Thèses sur le sujet « Screening, Signalling »

This thesis focuses on public debt under asymmetry of information. The first part of the thesis deals with foreign debt while the second part with domestic debt. In the first part of this work (Chapter 1 and 2) a theoretical model is developed in which adoption of an IMF programme signals a country's productivity. It is assumed that there are two types of country, one with a high return on investment and the other with a low return. The country's type is known only to itself. In the presence of a debt overhang, the high productivity country may choose not to undertake the investment, despite it being socially efficient to do so. In this case the creditor would like to offer the country some debt reduction, but the low productivity type will also benefit from the debt reduction. This problem can be avoided if the country has sufficient resources to engage in a debt buyback in order to gain the debt relief: only the high productivity type would be prepared to sacrifice current resources for the debt reduction, and thus a separation of the two types is achieved. (This is similar to an argument found in Acharya and Diwan, 1993). We argue, however, that it is unlikely that a heavily indebted country will have sufficient resources for a buyback. If the country is credit constrained, an alternative screening mechanism is to undertake an IMF programme in return for debt reduction and possibly an IMF loan. This mechanism can be the equilibrium outcome even if the programme creates only disutility for the country's policy makers. The existence of an empirical relationship between the adoption of an IMF programme and the concession of a debt rescheduling by commercial and official creditors is tested in Chapter 3 using a bivariate probit model (to control for the endogeneity of the choice "IMF adoption"). If countries who have arrangements with the IMF are more likely than others to obtain a rescheduling of their external debt we could conclude that the adoption of an IMF programme could work as a sort of signal of a country's "good intent", which is thus rewarded with the debt relief. The results confirm the existence of a significant effect of the adoption of an IMF programme on the subsequent concession of a debt rescheduling by creditors. In the second part of the thesis we have investigated some of the consequences due to the existence of a large public debt (as it is the case in some of the European countries, e.g., Belgium, Greece, Ireland and Italy). More specifically, a theoretical model is developed, in Chapter 4, to examine whether buybacks of domestic debt may signal a government type. In the model it is assumed that the government could be of two types: a dry type and a wet type, according to its willingness to implement a fiscal stabilisation (in this model this basically means reducing fiscal spending). Asymmetry of information between the government and private investors is assumed. In particular interest rates are assumed to incorporate a risk premium which reflect the expectation that the inability to implement a stabilisation programme may result in more inflation and/or taxation, or debt default. In particular, during a fiscal stabilisation, private investors would lack confidence in the stabilisation programme and interest rates would be too high, reflecting this lack of credibility. Thus, a dry type who has to finance new spending may want to signal her resolution in order to lower the interest costs and one way to do that would be to repurchase a fraction of the outstanding debt. The wet type could also decide to buy-back some of his debt in order to pretend to be dry and to (possibly) lower his interest payments. It is showed that a critical amount of buyback exists such that the two types could be separated. Finally, in Chapter 5, evidence is provided in favour of the hypothesis that the repurchase of public debt is actually perceived as a good signal by private investors, consistently with the theoretical model. According to our results, the initial impact of the repurchase was to make the prices of the remaining bonds rise. This was consistent with our theory as we can interpret an increase in bonds price (and correspondingly a decrease in their rates of returns) as a signal that the buyback operation has positively affected the credibility of a government.

