L’activation d’oncogènes entraine une prolifération aberrante des cellules, un stress réplicatif et des cassures de l’ADN. Un lien a été établi entre l’instabilité génomique résultant des cassures et l’inhibition de checkpoints entrainant l’accumulation de mutations et finalement le cancer (Halazonetis et al. 2008). Cependant, les mécanismes liant ces différents évènements n’ont pas encore été caractérisés. Notre hypothèse est que la prolifération incontrôlée des cellules augmente les incidents dus aux conflits entre les polymérases responsables de la réplication et celles responsables de la transcription. Lors de la rencontre des deux polymérases, l’accumulation de surenroulements positifs de l’ADN induit un blocage des fourches de réplication. Ceci crée des zones de fragilité, notamment dues à l’exposition d’ADN simple brin, et pourrait être à l’origine des cassures observées chez les cellules tumorales. Pour valider cette hypothèse, les biologistes de l'équipe ont étudié plusieurs lignées de cellules HeLa dans lesquelles les conflits réplication-transcription sont augmentés et j'ai réalisé l'analyse bioinformatique des approches génomiques suivantes :-DRIP-seq pour la détection des R-loops, une structure double brin hybride ADN/ARN qui se forme lors de la transcription, exposant ainsi un brin d’ADN simple brin.- ChIP-seq de γ-H2AX, une marque d’histone indiquant les cassures de l’ADN.-ChIP-seq de phospho-RPA (S33), un substrat de la kinase ATR au niveau des fourches bloquées. Pour chaque expérience, nous avons utilisé une lignée contrôle et deux lignées dans lesquelles TOP1 et ASF/SF2 sont appauvries avec un shRNA inductible (shTOP1 et shASF). La Topoisomérase I (TOP1) est une enzyme qui relaxe les surenroulements de l’ADN. Le complexe ASF/SF2 est un facteur d’épissage responsable entre autres de l’assemblage des mRNP (ribonucleoprotein particles) au moment de la transcription, qui limitent la formation des R-loops. L’analyse bioinformatique de ces données, ainsi que d'autres données de la littérature, m'a permis d'identifier des régions à risque du génome, localisées en aval de gènes fortement transcrits et répliqués précocement en phase S par des fourches progressant en sens opposé à la transcription. J’ai également observé que les gènes impliqués dans le cancer sont surreprésentés dans ces régions à risque. / Oncogenes activation promotes aberrant cell proliferation, increasing replication stress and DNA damage. It has been proposed that genomic instability leads to checkpoints inhibition and promotes cancer development (Halazonetis et al. 2008). However, the link between aberrant proliferation, replication stress and DNA breaks is still unclear. We hypothesized that aberrant proliferation leads to more incident due to DNA and RNA polymerases encounter and stalling. When the two polymerases encounter, the accumulation of positive-supercoiled DNA between two polymerases induces fork stalling, resulting in the formation of fragile structures such as single-stranded DNA (ssDNA). These ssDNAs formed at stalled forks could be a source for DNA breaks, promoting the development of cancer cells. To validate this hypothesis, biologists from our team have worked on HeLa cell lines with increased replication-transcription conflicts. I perform the bioinformatics analysis of the following genomic data:- DRIP-seq: R-Loops positioning on genome using immunoprecipitation on DNA/RNA hybrids.-γ-H2AX ChIP-Seq: Gamma-H2AX is an histone mark found at DNA breaks.-pRPA ChIP-Seq : Positioning of stalled forks using the substrate of ATR kinase, phospho-RPA (S33) as a marker.Each data was produced on control cells and two cell lines where TOP1 and ASF/SF2 were depleted by as inducible shRNA (shTOP1 and shASF). Topoisomerase 1 is a topological enzyme that unwinds DNA when supercoiling accumulates. ASF/SF2 is part of the splicing complexes that processes mRNP (messenger ribonucleoprotein particles) to prevent the accumulation of R-loops during transcription. Using these data and others from literature, I determined that regions having higher risk to induce replication stress are located downstream of highly transcribed and early replicated genes, and preferentially with head-on collision between DNA and RNA polymerases. I also revealed that cancer-related genes are enriched in these regions of the genome.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-06T15:53:09Z (GMT). No. of bitstreams: 1 Chen_LuShi_D.pdf: 1749829 bytes, checksum: aa7f95ee1b7db09369cb88541ee02c93 (MD5) Previous issue date: 2006 / Doutorado / Quimica Organica / Doutor em Ciências

