The RAS-mitogen-activated protein kinase (MAPK) pathway is an essential signaling mechanism that regulates cellular processes and culminates in the activation of specific gene expression programs. Alterations in the RAS-MAPK signaling cascade can modify epigenetic programs and confer advantages in cell growth and transformation. In fact, deregulation of the cascade is a key event in tumour development with 30% of human cancers harbouring RAS mutations. In breast and pancreatic epithelial cancers, characterization of an aberrant RAS-MAPK pathway has focused on upstream mediators such as receptors and oncogenic RAS molecules but the impact of downstream targets remain poorly defined. Stimulation of the RAS-RAF-MEK-MAPK pathway leads to activation of mitogen- and stress-activated protein kinases 1 and 2 (MSK1/2) which are responsible for the phosphorylation of histone H3 on S10 and S28. We postulate that deregulation of the RAS-MAPK pathway produced by constitutive activation and/ or over-expression of upstream components or mitogen stimulation consequently leads to enhanced MSK1 activity and elevated histone H3 phosphorylation levels. We further hypothesize that MSK1-mediated H3 phosphorylation is critical for immediate early gene (IEG) expression, Ras-driven transformation and is associated with regulatory regions upon gene transcription. In mouse fibroblasts, we present evidence for the critical involvement of MSK1 and H3 phosphorylation as mediators that bridge the aberrant signals driven by the RAS-MAPK pathway with nucleosomal modifications, chromatin remodeling, IEG expression and malignant transformation. We then examined if activation of RAS-MAPK signaling in breast cancer cells elicits similar molecular events. We demonstrate that the RAS-MAPK pathway is induced and enhances the association of MSK1 and H3 phosphorylation on the IEG Trefoil Factor 1 resulting in transcriptional activation. We further observed that mutated K-RAS expression did not correlate with genomic instability or altered signaling in pancreatic cancer cell lines while overexpressed HER2 and EGFR breast cancer cell lines generally exhibit upregulated ERK1/2 and H3 phosphorylation levels. Taken together, our studies contribute to the further understanding of MSK-mediated transcriptional activation in response to RAS-MAPK signaling in oncogene-transformed and cancer cell lines. Inhibition of MSK activity may be an unexplored avenue for combination cancer therapy with abnormal RAS-MAPK signaling pathways.

Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy. Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging. The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors.

Στα έντομα, η κυτταροφαγία, διεργασία ανάλογη με αυτή των θηλαστικών, είναι μια σημαντική ανοσολογική απόκριση στην εισβολή παθογόνων και ρυθμίζεται από μια σειρά σηματοδοτικών μορίων. Στη μύγα της Μεσογείου έχει βρεθεί ότι συμμετέχουν οι κινάσες FAK, Src, MAPK και ο μεταγραφικός παράγοντας Elk-1. Στην εργασία αυτή, δείχτηκε με ανοσοκατακρήμνιση και συνεστιακή μικροσκοπία ότι η φωσφορυλιωμένη μορφή του Elk-1 συμπλοκοποιείται και συνεντοπίζεται μόνο με τη φωσφορυλιωμένη μορφή της FAK στη θέση Tyr925. Με ανάλογα πειράματα διαπιστώθηκε ότι οι κινάσες JNK και p38 συμπλοκοποιούνται και συγκατακρημνίζονται με την FAK, χωρίς όμως να συνεντοπίζονται. Με τον