Obesity is an adverse factor in the development and severity of breast cancer. Obesity is often accompanied by an increase in circulating insulin, which is also associated with poor BC prognosis. Although not expressed in normal breast tissue, the insulin receptor (IR) is highly expressed in BC, therefore insulin signaling in BC cells may be responsible for the negative prognostic effects associated with hyperinsulinemia. This thesis describes the development of a cell-based system to study how insulin affects BC. My work shows that MCF10A, untransformed human breast epithelial cells that express the IR, require insulin for normal proliferation and morphology. Interestingly, I discovered hyperactivation of ERK1/2 in MCF10A cells in response to insulin withdrawal, resulting in a loss of epithelial phenotype. Unexpectedly, while losing epithelial phenotype, MCF10A cells depleted of insulin failed to migrate. In conclusion, breast cells that express IR require insulin for migration and maintenance of epithelial characteristics.

A yeast-two hybrid screen with Lyn identified a novel 130 kDa multidomain protein with a 36% identity to Actin Filament Associated Protein (AFAP) 110 and similar domains, including PH domains, potential sites of tyrosine and serine/threonine phosphorylation, a leucine-zipper domain, a potential actin binding site and multimerization site. AFAP110 has been shown to have a role in modulating actin filament integrity and induce lamellipodia formation, and is known to interact with Src family kinases. The aim of this thesis was to characterize this novel protein named Lyn-Associated Cytoskeletal Modulator (LACM) and determine any molecular interactions in order to attempt to elucidate a role for the protein in cell signaling through Lyn. LACM is encoded by a gene consisting of 18 exons and is located on human chromosome 5q33.1 and mouse chromosome 18 E1. LACM protein is expressed through a number of cell types including the R11 erythroid cell line, and mouse tissues including brain, lung, heart and embryos. LACM was shown to multimerize, and subcellular localization of the protein was observed to concentrate around the cell membrane at sites of filamentous actin in filopodia, lamellipodia and stress fibres. The carboxy-terminus of LACM was observed to localize the protein to sites at the cell membrane and through the cytoplasm. Removal of this terminal region resulted in all LACM protein localizing to the nucleus in punctuate spots. LACM protein was observed in heart muscle and potentially has a role at sites of nerve junctions on cardiac myocytes. LACM was shown to interact with the SH3 domain of Lyn at a polyproline motif on LACM. LACM was observed to co-localize and co-immunoprecipitate with Lyn and was tyrosine phosphorylated by the kinase domain of Lyn. Interestingly, the consititutively active Lyn and LACM caused transfected cells to

The current work entails the development of a novel high content platform for the measurement of kinetic ligand responses across cell signalling networks at the single-cell level in distinct PBMC subtypes ex vivo. Using automated sample preparation, fluorescent cellular barcoding and flow cytometry the platform is capable of detecting 21, 840 parallel cell signalling responses in each PBMC sample. We apply this platform to characterize the effects of neuropsychiatric treatments and CNS ligands on the T cell signalling repertoire. We apply it to define cell signalling network abnormalities in PBMCs from drug-naïve first-onset schizophrenia patients (n=12) relative to healthy controls (n=12) which are subsequently normalized in PBMCs from the same patients (n=10) after a six week course of clinical treatment with the atypical antipsychotic olanzapine. We then validate the abnormal cell signalling responses in PBMCs from an independent cohort of drug-naïve first-onset schizophrenia patients (n=25) relative to controls (n=25) and investigate the specificity of the abnormal PBMC responses in schizophrenia as compared to major depression (n=25), bipolar disorder (n=25) and autism spectrum disorder (n=25). Subsequently we conduct a phenotypic drug screen using the US Food and Drug Administration (FDA) approved compound library, in addition to experimental neuropsychiatric drug candidates and nutraceuticals, to identify compounds which selectively normalize the schizophrenia-associated cell signalling response. Finally these candidate compounds are characterized using structure-activity relationships to reveal specific chemical moieties implicated in the putative therapeutic effect.

