Adult mammalian central nervous system (CNS) neurons are unable to extend axons after injury, partially owing to the inhibitory myelin and chondroitin sulfate proteoglycans (CSPGs) present in the environment. A neuron's intrinsic state is also important for determining its regenerative potential. Peripheral nervous system (PNS) neurons, unlike their CNS counterparts, have increased ability to regrow their axons after injury, even in the presence of inhibitory molecules. With the goal of discovering novel regeneration associated genes, we have isolated the genes differentially expressed by PNS neurons. We then developed a high throughput neuronal transfection method to test whether these genes were sufficient to modify neurite growth in vitro. Using high content screening, we measured the ability of cerebellar neurons to initiate neurite outgrowth on inhibitory and permissive substrates. This combination of technologies (subtractive hybridization, microarray, high throughput electroporation and high content screening) allowed phenotypic examination of neurons after the overexpression of over a thousand genes. Additionally, kinases and phosphatases were assayed for their ability to modify neurite outgrowth in hippocampal neurons. Results from both of these large unbiased screens confirmed many of the existing candidates for neurite growth during development and regeneration. We also discovered many novel genes which promoted neurite outgrowth such as GPX3, EIF2B5, RBMX, CHKA, IRF6, and PKN2. To accurately interpret the large volume of data, new methods of analysis were performed. Finally, we developed novel techniques that took advantage of public databases to cluster genes and determine whether those clusters produced robust changes in neurite growth. In summary, we have provided a vast repository of functional data to study axon development and regeneration after injury as well as developing the tools needed to interpret that data.

Como em diversos tumores sólidos, no câncer de cabeça e pescoço (HNSCC) a presença de metástases é um importante fator de mal prognóstico. Até o momento, estudos indicam que, no microambiente inflamatório tumoral, a estimulação com o Fator de Necrose Tumoral Alpha (TNF-?) leva à ativação de diferentes vias moleculares, como a via do Fator Nuclear Kappa-B (NF-kB) e PI3K/AKT, que inibem GSK3? e consequentemente, promovem a estabilização e translocação nuclear de SNAIL e betacatenina. De um modo geral, em diversos tipos de câncer, enquanto beta-catenina tem ação em promover a proliferação celular, membros da família SNAIL são capazes de induzir o processo de transição epitélio-mesenquimal (EMT). Sabe-se que os eventos de EMT estão envolvidos tanto na iniciação de metástases quanto na geração de célulastronco tumorais (CSCs), que por sua vez estão associadas à falha terapêutica e recidiva, devido à características que lhe conferem resistência aos tratamentos convencionais. Ao silenciar a expressão gênica de modo pós-transcricional, microRNAs (miRs) tem sido associados à regulação tanto da EMT quanto da geração de CSCs. Com uso da abordagem de High-Content Screening (HCS, análise celular multiparamétrica quantitativa por microscopia automatizada), buscamos investigar a capacidade de um grupo de 30 miRs humanos, muitos deles envolvidos em vias inflamatórias e na pluripotência, em modular aspectos relacionados a sobrevivência celular e EMT, em uma linhagem celular derivada de HNSCC (FADU) sob estímulo inflamatório. Inicialmente, avaliamos o potencial do TNF-? em modular parâmetros morfométricos, bem como a presença/localização de proteínas relacionadas com a EMT e capacidade migratória. Posteriormente, avaliamos o efeito de moléculas miméticas dos miRs em suprimir ou potencializar a sobrevivência celular e EMT em células estimuladas com TNF-?, seguido da identificação de transcritos alvos preditos (bem como das vias de sinalização enriquecidas para estes alvos) comumente alvejados por grupos de miRs que levaram a alterações multiparamétricas similares. De modo geral, miRs que alvejaram RELA e AKT2/AKT3 foram responsáveis pela redução na proliferação celular e EMT, enquanto o oposto foi observado em miRs que alvejaram GSK3B e ARHGAP5 (inibidor de RhoA). O silenciamento por siRNAs específicos contra RELA e CTNNB1, causou à redução na sobrevivência celular, enquanto que o silenciamento de AKT1 e CTNNB1 levou à redução na expressão proteica de SNAIL/SLUG. Finalmente, o silenciamento