Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 103-115).

La leucémie lymphoïde chronique (LLC) est la plus fréquente des leucémies qui touchent le monde occidental. Cette pathologie est toujours incurable et se caractérise par une hétérogénéité d’évolution clinique marquée par une survie globale oscillant de quelques mois à des dizaines d’années. Il est donc primordial de savoir à quel type d’évolution le patient sera confronté afin d’adapter au mieux le suivi de la maladie et la précocité ou non du traitement. Au cours de ce travail, nous avons comparé les facteurs pronostiques les plus courants (stade de Binet, statut mutationnel des IgVHs, expression de ZAP70, du CD38 et de la LPL) afin d’évaluer leur association avec la survie sans traitement (treatment-free survival ou TFS) et la survie globale (overall survival ou OS) tout en mettant au point une technique de mesure de ZAP70 par PCR quantitative en temps réel (qPCR). Cette étude a conclu que ZAP70 mesuré par qPCR est un puissant facteur pronostique et le meilleur facteur de substitution du statut mutationnel parmi l’ensemble des marqueurs testés. Nous avons également mis en évidence que le temps de doublement du CD23 soluble (TDCD23s), un nouveau facteur pronostique associé à la dynamique de la maladie, pouvait prédire le TFS, l’OS mais également affiner le pronostic des patients. Finalement, nous avons démontré que la diminution de deux microARNs, miR-29c et miR-223, était fortement corrélée à une LLC agressive, une masse tumorale élevée et à d’autres facteurs de mauvais pronostic. De plus, l’association du pouvoir pronostique de ces deux microARNs avec celui de ZAP70 et de la LPL a permis de générer un score qPCR (de 0 à 4 facteurs de mauvais pronostic) permettant littéralement de stratifier le risque de besoin de traitement et la survie des patients LLC. Dans le but de comprendre les différences géniques entre les patients de bon et de mauvais pronostic, nous avons comparé le transcriptome de patients différant fortement pour l’expression de ZAP70. 27 gènes ont ainsi été mis en évidence comme différentiellement exprimés avec un FDR<10%, 135 avec une P<0.001 et 932 sondes avec P<0.05. Parmi ces gènes, nombreux sont capables de prédire le TFS et l’OS. Une analyse bioinformatique de type « gene set enrichment » a révélé la sur-représentation de gènes impliqués dans l’adhésion et la migration. Nous avons donc évalué la capacité d’adhésion et de migration de cellules de LLC issues de patient à pronostic différent au sein d’un microenvironnement stromal et en réponse à du milieu conditionné par les cellules stromales. De cette manière, avons démontré les meilleures capacités d’adhésion, de migration et de réponse aux stimuli du microenvironnement des cellules ZAP70+. Ces observations nous ont amenés à conclure que la classification d’un patient en bon ou mauvais pronostic selon de nombreux marqueurs n’est qu’un reflet de la capacité des cellules leucémiques à interagir avec leur microenvironnement et que la faculté des cellules ZAP70+ à adhérer/migrer dans leur microenvironnement explique leur meilleure survie et donc l’agressivité de la maladie. Le troisième volet de cette thèse s’est intéressé à une nouvelle stratégie thérapeutique pour la LLC : nous avons observé, in vitro, que l’acide valproïque (VPA) induisait l’apoptose des cellules de LLC en ciblant de nombreux gènes anti- et pro-apoptotiques. De plus, nos expériences ont démontré que le VPA pouvait inhiber la prolifération des cellules leucémiques en perturbant l’expression des gènes du cycle cellulaire. Finalement, l’association du VPA à différentes drogues chimiothérapiques augmente la chimiosensibilité des cellules de LLC. L’ensemble de ces résultats supporte fortement l’utilisation du VPA dans le traitement de la LLC, en particulier en combinaison avec d’autres agents antileucémiques.