The PIK3R1 gene encodes three proteins - p85$\alpha$, p50$\alpha$ and p55$\alpha$ - that are regulatory subunits of Class IA phosphoinositide 3-kinases (PI3Ks). These regulatory subunits heterodimerise with one of three catalytic subunit isoforms, namely p110$\alpha$, p110$\beta$, or p110$\delta$. Class IA PI3Ks are critical enzymes involved in fundamental metabolic and mitogenic signalling pathways. This thesis describes the delineation of biochemical and molecular mechanisms whereby PIK3R1 mutations cause diverse disease phenotypes observed in SHORT syndrome (defined by Short stature, Hyperextensibility, Ocular depression, Rieger anomaly and Teething delay), the primary immunodeficiency Activated PI3K-$\delta$ Syndrome 2 (APDS2), and cancer. Initial studies of purified wildtype or mutant PI3K complexes, utilising a modified PI3K fluorescence polarisation lipid kinase assay, established that SHORT syndrome-associated p85$\alpha$ mutations impaired phosphotyrosine peptide-stimulated PI3K activity when heterodimerised with either of the Class IA catalytic subunit isoforms. Two cancer-associated mutations assessed using the same assay demonstrated differential effects on PI3K function, causing either basal activation or impaired phosphotyrosine peptide-stimulated PI3K activity. To examine the effect of SHORT syndrome-associated p85$\alpha$ mutations in insulin-responsive cell types, 3T3-L1 preadipocyte models with conditional overexpression of p85$\alpha$ Y657X or p85$\alpha$ R649W were generated. Doxycycline-induced overexpression of mutant p85$\alpha$ attenuated insulin-stimulated Akt phosphorylation due to reduced insulin-stimulated association of p85$\alpha$/p110$\alpha$ heterodimers with either IRS1 or IRS2. This in turn resulted in impaired downstream signalling as indicated by low adipogenic efficiency. Cells and tissues isolated from Pik3r1$^{WT/Y657X}$ knock-in mice also demonstrated decreased insulin-stimulated Akt phosphorylation. Observations from a system with endogenous expression of mutant p85$\alpha$ Y657X supported the results obtained in the 3T3-L1 p85$\alpha$ overexpression models. The final part of this thesis focussed on a PIK3R1 exon skipping mutant (p85$\alpha$ $\Delta$Ex11) that confers PI3K activation in lymphocytes and causes APDS2. APDS2 patients have an immune-restricted phenotype, even though the mutation occurs within the ubiquitously expressed PIK3R1. To investigate this phenomenon, the doxycycline-inducible system was used to model overexpression of p85$\alpha$ $\Delta$Ex11, as well as an activating p110$\alpha$ H1047R mutation associated with cancer, in 3T3-L1 preadipocytes. Surprisingly, given that APDS2 is not normally associated with metabolic or growth problems, high overexpression of p85$\alpha$ $\Delta$Ex11 severely attenuated insulin-stimulated Akt phosphorylation and adipocyte differentiation. There was also reduced insulin-stimulated recruitment of p110$\alpha$ to either IRS1 or IRS2, and impaired heterodimerisation of p85$\alpha$ $\Delta$Ex11 with p110$\alpha$. Collectively, the data presented in this thesis contributes to the developing knowledge of PIK3R1-related diseases. In particular, these studies provided novel insights into the biochemical and molecular mechanisms of SHORT syndrome-associated p85$\alpha$ mutations. Additionally, these data delivered further understanding of potential mechanisms underlying the immune-specific phenotype of APDS2 caused by PIK3R1 mutations.