de RELA, AKT1, GSK3B e CTNNB1 levou a redução na sobrevivência celular e indução a apoptose mesmo na ausência de estimulação com TNF-?. Como um todo, nós demonstramos que a abordagem de HCS permitiu a identificação de miRs com efeitos fenotípicos similares (no contexto de proliferação e EMT) e que, a predição de alvos compartilhados por estes miRs, levou à identificação de alvos e vias de sinalização relevantes do ponto de vista terapêutico. / Like many solid cancers, in head and neck cancer (HNC) the presence of metastases is an important factor of poor prognosis. To date, studies indicate that, in the tumor inflammatory microenvironment, stimulation with Tumor Necrosis Factor Alpha (TNF-?) leads to the activation of different molecular pathways, such as the Nuclear Factor-Kappa B (NF-kB) and PI3K/AKT pathway, inhibiting GSK3? and the degradation of SNAIL and beta-catenin, stabilizing them promoting their nuclear translocation. In general, in several types of cancer, while beta-catenin acts to promote cell proliferation, members of the SNAIL family induce the epithelial-mesenchymal (EMT) transition process. It is known that EMT events are involved both in the initiation of metastases and generation of cancer stem cells (CSCs), which in turn are associated with therapeutic failure and relapse, due to its properties that confer resistance to conventional treatments. By silencing gene expression in a post-transcriptional fashion, microRNAs (miRs) have been associated with the regulation of both EMT and CSCs generation. Using the HighContent Screening (HCS) approach, we sought to investigate the ability of a group of 30 human miRs, many of them involved in inflammatory pathways and pluripotency, to modulate aspects related to cell survival and EMT, in a HNSCC-derived cell line (FADU) under inflammatory stimuli. Initially, we evaluated the potential of TNF-? in modulating morphometric parameters, as well as the presence/location of EMT-related proteins and migratory capacity. Subsequently, we evaluated the effect of miRs mimetic molecules on suppressing or potentiating cell survival and EMT in TNF-?-stimulated cells, followed by the identification of predicted target transcripts (as well as signaling pathways enriched for these targets) commonly targeted by groups of miRs that led to similar multiparametric changes. Overall, miRs that targeted RELA and AKT2/AKT3 were responsible for the reduction in cell proliferation and EMT, while the opposite was observed in miRs that targeted GSK3B and ARHGAP5 (RhoA inhibitor). Gene silencing by specific siRNAs against RELA and CTNNB1 caused a reduction in cell survival, while silencing of AKT1 and CTNNB1 led to reduced protein expression of SNAIL/SLUG. Finally, the silencing of RELA, AKT1, GSK3B and CTNNB1 led to a reduction in cell survival and induction of apoptosis even in the absence of TNF-? stimulation. As a whole, we demonstrated that the HCS approach allowed the identification of miRs with similar phenotypic effects (in the context of proliferation and EMT) and that the prediction of targets shared by these miRs led to the identification of relevant targets and signaling pathways from the therapeutic point of view.

Como em diversos tumores sólidos, no câncer de cabeça e pescoço (HNSCC) a presença de metástases é um importante fator de mal prognóstico. Até o momento, estudos indicam que, no microambiente inflamatório tumoral, a estimulação com o Fator de Necrose Tumoral Alpha (TNF-?) leva à ativação de diferentes vias moleculares, como a via do Fator Nuclear Kappa-B (NF-kB) e PI3K/AKT, que inibem GSK3? e consequentemente, promovem a estabilização e translocação nuclear de SNAIL e betacatenina. De um modo geral, em diversos tipos de câncer, enquanto beta-catenina tem ação em promover a proliferação celular, membros da família SNAIL são capazes de induzir o processo de transição epitélio-mesenquimal (EMT). Sabe-se que os eventos de EMT estão envolvidos tanto na iniciação de metástases quanto na geração de célulastronco tumorais (CSCs), que por sua vez estão associadas à falha terapêutica e recidiva, devido à características que lhe conferem resistência aos tratamentos convencionais. Ao silenciar a expressão gênica de modo pós-transcricional, microRNAs (miRs) tem sido associados à regulação tanto da EMT quanto da geração de CSCs. Com uso da abordagem de High-Content Screening (HCS, análise celular multiparamétrica quantitativa por microscopia