Considerable development in the field of nanotechnology is increasingly yielding novel applications of nanoparticles. The unique properties of nanoparticles in particular their high aspect ratio (length : width ratio), however could pose potential risks to the user. A high throughput genetic screening platform, RISCI (robotic single cDNA investigation), was previously established for the systematic evaluation of single gene activities. Here, RISCI was utilised to identify pro-apoptotic genes as well as genes involved in the positive and negative regulation of silica nanoparticle-induced cell death. This project describes the further development of the screening platform by harnessing its capability to screen a cDNA library comprising approximately 30,000 full length, completely annotated, and sequenced human genes for novel regulators of apoptosis. It integrates an extensive skill sets and is broadly organised into three major phases: Setup, Screen and Analysis. The integration of a pro-apoptosis treatment to screen for inhibitors and sensitizers is a novel aspect of the current experimental setup, along with the low redundancy library. The extensive setup phase focused on technical aspects. The cDNA library, acquired as plasmid DNA, was transformed into a bacterial host for replication and subsequent DNA isolation. A new high-throughput process was developed encompassing the production of competent bacteria and a heat shock transformation protocol, which was subsequently transferred onto the robotic platform. In parallel, the software controlling the robots was redeveloped to allow for execution of user-defined protocols while novel transfection protocols were adapted for automation. The screen identified 699 apoptosis inducers, 1,141 inhibitors and 626 sensitizers. Bioinformatics analysis revealed that the inducers were highly enriched for cell death associated terms, while the inhibitors were strongly associated with cancer profiles. Both inducers and sensitizers were predominantly achieving the functional effect on the protein level, but inhibitors were mainly transcription based. Enriched metal response genes also suggest that the silica nanoparticles were causing their toxicity through reactive oxygen species generation. Intriguingly, the screen identified many noncoding sequences as being functionally capable of regulating apoptosis. These noncoding candidates are capable of regulating the protein coding counterparts identified from the screen. The truly interesting part of the project outcome remains those unknown candidates that were implicated in apoptosis regulation for the first time. Dissemination of the consolidated candidate list would help accelerate the experimental validation of these candidates and aid other researchers in deriving novel hypotheses when the candidates are placed in their research context. [For supplementary files please contact author].

Beta-catenin is a crucial component of the Wnt signalling pathway, which is imperative in many developmental processes and aberrantly regulated in several different cancers. The standard model of Wnt/Beta-catenin signalling states that, upon stimulation by Wnt ligand, Beta-catenin accumulates and subsequently translocates to the nucleus to activate TCF-dependent transcription of a variety of target genes, including oncogenes. However, the mechanisms regulating the nuclear localisation of Beta-catenin and its correlation with TCF-dependent transcription are poorly understood. In order to identify novel regulators of Beta-catenin levels and localisation in Wnt signalling imaging-based high-throughput knockout screens were developed in a Wnt inducible cell line, in addition to a cancer cell line in the presence of normal and downregulated APC. Results from the screens show that, in addition to known Wnt signalling components, genes not previously ascribed to the pathway appeared to modulate Beta-catenin. The study has provided sources of possible mechanistic insights into a number of areas of biology that may be involved in β-catenin regulation. Furthermore, it reveals an unprecedented degree of cross talk between Wnt and many other major signalling pathways. Moreover, the data indicated a degree of cell-type specificity in the regulators identified and, significantly, a lack of correlation between β-catenin levels and transcriptional activity.

Wnt/β-catenin signalling plays a central role in the regulation of multicelluar organism development and in the maintenance of tissue homeostasis in adults. Dysregulation of Wnt signalling resulting in aberrant pathway activation is a key initiating step in the development of a diverse range of cancers, including colorectal cancer, and as such is an important target for therapeutic intervention. A novel Wnt pathway inhibitor, ‘MSC’, has been identified as blocking activated Wnt signalling, specifically through inhibiting the ability of CDK8 and CDK19 to activate nuclear β-catenin/TCF-dependent transcription. However, despite potently inhibiting Wnt-dependent transcription, the ability of MSC to reduce cellular viability was limited. This study aimed to determine genes that whose loss operated with MSC to reduce cell survival. A whole-genome RNAi chemical sensitisation screen identified 3 genes whose depletion in combination with MSC treatment conditionally reduced the viability of HCT116 cells in vitro. The outstanding hit of this screen was Histidyl Aminoacyl tRNA Synthetase (HARS). The identification of this enzyme as an MSC ‘interactor’ suggested links between Wnt signalling and the regulation of translation. BRAF and MED11 RNAi also conferred conditional sensitivity to MSC. Interestingly, MED11 is a component of the Mediator complex, a multiprotein transcription regulatory complex in which CDK8 functions to regulate β-catenin/TCF-dependent transcription, suggesting that mediator complex may be a key target of MSC action. A parallel overexpression screen was initiated to identify novel Wnt pathway activators, and subsequently used to map MSC resistance. Expression of the transcription factors GBX1 and HMGB2, determined to be novel regulators of TCF-dependent transcription, blocked MSC-mediated disruption of Wnt signalling. Overexpression of either gene in a clinical context might therefore be regarded as a contra-indication for MSC-class therapies. These studies have highlighted potential avenues for broadening the scope of MSC activity through the determination of survival and resistance mechanisms, thus the rational design of MSC-combination therapies could be of huge clinical benefit for the treatment of colorectal cancer.