Osteoblasts are highly active cells that are responsible for secreting bone forming components such as collagen type I and matricellular proteins that mediate collagen deposition and mineralisation. SPARC and osteopontin are matricellular proteins that are involved in bone regulation and cell-matrix interactions and are also upregulated in metastatic disease. Secretion of these proteins results in changes to the stromal environment that includes cell migration, angiogenesis, matrix degradation, matrix deposition, bone mineralisation and bone resorption. Signalling pathways not only lead to the expression of target proteins, but also have immediate early effects, for example, on cell adhesion. We asked if the ERK 1 and 2 module of the MAPK pathway was involved in the intracellular trafficking of SPARC and Osteopontin. Membrane trafficking is an essential process that ensures newly synthesised proteins pass from their site of synthesis to the extracellular environment. Using an inhibitor of ERK 1 and 2 activation (U0126), as well as siRNA directed against ERK 1 or 2 individually, a change in intracellular localisation of SPARC and osteopontin was observed in cells treated with U0126 and siRNA against ERK 2 alone, likely in or around the Golgi apparatus. Consistent with the observation above, analysis of protein secretion showed that there was a reduction of total protein secreted (30% reduction) when ERK 1 and 2 activation was prevented together or knock down of ERK 2 alone. A mechanism is proposed where ERK 2 is likely activating a substrate that is allowing SPARC and osteopontin to continue along the secretory pathway. This directly implicates ERK 2 as an important regulator of matricellular protein secretion in osteoblasts. In cancer, Ras mutations can lead to permanent activation of the MAPK pathway leading to cancer cell proliferation and survival, however, we propose another mechanism important in metastasis whereby ERK 2 activation is manipulated to facilitate secretion of matricellular proteins which can then mediate changes to the stromal environment that allow the tumour to metastasise successfully.

Neurofibromatosis type 2 (NF2) is an inherited, multiple tumour disease caused by loss of the tumour suppressor protein, Merlin. There are several tumours associated with NF2 including; ependymomas, meningiomas and schwannomas. Merlin loss can also occur sporadically in all of these tumours and is associated with upregulation of various growth factor receptors and their relevant signalling pathways. At present the only treatment options for NF2 are surgery or radiosurgery, both of which incur serious morbidity and are unable to prevent recurrence of tumours. Either new drug treatments, or re-profiling of other drugs already commercially available, are urgently needed to improve outcome for NF2 patients. Cellular prion protein (PrPC), encoded by PRNP gene, is involved in tumour development by altering proliferation, adhesion, and survival in some cancers via focal adhesion kinase (FAK) /Src/ NFκB, cyclin D1 and p53 -proteins. Our group previously showed a strong elevation of PRNP gene activity in schwannoma. I hypothesise that PrPC may contribute to schwannoma development. To study the role of PrPC in schwannoma development I have used the well-established in vitro model of schwannoma that comprises primary human Schwann and schwannoma cells. I show that PrPC is upregulated in schwannoma as well as in Merlin-deficient meningiomas and human malignant mesotheliomas. In schwannoma PrPC is released both via exosomes and by α-cleavage which forms biologically active N- and C-terminal portions of the protein. PrPC contributes to pathological proliferation, adhesion and survival of schwannoma cells by activating ERK1/2, PI3K/AKT, cyclin D1, FAK, p53 pathways via the 37/67kDa non-integrin laminin receptor (LR/37/67kDa) and CD44. Furthermore, schwannoma cells appear to be intrinsically drug-resistant due to upregulation of MDR1 protein p-glycoprotein (p-gp) expression. P-gp expression is dependent on PrPC thus, inhibiting PrPC may be a good potential new therapeutic option for schwannoma patients, either alone or in combination with Sorafenib and p-gp inhibitor Valspodar (PSC833). An inhibitor of LR/37/67kDa/PrP interaction, NSC47924, or Bortezomib, a proteasome/NFκB inhibitor which has been approved for the treatment of multiple myeloma, could also be of beneficial therapeutic effect and is something to investigate in future work. I conclude that PrPC is an interesting new therapeutic target through its involvement with schwannoma patholgenesis and resistance to drug treatments PrPC may prove to be a good therapeutic target in other NF2-related tumours like meningiomas and schwannomas.

