Tesis sobre el tema "Phenotypic assays"

Anti-HIV therapy has dramatically progressed from palliative care to high effectiveness, with virus replication and disease progression halted in most patients yet not eradicated. However, the need for lifelong therapy has kept anti-HIV drug development at high pace and novel strategies, drugs and drug classes have been regularly introduced over time. Issues to be tackled during prolonged antiretroviral therapy include adherence, toxicity, drug-drug interactions and the development of drug resistance. During my PhD course at the HIV Monitoring Laboratory (HML) of the Department of Medical Biotechnology at the University of Siena, I have been given the opportunity to focus my efforts in this area along two different lines. The first activity has been done in the field of drug discovery within the EU FP7 funded THINPAD project. THINPAD is the acronym of “Targeting the HIV-1 Nucleocapsid Protein to fight Antiretroviral Drug Resistance”, thus the objective of this project was to discover and develop a novel class of anti-HIV agents targeting NCp7, a small nucleocapsid protein that plays a number of different roles in HIV replication. The project used a funnel strategy from virtual screening to biochemical testing, then cell based assays and finally humanized mice as the animal model to validate the candidate NCp7 inhibitors (NCIs). My unit measured the antiviral activity of the candidate NCIs through cell based assays developed at the HML. The compounds with the highest selectivity indexes were next demonstrated to retain comparable efficacy against wild-type virus and virus resistant to licensed drug classes, thus confirming NCp7 as a specific target suitable to block the replication of currently circulating drug resistant viruses. The same compounds were used to perform experiments aiming at the definition of the mechanism of action through the measurement of viral nucleic acids intermediates produced in infected cells in presence of inhibitory concentration of NCIs at different time points. The results of this analysis were in agreement with antiviral activity exerted at the early and/or late steps of viral replication, suggesting that NCIs are able to interfere with variable impact on the different functions of NCp7 during HIV life cycle. While preliminary efficacy tests in the humanized mouse model were not successful, in vitro data support further NCI development. The second activity has investigated the role of natural HIV-1 variability or specific HIV-1 mutants in the susceptibility or genetic barrier to resistance to licensed or upcoming antiretrovirals. Completed studies of this kind have shown (i) a role for the natural polymorphisms E138A in the reverse transcriptase in lowering the genetic barrier to resistance to the non-nucleoside reverse transcriptase inhibitor etravirine, (ii) a minimal impact of the integrase E157Q polymorphism in resistance to integrase inhibitors and (iii) the infrequent occurrence of natural resistance to the novel entry inhibitor fostemsavir in the HIV-1 CRF02_AG, an evolutionary lineage of HIV-1 originated in Africa and substantially represented in Italy. Although different, the three topics shared the need to clarify uncertain areas and derive indications for optimal drug use in the clinic. Both activities have been using cell based systems developed at the HML and further refined for the specific application. Phenotypic drug susceptibility testing is a fairly complex task, usually assigned to specialized companies in collaborative research projects and not used at all in the clinic. Among academic systems proposed over time, our assay has been uniquely validated through comparison with the de facto reference commercially available Phenosense assay from Monogram Biosciences. Overall, contributing to expanding and using the laboratory portfolio of systems required for advanced investigation of anti-HIV drug was perfectly in line with my expectations as a biotechnologist.

The licensure of recombinant protein-based meningococcal vaccines has increased the complexity of strain coverage assessments. In 2015, the 4CMenB vaccine was introduced into the UK national infant immunisation schedule and an Enhanced Surveillance programme was launched by Public Health England’s Meningococcal Reference Unit. Meningococcal isolates, representing ~50% of laboratory-confirmed cases, are comprehensively characterised using whole genome sequencing and 4CMenB strain coverage is assessed phenotypically using the Meningococcal Antigen Typing System. For the remaining cases, which are confirmed using PCR only, strain characterisation was until recently restricted to geno-grouping and geno-subtyping. The purpose of this research was to establish new genotypic assays to improve strain coverage assessment among non-culture cases, as well as introduce the MEASURE assay to predict coverage of a second sub-capsular vaccine, rLP2086, among isolates. A PCR sequencing assay targeting the Factor H-Binding Protein antigen gene (fHbp) was developed and had an estimated analytical sensitivity limit of between 600ag/μL and 6fg/μL. Using this assay, fHbp was successfully sequenced from 1510 of the 1661 PCR-positive clinical samples tested (91%). The distributions of fHbp peptide variants among culture and non-culture strains were compared and, whilst differences were observed for a small number of predominant variants, the distribution was very similar within each capsular group. The prospect of performing WGS directly from non-culture specimens was investigated using the Agilent SureSelectXT system. Eight of ten clinical specimens yielded genomes of acceptable quality. It was estimated that up to 54% of non-culture cases could be sequenced using this technique, however, the financial cost is currently prohibitive. Finally, the MEASURE assay was risk assessed and overnight formaldehyde incubation was introduced to ensure cells were fully fixed. The assay results were similar to those generated in a collaborating laboratory, however, further standardisation may be required. These assays will help to increase the accuracy of strain coverage predictions of the currently-licenced and future sub-capsular meningococcal vaccines.

Background: MicrO is an ontology of microbiological terms, including prokaryotic qualities and processes, material entities (such as cell components), chemical entities (such as microbiological culture media and medium ingredients), and assays. The ontology was built to support the ongoing development of a natural language processing algorithm, MicroPIE (or, Microbial Phenomics Information Extractor). During the MicroPIE design process, we realized there was a need for a prokaryotic ontology which would capture the evolutionary diversity of phenotypes and metabolic processes across the tree of life, capture the diversity of synonyms and information contained in the taxonomic literature, and relate microbiological entities and processes to terms in a large number of other ontologies, most particularly the Gene Ontology (GO), the Phenotypic Quality Ontology (PATO), and the Chemical Entities of Biological Interest (ChEBI). We thus constructed MicrO to be rich in logical axioms and synonyms gathered from the taxonomic literature. Results: MicrO currently has similar to 14550 classes (similar to 2550 of which are new, the remainder being microbiologically-relevant classes imported from other ontologies), connected by similar to 24,130 logical axioms (5,446 of which are new), and is available at (http://purl.obolibrary.org/obo/MicrO.owl) and on the project website at https://github.com/carrineblank/MicrO. MicrO has been integrated into the OBO Foundry Library (http://www.obofoundry.org/ontology/micro.html), so that other ontologies can borrow and re-use classes. Term requests and user feedback can be made using MicrO's Issue Tracker in GitHub. We designed MicrO such that it can support the ongoing and future development of algorithms that can leverage the controlled vocabulary and logical inference power provided by the ontology. Conclusions: By connecting microbial classes with large numbers of chemical entities, material entities, biological processes, molecular functions, and qualities using a dense array of logical axioms, we intend MicrO to be a powerful new tool to increase the computing power of bioinformatics tools such as the automated text mining of prokaryotic taxonomic descriptions using natural language processing. We also intend MicrO to support the development of new bioinformatics tools that aim to develop new connections between microbial phenotypes and genotypes (i.e., the gene content in genomes). Future ontology development will include incorporation of pathogenic phenotypes and prokaryotic habitats.

Objective: The human immunodeficiency virus type 1 (HIV-1) currently infects over 30 million people worldwide. It uses one of two main co-receptors to infect cells. The primary objective of this thesis is to evaluate genotypic and phenotypic assays for co-receptor usage in the clinical setting and investigate approaches for improvement of these assays. Methods: The concordance of recombinant co-receptor phenotyping assays and the predictive ability of genotype-based methods including the ‘11/25’ rule, position specific scoring matrices (PSSMs), and support vector machines (SVMs) were evaluated in the clinical setting using patient-derived plasma samples. Samples and patient data were evaluated in cross-sectional analyses from a retrospective population-based cohort of HIV-infected individuals enrolled in the HIV/AIDS Drug Treatment Program in British Columbia, Canada. Results: Current implementations of HIV V3 region-based predictors for HIV co-receptor usage tested on patient derived samples are inadequate in the clinical setting, primarily due to low sensitivities as a result of difficult to detect minority species. Recombinant phenotype assays also show discordances when tested against each other on the same set of patient derived samples, raising doubts if any of these assays can truly be considered a ‘gold standard’. Significant associations between clinical progression, viral sequence-based predictors of co-receptor usage and the output of recombinant assays are observed, suggesting that sensitivity can be improved by incorporating CD4% into genotype-based predictors. This is verified with a SVM model which showed a 17% increase in sensitivity when CD4% was incorporated into training and testing. Conclusion: This work in this thesis has exposed the difficulty in determining the co-receptor phenotype in the clinical setting, primarily due to minority species. Although genotypic methods of screening for HIV co-receptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turn-around time over phenotypic methods, they are currently inadequate for use in the clinical setting due to low sensitivities. Although the addition of clinical parameters such as CD4 count significantly increases the predictive ability of genotypic methods, the presence of low-levels of X4 virus continues to reduce the sensitivity of both genotypic and phenotypic methods.

Background: The global assays assess the interaction of procoagulants and anticoagulants on the generation of thrombin. These assays, as functional tests, may reflect the bleeding tendency in patients with haemophilia better than the conventional assays. Design and Methods: In this study the use of three global assays including rotational thromboelastometry (ROTEM®), thrombin generation test (TGT), and clot waveform analysis were evaluated on a cohort of haemophilia A (HA) and haemophilia B (HB) patients. The global assay parameters were compared to the conventional assays and the most sensitive parameters correlated with the clinical phenotype of haemophilia patients. Results and discussion: The modified ROTEM® analysis initiated by a minute amount of tissue factor proved to be useful in assessing haemophilia patients. A strong correlation was found between the Maximum Velocity (MaxVel) parameter of ROTEM® and the factor VIII level of haemophilia individuals. The use of corn trypsin inhibitor (CTI) to inhibit contact pathway improved the sensitivity and specificity of the ROTEM® analysis. A significant difference between the TGT parameters of patients groups and the control was noted. The use of CTI improved the sensitivity and specificity of the test, in particular peak height in platelet rich plasma (PRP) samples. The results in PRP+CTI group showed that platelets play an important role in heterogeneity of thrombin generation amongst severe haemophilia patients where the relevant deficient factor is < 1.0 IU/dL compared to conventional clotting assays. In clot waveform analysis, this study showed a better correlation between the velocity indices of the APTT test (Min1 and Min2) and the FVIII activity of HA individuals compared to the correlation between the APTT test itself and the FVIII activity level. The Min1 and Min2 appear to be more sensitive, simple, fast and cost effective in the diagnosis of hypocoagulability, and monitoring of haemophilia patients compared to the conventional tests. The area under the thrombin generation curve (AUC), peak height (PH), and time to peak (TP) parameters of the in-house TGT in PRP+CTI test category showed a strong correlation with patient age at first joint bleed (r = 0.693 p = 0.003, r = 0.718 p = 0.002, and r = -0.703 p = 0.002 respectively). The type of F8 mutation was also correlated with the patient’s thrombin generation capacity. Based on two subgroups of null mutations and non-null mutations, the PH and AUC in PPP+CTI category showed a significant increase in the non-null mutation group. The time to the maximum velocity of ROTEM® (tMaxVel) was also shorter in the non-null mutation group. Conclusion: The use of global assays in diagnosing hypocoagulability, and monitoring haemophilia patients proved to be useful, especially in areas where conventional methods are not responsive. However, the multifactorial dependency of global assays make them difficult to interpret on their own, therefore, these assays should be used in parallel with the conventional tests in order to be more useful.

