The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Parkinson's Disease (PD) is the second most common neurodegenerative disorder, clinically characterized by severe motor impairment and pathologically characterized by progressive loss of the dopaminergic neurons of the substantia nigra pars compacta (SNpc) as well as the formation of cellular aggregate deposits called Lewy Bodies. While some advances have been made in understanding the molecular underpinnings of the disorder, the molecular implications of common genetic factors increasing risk for PD have not been adequately studied. First identified by GWA studies in 2009, the GAK/DGKQ/IDUA region on chromosome 4p16.3 shows significant genetic association to risk for PD, and the GAK protein has been shown to be associated with the primary component of Lewy Bodies, a-synuclein. In order to determine which gene in the 4p16.3 region may account for the genetic association to PD and to understand the molecular consequences of that association, post-mortem cortical brain tissue from 29 PD and 49 control patients was RNA-sequenced and differential exon usage in the context of disease and risk variant carrier status was analyzed. Exons in the 3' region of GAK were found to be associated to case status, and notably exon 25 expression in GAK was associated with both case status and the risk variant. This exon was further observed to be associated to several genes previously shown to interact with GAK, including SNCA, which codes for a-synuclein. As a proxy for expression of the 3' region of GAK, exon 25 was assessed for genome-wide association, and genes showing association to the exon were involved in pathways related to synaptic transmission and neuronal function. In order to validate these findings, microarray analysis of primary rat cortical neurons in which GAK expression was reduced by shRNA transduction was performed. GAK expression in rat neurons was significantly inversely correlated to endogenous SNCA expression, and also exhibited association to pathways involved in synaptic transmission and mitochondrial function. Together, these findings suggest aberrant GAK expression related to genetic risk to be an important factor in the pathogenesis of PD through GAK's influence on SNCA expression and through dysregulation of important neuronal pathways.

Weak Affinity Chromatography (WAC) is a technology that was developed to analyse weak (KD > 10-5 M) although selective interactions between biomolecules. The focus of this thesis was to develop this method for various applications in the drug development process.   Fragment Based Drug Discovery is a new approach in finding new small molecular drugs. Here, relatively small libraries (a few hundreds to a few thousands of compounds) of fragments (150 – 300 Da) are screened against the target. Fragment hits are then developed into lead molecules by linking, growing or merging fragments binding to different locations of the protein’s active site. However, due to the weakly binding nature of fragments, methods that are able to detect very weak binding events are needed. In this thesis, WAC is presented as a new robust and highly reproducible technology for fragment screening. The technology is demonstrated against a number of different protein targets – proteases, kinases, chaperones and protein-protein interaction (PPI) targets. Comparison of data from fragment screening of 111 fragments by WAC and other more established technologies for fragment screening, such as surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR), validates WAC as a screening technology. It also points at the importance of performing fragment screening by multiple methods as they complement each other.   