Proteomic analysis of a neurodegeneration cell model, treated with plant extracts with potential neuroprotective activity

Nowadays, a significant increase in chronic diseases is observed. Epidemiological studies showed a consistent relationship between the consumption of fruits and vegetables and a reduced risk of certain chronic diseases, namely neurodegenerative disorders. One factor common to these diseases is oxidative stress, which is highly related with proteins, lipids, carbohydrates and nucleic acids damage, leading to cellular dysfunction. Polyphenols, highly abundant in berries and associated products, were described as having antioxidant properties, with beneficial effect in these pathologies. The aims of this study were to evaluate by proteomic analyses the effect of oxidative insult in a neuroblastoma cell line (SK-N-MC) and understand the mechanisms involved in the neuroprotective effects of digested extracts from commercial and wild blackberry (R. vagabundus Samp.). The analysis of the total proteome by two-dimensional electrophoresis revealed that oxidative stress in SK-N-MC cells resulted in altered expression of 12 protein spots from a total of 318. Regarding some redox proteomics alterations, particularly proteins carbonylation and glutathionylation, protein carbonyl alterations during stress suggest that cells produce an early and late response; on the other hand, no glutathionylated polypeptides were detected. Relatively to the incubation of SK-N-MC cells with digested berry extracts, commercial blackberry promotes more changes in protein pattern of these cells than R. vagabundus. From 9 statistically different protein spots of cells incubated with commercial blackberry, only β-tubulin and GRP 78 were until now identified by mass spectrometry. Further studies involving the selection of sub proteomes will be necessary to have a better understanding of the mechanisms underlying the neuroprotective effects of berries.

Development of hyaluronic acid, dextrin and extracellular matrix hydrogels for cell expansion

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Engenharia Clínica)

The impact of carbon source in Candida albicans virulence: participation of RLM1 in pathogen host interaction

Dissertação de mestrado em Genética Molecular

Interacciones inmuno-endócrinas: participación de tlr-4 y citoquinas pro-inflamatorias en la regulación de la proliferación de células lactotropas. Immuno-endocrine interactions: Contribution of tlr-4 and pro-inflammatory cytokines in the regulation of lactotroph cell proliferation.

El presente proyecto tiene como objetivo estudiar, a nivel celular y molecular, los mecanismos inmuno-endócrinos que participan en la proliferación de células lactotropas normales y tumorales frente a procesos inflamatorios inducidos experimentalmente. Una particular atención se pondrá al evaluar la contribución de IL-6 como citoquina intrahipofisaria durante el desarrollo tumoral y su rol como señal paracrina/autocrina en la senescencia hipofisaria. Debido a que agentes inflamatorios y anti-inflamatorios pueden inducir alteraciones en el crecimiento y la función hipofisaria, no se descartaría que, en el curso de una inflamación, como la inducida por el lipopolisacárido bacteriano LPS, puedan ocurrir modificaciones en el índice proliferativo de las células lactotropas y/o en la secreción de su producto hormonal, la prolactina. Dado el auge en las investigaciones referidas al campo de la modulación inmuno-endócrina, es que planteamos investigar la participación de TLR4, componente crucial del complejo proteico que inicia la señal LPS, en hipófisis normales y tumorales inducidas por estrógeno así como también en la línea celular somatolactotrópica GH3B6. Dentro de las vías de transducción de señales involucradas se determinará la participación de MAPK-ERK1/2 y de PI3K asi como la contribución de NF-kB en la regulación del crecimiento celular inducido por IL-6/LPS mediante el uso de inhibidores específicos. La microscopía electrónica y confocal, resultarán de fundamental importancia para valorar los procesos de translocación nuclear de NF-kB como así también para definir la localización ultraestructural de los mediadores mencionados. Además, se valorará el mecanismo de senescencia celular hipofisaria mediante parámetros morfológicos, bioquímicos y ultraestructurales durante el desarrollo de prolactinomas inducidos experimentalmente. Finalmente dilucidar las posibles vías de transducción de señales que se desencadenan frente a estímulos inflamatorios/proliferativos podría explicar algunos aspectos moleculares sobre la función de control del ciclo celular y las limitaciones de crecimiento en adenomas hipofisarios que subyacen en la falta de progresión de estos tumores a la malignidad. The aim of the present project is to study the immuno-endocrine mechanisms involved in the proliferation of normal and tumoral lactotrophs experimentally induced by inflammatory factors. Also, the contribution of IL-6 as a paracrine / autocrine signal in the pituitary senescence will be assessed along tumor development induced by estrogen treatment. Considering that both, inflammatory and anti-inflammatory agents can modify the pituitary function, it is possible that in the course of inflammation, as induced by bacterial lipopolysaccharide LPS, some alteration may occur in the proliferative index of lactotrophs and / or in the PRL secretion. Our main objective is to investigate the cellular and molecular mechanisms involved by the activation of TLR4, a crucial component of the protein complex initiated by LPS, in normal and pathological pituitaries induced by estrogen as well as in the GH3B6 cell line. The participation of MAPK-ERK1 / 2 and PI3K signaling pathway and the contribution of NF-kB in the proliferative responses triggered by IL-6/LPS will be analyzed by using specific inhibitors. Confocal microscopy analysis is essential to assess the process of nuclear translocation of NF-kB as well as the use of electron microscopy to define the ultrastructural localization of the above mentioned mediators. In addition, the mechanisms of pituitary cell senescence will be evaluated through morphological, biochemical and ultrastructural approaches during the development of experimental prolactinomas. Finally, the elucidation of possible signal transduction pathways which are triggered by inflammatory / proliferative stimuli, would explain some molecular aspects of cell cycle control and limitations in pituitary tumor growth that underlie the lack of progress in these pituitary tumors to malignancy.

