Coral reef biodiversity is threatened by rapidly changing anthropogenic activities and natural perturbations, leading to massive ecological and economic consequences ranging from the loss of fisheries to coastal erosion. It is necessary to understand corals responses to environmental changes in order to determine management programs on appropriate spatial and temporal scales to address these issues. Coral larvae are the product of sexual reproduction, have the potential to recruit to new areas, and are fundamental in maintaining genetic diversity. These larvae are subjected to variations in local environmental conditions until they settle, inducing specific larval molecular response patterns. One factor that influences coral health is salinity. Low salinities can alter cell homeostasis creating stress in cells. In the natural environment larvae may be exposed to low salinities due to heavy rainfall or run-off. This study investigated larvae responses to low salinity and characterized gene expression in the reef-building coral Porites astreoides using a coral stress-focused microarray. Nine batches of 250+ larvae from three different colonies were collected and immediately exposed in an acute hyposalinity experiment. Samples from two treatments of 25 and 30 ppt, and a control at 35 ppt were used in this study. After experimental exposure these samples were stored in RNAlater® and molecular analysis was performed. The RNA from the samples was extracted, purified and hybridized to a coral stress-focused microarray. Statistical analysis indicates 72 genes were differentially expressed across treatments (p<0.003, analysis of variance). The hierarchical cluster analysis groups together the larvae exposed to salinities of 30 and 35 ppt indicating both treatments induced similar patterns of gene expression. Larvae responses to 30 ppt are minimal, suggesting larvae can tolerate acute exposures to 30 ppt salinity levels. In contrast, the lower salinity (25 ppt) induced a strong response in both the coral and zooxanthellae. The coral larvae up-regulated stress response genes and down-regulated genes associated with normal cell functioning. Additionally, the zooxanthellae down-regulated genes associated with photosynthesis. These results suggest larvae may be vulnerable to bleaching, which may affect the ability of larvae to successfully undergo metamorphosis and survive at low salinities. However, this has yet to be confirmed with complementary techniques. Long-term studies are recommended to examine the effects of hyposalinity on larvae at different time scales and life history stages.

Abstract For decades, the mammalian kidney has served as a model system for studying developmental processes, such as induced epithelialization, branching morphogenesis, and cell differentiations. The possibility to recapitulate and follow the renal organogenesis ex vivo in organ culture set-ups has provided a large amount of molecular and cellular information about sequential events during development. However, certain limitations remain when combining traditional organ culture set-ups with modern molecular technology. This thesis seeks to address these disadvantages. In the experimental part of the thesis, the traditional organ culture set-ups were studied, modified, and optimized to meet the needs of functional genetic screening. First, the traditional transfilter- induced nephrogenesis was characterized with a panel of nephron segment specific markers to reveal the differentiation level of in vitro developing mouse renal tissue. A comprehensive genome wide time course microarray analysis was also performed to in vitro- induced metanephric mesenchyme. Next, to improve the accessibility of genetic tools into the three- dimensional organ in culture, the classic kidney culture set-ups were modified to tolerate dissociation and re-aggregation before the induction of nephrogenesis. This step was achieved with the aid of preservative growth factors offering a 24- hour window to manipulate the genetic and cellular composition of the explant. The dissociation and re-aggregation per se had not particular effect on the progress of the nephron differentiation. Demonstrations of the addition and removal of cells, as well as a virus vector mediated gene knock in and knock down are presented. The gene expression data, together with the novel organ manipulation and culture techniques presented in this thesis, provide a useful guide and specific tools to further characterize the details of nephron development and differentiation in functional manner. / Tiivistelmä Nisäkkäiden munuainen on toiminut vuosikymmeniä mallielimenä tutkittaessa kehitysbiologisia tapahtumasarjoja, kuten epitelisaatiota, haaroittumismorfologiaa sekä solujen erilaistumista. Munuaisaihioita voidaan viljellä