Le phénotypage computationnel est un ensemble de technologies émergentes permettant d’étudier systématiquement le rôle du génome dans l’obtention de phénotypes, les caractéristiques observables d’un organisme et de ses sous- systèmes. En particulier, les essais cellulaires permettent de cribler des panels de petites molécules ou de moduler l’expression des gènes, et de quantifier les effets sur les caractéristiques phénotypiques allant de la viabilité à la morphologie cellulaire. Le criblage à haut contenu étend les méthodologies des criblages cellulaires à une lecture à haut contenu basée sur des images, en particulier les canaux multiplexés de la microscopie à fluorescence. Les cribles basés sur de multiples lignées cellulaires sont aptes à différencier les phénotypes de différents sous-types d’une maladie, représentant l’hétérogénéité moléculaire concernée dans la conception de thérapies médicales de précision. Ces modèles biologiques plus riches sous-tendent une approche plus ciblée pour le traitement de maladies mortelles telles que le cancer. Un défi permanent pour le criblage à haut contenu est donc la synthèse des lectures hétérogènes dans les cribles à multiples lignées cellulaires. Parallèlement, l’état de l’art établi en matière d’applications d’analyse d’images et de vision par ordinateur est l’apprentissage profond. Cependant, son rôle dans le criblage à haut contenu ne fait que commencer à être réalisé. Cette thèse aborde deux problématiques de l’analyse à haut contenu des lignées cellulaires cancéreuses. Les contributions sont les suivantes : (i) une démonstration du potentiel d’apprentissage profond et de modèles générateurs dans le criblage à haut contenu ; (ii) une solution basée sur l’apprentissage profond au problème de l’hétérogénéité dans un criblage de médicaments sur plusieurs lignées cellulaires ; et (iii) de nouvelles applications de modèles de traduction d’image à image comme alternative à la microscopie à fluorescence coûteuse actuellement nécessaire pour le criblage à haut contenu
Computational phenotyping is an emergent set of technologies for systematically studying the role of the genome in eliciting phenotypes, the observable characteristics of an organism and its subsystems. In particular, cell-based assays screen panels of small compound drugs or otherwise modulations of gene expression, and quantify the effects on phenotypic characteristics ranging from viability to cell morphology. High content screening extends the methodologies of cell-based screens to a high content readout based on images, in particular the multiplexed channels of fluorescence microscopy. Screens based on multiple cell lines are apt to differentiating phenotypes across different subtypes of a disease, representing the molecular heterogeneity concerned in the design of precision medicine therapies. These richer biological models underpin a more targeted approach for treating deadly diseases such as cancer. An ongoing challenge for high content screening is therefore the synthesis of the heterogeneous readouts in multi-cell-line screens. Concurrently, deep learning is the established state-of-the-art image analysis and computer vision applications. However, its role in high content screening is only beginning to be realised. This dissertation spans two problem settings in the high content analysis of cancer cell lines. The contributions are the following: (i) a demonstration of the potential for deep learning and generative models in high content screening; (ii) a deep learning-based solution to the problem of heterogeneity in a multi-cell-line drug screen; and (iii) novel applications of image-to-image translation models as an alternative to the expensive fluorescence microscopy currently required for high content screening

The lack of our understanding about Parkinson’s Disease (PD), the second most common and incurable neurodegenerative disorder, has caused a great impact in both treatment and research for this disease. Although not a few intensive studies have been conducted with the hope of gaining more knowledge about the complex biology underlying this debilitating condition, it seems that our efforts have not completely uncovered the molecular mechanisms related to the neurodegeneration, which is critical in PD. Phenotypic assay, particularly cytological profiling (CP), has recently emerged as a powerful unbiased strategy to study the mechanisms of action in many biological systems. CP is a cell-based and image-based approach that evaluates the effects of small molecules on several organelles within the cell, enabling the assessment of multidimensional phenotypic functions instead of a single interaction that is usually seen in target-based approaches. Previous research in our institute utilizing CP has identified quite a few compounds that interacted with PD patient-derived cells. It is therefore worth believing that CP could be a future approach for combating PD. This project was conducted as part of our ongoing research intending to discover molecules from nature to study the biology of PD. Our aim was to use 1H NMR guidance to discover natural products from a traditional Chinese medicinal plant. Cytological profiling on a patient-derived cell line (human olfactory neurosphere-derived (hONS) cells) was performed for the isolated compounds to determine if they are potentially qualified as chemical probes to serve the research of PD. This thesis will demonstrate the study that integrates chemistry and biology to identify small molecules and the effects they influenced to the cell model, revealing their potential value as molecular tools for a better interrogation of the disease mechanism. The thesis was initiated with an introduction that covered several concepts, including traditional Chinese medicine (TCM), PD, CP, hONS cell model and chemical probes. Starting with TCM – a multi-century health care system in China, the introduction acknowledged that TCM herbal plants are invaluable sources of bioactive small molecules with respect to the traditional experience accumulated for thousands of years. It then moved on to a brief discussion of Parkinson’s Disease ‒ an irremediable neural degenerative disorder, and some current therapeutic treatments for this disease, as well as giving rationale as to why this condition should be researched. The introduction went on with a review on CP and its application in drug discovery, followed by a description of current disease models for PD, as well as the hONS cell model used in this project. It was noted that hONS cells derived from PD patients can recapitulate some functional aspects of this disease, and thus coupling CP with hONS cell model could shed light on the biological studies of PD. The chapter continued with a description of chemical probes, their importance in medicinal research and that these small molecules are valuable tools for interrogating the pathways of PD. Finally, the durability of NMR fingerprinting in identify compounds was demonstrated in the last part of this chapter. Chapter 2 provided details of the equipment and procedures involved in this project to assist the chemical purification and biological characterization of the compounds isolated from the selected herbal plant. Detailed spectroscopic data were also present in this chapter. Chapter 3 first gave in introduction on the selected biota, Macleaya cordata (Willd.) R.Br., including how it was initially chosen in the previous PhD project and its traditional medicinal use. The chapter then moved on to the targeted isolation of natural product from this species by utilizing the robustness of 1H NMR spectroscopy and mass spectrometry (MS), which resulted in the isolation of two new compounds and fourteen known metabolites. The in-depth structural elucidation of the two new compounds, (6R)- 10-methoxybocconoline and 6-(1-Hydroxyethyl)-10-methoxy-5,6-dihydrochelerythrine, were also mentioned in this chapter, followed by the use of density functional theory (DFT) to assign the absolute configuration (AC) of one new molecule. The chapter concluded with discussion on the validity of NMR fingerprinting and future directions on the remaining work which involved AC assignment for the other new compound. Chapter 4, the final chapter, presented the intensive data analysis for the CP screening of the isolated compounds. It was revealed that four out of sixteen metabolites showed significant perturbations to the hONS cellular parameters. Those compounds included bocconoline which impacted EEA1- and mitochondria-associated features, 6- (1-hydroxyethyl)-5,6-dihydrochelerythrine, 3-O-feruloylquinic acid and ferulic acid 4-Oglucoside which influenced LC3b-related features. These compounds can potentially be used as molecular tools to probe the biological pathways of PD. The last part of the chapter was the discussion on the CP platform used to identify the above potential probes, as well as future directions on the biology cohort. It was concluded that more justifications should be made in order to verify the potency of the compounds being qualified as useful probes. Dose dependence, mechanisms of action and drug-like properties for the candidate probes were among the future follow-up research to have a better understanding in the exploration of complex molecular mechanisms underlying PD.
Thesis (Masters)
Master of Science (MSc)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Abstract Phenotypic characterization of novel antivirals for the treatment of multidrug resistant HIV-1 and emerging viruses Doctoral Research School of Medical Biotechnologies – Cycle XXXV Supervisor: Maurizio Zazzi; Candidate: Federica Giammarino Background The need for new antiviral drugs has increased overtime due to the worldwide circulation of different viruses together with the increased frequency and diversity of new outbreaks. The ideal option for a prompt response against both emerging and re-emerging viruses is represented by the use and the development of direct acting antiviral agents. During my PhD I was involved in several projects focused on the evaluation of the antiviral activity of licensed and investigational antiviral drugs against Human Immunodeficiency (HIV-1), West Nile (WNV), Dengue (DENV) and SARS-CoV-2 viruses. Results and discussion Doravirine The antiviral activity of the NNRTI doravirine was evaluated against viruses harbouring different patterns of NNRTI resistance mutations in two studies. Globally, our data confirmed that the antiviral activity of doravirine may be compromised by the presence of multiple NNRTI resistance mutations, even in the absence of specific doravirine mutations. A third study was focused on the role of the natural polymorphism of the reverse transcriptase V106I. Our results indicate that it minimally affects the susceptibility to doravirine in clinical isolates and that it does not impact the genetic barrier to resistance as compared to reference wild-type virus, while viruses including the NNRTI resistant mutation V106A or V106M rapidly showed viral breakthrough under doravirine pressure due to the reduced susceptibility. Islatravir Our study confirmed the decrease of susceptibility of the investigational NRTTI islatravir due to the presence of M184V mutation. The clinical impact of NRTI mutations in the activity of islatravir has still to be defined and the threshold of fold-change values associated to reduced activity in vivo remains to be established. Ibalizumab The combinatorial activity of ibalizumab together with other antivirals, both approved and investigational, was evaluated through a newly developed cell-based assay consisting in the infection of the MOLT4-R5 cell line with the wild-type strains NL4-3 and AD8, and by the analysis of the results using the innovative software SynergyFinderPlus. Our data suggest that ibalizumab positively interacts with other antivirals with possible synergistic effects in select cases. Further studies are needed to determine the impact of Env variability and viral tropism in combination with other entry inhibitors. Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication An easy-to-perform and fast flavivirus immunodetection assay (IA) was developed to determine antiviral activity of promising compounds against ZIKV and DENV. The system, validated with references compounds against both viruses, was able to distinguish between the inhibitory effect of molecules targeting the early and the post-budding phase of viral replication cycle. Evaluation of sofosbuvir activity and resistance profile against West Nile virus in vitro Since the activity of sofosbuvir has been documented against different flaviviruses, we investigated whether it may exert an activity also against WNV. In both cell-based and enzymatic assays sofosbuvir was able to inhibit WNV replication in the low micromolar range. Moreover, in vitro selection and molecular docking experiments indicated that HCV and WNV share a similar sofosbuvir resistance pattern. ORIGINALE CHEMIAE in Antiviral Strategy - Origin and Modernization of Multi-Component Chemistry as a Source of Innovative Broad Spectrum Antiviral Strategy The “ORIGINALE CHEMIAE in Antiviral Strategy” project aims to identify promising broad-spectrum antivirals by taking advantage of the Multi-Component Chemistry strategy. Following the synthetization of molecules, their antiviral activity was determined in in vitro standardized virus-cell systems against DENV, WNV, HIV-1 and SARS-CoV-2. We identified eight molecules able to inhibit at least one of the viruses tested. However, their low selectivity indexes indicate the need to further improve the design of these molecules to increase the antiviral activity and/or reduce the cell toxicity in order to identify candidates for preclinical testing in animal models. Monoclonal antibodies and antivirals vs. SARS-CoV-2 After the development of a quantitative live-virus microneutralization assay, we evaluated the efficacy of licensed monoclonal Antibodies (mAbs) and the antiviral drugs remdesivir, nirmaltrevir and molnupiravir against different circulating SARS-CoV-2 variants. Our results showed that these drugs, contrary to the mAbs, retained activity against all tested variants. Conclusions A continuous challenge for public health is represented by the control of viral infections. Both vaccines and antiviral drugs may synergistically help to reduce the spread and the fatality of acute viral diseases and chronic infections. All the studies described in this thesis emphasize the role of the laboratory of virology within all the steps of the in vitro investigation of antiviral drugs, from the identification of molecules endowed with antiviral activity to the definition of the mechanism of action.