Other applications of WAC in drug development are also presented. The method can be used for chiral separations of racemic mixtures during fragment screening, which enables affinity measurements of individual enantiomers binding to the target of interest. Further, analysis of crude reaction mixtures is shown. By these procedures, the affinity of the product can be assessed directly after synthesis without any time-consuming purification steps. In addition, a high performance liquid chromatography (HPLC) system for highly efficient drug partition studies was developed by stable immobilization of lipid bilayer disks – lipodisks – on a high performance silica support material. These lipodisks are recognized model membranes for drug partition studies. A WAC system with incorporated membrane proteins into immobilized lipodisks has also been produced and evaluated with the ultimate objective to study affinity interactions between ligands and membrane proteins. / Ett läkemedel utövar sin funktion genom att påverka aktiviteten hos ett protein i kroppen då det binder till dess aktiva säte. Förändringen i aktivitet leder till fysiologiska förändringar i kroppen beroende på vilken funktion proteinet har. Med läkemedelsmolekyl avses här en liten organisk molekyl. Fragment-baserad läkemedelsutveckling är en ny metod for att ta fram nya läkemedel. Metoden fungerar genom att man bygger läkemedelsmolekyler utifrån mindre fragment som binder till målproteinet. Fragmenten hittar man genom att screena hela bibliotek av olika fragment mot samma målprotein för att urskilja de som binder till proteinets aktiva säte. Fördelen med den här metoden är bl. a. att med mindre molekyler som utgångspunkt kan en större del av antalet möjliga kombinationer av atomer representeras med ett mindre antal fragment än för större molekyler. Normalt utgörs ett fragmentbibliotek enbart av några hundra till några tusen substanser. Eftersom fragmenten är små har de få interaktionspunker och binder relativt svagt. De svaga bindningarna är svåra att se och mycket känsliga metoder behövs.   Svagaffinitetskromatografi är en vätskekromatografisk metod som utvecklades för att studera svaga men mycket selektiva bindningar mellan biomolekyler. Den här avhandlingen syftar till att utveckla metoden för olika användningsområden inom läkemedelsutveckling, främst som en ny metod för fragment-screening. Här mäter man interaktionen mellan ett protein och ett fragment. Proteinet kopplas till ett material som sedan packas i en kolonn i formen av en cylinder. När provet pumpas igenom kolonnen kommer de analyter med affinitet till proteinets aktiva säte att fördröjas på kolonnen i relation till hur starkt de interagerar med målproteinet.   I den här avhandlingen presenteras fragment-screening med svagaffinitetskromatografi gentemot ett antal olika typer av målproteiner. Resultatet överensstämmer väl med andra metoder för fragment-screening. Analys av reaktionsblandningar med svagaffinitetskromatografi demonstreras också. Därmed kan bindningen mellan en produkt i en reaktionsblandning och ett målprotein mätas direkt utan föregående uppreningssteg av reaktionsblandningen. Lipodiskar är små diskformade modellmembran som kan användas för att bl. a. mäta hur effektivt läkemedlet tas upp i kroppen vid behandling. Ett system med immobiliserade lipodiskar i en kolonn utvecklades med det framtida målet att kunna arbeta med membranproteiner med svagaffinitetskromatografi.   Detta arbete utgör en del i att utveckla svagaffinitetskromatografi som en lättillgänglig och relativt billig metod för användning inom industrin och akademin för läkemedelsutveckling.

O efeito dos hormônios glicocorticóides (GC) de suprimir o crescimento celular é exercido através de cascatas celulares nas quais a transcrição de genes de resposta primária, mediada direta ou indiretamente pelo receptor de GC, regulam a transcrição e a atividade de um conjunto de genes, incluindo fatores importantes para a progressão no ciclo celular. Entretanto, a conexão funcional entre diversos dos genes ativados ou inibidos por GC e a inibição da proliferação de determinados tipos celulares ainda não é completamente conhecida. Nosso laboratório isolou a variante ST1, a partir da linhagem C6 de glioma de rato, utilizando-a como modelo de estudo do mecanismo de ação de GC como agentes anti-tumorais. O tratamento com GC confere, às células ST1, um crescimento totalmente dependente de soro e ancoragem, morfologia em cultura semelhante à de fibroblastos normais e incapacidade de gerar tumor em camundongos da linhagem \"nude\", caracterizando uma completa reversão fenotípica tanto in vitro como in vivo. Como abordagens para o entendimento do mecanismo molecular da ação de GCs, o laboratório vem buscando a clonagem de produtos gênicos diferencialmente expressos através do uso de técnicas que permitam a análise da abundância relativa dos mRNAs, e mais recentemente, empregando-se