ίδιο τρόπο διαπιστώθηκε ότι η JNK και η p38 συμπλοκοποιούνται με την ERK1/2, χωρίς όμως να συνεντοπίζονται. Προφανώς τα μόρια αυτά δεν βρίσκονται σε άμεση επαφή μεταξύ τους και συμπλοκοποιούνται είτε μέσω της FAK, είτε μέσω τρίτων μορίων και όλα μαζί προσδένονται στην FAK. Τα φαγοκύτταρα των θηλαστικών, μακροφάγα και ουδετερόφιλα, καθώς και τα αιμοκύτταρα των εντόμων, παράγουν δραστικές μορφές οξυγόνου κατά την κυτταροφαγία, οι οποίες δρουν ως δεύτερα μηνύματα. Με κυτταρομετρία ροής διαπιστώθηκε ότι η E. coli επάγει τη σύνθεση Η2Ο2 από τα αιμοκύτταρα της μύγας της Μεσογείου. Ο ρυθμιστικός ρόλος του Η2Ο2 επιβεβαιώθηκε με τη χρήση αναστολέων των ενζύμων παραγωγής Ο2- και Η2Ο2, όπου παρατηρήθηκε αύξηση της κυτταροφαγίας. Με πειράματα ανοσοκατακρήμνισης, ανοσοαποτύπωσης και ανοσοφθορισμού φάνηκε ότι το ένζυμο σύνθεσης του Η2Ο2, η δεσμουτάση του υπεροξεικού ανιόντος (SOD), υπάρχει στο κυτταρόπλασμα και στην πλασματική μεμβράνη. Η αποσιώπηση της SOD με siRNA, αύξησε την κυτταροφαγία. Με ανοσοαποτύπωση διαπιστώθηκε ότι η αναστολή της παραγωγής του Η2Ο2 αύξησε τη φωσφορυλίωση της ERK1/2. Τέλος, με συνεστιακή μικροσκοπία φάνηκε ότι η SOD δεν συνεντοπίζεται με τη β υπομονάδα των ιντεγκρινών. Φαίνεται λοιπόν το Η2Ο2 να εμποδίζει την κυτταροφαγία μέσω ελέγχου της φωσφορυλίωσης της ERK1/2. / Phagocytosis is an important innate immune response against pathogen, with similar mechanisms in insects and mammals and is regulated by many different signalling molecules. In medfly Ceratitis capitata, focal adhesion kinase (FAK), Src, MAP kinases and Elk-1 transcription factor regulate this process. Co-immunoprecipitation and confocal microscopy analysis showed that pTyr925FAK associates and co-localizes with pElk-1, during phagocytosis of E. coli and S. aureus, by medfly haemocytes. Moreover, the physical association between JNK and p38 MAP kinases with FAK, was confirmed by immunoprecipitation, but confocal analysis revealed no co-localisation. Similar experiments for JNK and p38 with ERK1/2, revealed an association between JNK, p38 and ERK1/2 and no co-localisation. Apparently, these molecules do not interact directly but they appear to associate with each other indirectly, via FAK molecule or a third molecule and thus all together associate with FAK. Hydrogen peroxide (Η2Ο2) participates as a second messenger in cell signalling in either macrophages and polymorphonuclear cells or insect haemocytes. In this work, the role of Η2Ο2 was investigated, in E. coli phagocytosis by the medfly haemocytes. Block of H2O2 synthesis by specific enzymic inhibitors, namely N-ethylmaleimide (ΝΕΜ) for NADPH oxidase and diethyldithiocarbamate (DDC) for SOD, resulted in the increase of E. coli phagocytosis. Immunoblot analysis, flow cytometry and confocal microscopy, revealed the constitutive expression of SOD, in the medfly haemocytes. Phagocytosis increased by small interfering RNA (siRNA) for SOD, revealing the active involvement of SOD and Η2Ο2. Immunoblot analysis showed an increase of the ERK1/2 phosphorylation, in the presence of the above H2O2 synthesis enzymic inhibitors. In addition, confocal microscopy showed no co-localization of SOD with β integrin subunit. It appears that SOD participates in the regulation of bacterial phagocytosis, due to involvement of the produced Η2Ο2 in the differential phosphorylation of MAP kinases.