<p>In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised.</p>

Al llarg de les dues darreres dècades hem tingut el privilegi d’assistir a un dels esdeveniments més importants en la història de la biomedicina: el desenvolupament del Projecte Genoma Humà. Conjuntament amb aquest fet, les tècniques de laboratori a gran escala, cada dia més potents i fiables, s’utilitzen actualment de manera rutinària en el camp de la recerca biomèdica. Tot i que encara queda un gran camí per recórrer, aquests fets indubtablement han sembrat la llavor per desenvolupar un nou paradigma de recerca en càncer, així com per millorar els futurs tractaments dels pacients. Ens estem movent progressivament d’un escenari on els diagnòstics i els tractaments es basen principalment en criteris patològics a un altre completament personalitzat, on cada pacient serà diagnosticat i tractat d’una manera especialitzada en funció de criteris moleculars. Tot i així, per assolir una medicina del càncer totalment eficient i personalitzada és essencial obtenir una imatge acurada de tots els processos moleculars involucrats en el desenvolupament d’aquesta malaltia complexa. Per obtenir aquesta imatge necessitarem, doncs, obtenir informació a gran escala de la cèl•lula tumoral a nivell de genoma, epigenoma, transcriptoma i proteoma. Un cop tota la información està disponible, s’hauran d’aplicar les tècniques analítiques integratives per detectar les alteracions moleculars que tenen un paper primordial el procés tumoral. Els estudis presentats en aquesta tesi pretenen ser una mostra d’aquests tipus d’anàlisis integratives. Aquesta tesi es basa en una col•lecció de tres articles que són el resultat de la feina duta a terme a la Unitat de Biomarcadors i Susceptibilitat de l’Institut Català d’Oncologia. / Over the last two decades we have been given the privilege of witnessing one of the most relevant breakthroughs in the history of biomedicine: the development and completion of the Human Genome Project. Along with it, large-scale laboratory techniques, every day more powerful and reliable, are now routinely applied in biomedical research. Although there is still a long way to go, this fact has undoubtedly set the seed for a new paradigm in cancer research and the future treatment of patients. We are progressively shifting from a scenario where diagnoses and treatments are mainly based on pathological criteria to a completely personalized one, where every single patient will be diagnosed and treated in a specialized manner according to molecular criteria. Nonetheless, to achieve a fully efficient and personalized cancer medicine, it is essential to obtain an accurate picture of all the molecular processes involved in the development of such a complex pathology as cancer. This picture can only be obtained if we can have a detailed view of a tumor cell’s status at a whole genome, epigenome, transcriptome and proteome levels. Once all the information is available, suitable analytical integrative techniques must be applied to detect the molecular alterations that play a driver role in the tumorigenic process. The studies presented in this thesis aim to be examples of such integrative analyses. This thesis is based on a collection of three articles, which are the result of the work done at the Unit of Biomarkers and Susceptibility at the Catalan Institute of Oncology.