a análise da abundância relativa das proteínas através da eletroforese bidimensional (2D-PAGE). As seqüências isoladas por estas metodologias são alvos potenciais para uma posterior análise funcional. Os objetivos gerais deste trabalho foram: a) identificar genes que estão diferencialmente expressos durante a reversão fenotípica das células ST1 induzida por GC, através da análise proteômica (2D-PAGE e espectrometria de massas) e b) determinar a função de um dos genes (Ciclina G) identificado anteriormente no laboratório como sendo induzido durante o tratamento das células ST1 com GC. A metodologia empregada se baseou na comparação dos perfis 2D de proteínas nucleares das células ST1 tratadas ou não (controle) com GC por 5 e 24h. Após análise de imagem, 33 polipeptídios foram considerados como diferencialmente representados após 5h de tratamento, 16 dos quais foram também identificados após 24h de tratamento. Seis destes polipeptídios foram identificados através da análise dos seus perfis de digestão tríptica (PMF). Evidências obtidas por ensaios de Western blot sugerem que um destes polipeptídeos, a Anexina 2 (ANX2), possui a sua localização sub-celular modulada pela ação de GC nas células ST1. A análise do papel da Ciclina G na reversão fenotípica tumoral-normal das células ST1 induzida por GC, foi feita através da sua super-expressão nestas células, utilizando um sistema retroviral, e avaliando-se os efeitos desta super-expressão sobre a resposta das células ST1 a GC, através de ensaios de curva de crescimento e citometria de fluxo. Os dados sugerem que a super-expressão da Ciclina G nas células ST1 intensifica e prolonga o efeito de GC nestas células. / The glucocorticoid (GC) growth suppression response is controlled through cellular cascades in which the transcription of primary response genes regulates the expression and activity of a diverse set of genes including important factors for cell cycle progression. However, the functional connection between the GC-regulated transcriptional events and cell cycle arrest of determined cells is poorly understood at the molecular level. In this context, our lab has isolated the ST1 variant of the C6 rat glioma cell line to study the mechanism of action of GC as anti-tumor agents. GC treatment leads ST1 cells to a dramatic tumoral to normal phenotypic reversion, characterized by inhibition of their growth rate in monolayer, loss of their ability to form colonies in semi-solid medium and to induce tumor formation in nude mice, and morphological changes (flattening). As part of the strategy to understand the anti-tumor action of GC, differentially represented proteins, associated with the phenotypic reversion displayed by ST1 cells have been isolated through mRNA-based blind cDNA cloning and, more recently, by two dimensional electrophoresis (2D-PAGE). The sequences isolated by these two methodologies are potential targets for functional analysis. In the present study, we aimed at a) identifying genes which are differentially expressed during the GC-induced phenotypic reversion of ST1 cells, using the proteomic approach (2D-PAGE and mass spectrometry) and b) determining the functional role of a gene (Cyclin G) which had previously being identified as being induced during GC-treatment of the ST1 cells. The analytical methodology used relied on the comparison of sets of 2D nuclear protein profiles of ST1 cells, maintained in the absence (control) and in the presence of GC for 5 and 24h. After image analysis and visual validation, 33 polypeptides were considered as differentially represented 5 h after treatment, 16 of which were also differentially represented after 24 h. Six of those polypeptides were identified by peptide mass fingerprinting (PMF). Evidence obtained by Western blot analysis indicates that one of those polypeptides, Annexin 2 (ANX2), has its sub-cellular location modulated by GC-treatment of ST1 cells. The role of Cyclin G in the tumoral to normal phenotypic reversion induced by GC in ST1 cells was analyzed by over-expression of Cyclin G using a retroviral system and evaluation of the effects of this over-expression over ST1 cells response to GC, by growth curve and flow cytometry assays. The data suggest that Cyclin G over-expression leads to a more intense and prolonged effect of GC on ST1 cells.