laboratorio-olosuhteissa, jolloin kehityksen aikaisia muutoksia päästään seuraamaan lähes reaaliaikaisesti. Perinteisten kudosviljelytekniikoiden tarjoamat mahdollisuudet solujen molekulaariseen muokkaukseen ovat kuitenkin varsin rajalliset. Tässä väitöskirjassa esitettävät tulokset pyrkivät osaltaan vähentämään näitä rajoitteita. Väitöskirjan kokeellisessa osassa tarkastellaan lähemmin klassista munuaiskudosviljelyä sekä esitetään siihen tehtyjä optimointeja, joiden avulla kudosviljelyä pyritään hyödyntämään geenien toiminnan tutkimuksessa. Aluksi perinteisellä tavalla reikäisen kalvon läpi indusoitu nefroni karakterisoitiin tarkasti hyödyntäen useita erilaistumista osoittavia merkkimolekyylejä. Lisäksi samalla tekniikalla tuotettujen munuaiskudosviljelmien geeniekspressiota tutkittiin mikrosiruanalyysillä. Klassisia kudosviljelytekniikoita muokattiin soveltuvammaksi moderneille geneettisille työkaluille. Munuaiskudos hajotettiin ensin solususpensioksi, jonka jälkeen solut muodostivat uudelleen kolmiulotteisen, kudosmaisen rakenteen. Hyödyntämällä suojaavia kasvutekijöitä, hajotus kyettiin tekemään jo ennen nefronien muodostumisen alkua. Näin saavutettin 24 tunnin aikaikkuna indusoimattoman kudoksen geneettiselle muokkaukselle. Väitöskirjassa esitelläänkin demonsrtaatiot solujen lisäämisestä ja poistamisesta sekä virusvälitteisestä geenin aktivoinnista ja hiljennyksestä hyödyntäen uutta kudosmanipulaatio ja –vilejelytekniikkaa. Nefronin kehityksen aikaisen geeniekspression kartoitus sekä tässä tutkimuksessa kehitetyt uudet kudosmanipulaatio ja -viljelytekniikat tarjoavat yhdessä työkaluja molekyylitason yksityiskohtaiseen tutkimiseen.

Background: The biological mechanisms underlying early-and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin-paclitaxel treatment. Methods: Tumour epithelia were isolated from two independent sets of primary EOC (n-72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1-AMPK and AKT-mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin-paclitaxel-sensitive and -resistant tumours. Results: Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK-AKT-mTOR axis compared to early EOC (P<0.05 for AMPK alpha T172, AMPK alpha 1 S485, AMPK beta 1 S108, AKT S473 and T308, mTOR S2448, p70S6 S371, 4EBP1 S65, GSK-3 alpha/beta S21/9, FOXO1 T24/FOXO3 T32, and FOXO1 S256). Advanced tumours with low relative activation of the metabolic signature and increased FOXO1 T24/FOXO3 T32 phosphorylation (P=0.041) were associated with carboplatin-paclitaxel resistance. Conclusions: If validated in a larger cohort of patients, the decreased AMPK-AKT-mTOR activation and phosphorylation of FOXO1 T24/FOXO3 T32 may help identify carboplatin-paclitaxel-resistant EOC patients.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Phloem-feeding insects like aphids feed on a variety of crop plants and limit plant productivity. In addition they are vectors for important plant viruses. Efforts to enhance plant resistance to aphids have been hampered by lack of sufficient understanding of mechanisms of plant defense against aphids. I have utilized a plant-aphid system consisting of the model plant Arabidopsis thaliana and the generalist aphid, Myzus persicae Sulzer (green peach aphid [GPA]), to study plant response to aphids. These studies have demonstrated an important role of premature leaf senescence in controlling aphid growth in Arabidopsis. Molecular and physiological studies suggest that the Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT 4) gene modulates the GPA feeding-induced senescence process. Furthermore, in comparison to the wild type plants, GPA growth was higher on pad4 mutant plants, suggesting an important role for PAD4 in plant defense against GPA. In contrast, constitutive expression of PAD4 in transgenic Arabidopsis enhanced basal resistance against GPA. Unlike its involvement in plant defense against pathogens, the role of PAD4 in Arabidopsis resistance to GPA is independent of its involvement in phytoalexin biosynthesis and of its interaction with EDS1, a PAD4-interacting protein. Instead, the heightened resistance to GPA in these PAD4 constitutively expressing plants was associated with the rapid activation of leaf senescence. The association of premature leaf senescence in basal defense against GPA is supported by our observation that in comparison to the wild type plant, GPA growth was restricted on the Arabidopsis hypersenescence mutants, ssi2 and cpr5. Gene expression studies suggested some overlap between plant responses to pathogens and aphids, for example, activation of genes associated with the salicylic acid (SA) signaling pathway. However, the characterization of aphid performance on Arabidopsis SA biosynthesis and signaling mutants have ruled out the involvement of SA signaling in controlling aphid growth.