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.

Hedgehog (Hh) morphogens play fundamental roles in development whilst dysregulation of Hh signalling leads to disease. Multiple receptors are involved in the modulation of Hh morphogens at the cell surface. Among these, the interactions of Hh ligands with glycosaminoglycan (GAG) (for example heparan or chondroitin sulphate) chains of proteoglycans in the extracellular matrix play a key role in shaping morphogen gradients and fulfil important functions in signal transduction. Several high resolution crystal structures of Sonic Hh (Shh)-GAG complexes have been determined. The interaction determinants, confirmed by binding studies and mutagenesis reveal a novel Hh site for GAG interactions, which appears to be common to all Hh proteins. This novel site is supported by a wealth of published functional data, and resides in a hot spot region previously found to be crucial for Hh receptor binding. Crystal packing analysis combined with analytical ultracentrifugation on Hh-GAG complexes suggest a potential mechanism for GAG-dependent multimerisation. A key step in the Hh pathway is the transduction of the Hh signal into the receiving cell. The Hh signal transducer, Smoothened, is a key target drug target in the pathway with several modulators in clinical trials, despite an absence of structural data. Smoothened is required to activate all levels of Hh signalling. Recent evidence points to the conserved N-terminal ectodomain (ECD) in regulating Smo activity, from vertebrates to invertebrates. Despite the central importance of the ECD, its precise function remains elusive. A crystal structure of the ECD at 2.2 Å resolution is reported here. Structural analysis and biophysical experiments are discussed with reference to the potential function of this intriguing domain.

ESCRT (Endosomal Sorting Complex Required for Transport) proteins regulate cell surface receptor degradation by sorting and packaging ubiquitinated cargoes into the intraluminal vesicles of multivesicular bodies (MVBs). A range of human diseases including cancer, and neurodegeneration display altered expression or are caused by mutations of ESCRT subunits. Studies have shown that Drosophila tissues lacking ESCRTs display neoplastic-like features like overproliferation and polarity defects, partly due to aberrant signalling including Notch signalling. To understand ESCRT-regulated processes in vivo, we utilised modification of a deformed wing phenotype specifically caused by knockdown (RNAi) of Vps25, an ESCRT-II subunit. We systematically screened chromosomal regions and identified 204 genetic interactors of Vps25 that enhanced/suppressed the phenotype. They include genes that function in trafficking, signalling, transcription, ion transport and many other biological processes; suggesting that ESCRTs influence a wide range of biological processes. We have focused on a subset of these hits that regulate tissue growth with a secondary screen based on modification of a Delta-driven eye overgrowth phenotype, isolating a subset of 43 genes involved in regulating tissue growth, some of which are novel and uncharacterised. Human orthologues of some of these genes are important in cancers; dropout (dop), whose mammalian orthologues are the MAST kinases, have been shown to contribute towards breast cancer development. dop mediates Delta-driven eye overgrowth possibly by upregulating Delta expression. In human cells, MAST2 does not affect Notch signalling but might contribute to tumorigenesis by regulating the NFκB pathway. We have also characterised another interactor, CG12163 which is the homologue of mammalian Cathepsin F. Mutations in Cathepsin F cause a rare form of neuronal ceroid lipofuscinosis (NCL) called Type B Kufs disease. Our Drosophila model which recapitulates aspects of the human disease phenotype suggests that defects in autophagy might underlie the pathogenesis of NCLs.

<strong>Chapter 1</strong> Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. <strong>Chapter 2</strong> Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. <strong>Chapter 3</strong> Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. <strong>Chapter 4</strong> Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. <strong>Chapter 5</strong> Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB₂R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB₂R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.