Submitted by RACHEL SANTINI PAULINO null (rachel_santini@hotmail.com) on 2018-04-04T13:58:29Z No. of bitstreams: 1 Dissertação Mestrado 2.pdf: 1550212 bytes, checksum: 709a7ceb2f2c96479c741d61c71f0143 (MD5) / Approved for entry into archive by Alexandra Maria Donadon Lusser Segali null (alexmar@fcav.unesp.br) on 2018-04-04T14:13:00Z (GMT) No. of bitstreams: 1 paulino_rs_me_jabo.pdf: 1550212 bytes, checksum: 709a7ceb2f2c96479c741d61c71f0143 (MD5) / Made available in DSpace on 2018-04-04T14:13:00Z (GMT). No. of bitstreams: 1 paulino_rs_me_jabo.pdf: 1550212 bytes, checksum: 709a7ceb2f2c96479c741d61c71f0143 (MD5) Previous issue date: 2018-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O estresse oxidativo causado pela produção de espécies reativas de oxigênio (EROs) é uma das respostas que as plantas apresentam frente a um estresse ambiental. Nosso objetivo foi avaliar o estresse oxidativo das macrófitas aquáticas Crinum americanum e Spartina alterniflora em duas condições: salinidade e nutrientes que ocorrem no estuário do rio Itanhaém (SP). Raízes e folhas (5 plantas) de C. americanum foram coletadas em cada área (5 amostras/área). O material vegetal para análises de clorofila (a+b), carotenóides, peróxido de hidrogênio (H2O2), malonaldeído (MDA) e enzimas antioxidantes SOD e CAT foram acondicionado em N2 líquido no campo e posteriormente armazenado em freezer a -80 °C. Também foram coletados em cada réplica material vegetal para avaliar o teor de N e P totais da planta e do sedimento Os dados obtidos foram submetidos à Análise de Variância (ANOVA) e comparados pelo teste de Tukey (p < 0,05). Os resultados obtidos mostraram que: C. americanum e S. alterniflora sofreram maior estresse oxidativo em alto e médio estuário, respectivamente; Os indivíduos de S. alterniflora sofreram maior estresse na área de lançamento de esgoto. / The oxidative stress caused by the production of reactive oxygen species (ROS) is one of the responses that plants exhibit facing environmental stress. Our objective was to evaluate the oxidative stress of the aquatic macrophytes Crinum americanum and Spartina alterniflora under two conditions: salinity and nutrients that occur in the estuary of the Itanhaém river (SP). Roots and leaves (5 plants) of C. americanum were collected in each area (5 samples / area). The plant material for analysis of chlorophyll (a + b), carotenoids, H2O2, MDA and antioxidant enzymes SOD and CAT with platform in N2 without field and liquid in freezer at -80 ° C. Vegetable material was also collected in each replicate to evaluate the total N and P content of the plant and of the sediment. The data were submitted to Analysis of Variance (ANOVA) and compared by the Tukey test (p <0.05). The results showed that: C. americanum and S. alterniflora suffered higher oxidative stress in upper and middle estuary, respectively; The State of São Paulo changes have suffered greater stress in the area of sewage / 2018SLR05032

Nano-sized materials are being used to address some of humanities greatest challenges— cancer therapy, food and water security, and environmental remediation. While extremely promising for these applications, the production, use, and disposal of nanomaterials have resulted in their release into environmental compartments. One major concern of any novel contaminant is how it interacts with bacteria. Bacteria play essential roles in human health, engineered systems, and ecological functioning. Bacteria are capable of macro-scale influence because they have evolved communication systems that enable coordinated behaviors. Communication among cells involves chemical signals that enter the environment, where they are subjected to its biogeochemistry, which now includes novel nanomaterials. The overall goal of this thesis was to improve understanding of the relationship between nanoparticles and cell-to-cell signaling behavior in bacteria focusing on two population-level behaviors: autolysis and quorum sensing. Specifically, this project sought to: (1) improve our understanding of how metal-oxide nanoparticles affect the autolytic process in Bacillus