Orientador: Anita Jocelyne Marsaioli / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T20:13:36Z (GMT). No. of bitstreams: 1 Lima_MariaLairSaboiadeOliveira_M.pdf: 6354441 bytes, checksum: 7cd17cb17f36613aa4366697163fbb5a (MD5) Previous issue date: 2013 / Resumo: O uso de catalisadores biológicos é importante na academia e na indústria, pois une o ecologicamente correto com o ecologicamente viável. Assim, a busca por novas rotas reacionais viáveis e menos poluentes se enquadra no contexto da química verde, onde o ambiente e os rejeitos são levados em consideração. Com base nestes princípios o presente trabalho visa avaliar o perfil enzimático de micro-organismos provenientes de rejeitos de mineração da Mina do Sossego (Canaã dos Carajás ¿ PA), pertencente à "VALE". Após o isolamento e preservação, as cepas foram caracterizadas por espectrometria de massas, com a técnica MALDI-TOF, onde foram encontradas bactérias dos gêneros Pseudomonas sp., Bacilus sp., Acinetobacter sp., Delfitia sp. e Escherichia sp. As atividades enzimáticas foram avaliadas através de uma triagem rápida e eficiente (HTS, high throughput screening) utilizando sondas fluorogênicas não comerciais baseadas na umbeliferona. Dos 228 micro-organismos isolados, 156 tiveram suas atividades enzimáticas avaliadas das quais 70 apresentaram atividade para esterases, 80 para lipases, 11 para epóxi-hidrolases e 53 para mono-oxigenases. Ainda com base no princípio da triagem enzimática de alto desempenho, foram sintetizadas sondas fluorogênicas quirais e um composto competidor para implementação de uma metodologia conhecida como Quick-E. Esta metodologia visa uma avaliação rápida da enantiosseletividade de esterases em células íntegras através de medidas das velocidades iniciais das biorreações com as sondas fluorogênicas quirais avaliadas separadamente / Abstract: Biocatalysts are important in academia and industry joining the ecological friendly to the ecological safe and efficient processes. Thus, the search for new synthetic routes and reactions producing less hazardous residues fits into the principles of green chemistry as environment and residues are taken into consideration. Based on these principles, present study aims at the enzymatic profiling of microorganisms (156 strains) from cooper mine (Sossego¿s mine, Canaã dos Carajás- PA), belonging to VALE. After isolation (228 microorganisms) and preservation, the strains were characterized by mass spectrometry (MALDI-TOF, 62 strains), being characterized bacterias Pseudomonas sp., Bacilus sp., Acinetobacter sp., Delfitia sp. and Escherichia sp.. The enzymatic activities were evaluated by a high throughput screening using fluorogenic probes based on umbelliferone. Outstanding enzymatic activities of these 156 micro-organisms were: esterases (70), lipases (80), epoxy hydrolases (11) and monooxygenases (53). Additionally the implementation of a novel Quick-E methodology with chiral fluorogenic probes and a competitor will allow the rapid evaluation of esterases enantioselectivities in whole cells by measuring inicial rates of bioreactions in micro scale reactions / Mestrado / Quimica Organica / Mestra em Química

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T09:30:03Z (GMT). No. of bitstreams: 1 Silva_CarlaPortoda_D.pdf: 8471917 bytes, checksum: 298adab03875869cd5d11c7dac0a7b90 (MD5) Previous issue date: 2012 / Resumo: Estima-se que o corpo humano que contém cerca de 10 trilhões de células seja portador de aproximadamente 100 trilhões de micro-organismos. Fatores ambientais como temperatura, umidade e exposição à luz, além de fatores do hospedeiro, como gênero, genótipo, status imune e uso de cosméticos, podem afetar a composição e a distribuição microbiana da pele. Inúmeras pesquisas indicam que a microbiota desempenha um papel importante no sistema imune da pele. Contudo, pouco é conhecido sobre os conjuntos de espécies presentes em amostras cutâneas bem como suas atividades