La terapia antiretrovirale purtroppo può indurre la selezione di ceppi virali resistenti ai farmaci, che possono essere trasmessi ad altre persone. In questo studio è stato messo a punto un saggio fenotipico per comprendere la dinamica evolutiva delle resistenze trasmesse, confrontando la capacità replicativa (RC), e quindi il potenziale epidemico, conferiti dai geni della proteasi (PR) e della trascrittasi inversa (RT) di ceppi virali provenienti da pazienti con infezione recente. Inoltre il saggio fenotipico è stato impiegato per studiare l'influenza del background genetico del gene dell’integrasi (IN) nella selezione di uno specifico percorso di resistenza. I geni dei pazienti sono stati clonati in specifici cloni molecolari appartenenti alla famiglia dei pNL4-3mod dotati di gene reporter (Green Fluorescent Protein) e cassetta di clonaggio per i geni menzionati, provenienti dal virus dei pazienti. La RC è stata valutata misurando la fluorescenza emessa da cellule infettate in coltura. Nelle infezioni recenti (quindi con resistenze trasmesse), gli isolati con mutazioni di resistenza nella RT hanno mostrato una RC paragonabile agli isolati non mutati e potrebbero avere lo stesso potenziale epidemico. È interessante notare che gli isolati in tutte le infezioni recenti hanno mostrato una RC significativamente inferiore rispetto agli isolati nelle infezioni non trattate di vecchia data, suggerendo un ruolo attivo del sistema immunitario, in maggiore salute nelle infezioni recenti, nella selezione di virus con una RC inferiore. I dati inoltre hanno rilevato che l’impatto sulla RC delle mutazioni di resistenza nella PR è maggiore rispetto a quello delle mutazioni nella RT. Per quanto concerne l’IN, la combinazione G140S/Q148H in tutti i cloni ha determinato una RC più alta rispetto agli altri percorsi di resistenza, suggerendo che il background genetico del gene IN non è determinante per la selezione di specifici percorsi di resistenza osservati in vivo.
Antiretroviral therapy induces the selection of HIV drug resistance strains, which occasionally are transmitted to other patients. A recombinant phenotypic assay was developed to understand the evolutionary dynamic of transmitted drug resistance, analyzing the replicative capacity (RC) conferred by protease (PR) and reverse transcriptase (RT) of viral strains from recently infected patients. In addition, it was used to investigate the influence of the viral genetic background of the integrase (IN) gene in selecting a specific resistance pathway, comparing the RC of the resistance mutations spontaneously selected in vivo to those of alternative resistance pathways introduced by site-direct mutagenesis. Selected genes were cloned in the pNL4-3mod HIV molecular clone, containing the GFP reporter gene and a cloning cassettes for the mentioned genes, which allow the cloning of the respective genes from clinical HIV strains. The RC was evaluated by measuring the fluorescence in cells infected in vitro. In recent infections, isolates bearing RT resistance mutations showed a RC comparable to that of wild type isolates and might have the same epidemic potential. Interestingly, wild type isolates from recent infections displayed a significantly lower RC than isolates from non-recent infections, suggesting an active role of the immune system (in greater health in recent infections) in selecting virus with lower RC. The data also revealed that the impact of PR resistance mutations on RC is much deeper than that of RT resistance mutations. Concerning the IN gene, clones with the G140S/Q148H combination showed a higher RC, compared to alternative mutational pathways, suggesting that the genetic background of HIV IN gene at baseline is not determinant for the selection of the naturally observed INSTI mutations. The results also confirmed that dolutegravir (DTG) has a high barrier against the development of resistance providing an explanation for its higher clinical efficacy.

Clinical trials seeking to improve cognitive performance are in dire need of biochemical biomarkers that reflect the processes taking place in the brain. In Down syndrome, this information is crucial to understand the progression of cognitive decline, or to evaluate the beneficial effects of a treatment, but there are few available biomarkers for this condition. We have analyzed the association between biomarkers (biochemical, genetic), executive functions, and cognitive decline, in the context of a clinical trial, taking into account extrinsic factors (education, diet) that may obscure the interpretation of the obtained results. We found that altered lipid profile or increased homocysteine concentrations in plasma are associated to worse executive functions. Additionally, increased plasma concentrations of amyloid peptides are associated to early dementia signs. I also optimized a new technique of tissue and cell culture to obtain neuronal precursors from the nasal olfactory epithelium for biomarker studies. The results of this study should provide with new tools to evaluate treatment efficacy and cognitive decline risk in the context of clinical trials in Down syndrome.
Els assajos clínics que cerquen la millora del rendiment cognitiu tenen la necessitat de disposar de biomarcadors que reflecteixin els processos que tenen lloc en el cervell. En la síndrome de Down aquesta informació és crucial per a entendre la progressió del declini cognitiu o per a avaluar els efectes beneficiosos d’un tractament però encara hi ha pocs biomarcadors disponibles per a aquesta afectació. Hem avaluat l’associació entre biomarcadors (bioquímics, genètics), funcions executives i declini cognitiu, en el context d’un assaig clínic, tenint en compte factors extrínsecs (educació, dieta) que poden afectar la interpretació dels resultats. Els nostres demostren que un perfil lipídic alterat o unes concentracions incrementades d’homocisteïna en plasma estan associats a pitjors funcions executives. També observem una associació entre concentracions incrementades de pèptids amiloides i signes primerencs de demència. També he optimitzat una nova tècnica de cultiu tissular i cel·lular per a obtenir precursors neuronals de l’epiteli olfactiu per a l’estudi de biomarcadors. Els resultats d’aquest estudi podrien proporcionar noves eines per a avaluar l’eficàcia de tractament i el risc de declini cognitiu en el context d’assaigs clínics en la síndrome de Down.

Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.

Yersinia ruckeri is the causative agent of enteric redmouth (ERM) disease in farmed salmonids. ERM disease is traditionally associated with rainbow trout (Oncorhynchus mykiss¸ Walbaum), but the incidence of the disease in Atlantic salmon (Salmo salar) has increased in recent years. Historically, motile (biotype 1), serotype O1 isolates of Y. ruckeri have been mostly responsible for ERM in rainbow trout worldwide but non-motile (biotype 2), serotype O1 isolates have become increasingly prevalent in this species over wide geographic areas since their first isolation in the UK in the 1980s. Yersinia ruckeri isolates responsible for infection of salmon have been less well characterised than those from rainbow trout and little is known about their diversity. The emergence of new pathogenic strains, together with vaccine breakdown in the field, has emphasised the need for greater knowledge about strain diversity which may lead to the development of improved vaccines for both species. In the present study, a unique and extensive strain collection encompassing 135 isolates of Y. ruckeri were characterised using complementary phenotypic, proteomic and genomic approaches. In the initial part of this thesis, 135 isolates recovered over a 14 year period in the UK from infected Atlantic salmon (109 isolates) and rainbow trout (26 isolates) were phenotypically characterised through biotype, serotype, and outer membrane protein (OMP) -type analysis. Atlantic salmon isolates represented a wider range of O-serotypes and associated lipopolysaccharide (LPS) types, and had more diverse OMP profiles than those from rainbow trout. Most significantly, a new O-serotype/LPS type was identified in 56 Atlantic salmon isolates; other Atlantic salmon isolates were represented by serotypes O1 (five isolates), O2 (34 isolates) and O5 (14 isolates). This new LPS type comprises a core polysaccharide region similar to that of serotype O1 but has a unique, previously unidentified O-antigen region. Atlantic salmon isolates could be assigned to one of four major OMP-types and to one of 11 OMP-sub types. Isolates recovered from rainbow trout were represented by the same non-motile clone that is responsible for the majority of ERM outbreaks in this species within the UK. This clone was not associated with any infected salmon. However, two isolates of the novel serotype O1/O5 were recovered from rainbow trout in 2010 and 2011. These data suggest that different Y. ruckeri strains are specifically adapted to cause disease in either Atlantic salmon or rainbow trout. The efficacy of current vaccine formulations against different clonal groups must be examined. Subsequently, an in-depth characterisation of the outer membrane (OM) proteome of isolates recovered from Atlantic salmon and rainbow trout was conducted. Outer membrane proteins are at the interface of host pathogen interactions, with important roles in adherence, evasion of host immune response, and transport. Using a bioinformatic prediction pipeline and four publicly available genomes, 141 proteins were confidently predicted to be OM associated. Subsequently, the OM proteomes of eight representative isolates (four from rainbow trout; four from Atlantic salmon) were analysed using a combination of gel-based and gel-free proteomic approaches. In total, 66 OMPs were identified through this combined approach, of which 28 were unique to the gel-free approach and 13 were unique to the gel-based approach. Further to this, the OM proteomes of these eight representative isolates were examined when cells were grown under conditions that aimed to mimic the in vivo and environmental conditions of Y. ruckeri. These included growing cells aerobically at 22°C and 28°C, anaerobically, under iron-depletion and in an artificial seawater medium at 22°C. In total, 76 OMPs were identified in all eight isolates under these growth conditions. Finally, a phylogenetic study was undertaken whereby the genomes of 16 representative isolates encompassing a range of biotypes, serotypes, host species (eight from rainbow trout, seven from Atlantic salmon and one from European eel), geographic locations and dates of isolation were considered. A phylogenetic species tree based on the concatenated sequences of 19 housekeeping genes revealed host specific lineages suggesting an earlier host-associated evolutionary split within Y. ruckeri. Subsequent analysis of the presence, absence and variation of the nucleotide and amino acid sequences of the 141 predicted OMPs revealed high levels of conservation (with 120 OMPs showing less than 1% nucleotide variation). One hundred and thirty proteins were identified in all 16 genomes examined. However, 11 proteins were not, and these included invasins, OmpE and proteins involved in pilus biogenesis. Further examination of the OMPs OmpA and OmpF, which were identified in the genomes of all 16 isolates, revealed variation in the surface exposed loop regions which may play a role in pathogenicity and/or host specificity. This study represents a comprehensive characterisation of Y. ruckeri isolates recovered from Atlantic salmon and rainbow trout using a range of molecular techniques, and reveals important adaptations that the bacteria may make in order to survive both inside and outside of the host. Importantly, this study provides comprehensive support for future work involving this fish pathogen.