subtilis, by elucidating the biological response of the interactions between titanium dioxide nanoparticles and biomolecules; (2) reveal the interactions between quorum sensing signaling molecules and metal cations commonly used in antimicrobial nanomaterials, silver and copper; and (3) demonstrate the potential of quorum sensing-regulated cyanide production to affect oxidation and dissolution of gold nanoparticles in an environmentally relevant system. By addressing these objectives, the work demonstrated that: 1. TiO2 nanoparticles disrupt the autolytic process by delaying the onset of autolysis, and intercepting released autolytic enzymes, preventing the enzymes from degrading peptidoglycan in neighboring cells. 2. Quorum sensing signaling molecules form complexes with Ag+ and Cu2+, removing the most bioavailable form (free HHL, Ag+, and Cu2+) from the cells’ environment. 3. Quorum sensing-regulated cyanide production induces oxidative dissolution in Au nanoparticles, which were previously assumed to be inert in environmental systems. Taken together, this body of work highlights the relationship between nanoparticles and population-level behavior in bacteria. The presence of nanoparticles can have significant effects on population-level behaviors, and the activity of population-level behaviors can have significant effects on nanoparticles behavior. This inter-connected relationship, where the nanoparticles are both acted on and act upon their environment, must be considered in nanoparticle-based studies and applications.

Calcium is a ubiquitous intracellular signal responsible for controlling numerous cellular responses including development and proliferation. Calcium (Ca²⁺) is stored in both neutral and acidic stores and its release through gated channels has been implicated in regulating development in Dictyostelium discoideum. This thesis aims to understand the roles of the calcium channel proteins, in particular the two-pore channel proteins (TPCs), found on acidic stores in Ca²⁺ signalling during Dictyostelium development. Bioinformatic analysis indicates conservation of a gene encoding an orthologue of TPC2 in Dictyostelia and, similar to plant TPCs, a Ca²⁺ sensing domain is predicted along with a novel potential calmodulin binding site. To investigate the role of intracellular Ca²⁺ channels, a series of strains was generated, disrupted in one or more of genes encoding the channels TPC2 and mucolipin (TRP-ML), predicted to be located on acidic stores, and IplA, located on neutral stores. All disrupted strains, including one lacking all three channels, are able to complete development. However, strains lacking TPC2 show a pronounced delay in early development, correlating with reduced expression of some early developmental genes. Vesicles derived from tpc2-null cells show normal Ca²⁺ release compared to parental cells but an increased rate of Ca²⁺ uptake. During early development, the pH of acidic vesicles is increased in the absence of TPC2. However development of tpc2-null cells showed increased sensitivity to weak bases in producing fewer aggregates but resistance to sodium chloride and weak bases in later development suggesting a complex role for TPC during development. In vivo cytosolic Ca²⁺ responses were analysed in strains expressing an ultra-sensitive Ca²⁺ indicator YC-Nano 15. Growing tpc2^- and iplA^- cells have lower basal cytosolic Ca²⁺ than parental Ax2 cells. Intercellular Ca²⁺ waves were observed in aggregates from Ax2, mcln^- and tpc2^- cells but were greatly reduced in iplA- aggregates, as was the increase in cytosolic Ca²⁺ in response to extracellular cAMP. In tpc2- aggregates, wave frequencies were reduced and the response to cAMP addition abolished after treatment with caffeine, a known adenylyl cyclase inhibitor in Dictyostelium. This work demonstrates that TPC2 plays a role in the early stages of Dictyostelium development. TPC2 is important for pH regulation in acidic vesicles and cytosolic Ca²⁺ levels, either or both of which could influence development either directly or via changes in early developmental gene expression.