enzimáticas. Esta tese visou realizar o estudo do potencial enzimático da microbiota da pele humana, vinculando este potencial às principais reações de degradação de formulações de cosméticos, mais especificamente, insumos de fragrâncias. O recrutamento dos voluntários levou em conta parâmetros como idade, sexo e fototipo de pele. As principais atividades enzimáticas das microbiotas coletadas foram avaliadas através de técnicas de triagem de alto desempenho, a fim de detectar proteases, lipases, amilases, esterases, epóxido hidrolases e mono-oxigenases, num total de 2.160 experimentos. Através dos resultados obtidos das triagens enzimáticas, algumas amostras foram selecionadas para a realização de ensaios de degradação de insumos de fragrâncias através de ensaios de multibiorreação. Todos os resultados obtidos foram avaliados com intuito de relacionar o tipo da microbiota coletada com reações de degradação de componentes de fragrância, levando em conta as diferenças intrínsecas de cada voluntário. Além disso, observou-se uma grande diversidade fúngica, ainda pouco descrita na literatura, onde diversos representantes foram isolados e identificados. Os dados obtidos demonstraram que os tipos de pele devem ser levados em consideração nas formulações de uso tópico a fim de atingir alvos específicos, tendo em vista que a pele humana não é um ambiente estéril, mas sim um microbioma complexo. Desta forma, o potencial de biotransformação de insumos cosméticos pela microbiota da pele é um fator relevante e poderá auxiliar na busca de produtos mais eficazes, seguros e versáteis / Abstract: The human body contains about 10 trillion cells and carries approximately 100 trillion microorganisms¿ cells. Environmental factors, such as temperature, humidity and light exposure and host factors such as gender, genotype, immune status and use of cosmetics, can affect the composition and distribution of skin microbes. Numerous studies indicate that skin microbiota plays an important role in the human skin immune system. However, little is known about the species present in skin samples and their enzymatic activities. Therefore, the aim of this thesis was to evaluate the enzymatic potential of the human skin microbiota, establishing a link between this potential and the main fragrance degradation of cosmetic formulations, more specifically, fragrance ingredients. The recruitment of volunteers (55) took into account some parameters such as age, gender and skin phototype. The main enzymatic activities of the collected microbiota were assessed by high throughput screening techniques in order to detect protease, lipase, amylase, esterase, epoxide hydrolase and monooxygenase, in a total of 2160 experiments. These enzymatic profiles were applied in the selection of microorganisms to probe the biodegradability of fragrance ingredients using the multibioreaction protocol. The results linking microbiota type and degradation reactions of fragrance ingredients took into consideration the intrinsic differences between volunteers. In addition, a great fungal diversity, still poorly described in the literature, was observed and several representative entities of this diversity were isolated and identified. The obtained data showed that skin types must be considered in topical formulations to achieve specific biological targets and , taking into consideration that the human skin is not a sterile environment, but rather consists of a complex microbiome. Consequently the biotransformation susceptibility of cosmetic ingredients to human skin microbiota is a relevant factor to consider in formulations / Doutorado / Quimica Organica / Doutor em Ciências