automatizada), buscamos investigar a capacidade de um grupo de 30 miRs humanos, muitos deles envolvidos em vias inflamatórias e na pluripotência, em modular aspectos relacionados a sobrevivência celular e EMT, em uma linhagem celular derivada de HNSCC (FADU) sob estímulo inflamatório. Inicialmente, avaliamos o potencial do TNF-? em modular parâmetros morfométricos, bem como a presença/localização de proteínas relacionadas com a EMT e capacidade migratória. Posteriormente, avaliamos o efeito de moléculas miméticas dos miRs em suprimir ou potencializar a sobrevivência celular e EMT em células estimuladas com TNF-?, seguido da identificação de transcritos alvos preditos (bem como das vias de sinalização enriquecidas para estes alvos) comumente alvejados por grupos de miRs que levaram a alterações multiparamétricas similares. De modo geral, miRs que alvejaram RELA e AKT2/AKT3 foram responsáveis pela redução na proliferação celular e EMT, enquanto o oposto foi observado em miRs que alvejaram GSK3B e ARHGAP5 (inibidor de RhoA). O silenciamento por siRNAs específicos contra RELA e CTNNB1, causou à redução na sobrevivência celular, enquanto que o silenciamento de AKT1 e CTNNB1 levou à redução na expressão proteica de SNAIL/SLUG. Finalmente, o silenciamento de RELA, AKT1, GSK3B e CTNNB1 levou a redução na sobrevivência celular e indução a apoptose mesmo na ausência de estimulação com TNF-?. Como um todo, nós demonstramos que a abordagem de HCS permitiu a identificação de miRs com efeitos fenotípicos similares (no contexto de proliferação e EMT) e que, a predição de alvos compartilhados por estes miRs, levou à identificação de alvos e vias de sinalização relevantes do ponto de vista terapêutico. / Like many solid cancers, in head and neck cancer (HNC) the presence of metastases is an important factor of poor prognosis. To date, studies indicate that, in the tumor inflammatory microenvironment, stimulation with Tumor Necrosis Factor Alpha (TNF-?) leads to the activation of different molecular pathways, such as the Nuclear Factor-Kappa B (NF-kB) and PI3K/AKT pathway, inhibiting GSK3? and the degradation of SNAIL and beta-catenin, stabilizing them promoting their nuclear translocation. In general, in several types of cancer, while beta-catenin acts to promote cell proliferation, members of the SNAIL family induce the epithelial-mesenchymal (EMT) transition process. It is known that EMT events are involved both in the initiation of metastases and generation of cancer stem cells (CSCs), which in turn are associated with therapeutic failure and relapse, due to its properties that confer resistance to conventional treatments. By silencing gene expression in a post-transcriptional fashion, microRNAs (miRs) have been associated with the regulation of both EMT and CSCs generation. Using the HighContent Screening (HCS) approach, we sought to investigate the ability of a group of 30 human miRs, many of them involved in inflammatory pathways and pluripotency, to modulate aspects related to cell survival and EMT, in a HNSCC-derived cell line (FADU) under inflammatory stimuli. Initially, we evaluated the potential of TNF-? in modulating morphometric parameters, as well as the presence/location of EMT-related proteins and migratory capacity. Subsequently, we evaluated the effect of miRs mimetic molecules on suppressing or potentiating cell survival and EMT in TNF-?-stimulated cells, followed by the identification of predicted target transcripts (as well as signaling pathways enriched for these targets) commonly targeted by groups of miRs that led to similar multiparametric changes. Overall, miRs that targeted RELA and AKT2/AKT3 were responsible for the reduction in cell proliferation and EMT, while the opposite was observed in miRs that targeted GSK3B and ARHGAP5 (RhoA inhibitor). Gene silencing by specific siRNAs against RELA and CTNNB1 caused a reduction in cell survival, while silencing of AKT1 and CTNNB1 led to reduced protein expression of SNAIL/SLUG. Finally, the silencing of RELA, AKT1, GSK3B and CTNNB1 led to a reduction in cell survival and induction of apoptosis even in the absence of TNF-? stimulation. As a whole, we demonstrated that the HCS approach allowed the identification of miRs with similar phenotypic effects (in the context of proliferation and EMT) and that the prediction of targets shared by these miRs led to the identification of relevant targets and signaling pathways from the therapeutic point of view.