Le cancer de la prostate (CaP) a l'incidence la plus élevée parmi les cancers masculins dans les pays européens et américains. Dans les stades avancés de la maladie, le développement des métastases (osseuses dans 80% des cas) entraîne un taux de mortalité important. Le récepteur MET et les fusions de gènes ETS avec un promoteur hormono-dépendant sont des acteurs importants dans la progression du cancer de la prostate. Parmi les membres de la famille de facteurs de transcription ETS, ERG est retrouvé dans 60% des cas des fusions et ETV1 dans 10% des cas. MET est un récepteur à activité tyrosine kinase exprimé dans les stades avancés de la maladie, dans les tumeurs hormono-résistantes et les métastases osseuses. Les fusions ERG et ETV1 sont retrouvées tout au long de la maladie, allant des stades d'initiation jusqu'aux stades métastatiques. De façon intéressante, il existe de nombreux liens fonctionnels entre MET et ERG/ETV1 suggérant leur appartenance à la même voie de régulation. Le but de notre étude est de comprendre le rôle individuel du récepteur MET et des fusions ETS ainsi que leur collaboration dans la progression du CaP.Pour ce faire, nous avons mis en place des modèles cellulaires de CaP hormono-indépendants dans lesquels l'expression et l'activité du récepteur MET sont effectives et pour lesquels une surexpression de ERG ou ETV1 a été induite via une infection rétrovirale. Ces modèles ont été utilisés pour réaliser des tests phénotypiques de prolifération, migration ou encore invasion, une analyse transcriptomique comparative (RNAseq) et des tests in vivo chez des souris humanisées exprimant l'HGF humain.Les résultats que nous avons obtenus montrent que les facteurs de transcription ERG et ETV1 induisent des capacités migratoires et invasives plus importantes in vitro et que l'activation de la voie de signalisation du récepteur amplifie les effets. In vivo, ERG et ETV1 entraînent des volumes tumoraux plus importants après injection des cellules en sous-cutanée et l'application d'un traitement par un inhibiteur spécifique de MET réverse ces effets. Finalement, la réalisation d'une analyse transcriptomique, comparant les différents modèles, a permis d'identifier des gènes différentiellement exprimés selon la surexpression de ERG, de ETV1 et/ou de l'activation de la voie MET, gènes cibles signature pouvant potentiellement être impliqués dans la progression tumorale.Ainsi, les données obtenues montrent pour la première fois une collaboration entre le récepteur MET et les facteurs ERG/ETV1 pour induire des caractéristiques plus agressives dans des modèles de CaP. A terme, le projet vise à identifier des signatures moléculaires de cette coopération afin de mettre en lumière des outils de pronostic, diagnostic ou encore de thérapie ciblée
Prostate cancer (PCa) has the highest incidence of all male cancers in Europe and the USA. In the advanced stages of the disease, the development of metastases (bone metastases in 80% of cases) leads to a high mortality rate. MET receptor and ETS gene fusions with a hormone-dependent promoter are important actors in the progression of prostate cancer. Among the members of the ETS family of transcription factors, ERG is found in 60% of fusions and ETV1 in 10%. MET is a tyrosine kinase receptor expressed in the advanced stages of the disease, in hormone-resistant tumours and in bone metastases. ERG and ETV1 fusions are found throughout the disease, from initiation to metastatic stages. Interestingly, there are many functional links between MET and ERG/ETV1, suggesting that they belong to the same regulatory pathway. The aim of our study is to understand the individual roles of MET receptor and ETS fusions and their collaboration in PCa progression.To this end, we built hormone-independent CaP cellular models in which MET receptor expression and activity are effective and ERG or ETV1 overexpression has been induced via retroviral infection. These models were used to perform phenotypic tests of proliferation, migration and invasion, comparative transcriptomic analysis (RNAseq) and in vivo tests in humanised mice expressing human HGF. The results we obtained show that the transcription factors ERG and ETV1 induce greater migratory and invasive capacities in vitro and that activation of the receptor signalling pathway amplifies the effects. In vivo, ERG and ETV1 induce larger tumour volumes after subcutaneous injection of the cells, and treatment with a specific MET inhibitor reverses these effects. Finally, a transcriptomic analysis comparing the different models, permits to identify genes differentially expressed according to overexpression of ERG, ETV1 and/or activation of MET pathway, signature target genes potentially involved in tumour progression.The data obtained show, for the first time, a collaboration between MET receptor and ERG/ETV1 factors to induce more aggressive characteristics in PCa models. The project aims to identify the molecular signatures of this cooperation in order to highlight prognostic, diagnostic and targeted therapy tools

The development of specific antiviral drugs directly targeting the hepatitis C virus (HCV) is clinically important, as the current standard interferon/ribavirin combination treatment is only partially effective, expensive and often associated with severe side effects. Inhibitors of the NS3 protease (PI) therefore represent a promising alternative or additional therapy. To date, the development and in vitro evaluation of PIs is restricted to the genotype 1/2 based replicon and the genotype 2a full length viral cell culture system. However, proteases of the different HCV genotypes vary substantially in their amino acid sequence and secondary structure and require separate evaluation of their efficacy before they go into clinical trials. To address this issue, a panel of intra- and intergenotypic recombinants based on the recombinant infectious clone Jc1 (pFK JFH1/J6/C-846) was developed in this work. The viability of these recombinants was assessed in the Huh7.5 cell culture system, where replicating viruses were detected by HCV-NS5A immunostaining. Intergenotypic recombinants containing genotype 1a, 1b, 3a, 4a and 6a derived proteases were replication defective, whereas the recombinant with genotype 5a derived protease replicated efficiently after acquiring cell culture adaptive mutations. The replacement of not only the NS3 protease gene region, but also its cofactor NS4A, allowed the generation of replication competent intra- and intergenotypic recombinants for all 6 major genotypes. Replacing the NS3 protease of the recombinants with that of patientderived proteases also generated replicating recombinants, greatly expanding the panel of intergenotypic recombinants available for phenotyping and PI evaluation. However, intra- and intergenotypic recombinants showed substantial differences in their replication kinetics, which may be influenced by naturally occurring polymorphism between genotypes and the differential requirement of adaptive/attenuating cell culture mutations. Genotype 1a recombinants replicated very poorly, which may be due to incompatibilities between the type 1a NS3/4A protease and the type 2a backbone. 50% inhibitory concentrations (IC50) of different PIs were measured using Foci Forming Units/ml (FFU/ml) reductions and replication inhibition assays. The different recombinants showed consistent, genotype-associated differences in their susceptibility to the PI BILN 2061, with genotypes 2a, 3a and 5a derived recombinants showing approximately 100-fold lower susceptibility than genotype 1b, 4a and 6a derived recombinants. These observations are consistent with major differences in response rates found in recent treatment trials of genotype 1, 2 and 3 infected patients. Differences in susceptibility were also observed for VX-950, with genotype 1b, 2a and 6a derived recombinants being twice as susceptible than genotype 3a, 4a and 5a derived recombinants. Passaging the intra- and intergenotypic recombinants under increasing concentrations of PI allowed the identification of PI resistance mutations. Resistance mutations to BILN 2061 mapped to the previously identified positions 156 and 168 within the NS3 protease, with a great diversity of amino acid substitutions observed within each genotype. Reintroduction of the identified resistance mutations into the original recombinant viruses conferred increased resistance towards BILN 2061 and some mutations also affected replication kinetics of the recombinants. The developed system will be of major value for the phenotypic characterisation of naturally occurring and treatment induced resistance mutations within all 6 major HCV genotypes towards different PIs. This will allow treatment response predictions for newly developed PIs before they enter clinical trials and the development of individually tailored antiviral treatment regimes.