The E3 ubiquitin ligase RNF43 (RING finger protein 43) is an important negative modulator of the WNT signalling pathway that acts at the plasma membrane by targeting Frizzled and its co-receptor LRP for degradation. In the small intestine, this prevents uncontrolled expansion of the stem cell compartment and so it is essential to the maintenance of normal tissue homeostasis. However, despite its crucial role in fine-tuning the WNT pathway and its role as a tumour suppressor, it is unclear whether RNF43 has further binding partners and what their functional relevance is to the modulation of WNT signalling. Here, I describe the development of a new screening strategy which combines CRISPR/Cas9 technology with 3D-intestinal organoid culture for the identification of novel molecular interactors of RNF43. Overall, this study and the technology developed provide a tool to enable the detailed description of the mechanism of action of RNF43, which is important not only in order to increase our understanding of WNT pathway regulation but also to gain potential new insights into RNF43 paralogs, by analogy. The investigation of paralogs is crucial as RNF43 belongs to a newly identified family of E3 ubiquitin ligases, named the PA-RING family, whose members are still poorly characterised. The majority of PA-RING family members have not been linked to any signalling pathway, most of their targets are still unknown and in many cases their in vivo function has not been addressed. In this context, my work has specifically focused on the investigation of the potential involvement of additional PA-RING family members in WNT pathway modulation and also on target identification for selected members. The results summarised in this dissertation show that no other PA-RING family member plays a prominent role in WNT pathway modulation aside from Rnf43 and its homologue Znrf3, however, different classes of adhesion molecules are likely to be regulated by certain of these E3 ligases. In conclusion, my work has contributed to unravelling previously unexplored aspects of this protein family, with particular regard to RNF43 and its mechanism of action. Thanks to this original approach, it was possible to identify potential new players involved either in membrane clearance of Frizzled or in RNF43 maturation. In particular, my thesis focuses on the characterisation of the role of DAAM in RNF43-mediated Frizzled internalisation.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disease resulting in episodic but progressive extraskeletal bone formation. FOP is caused by missense mutations in the cytoplasmic domain of the type I bone morphogenetic protein (BMP) receptor ACVR1, leading to dysregulated activation. Currently there are no available drug treatments and the structural mechanism of mutant activation is still poorly characterised. To address this, a number of BMP and TGFβ receptors, including FOP mutants of ACVR1 were cloned, expressed and purified for both structural and biophysical experiments. The arginine at the site of most recurrent FOP mutation, R206H, is common across all type I receptors except BMPR1A and BMPR1B which have a lysine at this site. The novel structure of BMPR1B differed to wild-type ACVR1 showing some of the conformational changes expected of the active conformation. However, a variety of disease related ACVR1 mutant structures, including ACVR1 R206H, revealed a surprisingly persistent inactive conformation in the kinase domain. Some conformational changes suggestive of activation were observed in the mutant Q207D affecting the ATP pocket, the β4–β5 hairpin and the activation loop. Additionally, the structure of the Q207E mutant showed a slight release of the regulatory glycine-serine rich domain from its inhibitory position. These subtle changes suggest that the mutant inactive conformation is destabilised and potentially more dynamic. In agreement, all of the ACVR1 mutants showed reduced binding to the inhibitory protein FKBP12. However, mutant phosphorylation of the substrate Smad1 was not constitutive, but dependent on the co-expression of the partner ACVR2, consistent with recent evidence from transgenic knock-out mice. A novel 2-aminopyridine inhibitor scaffold with favourable specificity for ACVR1 was identified using a fluorescence-based thermal shift assay. Further derivatives were characterised with improved potency and selectivity. The crystal structures of ACVR1 bound to these inhibitors showed exquisite shape complementarity, contributing to their favourable specificity. This work has increased the understanding of FOP-associated mutant activation and provided a novel starting scaffold for potential drug development.

All viruses encode for at least one viral interferon (IFN) antagonist, which is used to subvert the cellular IFN response, a powerful antiviral innate immune response. Numerous in vitro and in vivo studies have demonstrated that IFN antagonism is crucial for virus survival, suggesting that viral IFN antagonists could represent promising therapeutic targets. This study focuses on Respiratory Syncytial Virus (RSV), an important human pathogen for which there is no vaccine or virus-specific antiviral drug. RSV encodes two IFN antagonists NS1 and NS2, which play a critical role in RSV replication and pathogenicity. We developed a high-throughput screening (HTS) assay to target NS2 via our A549.pr(ISRE)GFP-RSV/NS2 cell-line, which contains a GFP gene under the control of an IFN-stimulated response element (ISRE) to monitor IFN- signalling pathway. NS2 inhibits the IFN-signalling pathway and hence GFP expression in the A549.pr(ISRE)GFP-RSV/NS2 cell-line by mediating STAT2 degradation. Using a HTS approach, we screened 16,000 compounds to identify small molecules that inhibit NS2 function and therefore relinquish the NS2 imposed block to IFN-signalling, leading to restoration of GFP expression. A total of twenty-eight hits were identified; elimination of false positives left eight hits, four of which (AV-14, -16, -18, -19) are the most promising. These four hit compounds have EC₅₀ values in the single μM range and three of them (AV-14, -16, -18) represent a chemically related series with an indole structure. We demonstrated that the hit compounds specifically inhibit the STAT2 degradation function of NS2, not the function of NS1 or unrelated viral IFN antagonists. At the current time, compounds do not restrict RSV replication in vitro, hence hit optimization is required to improve their potency. Nonetheless, these compounds could be used as chemical tools to determine the unknown mechanism by which NS2 mediates STAT2 degradation and tackle fundamental questions about RSV biology.