Palmitoylation refers to the covalent attachment of fatty acids, such as palmitate, onto the cysteine residues of proteins. This process may subsequently alter their localization and function. Nearly all of the enzymes that catalyze palmitoylation, zDHHC protein acyl transferases (PATs), are implicated in neurological disorders, infectious diseases, and cancer in humans. Of particular interest to those who study palmitoylation are Ras family GTPas and zDHHC9-GCP16, the zDHHC PAT that palmitoylates Ras proteins. Erf2-Erf4 is the zDHHC PAT that palmitoylates Ras proteins in Saccharomyces cerevisiae. Currently, there are no methods to therapeutically target palmitoylation for the treatment of disease. One of the barriers to identifying a modulator of palmitoylation is the lack of a reliable high-throughput screening system. To date, few assay systems have been developed to examine the kinetics and mechanism of that palmitoylation reaction. This lab has developed a fluorescence-based coupled assay to gain insight into the enzymology, biochemical mechanism, and kinetics of the palmitoylation reaction. This assay may be used to identify specific inhibitors of autopalmitoylation. In the first step of this reaction, the palmitoyl-moiety from palmitoyl-CoA is transferred to the zDHHC9 PAT cysteine side chain to form a palmitoyl:enzyme intermediate. The second step of palmitoylation is the subsequent transfer of the palmitoyl-moiety from the palmitoyl:enzyme intermediate to the cysteine residue of the substrate protein. This fluorescence-based coupled assay was utilized to screen a natural products library and a unique synthetic compound library for inhibitors of Erf2 autopalmitoylation. These screens led to the identification of fungal metabolite extracts and ten bis-cyclic piperazine compounds that inhibit Erf2 autopalmitoylation in the low micromolar range. This effect is similar to known inhibitors of palmitoylation that lack specificity for the palmitoylation reaction itself.

Les codes correcteurs d'erreurs sont une composante essentielle de tout système de communication, capables d’assurer le transport fiable de l’information sur un canal de communication bruité. Les systèmes de communication de nouvelle génération devront faire face à une demande sans cesse croissante en termes de débit binaire, pouvant aller de 1 à plusieurs centaines de gigabits par seconde. Dans ce contexte, les codes LDPC (pour Low-Density Parity-Check, en anglais), sont reconnus comme une des solutions les mieux adaptées, en raison de la possibilité de paralléliser massivement leurs algorithmes de décodage et les architectures matérielles associées. Cependant, si l’utilisation d’architectures massivement parallèles permet en effet d’atteindre des débits très élevés, cette solution entraine également une augmentation significative du coût matériel.L’objectif de cette thèse est de proposer des implémentations matérielles de décodeurs LDPC très haut débit, en exploitant la robustesse des algorithmes de décodage par passage de messages aux imprécisions de calcul. L’intégration dans le décodage itératif de mécanismes de calcul imprécis, s’accompagne du développement de nouvelles approches d’optimisation du design en termes de coût, débit et capacité de correction.Pour ce faire, nous avons considéré l’optimisation conjointe de (i) le bloc de quantification qui fournit l'information à précision finie au décodeur, et (ii) les unités de traitement imprécis des données, pour la mise à jour des messages échangés pendant de processus de décodage. Ainsi, nous avons tout d’abord proposé un quantificateur à faible complexité, qui peut être optimisé par évolution de densité en fonction du code LDPC utilisé et capable d’approcher de très près les performances d’un quantificateur optimal. Le quantificateur proposé a été en outre optimisé et utilisé pour chacun des décodeurs imprécis proposés ensuite dans cette thèse.Nous avons ensuite proposé, analysé et implémenté plusieurs décodeurs LDPC imprécis. Les deux premiers décodeurs sont des versions imprécises du décodeur « Offset Min-Sum » (OMS) : la surestimation des messages des nœuds de contrôle est d’abord compensée par un simple effacement du bit de poids faible (« Partially OMS »), ensuite le coût matériel est d’avantage réduit en supprimant un signal spécifique (« Imprecise Partially OMS »). Les résultats d’implémentation sur cible FPGA montrent une réduction importante du coût matériel, tout en assurant une performance de décodage très proche du OMS, malgré l'imprécision introduite dans les unités de traitement.Nous avions ensuite introduit les décodeurs à alphabet fini non-surjectifs (NS-FAIDs, pour « Non-Surjective Finite Alphabet Iterative Decoders », en anglais), qui étendent le concept d’« imprécision » au bloc mémoire du décodeur LDPC. Les décodeurs NS-FAIDs ont été optimisés par évolution de densité pour