Atlantic salmon (Salmo salar L.) has been domesticated since the 1960s and has undergone over 10 generations of artificial selection for economically important traits. As a result, domesticated salmon have diverged with respect to a number of phenotypic, genotypic and behavioural traits from their wild counterparts. Since the selection pressures that are present in the wild differ greatly from the ones that shape salmon under culture conditions, domesticated salmon stocks are considered to be maladapted to natural conditions. Despite strict regulations, insoluble issues pertaining to large-scale cage rearing of farmed fish mean that there is a continuous presence of farm escapees in the wild. Gene flow from escapees has been perceived as a factor in the decline of wild populations, suggested to occur through disruption of local adaptation. This study aims to improve understanding of the genetic differences between wild and domesticated stocks by comparing the transcriptomes of Figgjo (wild) and Mowi (domesticated) strains. A series of common garden experiments have been performed, utilizing pure and reciprocal hybrid crosses of the wild and domesticated stocks, reared under two different conditions and sampled at four time points and three distinct life stages (embryo, sac-fry and feeding fry). Microarray interrogations were performed employing a 44K custom microarray design to identify genes and gene pathways that are differentially expressed between the stocks. KEGG-based functional analyses have been implemented using different gene set enrichment packages, and dominance and additive parameters were calculated from normalized expression values to predict the mode of heritability of the genes identified as differentially expressed between stocks. Most biological functions represented in wild and domesticated crosses were consistent across life stages and environments. The transcriptomic differences detected between stocks in multiple developmental stages likely reflected adaptations to selection pressures differing between natural and aquaculture environments. Down-regulated environmental information processing and immune and nervous system functions in domesticated vs. wild fish may be due to local adaptation to captivity. These included reduced information acquisition and processing systems, altered stress responsiveness and changes in feeding behaviour. In line with the resource allocation theory of production trait animals, reduced immune function was coupled with increased expression of growth and development related pathways in domesticated salmon, compared to wild counterparts. Although there is support for this trade-off in all life-stages, resource allocation showed a shift over time; possibly reflecting variation in the utilization of energy sources during the transition from endogenous to exogenous feeding. Differences in cell communication and signalling pathways between wild and domesticated stocks, associated with organogenesis during the embryo stage, reflect sampling time and are indicative of altered organ development in response to domestication. Stress responses common across stocks included the down-regulation of cellular processes, including cell cycle and meiosis, and genetic information processing, such as replication and repair, transcription and translation pathways, probably reflecting the reallocation of energy resources away from growth and towards the restoration of homeostasis. Moreover, the mobilization of energy to cover the increased demands of maintaining homeostasis was indicated by the up-regulation of some metabolic pathways, mostly involved in energy, lipid and carbohydrate metabolism in response to stress. The analysis also revealed cross-specific stress responses, including indicators of a non-additive stress response in hybrid crosses. Most differentially expressed transcripts exhibited additive (31-59%) or maternal dominant (19-33%) inheritance patterns, although maternal over-dominance (23-26%) was also significant in the embryo stage. The mode of heritability of some immune transcripts was suggestive of maternal environmental influence having been affected by aquaculture. This study has demonstrated that biological functions affected by domestication include those associated with allocation of resources, involve reduction of information acquisition and processing systems and may lead to loss of local adaptation to wild conditions. Since such changes may affect key systems, such as immunity and responsiveness to stress, they can potentially have serious negative consequences under natural conditions. Transcriptomic differences observed between wild and domesticated stocks primarily exhibited additive and maternal dominant inheritance modes. Since gene-flow from farmed fish can be frequent and primarily concerns farmed females, this suggests that introgression due to repeated large scale escape events has the capacity to significantly erode local adaptation.