La compréhension des mécanismes moléculaires qui permettent l'adaptation rapide des mollusques vecteurs de parasites à de nouveaux environnements est importante pour le contrôle des maladies. L'adaptation rapide est difficile à expliquer par la génétique mendélienne traditionnelle et il existe des preuves solides qui soutiennent que les mécanismes épigénétiques sont à l'origine des adaptations rapides chez plusieurs espèces. Je me suis focalisée sur une marque épigénétique appelée la méthylation de l’ADN, qui est modulée par l'environnement et joue un rôle dans la plasticité phénotypique chez de nombreuses espèces, principalement les plantes et les vertébrés. Néanmoins, le rôle de la méthylation de l'ADN dans la génération de variations phénotypiques chez les invertébrés a été très peu étudié. J'ai abordé la question du rôle de la méthylation de l'ADN dans la génération de la plasticité phénotypique et de son héritabilité chez l'escargot B. glabrata, l'hôte intermédiaire du parasite Schistosoma mansoni, l'agent pathogène de la schistosomiase, une maladie tropicale négligée. La méthylation de l'ADN chez B. glabrata est régulée par l'infection3du parasite S. mansoni et par le stress environnemental, de plus, il a été démontré que la méthylation de l'ADN affecte son expression génique, suggérant que la méthylation de l'ADN peut affecter la variation phénotypique et donc l'adaptation de l'escargot à de nouveaux environnements. Pour étudier le rôle de la méthylation de l'ADN dans la génération de la variation phénotypique, une manipulation expérimentale de la méthylation de l'ADN chez l'escargot était nécessaire. Par conséquent, deux approches ont été proposées dans cette thèse pour introduire des épimutations chez l'escargot B. glabrata: 1) Épi-mutagenèse aléatoire en utilisant des inhibiteurs chimiques des enzymes ADN methyltransferases (DNMT) et par ségrégation conséquente des épimutations dans des lignées d'autofécondation et 2) Par la méthylation des cytosines d'un locus ciblé avec un outil d'édition épigénétique qui consiste à l'utilisation d'une vecteur plasmidique codant pour l’ADN méthyltranférase (DNMT3) fusionnée avec l’enzyme dCas9 (Cas9 avec l’activité nucléase désactivé). Pour l’approche d’épimutagenèse aleatoire, un nouvel inhibiteur des enzymes DNMT a montré des effets d’inhibition de la méthylation dans deux générations consécutives, en montrant un effet épigénétique multigénérationnelle et sans montrer d’effet toxique ni dans la survie ni dans la fécondité de l’escargot B. glabrata. De plus l’inhibiteur Flv1 a montré être efficace dans deux autres espèces de mollusques, l’escargot d’eau douce Physa acuta et l’huître creuse Crassostrea gigas, ce qui suggère que cet inhibiteur représente un potentiel outil moléculaire pour moduler la méthylation de l’ADN chez d’autres mollusques. Dans le cas de l’approche ciblée, j’ai utilisé une méthode de transfection qui permet d’introduire deux vecteurs plasmidiques avec un promoteur viral SV40 de façon in vivo dans des embryons de l’escargot B. glabrata. La transfection a été effectuée au stade gastrula, ce qui a entrainé une incorporation mosaïque du vecteur dans les cellules transfectées. Toutefois, la méthode a permis de méthyler certains sites CpG du gène ciblé
The understanding of the molecular mechanisms that allows the rapid adaptation of mollusks that are vector of parasites, to new environments is important for disease control. Rapid adaptation is difficult to explain by traditional Mendelian genetics and there is strong evidence supporting that epigenetic mechanisms, are behind rapid adaptations in other species. I studied one epigenetic mark called DNA methylation that has demonstrated to be environmentally modulated and to play a role in phenotypic plasticity in many species, principally plants and vertebrates. Nevertheless, the role of DNA methylation in generating phenotypic variation in invertebrates has been poorly studied. I addressed the question of the role of DNA methylation in the generation of phenotypic plasticity and its heritability in the snail Biomphalaria glabrata, the intermediate host of the parasite Schistosoma mansoni, the causal agent of schistosomiasis, a neglected tropical disease. DNA methylation in B. glabrata has been found to be modulated by the infection of the parasite S. mansoni and by environmental stress, furthermore, it was demonstrated that DNA methylation affects its gene expression, suggesting that DNA methylation can affect phenotypic variation and therefore the adaptation of the snail to new environments. To study the role of DNA methylation in the generation of phenotypic variation, experimental manipulation of the DNA methylation in the snail was necessary. Therefore, two approaches were proposed in this thesis to introduce epimutations in the snail B. glabrata: 1) Random epi-mutagenesis using chemical DNA methyltransferase (DNMT) inhibitors and by consequent segregation of epimutations in self-fertilization lines and 2) Methylate the cytosines of a targeted locus with a targeted epigenome editing tool consisting in the use of the DNA methyltransferase (DNMT3) construct fused to the nuclease-inactivated dCas9. For the random epi-mutagenesis approach, a novel DNMT inhibitor has shown methylation inhibiting effects in two subsequent generations, showing a2multigenerational epigenetic effect and without showing toxic effects in either survival nor fecundity of the snail B. glabrata. In addition, the inhibitor Flv1 has been shown to be effective in other two mollusk species, the freshwater snail Physa acuta and the pacific oyster Crassostrea gigas, which suggests that this inhibitor represents a molecular tool to modulate the methylation of DNA in other mollusks. In the case of the targeted epimutagenesis approach, I used a transfection method that allows introducing two plasmid vectors with an SV40 viral promoter in vivo in embryos of the snail B. glabrata. The transfection was performed at the gastrula stage, which resulted in mosaic incorporation of the vector into the transfected cells. However, the method was able to methylate some CpG sites of the targeted gene

Fluoxetin är den aktiva substansen i många serotoninreglerande läkemedel som förs in i vattendrag. Substansen har visats påverka beteende av vattenlevande organismer som fiskar, mollusker och kräftdjur genom att öka deras djärvhet. I denna studie undersöktes fluoxetins akuta (på vildfångade individer) och kroniska (på labbuppfödda individer) effekter av koncentrationerna 0, 3 och 30 ng L-1 på Asellus aquaticus (sötvattengråsugga) antipredatorbeteende. Detta gjordes genom tre beteendetest: (1) tid att lämna refug, (2) spontan aktivitet samt (3) flyktbeteende under predationsrisk. Överlag hittades få eller inga effekter på A. aquaticus från fluoxetin. De effekter som dock påverkade individer signifikant visade att exponerade individer flydde en signifikant kortare (30 %) tidsperiod från en simulerad predatorattack. Utöver denna huvudeffekt av fluoxetin hittades även signifikanta skillnader i fluoxetins påverkan på de två grupperna, när individer blev utsatta för den högsta koncentrationen ökade vildfångade individer sin aktivitet (38 % fler stopp och 49 % mer rörelse) medan labbuppfödda individer sänkte sin aktivitet (43 % färre stopp och 37 % mindre rörelse). Individer som inte var exponerade visade signifikanta skillnader i alla beteendetest för de två grupperna. Det är troligt att beteendeskillnader är en följd av olika uppfödningsmiljöer, dock går det inte att utesluta att ändrade genfrekvenser uppkommit. Studien lyser sken på behovet av fler studier av långtidsexponering av läkemedelsrester, de är sällan akut giftiga men har däremot subletal påverkan i låga doser.
Fluoxetine is the active substance in many selective serotonin reuptake inhibitive pharmaceuticals that currently enters surface waters. The substance has been shown to affect behaviors of water living organism such as fish, molluscs and crustaceans by making them less cautious. This study investigated the acute (on wild caught individuals) and chronic (on lab reared individuals) effects of fluoxetine on the antipredator behavior of Asellus aquaticus for three concentrations; 0,3 and 30 ng L-1. Three tests were used to determine the effects: (1) time to leave a shelter, (2) spontaneous activity and (3) escape behavior under predation risk. Few statistically significant effects of fluoxetine on A. aquaticus were found. However, individuals exposed to fluoxetine had a significantly shorter (30 %) escape period. Besides this main effect of fluoxetine, significant interactions between the two groups and fluoxetine were also found. When exposed to the highest concentration wild caught individuals increased their spontaneous activity (38 % more stops and 49 % more movement), while lab reared individuals reduced their activity (43 % fewer stop and 37 % less movement). Furthermore, non-exposed individuals from the two groups behaved significantly different in all the tests. It is likely that the differences in behavior occurred due to environmental effects of laboratory rearing, although altered gene frequencies cannot be excluded. This study emphasizes the need for development of methods for more chronic testing of pharmaceuticals, especially considering that pharmaceuticals are seldom acutely toxic but often has sub lethal effects in low doses.

Cereals are the basis of the normal diet in most Mediterranean countries and it is estimated that they account for 35-50% of the regional populations’ dietary energy consumption. Water deficit is the main constraint limiting cereal productivity in the Mediterranean regions. Crop management and breeding may improve the performance of cereals under such stress conditions. However, the lack of efficient tools to monitor the performance of agronomical practices or to undertake appropriate phenotyping in breeding programs limits the efficiency of both avenues. Novel tools to monitor the cereals (durum wheat and maize) performance in the thesis are: 1. Stable isotopes In C3 plants the carbon isotope composition (δ13C) measured in plant tissues is considered as one of the most promising secondary traits in wheat (and other C3 cereals) when breeding for drought resilience. The δ13C has been reported to negatively correlate with Ci/Ca (the ratio of leaf intercellular to ambient CO2 concentration) and positively correlated with A/E (the ratio of net assimilation to water evaporated from the transpiring organs). Therefore, the δ13C is positively related to WUE, which is considered as the biomass produced per unit of water transpired. Correlations between δ13C and GY and/or aerial biomass (AB) may be either negative or positive according to the plant tissue sampled and environmental conditions tested. In the case of a C4 plant like maize, variations in the δ13C in response to water conditions are small in compared to C3 plants like wheat, but they are still adequate for use in maize as an indicator of water conditions during growth. The oxygen isotope composition (δ18O) of plant tissues is known to reflect the evaporative conditions throughout the crop cycle and thus it has been proposed as a proxy method for measuring transpiration as well as an indicator of genotypic differences in stomatal conductance (gs) in C3 and C4 plants. 2. Root traits Plant roots are the key organs in the plant responsible for the absorption of water and nutrients. Concerning root traits, their response to drought stress is still a challenging subject for research. The laborious work required for the study of the root system has prevented the adoption of root characteristics as routinely phenotyping traits for crop breeding. The root weight density (RWD) and root length density (RLD) are frequently used in root studies to describe the root weight and root length, respectively, within a soil volume and they reflect the capacity of roots to extract water and nutrients. The specific root length (SRL) is considered another of the most important and commonly measured morphological traits. Previous studies showed that a high SRL facilitates nutrient uptake in low-nutrient environments and makes plants more competitive for soil nutrient uptake. 3. The use of proximal (remote) sensing The assessment of AB is important for monitoring crop growth because it could reflect the effect of stresses on crop growth and senescence. Thus, a number of studies have revealed that spectral reflectance techniques have the potential to provide precise, non-destructive instantaneous quantitative estimates of AB. The Normalized Difference Vegetation Index (NDVI) has been used as an indicator of AB and GY in cereals. In recent years the use of digital Red-Green-Blue (RGB) images has been proposed as an alternative to develop vegetation indices that may replace spectroradiometrical based NDVI. The price, size, and the easy use of conventional digital cameras make them viable alternatives to assess AB and GY in cereals. A number of studies have used digital RGB imaging to measure different colour parameters such as: greenness; intensity of green, red and blue; and derived normalized indices from the green, red and blue bands.
Los cereales son la base de la dieta normal en la mayoría de los países del Mediterráneo y se estima que representan del 35 al 50 % del consumo de energía alimentaria de las poblaciones de la región. La falta de agua, a veces acompañada con una baja disponibilidad de nitrógeno, es el principal limitante de la productividad de los cereales en las regiones Mediterráneas. Una mejora genética y un manejo de cultivos más eficientes pueden mejorar el rendimiento de los cereales en estas condiciones de estrés. Sin embargo, la falta de herramientas eficaces para monitorear el estado fisiológico del cultivo, ya sea para su empleo en manejo agronómico, como herramientas de fenotipeado asociado a la mejora genética o incluso para la predicción del rendimiento limita la agricultura mediterránea. La composición en isótopos estables de carbono (δ13C) medida en los tejidos de la planta se considera como uno de los rasgos secundarios más prometedores del trigo (y otros cereales del C3 y C4) en la mejora genética para resistencia a la sequía. Las correlaciones entre la δ13C y el rendimiento en grano o la biomasa aérea pueden ser negativas o positivas debido las condiciones medioambientales del ensayo. Las raíces de las plantas son los órganos clave de la planta responsables de la absorción de agua y nutrientes. Cómo la arquitectura de la raíz responde a la sequía y que rasgos de las raíces son claves continúa siendo un área de investigación que no está cerrada. El laborioso trabajo requerido para el estudio del sistema radicular ha impedido determinar que rasgos de la raíz pueden emplearse como criterios de fenotipado en mejora genética de cultivos. La evaluación de la biomasa aérea es importante para monitorear el crecimiento del cultivo porque podría reflejar el efecto de las diferentes condiciones de estrés en el crecimiento y la senescencia del cultivo. Así, una serie de estudios han reportado que las técnicas de reflectancia espectral/ imágenes digitales rojo-verde-azul (RGB) tienen el potencial de facilitar una estimaciones cuantitativas, instantáneas, no destructivas y precisas de la biomasa aérea.