des codes LDPC réguliers et irréguliers. Les résultats d'optimisation révèlent différents compromis possibles entre la performance de décodage et l'efficacité de la mise en œuvre matérielle. Nous avons également proposé trois architectures matérielles haut débit, intégrant les noyaux de décodage NS-FAID. Les résultats d’implémentation sur cible FPGA et ASIC montrent que les NS-FAIDs permettent d’obtenir des améliorations significatives en termes de coût matériel et de débit, par rapport au décodeur Min-Sum, avec des performances de décodage meilleures ou très légèrement dégradées. / The increasing demand of massive data rates in wireless communication systems will require significantly higher processing speed of the baseband signal, as compared to conventional solutions. This is especially challenging for Forward Error Correction (FEC) mechanisms, since FEC decoding is one of the most computationally intensive baseband processing tasks, consuming a large amount of hardware resources and energy. The conventional approach to increase throughput is to use massively parallel architectures. In this context, Low-Density Parity-Check (LDPC) codes are recognized as the foremost solution, due to the intrinsic capacity of their decoders to accommodate various degrees of parallelism. They have found extensive applications in modern communication systems, due to their excellent decoding performance, high throughput capabilities, and power efficiency, and have been adopted in several recent communication standards.This thesis focuses on cost-effective, high-throughput hardware implementations of LDPC decoders, through exploiting the robustness of message-passing decoding algorithms to computing inaccuracies. It aims at providing new approaches to cost/throughput optimizations, through the use of imprecise computing and storage mechanisms, without jeopardizing the error correction performance of the LDPC code. To do so, imprecise processing within the iterative message-passing decoder is considered in conjunction with the quantization process that provides the finite-precision information to the decoder. Thus, we first investigate a low complexity code and decoder aware quantizer, which is shown to closely approach the performance of the quantizer with decision levels optimized through exhaustive search, and then propose several imprecise designs of Min-Sum (MS)-based decoders. Proposed imprecise designs are aimed at reducing the size of the memory and interconnect blocks, which are known to dominate the overall area/delay performance of the hardware design. Several approaches are proposed, which allow storing the exchanged messages using a lower precision than that used by the processing units, thus facilitating significant reductions of the memory and interconnect blocks, with even better or only slight degradation of the error correction performance.We propose two new decoding algorithms and hardware implementations, obtained by introducing two levels of impreciseness in the Offset MS (OMS) decoding: the Partially OMS (POMS), which performs only partially the offset correction, and the Imprecise Partially OMS (I-POMS), which introduces a further level of impreciseness in the check-node processing unit. FPGA implementation results show that they can achieve significant throughput increase with respect to the OMS, while providing very close decoding performance, despite the impreciseness introduced in the processing units.We further introduce a new approach for hardware efficient LDPC decoder design, referred to as Non-Surjective Finite-Alphabet Iterative Decoders (FAIDs). NS-FAIDs are optimized by Density Evolution for regular and irregular LDPC codes. Optimization results reveal different possible trade-offs between decoding performance and hardware implementation efficiency. To validate the promises of optimized NS-FAIDs in terms of hardware implementation benefits, we propose three high-throughput hardware architectures, integrating NS-FAIDs decoding kernels. Implementation results on both FPGA and ASIC technology show that NS-FAIDs allow significant improvements in terms of both throughput and hardware resources consumption, as compared to the Min-Sum decoder, with even better or only slightly degraded decoding performance.