Insulin secretion is regulated by the glucose-dependent production of islet beta cell macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF), originally identified as a cytokine secreted by T lymphocytes, was found recently to be both a pituitary hormone and a mediator released by immune cells in response to glucocorticoid stimulation. We report here that the insulin-secreting beta cell of the islets of Langerhans expresses MIF and that its production is regulated by glucose in a time- and concentration-dependent manner. MIF and insulin colocalize by immunocytochemistry within the secretory granules of the pancreatic islet beta cells, and once released, MIF appears to regulate insulin release in an autocrine fashion. In perifusion studies performed with isolated rat islets, immunoneutralization of MIF reduced the first and second phase of the glucose-induced insulin secretion response by 39% and 31%, respectively. Conversely, exogenously added recombinant MIF was found to potentiate insulin release. Constitutive expression of MIF antisense RNA in the insulin-secreting INS-1 cell line inhibited MIF protein synthesis and decreased significantly glucose-induced insulin release. MIF is therefore a glucose-dependent, islet cell product that regulates insulin secretion in a positive manner and may play an important role in carbohydrate metabolism.

CD8 kinetically promotes ligand binding to the T-cell antigen receptor.

The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation.

Asthma is a chronic inflammatory disease of the airways that involves many cell types, amongst which mast cells are known to be important. Adenosine, a potent bronchoconstricting agent, exerts its ability to modulate adenosine receptors of mast cells thereby potentiating derived mediator release, histamine being one of the first mediators to be released. The heterogeneity of sources of mast cells and the lack of highly potent ligands selective for the different adenosine receptor subtypes have been important hurdles in this area of research. In the present study we describe compound C0036E08, a novel ligand that has high affinity (pK(i) 8.46) for adenosine A(2B) receptors, being 9 times, 1412 times and 3090 times more selective for A(2B) receptors than for A(1), A(2A) and A(3) receptors, respectively. Compound C0036E08 showed antagonist activity at recombinant and native adenosine receptors, and it was able to fully block NECA-induced histamine release in freshly isolated mast cells from human bronchoalveolar fluid. C0036E08 has been shown to be a valuable tool for the identification of adenosine A(2B) receptors as the adenosine receptors responsible for the NECA-induced response in human mast cells. Considering the increasing interest of A(2B) receptors as a therapeutic target in asthma, this chemical tool might provide a base for the development of new anti-asthmatic drugs.

Drosophila Myc interacts with host cell factor (dHCF) to activate transcription and control growth.

The Myc proto-oncoproteins are transcription factors that recognize numerous target genes through hexameric DNA sequences called E-boxes. The mechanism by which they then activate the expression of these targets is still under debate. Here, we use an RNAi screen in Drosophila S2 cells to identify Drosophila host cell factor (dHCF) as a novel co-factor for Myc that is functionally required for the activation of a Myc-dependent reporter construct. dHCF is also essential for the full activation of endogenous Myc target genes in S2 cells, and for the ability of Myc to promote growth in vivo. Myc and dHCF physically interact, and they colocalize on common target genes. Furthermore, down-regulation of dHCF-associated histone acetyltransferase and histone methyltransferase complexes in vivo interferes with the Myc biological activities. We therefore propose that dHCF recruits such chromatin-modifying complexes and thereby contributes to the expression of Myc targets and hence to the execution of Myc biological activities.

Targeting mTORC2 inhibits colon cancer cell proliferation in vitro and tumor formation in vivo.

The mammalian target of rapamycin (mTOR), which exists in two functionally distinct complexes, mTORC1 and mTORC2 plays an important role in tumor growth. Whereas the role of mTORC1 has been well characterized in this process, little is known about the functions of mTORC2 in cancer progression. In this study, we explored the specific role of mTORC2 in colon cancer using a short hairpin RNA expression system to silence the mTORC2-associated protein rictor. We found that downregulation of rictor in HT29 and LS174T colon cancer cells significantly reduced cell proliferation. Knockdown of rictor also resulted in a G1 arrest as observed by cell cycle analysis. We further observed that LS174T cells deficient for rictor failed to form tumors in a nude mice xenograft model. Taken together, these results show that the inhibition of mTORC2 reduces colon cancer cell proliferation in vitro and tumor xenograft formation in vivo. They also suggest that specifically targeting mTORC2 may provide a novel treatment strategy for colorectal cancer.