O efeito dos hormônios glicocorticóides (GC) de suprimir o crescimento celular é exercido através de cascatas celulares nas quais a transcrição de genes de resposta primária, mediada direta ou indiretamente pelo receptor de GC, regulam a transcrição e a atividade de um conjunto de genes, incluindo fatores importantes para a progressão no ciclo celular. Entretanto, a conexão funcional entre diversos dos genes ativados ou inibidos por GC e a inibição da proliferação de determinados tipos celulares ainda não é completamente conhecida. Nosso laboratório isolou a variante ST1, a partir da linhagem C6 de glioma de rato, utilizando-a como modelo de estudo do mecanismo de ação de GC como agentes anti-tumorais. O tratamento com GC confere, às células ST1, um crescimento totalmente dependente de soro e ancoragem, morfologia em cultura semelhante à de fibroblastos normais e incapacidade de gerar tumor em camundongos da linhagem \"nude\", caracterizando uma completa reversão fenotípica tanto in vitro como in vivo. Como abordagens para o entendimento do mecanismo molecular da ação de GCs, o laboratório vem buscando a clonagem de produtos gênicos diferencialmente expressos através do uso de técnicas que permitam a análise da abundância relativa dos mRNAs, e mais recentemente, empregando-se a análise da abundância relativa das proteínas através da eletroforese bidimensional (2D-PAGE). As seqüências isoladas por estas metodologias são alvos potenciais para uma posterior análise funcional. Os objetivos gerais deste trabalho foram: a) identificar genes que estão diferencialmente expressos durante a reversão fenotípica das células ST1 induzida por GC, através da análise proteômica (2D-PAGE e espectrometria de massas) e b) determinar a função de um dos genes (Ciclina G) identificado anteriormente no laboratório como sendo induzido durante o tratamento das células ST1 com GC. A metodologia empregada se baseou na comparação dos perfis 2D de proteínas nucleares das células ST1 tratadas ou não (controle) com GC por 5 e 24h. Após análise de imagem, 33 polipeptídios foram considerados como diferencialmente representados após 5h de tratamento, 16 dos quais foram também identificados após 24h de tratamento. Seis destes polipeptídios foram identificados através da análise dos seus perfis de digestão tríptica (PMF). Evidências obtidas por ensaios de Western blot sugerem que um destes polipeptídeos, a Anexina 2 (ANX2), possui a sua localização sub-celular modulada pela ação de GC nas células ST1. A análise do papel da Ciclina G na reversão fenotípica tumoral-normal das células ST1 induzida por GC, foi feita através da sua super-expressão nestas células, utilizando um sistema retroviral, e avaliando-se os efeitos desta super-expressão sobre a resposta das células ST1 a GC, através de ensaios de curva de crescimento e citometria de fluxo. Os dados sugerem que a super-expressão da Ciclina G nas células ST1 intensifica e prolonga o efeito de GC nestas células. / The glucocorticoid (GC) growth suppression response is controlled through cellular cascades in which the transcription of primary response genes regulates the expression and activity of a diverse set of genes including important factors for cell cycle progression. However, the functional connection between the GC-regulated transcriptional events and cell cycle arrest of determined cells is poorly understood at the molecular level. In this context, our lab has isolated the ST1 variant of the C6 rat glioma cell line to study the mechanism of action of GC as anti-tumor agents. GC treatment leads ST1 cells to a dramatic tumoral to normal phenotypic reversion, characterized by inhibition of their growth rate in monolayer, loss of their ability to form colonies in semi-solid medium and to induce tumor formation in nude mice, and morphological changes (flattening). As part of the strategy to understand the anti-tumor action of GC, differentially represented proteins, associated with the phenotypic reversion displayed by ST1 cells have been isolated through mRNA-based blind cDNA cloning and, more recently, by two dimensional electrophoresis (2D-PAGE). The sequences isolated by these two methodologies are potential targets for functional analysis. In the present study, we aimed at a) identifying genes which are differentially expressed during the GC-induced phenotypic reversion of ST1 cells, using the proteomic approach (2D-PAGE and mass spectrometry) and b) determining the functional role of a gene (Cyclin G) which had previously being identified as being induced during GC-treatment of the ST1 cells. The analytical methodology used relied on the comparison of sets of 2D nuclear protein profiles of ST1 cells, maintained in the absence (control) and in the presence of GC for 5 and 24h. After image analysis and visual validation, 33 polypeptides were considered as differentially represented 5 h after treatment, 16 of which were also differentially represented after 24 h. Six of those polypeptides were identified by peptide mass fingerprinting (PMF). Evidence obtained by Western blot analysis indicates that one of those polypeptides, Annexin 2 (ANX2), has its sub-cellular location modulated by GC-treatment of ST1 cells. The role of Cyclin G in the tumoral to normal phenotypic reversion induced by GC in ST1 cells was analyzed by over-expression of Cyclin G using a retroviral system and evaluation of the effects of this over-expression over ST1 cells response to GC, by growth curve and flow cytometry assays. The data suggest that Cyclin G over-expression leads to a more intense and prolonged effect of GC on ST1 cells.