Multicomponent metal alloys play an essential role in many technologies and their properties must be optimized by rational selection of the alloy’s components and its fractional composition of each. High-throughput materials synthesis allows us to prepare Composition Spread Alloy Films (CSAFs), sample libraries that contains all possible compositions of a binary or ternary alloy. In our lab, a Rotatable Shadow Mask (RSM) – CSAF deposition tool has been developed for the creation of CSAFs. Such CSAFs can be prepared with composition gradients and/or thickness gradients in arbitrarily controlled directions and on a variety of substrates. Once prepared, the CSAF libraries can be characterized thoroughly using a variety of highthroughput spectroscopic methods. Their bulk composition is mapped across the library using Energy Dispersive X-ray spectroscopy (EDX). The near-surface compositions are mapped across composition space using X-ray Photoemission Spectroscopy (XPS). Finally, the electronic structure can be mapped using UV photoemission spectroscopy (UPS) and valence band XPS. Once characterized, these CSAFs are being used for high-throughput studies of alloy catalysis and thermal properties of the alloys and of alloy-substrate interfaces. First of all, PdzCu1-z CSAF was prepared to show that alloy nanoparticles (aNPs) and thin films can adopt phases that differ from those of the corresponding bulk alloy. The mapping of XPS-derived core level binding energy shifts across PdzCu1-z SCSNaP library shows a promising result that the FCC phase can be dimensionally stabilized over the composition range where B2 phase exists in the bulk. This observation can potentially improve the performance of PdzCu1-z NP catalysts in H2 separation. Secondly, the relationship between catalyst activity-electronic structure-composition has been investigated. A high throughput characterization of electronic structure (valence band energy) of binary PdxAg1-x and ternary PdxCuyAu1-x-y CSAFs were performed by XPS. This XPS-derived valence band center is compared with UPS-derived data across PdxCuyAu1-x-y CSAFs. In addition, H2-D2 exchange reaction was studied on PdxAg1-x CASF. A higher HD formation rate is experimentally observed but cannot be predicted by the Langmuir-Hinshelwood model when the surface coverage is saturated. However, the proposed H2-D2 exchange mechanism (breakthrough model) involved with surface and subsurface hydrogen reaction is investigated to produce a same reaction order as Langmuir-Hinshelwood mechanism, which cannot explain the experimental observation. Furthermore, the thermal interface conductance (G) was studied as a function of metal alloy composition. A high-throughput approach to preparation, characterization, and measurement of G was also demonstrated to study the thermal property of alloyed materials. Our result in studying the G across the AuxY1-x (Y = Pd and Cu) CSAFs-dielectric interfaces has shown a linear relationship with alloy composition, which monotonically increases with decreasing Au (at. %). Lastly, the effect of interdiffusion in metal films on G at metal-dielectric interface was also examined. The XPS depth profiling was designed to experimentally determine the temperature effect on compositional profiles in the Au-Cu system, and how to further influence G. This study provides fundamental understanding of stability of adhesion layer of Cu and the effect of interdiffusion in Cu-Au alloy on the heat dissipation. All in all, the key value to these CSAF libraries is that they enable measurement of important alloy properties across entire binary or ternary alloy composition spaces, without the need to prepare and characterize numerous discrete composition samples.