Les Tunneling nanotubes (TNT) sont des protrusions cellulaires à base d'actine qui médient la communication cellulaire en transférant des cargos cellulaires. Les différents types de communication intercellulaires sont de plus en plus considérés comme des cibles potentielles pour le traitement de différentes maladies, telles que les maladies infectieuses liées aux virus et bactéries, les cancers ou les maladies neurodégénératives. Des études récentes ont mis en évidence un mécanisme de propagation d'agrégats protéiques ressemblant à la propagation du prion dans diverses maladies neurodégénératives non infectieuses telles que la maladie d'Alzheimer (AD), la démence frontotemporelle (FTD), la maladie de Parkinson (PD) et la maladie de Huntington. Ces maladies se caractérisent par l'accumulation de protéines mal repliées dans le cerveau des patients. Ainsi, on peut envisager de nouvelles stratégies thérapeutiques pour bloquer la propagation des protéines anormales dans tout le cerveau. Il a été démontré que les TNT pourraient jouer un rôle essentiel dans la propagation des agrégats de prions au sein du système nerveux central (SNC) et périphérique. Par conséquent, l'étude du mécanisme de la formation de TNT pourrait fournir de nouvelles idées sur le mécanisme de propagation de la maladie et de nouvelles cibles thérapeutiques. L'objectif de ma thèse était d'étudier le rôle du transfert des agrégats de protéines par les TNT entre les cellules et d'étudier le mécanisme de formation des TNT. Dans notre laboratoire, nous avons déjà montré que les TNT permettent le transfert de prions entre les cellules. Dans la première partie de mon doctorat, j'ai confirmé que les transferts d'agrégats de prions entre les cellules de CAD neuronales se faisaient par les TNT à l'intérieur de vésicules endocytiques (Zhu et al., 2015). De plus, en collaboration avec un collègue, nous avons fourni des preuves que les agrégats de prions pourraient être transférés entre des astrocytes primaires et des neurones et que ce transfert était médié par un contact cellulaire (Victoria et al., 2016). J'ai également collaboré à une autre étude où nous avons montré que les agrégats d'α-synucléine (caractéristiques de la maladie de Parkinson) peuvent être transférés entre les cellules à l'intérieur des lysosomes, et que ce transfert intercellulaire est médié par les TNT (Abounit et al., 2016). Dans mon deuxième projet, afin d'étudier le mécanisme de la formation de TNT, j'ai effectué un crible à haut débit pour les Rab GTPase. J'ai trouvé que Rab8 et Rab11 peuvent favoriser la formation des TNT, et que les cascades Rab8-VAMP3, Rab11-ERM et Rab8-Rab11 sont impliquées dans la formation des TNT. Mes données suggèrent que la polymérisation de l'actine et le trafic de membranes sont impliqués dans la formation des TNT. Ces résultats permettent d'éclairer le mécanisme de la formation des TNT et de fournir des preuves moléculaires que les Rab GTPases régulent ce processus. / Tunneling nanotubes are actin-based cell protrusions that mediate cell-to-cell communication by transferring cellular cargos. The different types of intercellular communication are increasing by being considered as potential targets for the treatment of various diseases, such as infectious diseases linked to viruses and bacteria, cancers or neurodegenerative diseases. Recent studies have highlighted a prion-like mechanism of propagation of protein misfolding in a variety of common, non-infectious, neurodegenerative diseases such as Alzheimer’s disease (AD), Frontotemporal dementia (FTD), Parkinson’s disease (PD), and Polyglutamine (PolyQ) diseases, which are characterized by the accumulation of misfolded proteins in the brain of patients. Thus, new therapeutic strategies to block propagation of protein misfolding throughout the brain can be envisaged. It has been shown that TNTs might play a critical role in spreading of prion aggregates within the CNS and from the periphery. Therefore, the study of mechanism of TNT formation could provide new insights on the mechanism of disease propagation and novel therapeutic targets. The aim of my thesis was to study the role of TNT-mediate protein aggregates transfer between cells and to investigate the mechanism of TNT formation. In our lab, we already reported TNT mediate prion transfer between cells. In the first part of my PhD, I further confirmed that prion aggregates transfer between neuronal CAD cells through TNT inside endocytic vesicles (Zhu et al., 2015). Furthermore in collaboration with a colleague, we provided evidences that prion aggregates could transfer between primary astrocytes and neurons and the transfer was mediated by cell-to-cell contact (Victoria et al., 2016). I also collaborated to another study where we showed that α-synuclein aggregates (Parkinson’s disease) can transfer between cells inside lysosomes, and the intercellular transfer is mediated by TNTs (Abounit et al., 2016).In my second project, in order to investigate the mechanism of TNT formation, I performed a High-content screening of Rab GTPase. I found that Rab8 and Rab11 can promote TNT formation, that Rab8-VAMP3, Rab11-ERM and Rab8-Rab11 cascades are involved in TNT formation. My data suggests that both actin polymerization and membrane trafficking are involved in TNT formation. These results help to shed light on the mechanism of TNT formation, and provide molecular evidences that Rab GTPases regulate this process.