The researcher has a daughter who was born with an encephalocele and her neuropsychological assessment indicates a Nonverbal Learning Disability (NLD). The difficulties of the educational experiences that emerged over time, mainly because her learning profile was not understood, prompted reflection on the consequences for other students who present with this profile. A concern for the long-term implications for students and parents of the frequent misunderstandings of the NLD has inspired this study. A review of the literature suggested a need to raise educator awareness about the subtle but disabling nature of the NLD syndrome. This study explored the perceptions of teachers, teacher aides and parents involved with 5 students who showed hallmark signs of an NLD. The theoretical foundation rests in the understanding that a student's learning experiences are influenced by past and present school experiences, the attitudes of peers, and parental expectations. The purpose of this thesis is to help parents, teachers and others appreciate the school experiences of children at Level 1 risk of developing an NLD, those with a hydrocephalic condition. It does not purport to offer ultimate solutions or to contribute to diagnosis but rather to act as a starting point for a body of theory to guide development of suitable learning environments for such children. Of further importance is emphasis on the need for similar studies to be conducted into the learning experiences of other children who demonstrate specific syndromes or mosaic forms of those syndromes. Naturalistic Inquiry methodology was used to explore the educational experiences of five students who attended different Australian schools. After completion of all interviews, psychological testing assessed general intelligence and the NLD status of each student. All students were found to be severely learning disabled and all were high on the NLD parameter. Educators generally did not reveal understanding of the NLD syndrome "Nonverbal, what is it? So is it a visual ..." Some teachers devised innovative strategies to help the student cope in class while others expressed frustration ... if the traditional instruction "doesn't work either, what does?" What stood out was an absence of understanding about nonverbal deficits. Frustration about poor organisation, decision making, task completion and problem-solving was expressed and a mixture of concern and criticism was levelled at social incompetence. Students who could not work independently were perceived by some teachers and aides as "lazy" or "molly-coddled" and problems with everyday living skills were sometimes blamed on the student's family. Findings revealed a compelling need to raise educator awareness about the range of cognitive, learning and social problems associated with shunted hydrocephalus and spina bifida. They also highlighted a need for teachers to question "Why can't this student do things one would expect they could do" and demand answers that explicate the serious difficulties being experienced.

The researcher has a daughter who was born with an encephalocele and her neuropsychological assessment indicates a Nonverbal Learning Disability (NLD). The difficulties of the educational experiences that emerged over time, mainly because her learning profile was not understood, prompted reflection on the consequences for other students who present with this profile. A concern for the long-term implications for students and parents of the frequent misunderstandings of the NLD has inspired this study. A review of the literature suggested a need to raise educator awareness about the subtle but disabling nature of the NLD syndrome. This study explored the perceptions of teachers, teacher aides and parents involved with 5 students who showed hallmark signs of an NLD. The theoretical foundation rests in the understanding that a student's learning experiences are influenced by past and present school experiences, the attitudes of peers, and parental expectations. The purpose of this thesis is to help parents, teachers and others appreciate the school experiences of children at Level 1 risk of developing an NLD, those with a hydrocephalic condition. It does not purport to offer ultimate solutions or to contribute to diagnosis but rather to act as a starting point for a body of theory to guide development of suitable learning environments for such children. Of further importance is emphasis on the need for similar studies to be conducted into the learning experiences of other children who demonstrate specific syndromes or mosaic forms of those syndromes. Naturalistic Inquiry methodology was used to explore the educational experiences of five students who attended different Australian schools. After completion of all interviews, psychological testing assessed general intelligence and the NLD status of each student. All students were found to be severely learning disabled and all were high on the NLD parameter. Educators generally did not reveal understanding of the NLD syndrome "Nonverbal, what is it? So is it a visual ..." Some teachers devised innovative strategies to help the student cope in class while others expressed frustration ... if the traditional instruction "doesn't work either, what does?" What stood out was an absence of understanding about nonverbal deficits. Frustration about poor organisation, decision making, task completion and problem-solving was expressed and a mixture of concern and criticism was levelled at social incompetence. Students who could not work independently were perceived by some teachers and aides as "lazy" or "molly-coddled" and problems with everyday living skills were sometimes blamed on the student's family. Findings revealed a compelling need to raise educator awareness about the range of cognitive, learning and social problems associated with shunted hydrocephalus and spina bifida. They also highlighted a need for teachers to question "Why can't this student do things one would expect they could do" and demand answers that explicate the serious difficulties being experienced.

Introdução: estudos com Enfuvirtida (ENF) mostraram que mutações na HR1 da gp41 levam à resistência primária de cepas em pacientes sem tratamento prévio com inibidores de fusão (Derdeyn et al., 2000; Rimsky et. al., 1998; Sista et. al., 2002). Outros, que o uso contínuo de HAART leva à falha virológica (Shafer et. al., 1998; Shafer & Vuitton, 1999). Para a melhor escolha terapêutica recomenda-se usar a Genotipagem do HIV1 (Ministério da Saúde, 2008b; Perez-Elias et. al., 2003; Shafer et. al., 2001). Objetivos: avaliar o perfil de resistência do HIV1 ao ENF pelo sequenciamento da HR1 da gp41 em pacientes sob HAART, sem uso de inibidor de fusão. Realizar fenotipagem virtual da pol do HIV1 em trinta e duas cepas com resistência aos NRTI, NtRTI , NNRTI e PI para verificar indicação do ENF. Métodos: investigamos 877 prontuários de pacientes do CRT-DST/AIDS entre 5/2002 a 7/2005 e verificamos que 92 que haviam realizado o Teste de Genotipagem do HIV1 em nosso laboratório, sem tratamento prévio com inibidores de entrada poderiam participar do nosso estudo. O primeiro grupo, com 60 pacientes apresentou cepas sensíveis à pelo menos um antirretroviral (ARV) e o segundo com 32 cujas cepas apresentaram resistência parcial e completa às drogas. A região HR1/HR2 da gp41 foi seqüenciada com o kit Big Dye Terminator vs. 3.1. Alinhamos as seqüências e as comparamos com HXB2 através do programa BioEdit vs.7.0.9.0. As mutações de resistência ao ENF foram determinadas pelo algoritmo Francês (ANRS) e pelo da Universidade de Stanford. Submetemos as sequências ao Virtual Phenotype Assay (Virco, Bélgica) para obtermos a Fenotipagem Virtual. Os subtipos da HR1/HR2 da gp41 e do pol foram determinados por análises filogenéticas e os recombinantes, pelo Simplot v 3.5.1. Resultados: 89(97%) amostras foram seqüenciadas, 2 (2%) não amplificaram e 1 (1%) não tinha material suficiente. Destas, 82 (92%) eram do subtipo B, 6(7%) do F1 e 1(1%), do BF na gp41. No pol, 80(87%) eram B, 4(4%) eram F e 7(8%), eram FB. Três (3%) apresentaram resistência primária ao ENF, determinada pela N42D (1%), N43H (1%) e L44M (1%). Dez amostras tinham os polimorfismos Q39R(10%), N42H(10%), N42R(10%), N42N/S(10%) e N42S(60%). Na HR2, as mutações N126N/K, N126K/Q/E, E137E/K, E137K e S138S/A estavam presentes em 26% das cepas. Apesar das cepas apresentarem resistência aos ARV pelo teste de genotipagem, a fenotipagem virtual mostrou que 10% dos ARV ainda atuavam sobre elas, enquanto que 75% delas apresentaram resistência parcial e 15%, completa às drogas. Conclusão: nosso estudo sugere a introdução do teste de genotipagem da HR1/HR2 da gp41 antes de indicar do ENF em pacientes com falha terapêutica e que a Fenotipagem Virtual pode ser usada para a escolha do melhor esquema antirretroviral que permita uma supressão viral sustentada.
Background: studies using Enfuvirtide (ENF) have showed that mutations in the HR1 (gp41) have been associated with primary resistance in strains of the patients without previous treatment with fusion inhibitors. (Rimsky et. al.,1998; Derdeyn et al.,2000, Sista et. al.,2002). The widespread use of HAART may achieve maximal suppression of the viral load, but the viral rebound may occur, leading to virologic failure (Shafer et. al.,1998; Shafer & Vuitton,1999). To optimize therapeutic choices it is recommended to use the HIV1 Genotypic Assay. Objective: to evaluate the resistance profile in the HR1 in patients submitted to HAART, but naïve entry inhibitors. To make the Virtual Phenotypic Assay in the pol of the HIV1 strains isolated of the 32 patients with resistance to the antiretroviral (ARV) and to verify the possibility of using the ENF. Methods: we investigated 877 handbook patients of the CRT-DST/AIDS from May/2002 to July/2005. We identified 92 naïve patients to entry inhibitors included in our cohort to do genotypic assay; they were included in our research. The first group was composed by 60 persons with sensible strains to only one ARV. The second, with 32 presented strains with resistance to all ARV. HR1/HR2 was sequenced (ABIPrism BigDye Terminator vs 3.1). Bioedit Software vs.7.0.9.0 was used to compare sequences with the HIVHXB2. The resistance mutations to ENF were analyzed using French ANRS and Stanford HIV Drug Resistance Algorithm. The results by Virtual Phenotype Assay of 32 patients that presented regimen failure by Genotypic Assay predicted HIV phenotypic resistance. Subtype analysis were did by Phylogeny and the recombinants strains results, confirmed by Simplot v.3.5.1. Results: 89(97%) out of 92 samples were sequenced. Two of them (2%) could not be amplified and one (1%) we did not have enough material to use. In the HR1/HR2 82(92%) of the samples were typed as B, 6(7%), as F and 1(1%) as FB. In the pol 80(87%) were classified as B, 4(4%), as F and 7(8%) as FB. According to algorithms used, 3(3%) out of 89 ENF naïve patients presented complete resistance to this drug. One virus harbored the N42D mutation (1%), another, the N43H(1%) and the other one the L44M(1%). In 10 samples we observed the polymorphisms Q39R(1%), N42H(1%), N42R(1%), N42N/S(1%) and N42S(7%). In the HR2 domain the substitutions N126N/K, N126K/Q/E, E137E/K, E137K and S138S/A were present in 26% of the samples. Although strains presented resistance to the ARV by Genotypic Assay, in the Virtual Phenotypic Assay we verified that 10% of the drugs had some effect upon them. 75% of the strains presented partial and 15% completed resistance to ARV. Conclusion: it is important to introduce the HR1/HR2 genotyping test to verify resistance mutations to ENF before indicating this drug to patients with virologic failure. The virtual phenotypic assay can be used to select the better combination therapy to obtain viral suppression sustained response.