Le mélanome malin (MM) métastatique, un des cancers les plus intrinsèquement résistants aux agents anti-cancéreux et présentant une forte capacité à développer des résistances acquises, constitue un défi thérapeutique. La meilleure compréhension des mécanismes impliqués dans cette chimiorésistance permettrait d'identifier des cibles thérapeutiques ou de guider le choix du traitement pour une meilleure efficacité. Les travaux réalisés durant cette thèse se sont focalisés sur l'identification de nouveaux déterminants moléculaires de la résistance acquise du MM vis-à-vis (i) des vinca-alcaloïdes (VAs, chimiothérapie classique), (ii) des inhibiteurs de MAP kinases (iMAPK, thérapie ciblée). Pour la première étude, un modèle de lignées cellulaires de MM résistantes aux VAs (CAL1R-VAs) a été établi (exposition continue, 12 mois, de la lignée parentale CAL1-wt à la VCR, la VDS ou la VRB : CAL1R-VCR, CAL1R-VDS et CAL1R-VRB respectivement). La comparaison des profils d'expression a permis de distinguer deux groupes de lignées cellulaires (CAL1R-VCR et CAL1R-VDS ; CAL1R-VRB et CAL1-wt), suggérant une résistance différentielle du MM aux VAs : d'une part à la VCR et à la VDS, d'autre part à la VRB. L'analyse des données transcriptomiques par une démarche associant successivement trois méthodes - RMA (Robust Multi-array Average), RDAM (Rank Difference Analysis of Microarrays) et MGSA (model-based gene set analysis) – a permis d'identifier des fonctions cellulaires altérées lors de la sélection des lignées CAL1R-VAs, et donc potentiellement à l'origine de la résistance de ces lignées. Des analyses fonctionnelles in vitro ont permis de confirmer l'implication des lysosomes et de la réponse au stress du réticulum endoplasmique (RE) dans la résistance différentielle des cellules CAL1 aux VAs. Ainsi, une sous-expression des cathepsines B et L (bioinformatique) et une réduction du volume du compartiment acide (in vitro) ont été observées spécifiquement dans le premier groupe de lignées (CAL1R-VCR et CAL1R-VDS), suggérant une sensibilité réduite de ces lignées à la voie lysosomale de l'apoptose. Par ailleurs, l'inhibition de la voie de réponse au stress du RE par l'acide tauroursodésoxycholique (TUDCA) a induit une sensibilisation différentielle de l'ensemble des lignées CAL1 aux VAs, suggérant l'implication de cette voie dans la résistance différentielle primaire et acquise aux VAs. De plus, l'inhibition de la réponse au stress du RE a induit une sensibilisation d'une autre lignée cellulaire de MM, MDA-MB-435, à la VCR et à la VDS mais pas à la VRB. Ainsi, la voie de réponse au stress du RE semble impliquée dans la résistance différentielle du MM aux VAs. Ce mécanisme pourrait mettre en jeu l'autophagie, dont le flux était significativement augmenté dans le premier groupe de lignées. La même démarche d'analyse transcriptomique a été appliquée pour l'étude des mécanismes moléculaires de résistance acquise du MM aux iMAPK. Des lignées cellulaires de MM résistantes aux trois iMAPK majeurs ont été établies par exposition continue de la lignée parentale A375-wt, portant la mutation activatrice BRAF V600E, au vémurafenib (VMF, inhibiteur de BRAF), dabrafenib (DBF, inhibiteur de BRAF), et trametinib (TMT, inhibiteur de MEK): A375R-VMF, A375R-DBF et A375R-TMT respectivement. La comparaison des profils transcriptomiques n'a pas permis de regrouper les lignées résistantes entre elles, suggérant que les mécanismes de résistance au VMF, au DBF ou au TMT sont différents. Ces mécanismes ne seraient donc communs ni à la voie ciblée (MAPK), ni à la cible moléculaire (BRAF ou MEK). L'identification des fonctions cellulaires altérées procurera un rationnel pour l'étude mécanistique de nouveaux déterminants de la résistance du MM aux iMAPK. / Malignant melanoma (MM), one of the most intrinsically resistant cancers to anticancer agents and presenting a strong ability to develop acquired resistance, remains a therapeutic challenge. A better understanding of the mechanisms involved in MM chemoresistance should provide therapeutic targets or guide therapeutic choice for improved efficiency. This