This thesis explores roles of 2-oxoglutarate-dependent (2OG) oxygenases as interfaces that modulate steps in the flow of genetic information in cells in response to oxygen availability. Chapter 1 introduces mechanistic, biochemical and physiological aspects of major subfamilies of 2OG oxygenases, and their established regulatory roles in cells. In addition, structural and functional aspects of the ribosome and the translation process are discussed, with a focus on post-translational ribosome modifications. Chapter 2 investigates histone demethylases, which mediate chromatin-dependent regulation of gene expression and provides proof-of-concept for the rational, structure-guided design of small-molecules for selective inhibition of 2OG oxygenases with roles in cancer and inflammatory disease. Chapter 3 suggests regulatory roles for ten-eleven-translocation (TET)- catalysed DNA hydroxylation; calorimetric and thermal analyses reveal a duplex-stabilizing effect of the epigenetic 5-methylcytosine mark that is reversed upon conversion to 5- hydroxymethylcytosine (also termed the ‘sixth’ DNA base), raising the possibility that 2OG oxygenase catalysis might affect transcription via biophysical effects. Chapter 4 investigates fluoride release assays as a technology to enable medicinal chemistry studies on 2OG oxygenases with roles in fat mass regulation and obesity, cancer and inflammation; studies on the ALKBH5 enzyme show that it is a hypoxically upregulated 2OG oxygenase with a substrate preference distinct from previously characterized ALKBH enzymes. Chapter 5 identifies OGFOD1 as a 2OG-dependent ribosomal protein hydroxylase. OGFOD1 catalysis is conserved from yeast to humans. OGFOD1 catalyses formation of trans-3- hydroxy-L-proline in a highly conserved loop of ribosomal protein S23 proximal to the ribosomal decoding centre, possibly to modulate the interactions of eukaryotic ribosomes with tRNA, mRNA and translation factors in an oxygen-dependent manner. OGFOD1 is the functionally most well-conserved protein-modifying 2OG oxygenase; likewise, ribosomal protein S23 hydroxylation is the most well-conserved post-translational ribosome modification in eukaryotes. Some cell lines require OGFOD1 for proliferation, and scaffolds for OGFOD1- selective inhibitors are developed for use as potential antiproliferative agents and probes for cellular function. Chapter 6 shows the development of assays to investigate whether OGFOD1 catalysis affects ribosome assembly and function, including processivity, accuracy of initiation, elongation and termination, in yeast and mammalian cell lines. Chapter 7 concludes that ribosome hydroxylation might present an additional layer of regulatory complexity by which 2OG oxygenases could enable cells to respond to fluctuating oxygen levels.