abstract: In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

Orientadores: Hisroshi Aoyama, Carmen Verissima Ferreira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-12T15:31:45Z (GMT). No. of bitstreams: 1 Sousa_RobertaReginaRuelade_D.pdf: 21488471 bytes, checksum: e07168c1121fd17c1a2f6c9e820ff8eb (MD5) Previous issue date: 2008 / Resumo: A busca por compostos que atuem especificamente no tumor e causem menos efeitos colaterais é o grande desafio da pesquisa no tratamento contra o câncer. Muitos compostos naturais extraídos de plantas têm apresentado resultados promissores como agentes moduladores de vias de transdução de sinais em diversos tipos de tumores. Os flavonóides, compostos polifenólicos encontrados em diversos alimentos como frutas, chás e vinhos, têm recebido bastante atenção em função dos seus efeitos quimiopreventivos e quimioterapêuticos. Nos últimos anos, muitos estudos relacionados à ação dos terpenos em doenças foram desenvolvidos, decorrentes das suas propriedades anti-inflamatórias, antimicrobiana, gastroprotetora e antitumoral. Entretanto, poucos estudos com enfoque molecular foram desenvolvidos para entender o mecanismo de indução de morte de células tumorais por tais compostos. O objetivo deste trabalho foi estudar a ação dos flavonóides apigenina e fisetina e do diterpeno ferruginol, sobre as vias de transdução de sinal envolvidas na proliferação e morte em células de leucemia e câncer de próstata. Todos os três compostos estudados levaram à morte das células tumorais por apoptose. As células leucêmicas tratadas com fisetina apresentaram aumento da expressão de NFkB, ativação da MAPK p38 e aumento dos níveis de fosforilação de proteínas. A apigenina, por sua vez, mostrou-se um forte inibidor de quinases, especificamente as envolvidas com a proliferação celular, como PI3K, AKT, Src e MAPK, além de induzir apoptose através da via intrínsica. O ferruginol foi capaz de inibir e/ou regular negativamente PI3K, STAT3 e STAT5, proteínas tirosinas fosfatases e proteínas quinases envolvidas com a regulação do ciclo celular. Em ambiente redutor, o efeito citotóxico do ferruginol foi drasticamente impedido, indicando que sua atividade antitumoral pode estar relacionada com a modulação do status redox. O presente trabalho mostrou que os compostos naturais fisetina, apigenina e ferruginol apresentaram um grande potencial como agentes quimioterápicos. / Abstract: The search for compounds that specifically act on tumors, with low side effects, is a great aim in the studies related to cancer treatment. Many natural compounds isolated from plants have presented promising results as modulating agents of the signal transduction pathways in several types of tumors. Flavonoids, polyphenolic compounds largely distributed in foods such as fruits, teas and wines, have been greatly focused due to their chemopreventive and chemotherapeutic effects. In the last years, many studies about the action of terpenes in diseases have been done, due to their antiinflamatory, antimicrobial, gastroprotective and antitumoral properties. However, few molecular studies have been developed to understand the mechanism of cell death induction by these compounds. The aim of this work was to define the molecular action of apigenin, fisetin and ferruginol on the signal transduction pathways involved in the leukemic and prostate cancer cells proliferation and death. All the three compounds studied provoked the cell death by apoptosis. Leukemic cells treated with fisetin presented an increase of NF?B expression and protein phosphorylation levels and activation of p38 MAPK. Apigenin showed to be a strong inhibitor of kinases involved with cellular proliferation, such as, PI3K, AKT, Src, MAPK and additionally to induce apoptosis via the intrinsic pathway. Ferruginol was able to inhibit and/or to negatively regulate PI3K, STAT3 and 5, protein tyrosine phosphatases and protein kinases involved in the regulation of the cell cycle. In reducing environment, the cytotoxic effect of ferruginol was drastically impeded, which indicates that the anti-tumoral activity of ferruginol might be related to redox status modulation. The present work showed that the natural compounds fisetin, apigenin and ferruginol presented great potential as chemotherapeutic agents. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

7Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by accumulation and deposition of extracellular beta-amyloid peptides (Aβ) and intra-neuronal hyperphosphorylated tau in the brain. The insulin signalling pathway begins upstream at the insulin receptor (IR), where the intracellular insulin receptor substrate 1 (IRS1) is phosphorylated, thus propagating the signal downstream to the PI3K/Akt signalling pathway, which affects both the glycogen synthase kinase 3 beta (GSK3β), which is a tau kinase, and mTOR, which is a critical part of the mTORC1 and mTORC2 complexes that not only mediate a wide range of cell functions, but also feed back upstream to regulate Akt. Increasing evidence builds a strong case for the role of soluble Aβ oligomers (AβOs) in the impairment of insulin signalling in AD. Our in vitro studies with neuroblastoma 2a (N2a) cells stably transfected with human APP695 gene (N2a-APP), which secrete excess Aβ, show that the phosphorylation and expression of several but not all critical signalling proteins along the insulin signalling pathway are dysregulated in the cells in comparison to the parental N2a cells. N2a-APP cells were also found to be phenotypically insulin resistant. Subsequently, N2a-APP cells were treated with the Aβ binding peptide (ABP), which binds Aβ oligomers. The ABP treatment was observed to enhance insulin signalling response compared to untreated controls. The results suggest that Aβ may be responsible for inducing the insulin resistant phenotype in N2a-APP cells, and that the removal of Aβ oligomers is a potential treatment consideration for dysfunctional insulin signalling involved in Alzheimer’s disease.

La différenciation terminale des lymphocytes B qui se déroule dans les centres germinatifs des organes lymphoïdes secondaires est l’étape ultime de la réponse T dépendante et aboutit à la production de plasmocytes (PC) à longue durée de vie qui sécrètent des anticorps hautement affins spécifiques de l’antigène et caractéristiques de la réponse immune adaptative. La transition d’une cellule B naïve vers un PC est gouvernée par un réseau de régulation génique bien décrit et est largement influencée par l’intégration de stimuli externes qui contrôlent le devenir des cellules B tels que l’interaction BCR-antigène et les cytokines produites par les cellules T. La stimulation précoce des lymphocytes B humains activés par IL-2, induit la différenciation en PC via une signalisation ERK prolongée entraînant la baisse d’expression de BACH2, un facteur de transcription clef des cellules B. La répression transitoire de BACH2 est suffisante pour déclencher la différenciation en plasmablastes en l’absence d’IL-2, suggérant ainsi qu’il joue un rôle de « verrou moléculaire » de la différenciation en PC. Il est à noter que cette répression forcée de BACH2 aboutit à la production de plasmablastes caractérisés par un phénotype lymphoplasmocytaire. Ce travail de recherche s’est focalisé sur la caractérisation des mécanismes moléculaires régulant l’expression de BACH2 via la voie de signalisation ERK induite par IL-2. Nous avons identifié ELK-1 comme un médiateur de la répression de BACH2 par la voie IL-2/ERK, comme l’atteste sa capacité à se lier avec un élément de régulation d’un enhancer localisé dans l’intron 1 de BACH2, induisant ainsi la répression de l’enhancer et déverrouillant la différenciation en PC. La caractérisation de cet enhancer de BACH2 a confirmé qu’il est régulé de manière dynamique au cours de la différenciation terminale B et qu’il est localisé dans une région sujette aux mutations suggérant qu’il pourrait être impliqué dans la lymphomagenèse. / The terminal differentiation of B cells, which takes places within germinal centres of secondary lymphoid organs, is the ultimate step of a T cell dependent response and results in the generation of long-lived plasma cells (PCs) that secrete protective, antigen-specific, high-affinity antibodies as part of adaptive immunity. The transition of a naive B cell into a PC is governed by a well-characterised gene regulatory network and is heavily influenced by the integration of externally received signals, including BCR-antigen binding and T cell help, such as cytokines which guide B cell fate. The early IL-2 priming of human primary activated B cells triggers PC differentiation through sustained ERK signalling resulting in the down regulation of B cell transcription factor BACH2. Transient BACH2 repression is sufficient to trigger plasmablast differentiation in the absence of IL-2 suggesting that it acts as a key lock of PC differentiation. Importantly, this enforced BACH2 repression results in the generation of plasmablasts with a lymphoplasmacytic phenotype. The focus of this thesis was to characterise the molecular mechanisms regulating BACH2 expression via the IL-2 ERK transduction pathway. We identify ELK-1 as the mediator of IL-2 ERK induced BACH2 downregulation as it binds to a regulatory enhancer element located within intron 1 of BACH2 instigating its repression and unlocking the PC programme triggering differentiation. The characterisation of this BACH2 enhancer confirms that it is dynamically regulated during PC differentiation and is located within a region targeted for mutation suggesting that it may have a potential role in lymphomagenesis.