Image Super resolution is a widely-studied problem in computer vision, where the objective is to convert a lowresolution image to a high resolution image. Conventional methods for achieving super-resolution such as image priors, interpolation, sparse coding require a lot of pre/post processing and optimization. Recently, deep learning methods such as convolutional neural networks and generative adversarial networks are being used to perform super-resolution with results competitive to the state of the art but none of them have been used on microscopy images. In this thesis, a generative adversarial network, mSRGAN, is proposed for super resolution with a perceptual loss function consisting of a adversarial loss, mean squared error and content loss. The objective of our implementation is to learn an end to end mapping between the low / high resolution images and optimize the upscaled image for quantitative metrics as well as perceptual quality. We then compare our results with the current state of the art methods in super resolution, conduct a proof of concept segmentation study to show that super resolved images can be used as a effective pre processing step before segmentation and validate the findings statistically. / Image Super-resolution är ett allmänt studerad problem i datasyn, där målet är att konvertera en lågupplösningsbild till en högupplöst bild. Konventionella metoder för att uppnå superupplösning som image priors, interpolation, sparse coding behöver mycket föroch efterbehandling och optimering.Nyligen djupa inlärningsmetoder som convolutional neurala nätverk och generativa adversariella nätverk är användas för att utföra superupplösning med resultat som är konkurrenskraftiga mot toppmoderna teknik, men ingen av dem har använts på mikroskopibilder. I denna avhandling, ett generativ kontradiktorisktsnätverk, mSRGAN, är föreslås för superupplösning med en perceptuell förlustfunktion bestående av en motsatt förlust, medelkvadratfel och innehållförlust.Mål med vår implementering är att lära oss ett slut på att slut kartläggning mellan bilder med låg / hög upplösning och optimera den uppskalade bilden för kvantitativa metriks såväl som perceptuell kvalitet. Vi jämför sedan våra resultat med de nuvarande toppmoderna metoderna i superupplösning, och uppträdande ett bevis på konceptsegmenteringsstudie för att visa att superlösa bilder kan användas som ett effektivt förbehandling steg före segmentering och validera fynden statistiskt.

Gene therapy is the process of delivering genetic material, such as DNA (encoding for an important protein) into a patient’s cells in order to treat a particular disease such as a genetic disorder or heart disease. This process of DNA delivery into cells is known as “transfection” and it is important that the efficiency of transfection be optimized such that a patient can obtain maximum therapeutic benefit from such a treatment. DNA is susceptible to being destroyed by harsh physiological environments prior to reaching its target. This problem can be diminished with the use of vectors that not only protect against harsh conditions but also encourage entry into cells. By mixing 1) DNA with 2) positively charged polymers, “polyplexes” form which protect DNA from degradation and increase transfection efficiency. The development of effective polyplex formulations requires optimization. In the work presented here, it was discovered that when polyplexes contained specific sequences within the DNA called “CpG”, this lowered transfection efficiencies and increased inflammatory responses compared to DNA without CpG, as measured using a mouse lungs model. Thus, DNA composition played an important role in influencing DNA transfection efficiency of polyplexes. Another aspect to take into account is the degree of positive charge of the polymer. We tested a new polymer called poly(galactaramidoamine) or PGAA. We found that this PGAA can form polyplexes with DNA and could be used in gene therapy. At the present time, mechanisms by which the polyplexes get inside and transfect the cells are still unclear. We also introduced a new system called high-content screening to the gene delivery field. This system offers automated measurements of transfection efficiency and cytotoxicity and could be used to reveal the polyplexes trafficking inside cells.