Dissertação de mestrado em Genética Molecular<br>Most of our knowledge about basic cellular processes has originated from model organisms. Saccharomyces cerevisiae is consider a model system, representing the simplest eukaryotic organism, whose genome can be easily manipulated allowing suitable analysis and efficient assessment of gene products from humans. RAS genes encode low molecular weight, GTP-binding, GTP-hydrolysing proteins that are highly conserved throughout all eukaryotic species. RAS pathway has attracted attention because of its importance in malignant transformation of human cells. KRAS is a human RAS isoform, expressed in almost all cell types and essential for the normal cellular development, in addition it is the isoform most frequently mutated in many cancer types. The S. cerevisiae has two RAS genes, RAS1 and RAS2 that are essential, once their double deletion renders yeast unviable. These genes are structurally and functionally homolog of the mammalian RAS proto-oncogenes. This conservation allows the use of yeast genetics to the study of KRAS. Considering this, the first aim of this work was to build a set of S. cerevisiae strains (using haploid BY4741 wild type, ras1Δ, and ras2Δ as a basis) expressing the human KRASwt cDNA, using a plasmid-based expression. The second aim was the phenotype screening of these humanized yeasts, as well as of the correspondent recipient strains. The effect of KRASwt expression was evaluate on the cell stress response, growth, chronological aging, cell cycle progression and haploid invasive growth. According to the results, the KRASwt heterologous expression in yeast (1) had a negative or null effect on the resistance to the temperature, pH, osmotic and oxidative stresses; (2) decreased growth on non-fermentable carbon sources; (3) increased the adhesion capacity; (4) stimulated the haploid invasive growth in the ras2Δ strain; (5) modified the budding pattern of the wild type cells; (6) changed the cellular proliferation in a strain-dependent way; and (7) decreased the chronological lifespan. The results indicated that the expression of KRASwt in the wild type strain, possibly leads to a hyperactivaction of the RAS/cAMP/PKA signalling pathway, which in turn triggers a decrease of stress resistance and longevity. This study also highlighted the relevance of the yeast background, to the possible achievement of different results, regardless the functional conditions used. The present work contributed to gain insight into KRAS mechanism of action using yeast as a model organism. Furthermore, all obtained results will constitute part of the development of a yeast-based high throughput phenotype platform for future pharmacological testing.<br>Grande parte do nosso conhecimento acerca dos processos celulares fundamentais provêm de organismos modelo. A levedura Saccharomyces cerevisiae é considerada um sistema modelo, representando um organismo eucariota simples, cujo genoma pode ser facilmente manipulado, permitindo uma análise adequada e uma avaliação eficiente das proteínas humanas. Os genes RAS codificam proteínas de baixo peso molecular, de ligação e hidrólise de GTP, que estão amplamente conservadas em todas as espécies eucariótas. O estudo da via RAS tem atraído a atenção devido à sua importância na transformação maligna das células humanas. O KRAS é uma das isoformas RAS humanas, expressa em quase todos os tipos de células e essencial para o normal desenvolvimento celular, para além disso, é também a isoforma que se encontra frequentemente mutada em diversos tipos de cancro. S. cerevisiae tem dois genes RAS, RAS1 e RAS2, essenciais uma vez que a sua dupla deleção é letal para a levedura. Estes genes são estruturalmente e funcionalmente homólogos dos proto-oncogenes RAS dos mamíferos. Esta conservação permite a utilização da levedura para o estudo do KRAS. Deste modo, o primeiro objectivo deste trabalho foi construir um conjunto de estirpes de S. cerevisiae (BY4741 haplóide wild type, ras1Δ e ras2Δ como receptoras) a expressar o KRASwt humano, usando um plasmídeo de expressão. O segundo objectivo foi a realização de um screening fenotípico das leveduras humanizadas, bem como das estirpes não transformadas. O efeito da expressão do KRASwt foi avaliado na resposta ao stress celular, crescimento, envelhecimento cronológico, progressão do ciclo celular e no crescimento invasivo haplóide. De acordo com os resultados, a expressão heteróloga do KRASwt na levedura (1) teve um efeito negativo, ou nulo, na resistência aos diferentes stresses (temperatura, pH, osmótico e oxidativo); (2) reduziu o crescimento em fontes de carbono não fermentáveis; (3) aumentou a capacidade de adesão; (4) estimulou o crescimento invasivo haplóide na estirpe ras2Δ; (5) modificou o padrão de divisão das células wild-type; (6) alterou a proliferação celular; e (7) diminuiu a longevidade cronológica. Os resultados indicaram que a expressão do KRASwt na estirpe wild type, levou a uma possível hiperativação da via de sinalização RAS/cAMP/PKA, responsável pela diminuição da resistência ao stress e da longevidade. Este estudo também demonstrou a relevância do background da levedura para a possível obtenção de diferentes resultados, independentemente das condições usadas. O presente trabalho contribuiu para o conhecimento do mecanismo de ação do KRAS através da utilização da levedura como organismo modelo. Além disso, todos os resultados obtidos farão parte de uma plataforma de fenótipos de levedura para futuros testes farmacológicos.