In the central nervous system (CNS), oligodendrocytes (OLs) generate myelin to facilitate the rapid propagation of neuronal impulses. In multiple sclerosis (MS), chronic demyelination leads to irreversible neurodegeneration that eventually impairs physical and cognitive function. Much effort is directed at elucidating the mechanisms underlying OL development in hope to unveil therapeutic targets for promoting remyelination in MS. Many aspects of OL biology are regulated by the integrins, a large family of transmembrane extracellular matrix (ECM) receptors. ECM components such as laminin and fibronectin bind to OL integrin receptors and initiate downstream signaling cascades involved in survival, proliferation, differentiation/myelination and migration. Integrin-linked kinase (ILK), an adaptor protein that binds to integrin cytosolic tails, works to stabilize the ECM-integrin connection by indirectly targeting the actin cytoskeleton to ECM adhesion sites. We hypothesized that ILK played an important role in OL migration, differentiation and capacity to myelinate neuronal projections. To address this hypothesis, we developed three cell culture techniques to assess these cellular phenomena in vitro. Conditional knockout of Ilk compromised both the morphological and molecular differentiation of primary mouse OLs in vitro, and reduced their capacity to produce myelin-like membrane. ILK was required for proper OL ensheathment of neuronal extensions when co-cultured with primary neurons. Conditional ablation of Ilk in vivo produced a transient amyelination defect that was endogenously compensated for at later time points. Loss of ILK in primary OLs was associated with upregulated RhoA signaling, and pharmacological inhibition of the RhoA axis restored the morphology of a distinct subset of NG2+ OPCs. ILK depletion in OL precursor cells (OPCs) resulted in a substrate-dependent defect in migration velocity and migration initiation. Inhibition of the RhoA signaling pathway enhanced the migratory velocity of wild-type OPCs, an effect that was dependent on ILK expression. In sum, we established three primary mouse OL cell culture techniques, with which we defined roles for ILK in OL biology. Our work highlights the importance of integrin signaling in OLs and provides new experimental methods useful in MS research.

Many consumer and medical products contain engineered nanomaterials (ENMs) due to their unique properties arising from their small size of <100 nm in at least one dimension. Although ENMs could greatly improve the quality of daily life, concerns for their health and environmental safety emerged in recent years because the same properties that make ENMs beneficial may also render them toxic. The small size allows ENMs’ entrance into the cell where they may attach to biological molecules and membranes, disrupting their function and/or leading to oxidative stress and/or damage. This thesis focused on silver nanoparticles (AgNPs). Several articles demonstrated that during washing AgNPs are released from the AgNP-impregnated fabrics and could pose a risk to aquatic species. Given that the toxicity mechanisms of AgNPs are yet to be clearly understood this thesis investigated the effects of AgNPs from ‘oxidative stress’ and ‘endocrine disruption’ points of view, using both in vivo and in vitro model fish systems. A 4 d exposure of zebrafish (Danio rerio) embryos to AgNPs increased mortality, delayed hatching, and increased oxidative stress. The silver ion (Ag+) was more effective in eliciting these effects at equivalent silver concentrations. Moreover, the Ag-chelator cysteine reduced the toxicity of both Ag-types. Despite these effects AgNPs or Ag+ did not affect the ability of zebrafish larvae or adults (raised to adulthood in Ag-free water) to increase cortisol levels, but there were differential effects on the expression of corticotropin-releasing factor (CRF)-related genes, suggesting that other physiological processes regulated by CRF may be impacted. Furthermore, a 48 h exposure of rainbow trout (Oncorhynchus mykiss) erythrocytes and hepatocytes to AgNPs or Ag+ increased oxidative stress, but Ag+ was more potent. Moreover, AgNPs elevated lipid peroxidation, while Ag+ increased DNA damage, suggesting different modes of action for the two Ag-types. Cysteine treatment reduced the toxicity of Ag+ and AgNPs, while buthionine sulfoximine, which inhibits glutathione synthesis, increased it, suggesting the importance of glutathione in silver toxicity. Finally, AgNPs increased glycogenolysis in trout hepatocytes independently of the beta-adrenoreceptor or the glucocorticoid receptor.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear. Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative in vitro model is critical to elucidate the signaling pathways orchestrating CSF production in the CP. This research aims to characterize an in vitro culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.

No description available.