After an impressive growth at the end of the last century, the number of new molecular entities launched on the market dramatically decreased in recent years. Thus, there is a need for efficient synthetic processes capable of generating an high number of different molecules, which differ not only for the appendages, but also for the molecular skeleton. In this context, Diversity-Oriented Synthesis (DOS) has proved to be very effective in the achievement of high quality small molecules collections for high-throughput screening and phenotypic assays and chemical genetics studies, leading to the discovery of new lead compounds. The aim of this thesis work is to apply the principles of the DOS to obtain densely functionalized molecular scaffolds, with potential glyco- and/or peptidomimetic moieties. In particular, in a first part, the synthesis of six skeletally different compounds starting from D-mannose has been discussed, together with their application in a phenotypic screening. This cell-based assay, combined with follow up synthesis and further biological studies, led to the discovery of the hexahydro-2H-furo[3,2-b][1,4]oxazine structure as an active modulator of MDA-MB-231 cell growth, through cytostatic effect. Then, the synthesis of a dipeptide isoster, the dihydropyrazinone skeleton, through morpholine acetal rearrangement has been developed. Finally, during a secondment activity of this PhD in the group of Prof. Darren J. Dixon at the University of Oxford, the generation of α-amino nitriles from tertiary amides and lactams was studied and applied in the late stage functionalization of drugs and natural products.

Atlantic salmon aquaculture increasingly faces global sustainability and environmental challenges that influence fish growth, nutritional value of the final product and production efficiency. Proteomics has established itself as an exploratory approach to understand the impact of changes in environmental factors and to production strategies at a detailed biological level. This thesis aims to examine the use of label-free shotgun proteomics as a method to further our understanding of current and important key aspects of Atlantic salmon aquaculture production: triploidy, fish oil replacement and heat stress. Understanding diet- and environmentally-induced physiological changes in fish larvae is a major goal for the aquaculture industry. The application of proteomics to study larval fish is challenging due to the large dynamic range of protein expression in such complex biological matrix. Using sequential protein extraction to reduce sample complexity achieved proteome coverage of 40% greater than a conventional direct extraction method. Proteins and functional categories related to mitochondrial function, oxidative phosphorylation and antioxidant defense were particularly enriched, providing a platform for a better understanding of physiological changes. A considerable knowledge gap exists regarding the physiology of triploid fish. A multi-tissue (whole fish, muscle and liver) and time-series proteomics sampling-approach was combined with fatty acid analysis to explore triploid-specific physiological and phenotypic traits of freshwater Atlantic salmon under optimal growing conditions. The very high level of similarity between the triploid and diploid fish at the proteome level was paralleled by subtle differences in growth and body composition. This is the first proteome characterization of freshwater triploid Atlantic salmon and supports the idea of physiological similarity between ploidies under optimal growing conditions. The future use of novel docosahexaenoic acid (DHA)-enriched Camelina oil emerges as the most efficient and long-term strategy to reduce fish oil use in aquafeeds while improving the current nutritional value of salmon products for consumers. The formulation of an oil blend with similar fatty acid profile to DHA-enriched Camelina oil was combined with liver proteomics to understand tissue fatty acid deposition and the consequent dietary induced metabolic changes in Atlantic salmon smolt. This study demonstrated the suitability of this novel DHA-enriched oil to improve the fillet content of omega-3 long-chain (≥C20) polyunsaturated fatty acids (n-3 LC-PUFA) obtained with a current commercial blend oil. The liver proteome reflected lipid peroxidation-induced oxidative stress and the subsequent activation of the antioxidant and detoxification response, drawing attention to the need of additional antioxidant supplementation if DHA-enriched Camelina oil is used at high inclusion rates. Heat stress causes concerns regarding negative effects on performance and health of fish. Liver proteomics was used to identify the underpinning mechanisms of adaptation to chronic elevated temperature (21°C) in pre-harvest Atlantic salmon. The induction of oxidative stress was paralleled by the suppression of the protein turnover rates, with the latter and an increased dependence on amino acid catabolism as the main mechanisms to balance for the increased energy demand. This study corroborates candidate biomarkers of thermal stress and refines our understanding towards the development of salmon feeds for summer conditions. In conclusion, the studies presented in this thesis advance our understanding of some of the key factors pertinent to Atlantic salmon farming globally, that are fundamental to growth and production efficiency, nutritional value and industry sustainability. Identification of molecular mechanisms and stressors in parallel with phenotypic changes underlines the potential of proteomics to further advance animal production generally and for aquaculture specifically, and provide focus for further targeted studies towards the development of specific nutritional and husbandry strategies. This work supports the current industry standards to produce high quality triploid smolt and suggests revision of the requirements of antioxidant, amino acid and other supplements in Atlantic salmon under the present sustainability and environmental challenges.

Introduction: Dermal scarring affects 91% of burn patients every year and yet, there is currently no effective treatment for their prevention. Dermal scarring is often caused by the dysregulation in different stages of the wound healing process – particularly the sustained transformation of fibroblasts to alpha-smooth muscle actin (α-SMA) expressing myofibroblasts. The aim of this thesis was to identify novel drugs that can be re-purposed for the prevention of dermal scar formation, using a phenotypic, high-throughput screening (HTS) assay. Materials & Methods: Primary fibroblasts, derived from excised scar tissue, were exposed to transforming growth factor beta-1 (TGF-β1) to induce transformation to myofibroblasts. Using the In-Cell ELISA method, a HTS assay was optimised and validated before screening 1,954 approved drugs. Hits were defined as showing >80% inhibition of α-SMA expression, whilst maintaining >80% cell viability. Anti-myofibroblast activity of the candidate drugs was confirmed by construction of concentration response curves, before investigating their effects on cell viability, extracellular matrix (ECM) production and keratinocyte epithelial-mesenchymal transition (EMT). Results: A HTS assay measuring α-SMA expression was optimised and validated, yielding a Z-factor of 0.59 ± 0.03. The screening of 1,954 approved drugs identified 90 hits (4.6%) that successfully inhibited myofibroblast transformation. 10 hits were identified as having a desirable safety profile and could be used for topical application. The anti-myofibroblast activity of one drug class (hydroxypyridone anti-fungals) was confirmed – ciclopirox (IC50 = 16.7 ± 2.3 μM), ciclopirox ethanolamine (IC50 = 10.3 ± 0.8 μM) and piroctone olamine (IC50 = 1.4 ± 0.1 μM). Secondary assays showed that the hydroxypyridone anti-fungals could reduce ECM production and inhibit keratinocyte EMT, whilst maintaining cell viability. Conclusions: Using primary fibroblasts, a phenotypic HTS assay identified hydroxypyridone anti-fungals as being able to inhibit TGF-β1-induced myofibroblast transformation. These drugs exhibited further anti-fibrotic effect when measuring for ECM production and keratinocyte EMT. This is the first study to identify and investigate the anti-fibrotic effect of hydroxypyridone anti-fungals in dermal scarring, suggesting that these drugs could be re-purposed as medical therapy to prevent dermal scarring.

Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2020
Monócitos e macrófagos são células do sistema imune inato com papel conhecido não só na infeção aguda por VIH e subsequente estabelecimento de reservatórios virais, mas também na inflamação generalizada que determina a progressão da doença. As semelhanças entre VIH-1 e VIH-2, assim com diferenças descritas na interação entre vírus e células imunes inatas levou à hipótese de um distinto perfil de ativação e diferenciação contribuir para o melhor prognóstico associado à infeção por HIV-2. A compreensão do papel destas células na patogénese do VIH tem sido limitada pela sua reatividade à manipulação e complexidade da caracterização fenotípica. Consequentemente, com este trabalho pretendeu-se determinar o impacto do processamento no fenótipo e subsequente diferenciação monócito-macrófago, complementado por fenotipagem compreensiva por citometria de fluxo. Monócitos circulantes de doentes infetados por VIH-1 e VIH-2 foram adicionalmente avaliados para validação dos resultados. Apesar de maior pureza, este trabalho demonstrou que isolamento de PBMC por centrifugação por gradiente de densidade com Ficoll seguido de Percoll resultou numa maior expressão de marcadores de ativação e apresentação antigénica, com concomitante menor expressão de PD-L1, associado a imunossupressão. De salientar, extensa fenotipagem identificou CD163 como marcador diferenciador entre macrófagos M1 e M2 assim como amplificação do fenótipo pró-inflamatório com positividade para CD16. Relativamente à infeção por VIH, alguns marcadores correlacionaram-se com tipo de vírus enquanto outros com gestão da doença. Resultados preliminares sugerem uma associação entre a expressão de CD163 e infeção por VIH-1. Menor expressão de HLA-DR em monócitos clássicos e sobre-expressão de SLAN em monócitos não-clássicos foi observada em piores condições clínicas, particularmente se VIH-2-positivo. Este trabalho sugere um fenótipo monócito/macrófago e perfil de ativação distinto, com implicações terapêuticas no potencial desenvolvimento de fármacos imunomodeladores. Futuros estudos baseados nestes protocolos serão usados para aprofundar o conhecimento da complexa relação entre HIV e o sistema monócito/macrófago.
Monocytes and macrophages are innate immune cells known to contribute not only to acute HIV infection and reservoir establishment but also to the generalised inflammation that determines disease progression. The similarities between HIV-1 and HIV-2 and the reports of different interaction between each virus and innate immune cells prompted the hypothesis of a distinct monocyte/macrophage activation profile and differentiation contributing to the better prognosis and slow disease progression seen with HIV-2 infection. Understanding the contribution of these cells to HIV pathogenesis has been limited by their high reactivity to manipulation and complexities of phenotypic characterization. Consequently, we sought to determine the impact of experimental procedures in monocyte/macrophage phenotype and subsequent differentiation, complemented by comprehensive flow cytometric phenotyping. Furthermore, circulating monocytes from HIV-1 and HIV-2 infected individuals were evaluated for validation. Despite higher monocyte purity, the present study revealed that PBMC purification with Ficoll followed by Percoll density gradient centrifugation resulted in greater expression of molecules associated with activation and antigen presentation along with reduced PD-L1 expression, associated with immunosuppression. Noteworthy findings following comprehensive phenotyping include CD163 as a relevant differentiating marker between M1 and M2 macrophages as well as increased inflammatory phenotype and antigen presentation with CD16 positivity. Pertaining to HIV infection, some markers correlated with HIV-type whereas others with clinical disease control. Preliminary data suggest an association between CD163 expression and HIV-1 infection. Interestingly, lower HLA-DR expression in classical monocytes and greater SLAN expression in non-classical monocytes were seen with worse clinical conditions, particularly in HIV- 2 infected patients. Collectively, our data suggests a distinctive monocyte phenotype and activation profile in HIV- 1 compared to HIV-2 infection, with potential therapeutic implications for the development of immunomodulatory agents. Future data based on these optimised procedures will be used to further evaluate the complex relationship between HIV infection and the monocyte/macrophage system.