Despite active development during past years, the task of sequencing a genome still remains a challenge. Our current technologies are not able to read the whole genome in one piece. Instead, we shatter the target genome into a large amounts of small pieces that are then sequenced separately. The process of assembling these small pieces together, in order to obtain sequence of the whole genome, is painful and rsource-consuming. Multiple algorithms to solve the assembly problem were developed. This thesis presents yet another assembly algorithm, based on the usage of de Bruijn graphs, and focusing on sequencing short genome regions. The algorithm is compared to well-known solutions in the field. 1

Abstract The aim of this PhD-study was to address some of the overlapping bottlenecks in protein engineering and metagenomics by developing or applying new tools which are useful for both disciplines. Two enzymes were studied as an example: Triosephosphate Isomerase (TIM) and Uridine Phosphorylase (UP). TIM is an important enzyme of the glycolysis pathway and has been investigated via means of protein engineering, while UP is a key enzyme in the pyrimidine-salvage pathway. In this thesis TIM was used to address protein engineering aspects, while UP was used in regards to some metagenomic and bioinformatic aspects. The aspects of a structural driven rational design approach and its implications for further engineering of monomeric TIM variants are discussed. Process development based on a new technology, EnBase®, addresses the relative instability of new variants, compared to its ancestors, for further studies. EnBase® is then applied for the production of 15N isotope labeling of a monomeric TIM variant, A-TIM. Systematical function- and engineering studies on dimeric TIM and monomeric TIM in regards to the hinges of the catalytic loop-6 were conducted to investigate enzyme activity and stability. Both the A178L and P168A were proposed to induce loop-6 closure, a wanted feature for A-TIM variants. The P168A mutants are hardly active, but gave great insight into the catalytic machinery, while the A178L mutants did induce partial loop-6 closure, however in addition, monomeric A178L was destabilized. Homology driven genome mining and subsequent isolation- high throughput (HTP) overexpression of a thermostable UP from the Archaea Aeopyrum pernix was carried out as an example for the production of recombinant proteins. In addition an alternative kinetic method to study the kinetics of UP by means of NMR directly from cell lysate is discussed. The combination of expression libraries and EnBase® in a HTP manner may relieve up the gene-to-product bottleneck. The structural aspects of A. pernix UP are explored by means of simple bioinformatic tools in the last section of this thesis. A thermostable, truncated version of UP was created and its use for protein engineering in the future is explored. The long N-terminal and C-terminal ends of A. pernix UP seem to be involved in stabilizing the dimeric and hexameric structures of UP. However, deletion of the N-terminal end of A. pernix UP yielded a thermostable protein. Overall, the finding in regards to process optimization and HTP expression and optimization and the underlying methods used in the TIM studies and the UP studies are interchangeable.

Glioblastoma Multiforme (GBM) is a primary malignant brain cancer, affecting children and adults and has a very poor prognosis. Up to 30% of the tumour mass consists of host derived immune cells, and a better understanding of how these cells affect tumour behaviour is required. These cells, called ‘Tumour Associated Macrophages’ (TAM) have been shown to occur in peripheral solid organ cancers, where they can cause local immune suppression, increase invasiveness and aid tumour growth. In the brain however, TAMs can consist of centrally derived microglia and peripherally derived macrophages, and although these cells could be exerting different effects on the tumour, there is currently no reliable way of distinguishing between the two. Cancer Stem Cells (CSC) are a subpopulation of cells within the tumour mass with stem-like features, are capable of self-renewal, and can recapitulate a tumour in an immunocompromised mouse host. It is thought that these cells play a role in disease recurrence and hence represent a potential target for anti-GBM therapies. In the first project we investigate the interaction between Cancer Stem Cells and TAMs. We first describe a method of culturing CSCs and TAMs from a single human patient sample, followed by an investigation into the functional properties of these cell types. We found functional differences between established cell line pairings of U87-MG and CHME3 versus primary patient derived CSCs and TAM cell line pairings. Polarisation of microglia/TAMs with lipopolysaccharide caused significant decrease in proliferative capacity of the GBM cell lines. Next we used a Non-Myeloablative Conditioning System (NMCS) to selectively replace the peripheral bone marrow compartment of wild type animals with labelled bone marrow cells, without disturbing brain homeostasis. We demonstrate that peripheral cells home exclusively to the orthotopically implanted tumour, and that some of these cells are CD11b+ and Gr1+, suggestive of myeloid derived suppressor cells (MDSC). We evaluate current CD45 based gating strategies for distinguishing peripheral and central cells and show them to be inadequate. Finally we compared the chemosensitivity profiles of different patient derived CSC lines using high throughput content screening (HTCS), against currently approved chemotherapeutic drugs and rank these drugs in a response space, based on HTCS parameters including 2D and 3D culture with and without irradiation. Differential chemosensitivities were noted between different patient derived cell lines. Drugs not currently used in the treatment of GBM such as Actinomycin D and Mitoxantrone were also identified using HTCS, suggesting the potential utility of such an approach to personalised treatments in GBM.