EGF. The matrices AE also showed improved cell adhesion. Thus, these matrices are a possible strategy for optimizing the regeneration of neurologic lesions.