thesis has focused on the identification of new molecular determinants of MM acquired resistance to (i) vinca alkaloids (VAs, conventional chemotherapy), and to (ii) MAP kinases inhibitors (MAPKi, targeted therapy). In the first study, MM cell lines resistant to VAs (CAL1R-VAs) were established (continuous exposure, 12 months, of CAL.1-wt parental line to the VCR, VDS or VRB: CAL1R-VCR, CAL1R- VDS and CAL1R-VRB respectively). Comparison of expression patterns led to distinguish two groups of cell lines (CAL1R-VCR and CAL1R-VDS; CAL1R-VRB and CAL.1-wt), suggesting a differential resistance of MM to VAs: one the one hand to VCR and VDS, on the other hand to VRB only. The analysis of transcriptome data by a process involving successively three methods - RMA (Robust Multi-array Average), RDAM (Rank Difference Analysis of Microarrays) and MGSA (model-based gene set analysis) – allowed the identification of functions altered during the resistant cell line selection, and therefore potentially involved in resistance mechanisms of these cell lines. In vitro functional analyzes confirmed the involvement of the lysosomes and of the response to endoplasmic reticulum (ER) stress (unfolded protein response, UPR) in the differential resistance of CAL1 cells to VAs. Thus, an under-expression of cathepsins B and L (bioinformatics), and a reduction of the acidic compartment volume (in vitro) were specifically observed in the first cell group (CAL1R-VCR and CAL1R-VDS), suggesting a reduced sensitivity of these lines to the lysosomal pathway of apoptosis. Furthermore, UPR inhibition using tauroursodeoxycholic acid (TUDCA) induced a differential sensitization of all the CAL1 lines to VAs, suggesting the involvement of this pathway in the primary and acquired differential resistance to VAs. Moreover, TUDCA-inhibition of UPR induced sensitization another MM cell line, MDA-MB-435, to VCR and VDS but not to VRB. Thus, a UPR up-regulation could to be a significant mechanism of differential resistance of MM to VAs. This mechanism could involve autophagy, whose flow was significantly increased in the first group of lines. The same transcriptome analysis strategy was applied to study (ii) the molecular mechanisms of MM acquired resistance to MAPKi. MM cell lines resistant to the three major MAPKi were established by continuous exposure of the parental A375-wt line, carrying the activating mutation BRAF V600E, to vemurafenib (VMF, BRAF inhibitor), dabrafenib (DBF, BRAF inhibitor), or trametinib (TMT, MEK inhibitor): A375R-VMF, A375R-DBF and A375R-TMT, respectively. Comparison of transcriptomic profiles showed separate expression profiles, suggesting that the molecular mechanisms responsible for resistance to VMF, DBF or TMT were different. These mechanisms cannot therefore be common to the targeted pathway (MAPK) or to the molecular target (BRAF or MEK). The identification of the altered cellular functions will provide a rationale for mechanistic studies of new determinants of MM resistance to MAPKi.

Microarray platforms have been around for many years and while there is a rise of new technologies in laboratories, microarrays are still prevalent. When it comes to the analysis of microarray data to identify differentially expressed (DE) genes, many methods have been proposed and modified for improvement. However, the most popular methods such as Significance Analysis of Microarrays (SAM), samroc, fold change, and rank product are far from perfect. When it comes down to choosing which method is most powerful, it comes down to the characteristics of the sample and distribution of the gene expressions. The most practiced method is usually SAM or samroc but when the data tends to be skewed, the power of these methods decrease. With the concept that the median becomes a better measure of central tendency than the mean when the data is skewed, the tests statistics of the SAM and fold change methods are modified in this thesis. This study shows that the median modified fold change method improves the power for many cases when identifying DE genes if the data follows a lognormal distribution.