Tumor malignancies are the second leading cause of death worldwide. One of the reasons for the failure of oncological treatment are the uniformly set clinical guidelines, which neglect the effect of high intertumoral heterogeneity. The in vitro chemosensitivity and resistance (CSRA) assays allow for the stratification of patients prior to therapy. Therefore, the CSRA are a long-considered method for personalization of components of chemotherapy regime. Nevertheless, none of them is being routinely used in clinical practice. Certain chemotherapeutics used for their cytotoxic and cytostatic effect are also able to induce so-called immunogenic cell death (ICD) of tumor cells and activate an anti-tumor immune response. Monitoring of changes in the expression of molecules associated with the regulation of the innate immune system on the surface of dying tumor cells would enable to predict the patient's ability to respond to treatment involving modern immunotherapeutics. The feasibility of CSRA using flow cytometry and microscopy is critically evaluated in this thesis on a model of bladder cancer. Simultaneously, the correlation of the immunogenic phenotype of tumor cells and their sensitivity to selected chemotherapeutics is discussed.

Tese de doutoramento em Engenharia Biomédica, apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra
Osteosarcoma is the most common primary malignant bone tumor, afflicting mainly young patients. Since the inception of chemotherapy, survival rates augmented significantly, but are stagnated over the past decades, due to the absence of improved therapy. Most patients succumb to metastatic disease, which can occur even after apparent successful histological response to chemotherapy that precedes the surgical removal of the primary tumor. Recently, the cancer stem cell (CSC) model has been receiving attention and suggests that stem-like cells exist in many tumor types, form the clonogenic core of the tumor and actively contribute to chemoresistance. This theory was originally postulated as a somewhat rigid hierarchical model of tumor development, in which a self-renewing CSC originates differentiated progeny and is responsible for feeding the bulk tumor mass. However, evidence suggests that diverse pools of CSCs might exist, contributing to the intratumoral heterogeneity of solid tumors, including osteosarcoma. Besides, extrinsic factors, e.g. drug exposure, may also constitute a source of stemness within the tumors. In this thesis we performed a molecular and functional characterization of CSCs in a panel of cell lines representative of two histological subtypes of high-grade osteosarcoma, and investigated the central role of the regulatory Wnt/β-catenin pathway in the stemness properties and survival advantages of CSCs. In the first part, we demonstrated that different CSCs populations may co-exist in osteosarcoma cell lines exhibiting distinct functional properties. CSCs isolated from fibroblastic tumors are slowly-proliferating populations overexpressing Sox2 and Klf4 pluripotency-related markers, have enhanced tumorigenic potential and specific activation of the self-renewal-related Wnt/β-catenin pathway, as assessed by nuclear β-catenin positivity, AXIN2 expression and TCF/LEF transcriptional activity. The Aldefluor+ populations detected in the two histological osteosarcoma subtypes are SOX2+, but KLF4-, whereas the side-population subset phenotype is correlated with ABCG2 drug-efflux transporter expression. Altogether, these results suggest that distinct functional methods identify CSCs with dissimilar characteristics, which may have implications on the design of CSC-targeted therapies. In the second part, we evaluated the therapeutic potential of inhibiting Wnt/β-catenin against chemoresistant CSCs. The Wnt signaling antagonist IWR-1 was selectively cytotoxic for CSCs, by decreasing cell viability, proliferation and cell cycle progression. IWR-1 induced apoptosis of osteosarcoma CSCs and in combination with doxorubicin treatment elicited synergistic cytotoxicity, reversing CSCs intrinsic resistance to this drug. IWR-1 impaired CSC’ self-renewal capacity by compromising landmark steps of the canonical Wnt signaling, namely nuclear β-catenin translocation and subsequent TCF/LEF activation and expression of downstream targets. Wnt inhibition also hampered Aldefluor activity and expression of key pluripotency-related genes. We observed a remarkable anti-tumor effect of IWR-1 in osteosarcoma-xenografted models that potentiated the anti-tumor efficacy of doxorubicin, accompanied by down-regulation of TCF/LEF transcriptional activity, Sox2 and AXIN2 expression and nuclear β-catenin. In the third part, we explored the striking hypothesis that drugs used in osteosarcoma treatment induce stemness properties in differentiated cells. Doxorubicin, cisplatin and methotrexate induced a phenotypic stem-like cell transition, by increasing Aldefluor activity and ALDH, ABC transporters and pluripotency markers expression. Doxorubicin up-regulated stemness markers via Wnt/β-catenin activation, but co-treatment with IWR-1 prevented the drug-induced phenotype. Altogether, these results are consistent with the ability of doxorubicin to kill rapidly-dividing cancer cells, and of IWR-1 to eliminate chemoresistant CSCs populations and overcome acquired resistance. Translational significance of this study was conveyed by the pluripotency mRNA signature found in uncultured osteosarcoma patient samples and by the correlation of stemness-related markers expression with a worst prognosis in osteosarcoma patients who responded poorly to chemotherapy (EuroBonet dataset of whole genome expression). To conclude, our results suggest the existence of phenotypic heterogeneity in osteosarcoma CSCs, and revealed the Wnt/β-catenin as a key determinant of the stemness and chemoresistant profile of osteosarcoma. Targeting the Wnt pathway may simultaneously circumvent chemoresistance and the phenotypic differentiated-to-stem like cell transition induced by chemotherapeutics, and thus contribute to reduce chemotherapy doses and ameliorate the prognostic outcomes of osteosarcoma patients.
O osteossarcoma é o tumor ósseo primário maligno mais comum e afecta sobretudo jovens adolescentes. Com a introdução da quimioterapia, as taxas de sobrevivência aumentaram significativamente, mas estagnaram nas últimas décadas, devido à falta de terapias mais eficazes. Muitos doentes desenvolvem metástases e sucumbem à doença, mesmo após boa resposta histológica à quimioterapia que antecede a remoção cirúrgica do tumor primário. Recentemente, o modelo das células estaminais cancerígenas (CSCs) sugere a existência de células com propriedades estaminais que formam o núcleo clonogénico tumoral e contribuem para a quimio-resistência. Esta teoria foi inicialmente postulada como um modelo hierárquico rígido, no qual CSCs com capacidade de auto-renovação e de diferenciação sustentam o crescimento do tumor e dão origem à população heterogénea de células diferenciadas. No entanto, há evidências que sugerem a existência de diferentes subpopulações de CSCs, podendo assim contribuir para a marcada heterogeneidade intratumoral típica dos tumores sólidos, incluindo o osteossarcoma. Além disso, factores extrínsecos, como por exemplo a exposição à quimioterapia, podem também contribuir para a aquisição de um fenótipo estaminal nos tumores. No âmbito desta tese foi realizada uma caracterização molecular e funcional de CSCs num painel de linhas celulares representativas de dois subtipos histológicos de osteossarcoma de alto-grau, tendo-se investigado o papel central da via Wnt/β-catenina na regulação da estaminalidade e na sobrevivência de CSCs. Numa primeira parte, demonstrámos a existência de diferentes populações de CSCs no osteossarcoma, com propriedades funcionais distintas. As CSCs isoladas a partir de tumores fibroblásticos, caracterizam-se por uma baixa taxa de proliferação, expressão de marcadores de pluripotência (Sox2 e Klf4), elevado potencial tumorigénico e ativação da via Wnt/β-catenina, evidenciada pela localização nuclear de β-catenina, expressão da AXIN2 e actividade de transcrição do TCF/LEF. As populações Aldefluor+ detectadas nos dois subtipos histológicos de osteossarcoma, são SOX2+, mas KLF4-, enquanto o fenótipo side-population se correlaciona com a expressão do transportador de efluxo ABCG2. Estes resultados sugerem que diferentes métodos funcionais identificam CSCs com características distintas, o que pode ter implicações no desenho de terapias dirigidas às CSCs. Numa segunda parte, avaliámos o potencial terapêutico de inibição da via Wnt/β-catenina em CSCs quimio-resistentes. O inibidor IWR-1 mostrou seletividade citotóxica para as CSCs, como demonstrado pela diminuição na viabilidade, proliferação e progressão do ciclo celular e indução da apoptose. Demonstrou ainda sinergia em combinação com a doxorrubicina, revertendo a resistência intrínseca das CSCs a este fármaco. A inibição da via Wnt, demonstrada pela diminuição da translocação nuclear da β-catenina e repressão da atividade de transcrição do complexo TCF/LEF, comprometeu a capacidade de auto-renovação das CSCs e diminuiu a actividade do Aldefluor, assim como a expressão de genes de pluripotência. No modelo in vivo de osteossarcoma, o tratamento com o inibidor IWR 1 mostrou um efeito anti-tumoral pronunciado, acompanhado por uma diminuição da atividade de transcrição do TCF/LEF e da expressão de AXIN2, Sox2 e β-catenina nuclear. Na terceira parte, avaliámos os efeitos de agentes de quimioterapia utilizados no tratamento de osteossarcoma, nomeadamente a doxorrubicina, cisplatina e o metotrexato na aquisição de um fenótipo estaminal. Observou-se um aumento na atividade e expressão de ALDH, bem como da expressão de transportadores ABC e dos marcadores de pluripotência nas células expostas à quimioterapia, efeito que foi mediado pela ativação da via de sinalização Wnt/β-catenina. A inibição desta via com o IWR 1 preveniu a aquisição de um fenótipo estaminal induzido pela doxorrubicina, o que demonstra a capacidade do IWR 1 em eliminar CSCs e prevenir o desenvolvimento de resistência adquirida. O potencial translacional desta tese assenta na observação de uma potencial assinatura genética definida pela expressão de marcadores de pluripotência em amostras clínicas de osteossarcoma, e da correlação da expressão destes marcadores com um mau prognóstico, em doentes não-responsivos à quimioterapia, de acordo com a base de dados EuroBonet. Em conclusão, este trabalho sugere a existência de heterogeneidade fenotípica em CSCs de osteossarcoma e demonstrou que a via da Wnt/β-catenina constitui um fator chave associado à estaminalidade e à quimio-resistência das CSCs. O desenvolvimento de novas terapias dirigidas às CSCs, tendo como alvo a via da Wnt/β-catenina poderá contribuir para contornar a quimio-resistência intrínseca das CSCs e prevenir a aquisição de um fenótipo estaminal induzido pela exposição a fármacos. Esta abordagem poderá contribuir para reduzir as doses de quimioterapia e melhorar o prognóstico de doentes com osteossarcoma.
Centro de Investigação em Meio Ambiente, Genética e Oncobiologia da Faculdade de Medicina da Universidade de Coimbra” (NRC-LPCC/CIMAGO Grant 2014)