La resposta enfront a una situació d'estrès en microorganismes genera una resposta adaptativa que implica una remodelació en l'expressió gènica. Per estudiar la resposta transcripcional enfront a una situació d'estrès ens vàrem plantejar una solució atrevida que consistia en l'amplificació dels aproximadament 6000 ORFs del genoma sencer de Saccharomyces cerevisiae amb la col·laboració dels laboratoris dels Drs. José Enrique Perez-Ortín (Universitat de València) i Javier Arroyo (Universitat Complutense de Madrid) per fabricar nosaltres mateixos microxips de DNA. Un primer ús d'aquests microxips va ser l'estudi de la resposta transcripcional de S. cerevisiae enfront a una situació d'estrès per pH alcalí. Les respostes moleculars produïdes per variacions del pH extracel·lular han estat poc estudiades en Saccharomyces cerevisiae a diferència d'altres fongs com Aspergillus nidulans, Candida albicans o Yarrowia lipolytica. Quan es va iniciar aquest treball de recerca el nombre de gens de Saccharomyces cerevisiae que es sabia que presentaven una resposta a pH alcalí era sorprenentment petit: ENA1, que codifica un ATPasa de sodi essencial per la destoxificació de sodi, SHC1 i SCY1 dels quals no es coneixia pràcticament res. Per aquest motiu en aquest treball s'han utilitzat microxips de DNA fabricats en el nostre laboratori amb l'objectiu de realitzar una millor caracterització de la resposta transcripcional enfront a una situació d'estrès com és la exposició a pH alcalí.Recentment, s'ha observat en el nostre laboratori que la calcineurina podria estar involucrada en la tolerància enfront a pH alcalí. Es va demostrar que una part significativa de la resposta a pH alcalí del promotor ENA1 i pràcticament la totalitat de la resposta de PHO89 (codifica una permeasa Na+/Pi) estava bloquejada per l'inhibidor de la calcineurina FK506 i també en absència de Cnb1 o Crz1 suggerint que la resposta transcripcional d'una sèrie de gens enfront a l'acalinització del medi podria ser, com a mínim en part, depenent de la calcineurina. El fet que dos dels quatre gens estudiats més exhaustivament presentessin dependència a calcineurina en la seva resposta enfront a pH alcalí ens va fer pensar que un nombre important de gens de llevat podrien respondre a pH alcalí a través de la via de senyalització de la calcineurina. En aquest treball confirmem i ampliem aquesta hipòtesis definint, mitjançant la tecnologia de microxips de DNA, un conjunt de gens la resposta transcripcional dels quals està regulada per la via de la calcineurina. / Exposure of microorganisms to stress resulted in adaptative changes that involved remodeling the gene expression. To study the transcripcional response to stress we took the daring decision to amplificate the entire Saccharomyces cerevisiae genome (6000 ORFs approximately) in collaboration with Drs. José Enrique Perez-Ortín (Universitat de València) and Javier Arroyo (Universitat Complutense de Madrid) to generate home made microarrays. This microarrays were firstly used to study transcriptional response to alkaline conditions.The study of alkaline response in S. cerevisiae has received little attention compared with other fungi like Aspergillus nidulans, Candida albicans or Yarrowia lipolytica. When this work was initiate, the number of Saccharomyces genes known to be responsive to alkalinization of the medium was surprisingly small: ENA, encoding a P-type Na+-ATPase essential for sodium detoxification, SHC1 and SCY1 of unknown function. For this reason we used home made microarrays to study transcriptional response of yeast cells to an increase of external pH.Recent evidence in our laboratory suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. We showed that significant part of the alkaline response of the ENA1 promoter, and most of the PHO89 response, was blocked by the calcineurin inhibitor FK506 as well as by the absence of Cnb1 or Crz1/Tnc1 suggesting that the transcriptional response of certain genes to alkalinization of the medium could be, at least in part, dependent on calcineurin. These findings let us to speculate the possibility that calcineurin pathway could play an important role in transcriptional response to alkaline pH. In this work we confirm this hypothesis by defining, using DNA microarray technology, the subset of genes whose alkaline transcriptional response is mediated by the calcineurin pathway.

The physiological responses to exercise depend on intensity, duration, and type of exercise. The muscles in the body have complex regulation responses in order to create a certain resistance and adaptation to the exercise demands without fatigue. In the following study, we used the model of Alaskan sled dogs in order to analyze changes in gene expression within muscle tissue. Gene expression allows us to look more in depth into temporal or long term biological changes that take place in order for the muscle to adapt and maintain homeostasis. Eight dogs were used for the study; four biopsies from the femoris biceps were taken from each at different time points. Time point 1 (Tp1) untrained dogs, time point 2 (Tp2) after mid training, time point 3 (Tp3) fully trained and time point 4 (Tp4) were taken after dogs had completed a 400 mile run in 4 consecutive days. Time point one was used as a control ratio for the other three time points for analysis one, for the second analysis Tp1 was eliminated as a control. Analysis, one compared Tp2-Tp3 and Tp3-Tp4; the subsequent analysis looked at Tp1-Tp3. For Mid trained animals compared to fully trained, we looked at a total of 25 differentially expressed genes, for fully trained compared against acute exercise performance, we looked at total of 52 differentially expressed genes (based on a ≤0.01 p-value and fold change of ≥3), and untrained was compared to fully trained where we looked at a total of 26 differentially expressed genes. Known transcriptional regulators were mapped from these differentially expressed genes, such as exocyst complex, lysyl oxidase, protein tyrosine phosphatase, protein kinase C, creatine kinase, HSP40, cytochrome P450, ACSL6 gene responsible for Acyl-CoA synthesis, myosin chain, ATP binding, and ubiquitin, among others. These transcripts were linked to important biological pathways, and functional analysis of these pathways demonstrated that changes found in gene expression are responsible for muscle tissue remodeling, energy storage and metabolism changes, cardiovascular enhancement, and activation of elements that regulate metabolism via the nervous system. The following study of transcriptional regulation mechanisms helped identify specific responses to exercise stimuli in the organism that allow the athletes to adapt to the demands of exercise.

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains xiii, 180 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 138-151).

published_or_final_version / Microbiology / Master / Master of Philosophy

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.