The human genome contains tens of thousands of gene loci which code for an even greater number of protein and RNA products. The highly complex temporal and spatial expression of these genes makes possible all the biological processes of life. Altered gene expression by mutation or deregulation is fundamental for the development of many human diseases. The ultimate aim of this thesis was to identify gene expression changes relevant to cancer. The advent of genome-wide expression profiling techniques, such as microarrays, has provided powerful new tools to identify such changes and researchers are now faced with an explosion of gene expression data. Processing, comparing and integrating these data present major challenges. I approached these challenges by developing and assessing novel methods for cross-platform analysis of expression data, scalable subspace clustering, and curation of experimental gene regulation data from the published literature. I found that combining results from different expression platforms increases reliability of coexpression predictions. However, I also observed that global correlation between platforms was generally low, and few gene pairs reached reasonable thresholds for high-confidence coexpression. Therefore, I developed a novel subspace clustering algorithm, able to identify coexpressed genes in experimental subsets of very large gene expression datasets. Biological assessment against several metrics indicates that this algorithm performs well. I also developed a novel meta-analysis method to identify consistently reported genes from differential expression studies when raw data are unavailable. This method was applied to thyroid cancer, producing a ranked list of significantly over-represented genes. Tissue microarray analysis of some of these candidates and others identified a number of promising biomarkers for diagnostic and prognostic classification of thyroid cancer. Finally, I present ORegAnno (www.oreganno.org), a resource for the community-driven curation of experimentally verified regulatory sequences. This resource has proven a great success with ~30,000 sequences entered from over 900 publications by ~50 contributing users. These data, methods and resources contribute to our overall understanding of gene regulation, gene expression, and the changes that occur in cancer. Such an understanding should help identify new cancer mechanisms, potential treatment targets, and have significant diagnostic and prognostic implications. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Cancer is a serious threat to human health and is now one of major causes of death worldwide. However, the complexity of the cancer makes the development of new and specific diagnostic tools particularly challenging. A number of different strategies have been developed for biomarker discovery in cancer using microarray data. The problem that typically needs to be addressed is the scale of the data sets; we simply do not have (or are likely to obtain) sufficient data for classical machine learning approaches for biomarker discovery to be properly validated. Obtaining a biomarker that is specific to a particular cancer is also very challenging. The initial promise that was held out for gene microarray work for the development of cancer biomarkers has not yet yielded the hoped for breakthroughs. This work discusses the construction of a strategy for a systematic approach to biomarker discovery validation using lung cancer gene expression microarray data based around non-small cell cancer and in patients which either stayed disease free after surgery (a five year window) or in which the disease progressed and re-occurred. As a means of assisting the validation purposes we have therefore looked at new methodologies for using existing biological knowledge to support machine learning biomarker discovery techniques. We employ text mining strategy using previously published literature for correlating biological concepts to a given phenotype. Pathway driven approaches through the use of Web Services and workflows, enabled the large-scale dataset to be analysed systematically. The results showed that it was possible, at least using this specific data set, to clearly differentiate between progressive disease and disease free patients using a set of biomarkers implicated in neuroendocrine signaling. A validation of the biomarkers identified was attempted in three separately published data sets. This analysis showed that although there was support for some of our findings in one of these data sets, this appeared to be a function of the close similarity in experimental design followed rather than through specific of the analysis method developed.

Cardiomyocyte perinatal development is characterized by the transition from a hyperplastic to a hypertrophic growth. We hypothesize that genetic factors and processes in the cardiac perinatal transitional program can be identified by a systematic analysis of different stages in heart development. Microarray expression patterning of mRNAs and microRNAs uncovered a perinatal cardiogenomic switch between 5 and 7 days post-birth. Gene ontology analysis revealed cellular and metabolic processes as highly representative Biological Processes. Moreover, approximately 40% of known mice transcription factors are significantly (p<0.05) fluctuating between embryonic day 19 and 10 days post-birth. As the heart matures, cardiomyocytes progressively exit cell cycle with day 5 as a pivotal point. Hypertrophy entails cardiomyocyte binucleation which may be promoted by Protein Regulator of Cytokinesis (Prc1) and its interactors. Temporal cardiac transcription expression analysis provides insight into underlining effectors within the cardiac perinatal transitional program as well as cardiac pathology.