The beta1 and beta3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation.

Recognition by CD8+ cytotoxic T lymphocytes (CTLs) of antigenic peptides bound to major histocompatibility class (MHC) I molecules on target cells leads to sustained calcium mobilization and CTL degranulation resulting in perforin-dependent killing. We report that beta1 and beta3 integrin-mediated adhesion to extracellular matrix proteins on target cells and/or surfaces dramatically promotes CTL degranulation. CTLs, when adhered to fibronectin but not CTL in suspension, efficiently degranulate upon exposure to soluble MHC.peptide complexes, even monomeric ones. This adhesion induces recruitment and activation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the Src kinases Lck and Fyn in the contact site. The T cell receptor, by association with Pyk2, becomes part of this adhesion-induced activation cluster, which greatly increases its signaling.

Improved enzyme-linked immunoadsorbent assay (ELISA) for the study of Trypanosoma cruzi-host cell interaction in vitro

We herein present an improved assay for detecting the presence of Trypanosoma cruzi in infected cultures. Using chagasic human sera (CHS), we were able to detect T. cruzi infection in primary cultures of both peritoneal macrophages and heart muscle cells (MHC). To avoid elevated background levels - hitherto observed in all experiments especially in those using HMC - CHS were preincubated with uninfected cells in monolayers or suspensions prior to being used for detection of T. cruzi in infected monolayers. Preincubation with cell suspensions gave better results than with monolayers, reducing background by up to three times and increasing sensitivity by to twenty times. In addition, the continous fibroplastic cell line L929 was shown to be suitable for preadsorption of CHS. These results indicate that the high background levels observed in previous reports may be due to the presence of human autoantibodies that recognize surface and/or extracellular matrix components in cell monolayers. We therefore propose a modified procedure that increases the performance of the ELISA method, making it an useful tool even in cultures that would otherwise be expected to present low levels of infection or high levels of background

Disease-causing mutations improving the branch site and polypyrimidine tract: pseudoexon activation of LINE-2 and antisense Alu lacking the poly(T)-tail.

Cryptic exons or pseudoexons are typically activated by point mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements. Here we describe two cases of tetrahydrobiopterin deficiency caused by mutations improving the branch point sequence and polypyrimidine tracts of repeat-containing pseudoexons in the PTS gene. In the first case, we demonstrate a novel pathway of antisense Alu exonization, resulting from an intronic deletion that removed the poly(T)-tail of antisense AluSq. The deletion brought a favorable branch point sequence within proximity of the pseudoexon 3' splice site and removed an upstream AG dinucleotide required for the 3' splice site repression on normal alleles. New Alu exons can thus arise in the absence of poly(T)-tails that facilitated inclusion of most transposed elements in mRNAs by serving as polypyrimidine tracts, highlighting extraordinary flexibility of Alu repeats in shaping intron-exon structure. In the other case, a PTS pseudoexon was activated by an A>T substitution 9 nt upstream of its 3' splice site in a LINE-2 sequence, providing the first example of a disease-causing exonization of the most ancient interspersed repeat. These observations expand the spectrum of mutational mechanisms that introduce repetitive sequences in mature transcripts and illustrate the importance of intronic mutations in alternative splicing and phenotypic variability of hereditary disorders.

TAT-RasGAP317-326-mediated tumor cell death sensitization can occur independently of Bax and Bak.

The increase of cancer specificity and efficacy of anti-tumoral agents are prime strategies to overcome the deleterious side effects associated with anti-cancer treatments. We described earlier a cell-permeable protease-resistant peptide derived from the p120 RasGAP protein, called TAT-RasGAP317-326, as being an efficient tumor-specific sensitizer to apoptosis induced by genotoxins in vitro and in vivo. Bcl-2 family members regulate the intrinsic apoptotic response and as such could be targeted by TAT-RasGAP317-326. Our results indicate that the RasGAP-derived peptide increases cisplatin-induced Bax activation. We found no evidence, using in particular knock-out cells, of an involvement of other Bcl-2 family proteins in the tumor-specific sensitization activity of TAT-RasGAP317-326. The absence of Bax and Bak in mouse embryonic fibroblasts rendered them resistant to cisplatin-induced apoptosis and consequently to the sensitizing action of the RasGAP-derived peptide. Surprisingly, in the HCT116 colon carcinoma cell line, the absence of Bax and Bak did not prevent cisplatin-induced apoptosis and the ability of TAT-RasGAP317-326 to augment this response. Our study also revealed that p53, while required for an efficient genotoxin-induced apoptotic response, is dispensable for the ability of the RasGAP-derived peptide to improve the capacity of genotoxins to decrease long-term survival of cancer cells. Hence, even though genotoxin-induced Bax activity can be increased by TAT-RasGAP317-326, the sensitizing activity of the RasGAP-derived peptide can operate in the absence of a functional mitochondrial intrinsic death pathway.