<p><strong><em>Background</em></strong>: Chemotherapy and radiation resistance are major causes of failure in cancer treatment. The response to treatment in cancer cells depends on several mechanisms and pathways such as Janus kinases-signal transducers and activators of transcription JAK/STAT pathway. STAT1 was the first described transcription factor in the STAT family. STAT1 is activated by stimulation of signaling proteins such as type II interferon (IFN- γ) and the activated STAT1 translocates from cytoplasm to nucleus. The translocation of STAT1 would result in transcription and changes in the cell activity in terms of apoptosis, proliferation and angiogenesis. Overexpression of STAT1 is suggested to be involved in the development of resistance to chemotherapy and radiation. In this study, we were interested in finding an inhibitor of the STAT1 translocation. <strong><em>Material and methods</em></strong>: The cervix carcinoma cell line, HeLa, was exposed to test compounds for 2h and were then stimulated with IFN-γ to induce the translocation of STAT1. To detect STAT1-protein and the nucleus, the cells were stained with fluorescent antibodies and Hoescht 33324, respectively, using a STAT1 activator assay. The difference in fluorescence intensity between cytoplasm and nucleus was measured using a high-content microscope, ArrayScan^®. <strong><em>Results</em></strong>: β-lapachone and CRA-1 were found to be inhibitors of STAT1 translocation.</p>

Trauma to the central nervous system (CNS) results in an irreversible disruption of axon tracts, often leading to lifelong functional deficits. Despite a large body of research into the mechanisms that underlie the lack of axonal regeneration after CNS injury, there are currently no effective treatments. One major obstacle involves the presence at injury sites of CNS growth-inhibitory molecules, such as myelin proteins and astrocyte-derived chondroitin sulfate proteoglycans (CSPGs), which act as environmental barriers to axonal regeneration. Our lab recently described the identification and characterization of a novel compound, F05, which promotes growth on inhibitory substrates in vitro. I show that F05 improves regeneration in vivo after acute sensory axon transection as well as after optic nerve crush injury. F05 does not target known signaling molecules involved in CSPG or myelin mediated inhibition but does affect growth cone microtubule dynamics, suggesting a potentially novel mechanism of growth promotion. Using a protein microarray, I show that apoptotic signaling pathways may underlie glial-derived inhibition and its relief by F05. In addition, I employed a comparative gene microarray to show that F05 induces similar changes in gene expression as antipsychotics of the piperazine phenothiazine structural class (PhAPs). Indeed, PhAPs share F05’s ability to overcome glial-derived inhibition of cultured CNS neurons and do so through a mechanism dependent on antagonism of calmodulin. These studies have led to the identification of potentially novel clinical treatments for CNS injury as well as a better understanding of environmentally derived growth-inhibitory signaling mechanisms.

Background: Chemotherapy and radiation resistance are major causes of failure in cancer treatment. The response to treatment in cancer cells depends on several mechanisms and pathways such as Janus kinases-signal transducers and activators of transcription JAK/STAT pathway. STAT1 was the first described transcription factor in the STAT family. STAT1 is activated by stimulation of signaling proteins such as type II interferon (IFN- γ) and the activated STAT1 translocates from cytoplasm to nucleus. The translocation of STAT1 would result in transcription and changes in the cell activity in terms of apoptosis, proliferation and angiogenesis. Overexpression of STAT1 is suggested to be involved in the development of resistance to chemotherapy and radiation. In this study, we were interested in finding an inhibitor of the STAT1 translocation. Material and methods: The cervix carcinoma cell line, HeLa, was exposed to test compounds for 2h and were then stimulated with IFN-γ to induce the translocation of STAT1. To detect STAT1-protein and the nucleus, the cells were stained with fluorescent antibodies and Hoescht 33324, respectively, using a STAT1 activator assay. The difference in fluorescence intensity between cytoplasm and nucleus was measured using a high-content microscope, ArrayScan®. Results: β-lapachone and CRA-1 were found to be inhibitors of STAT1 translocation.

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