Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved process during which cells lose epithelial characteristics and gain a migratory phenotype. Although downregulation of epithelial cadherins by Snail and other transcriptional repressors is generally considered a prerequisite for EMT, recent studies have challenged this view. Here we investigate the relationship between E-cadherin and P-cadherin expression and localization, Snail function and EMT during gastrulation in chicken embryos. Expression analyses show that while E-cadherin transcripts are detected in the epiblast but not in the primitive streak or mesoderm, P-cadherin mRNA and protein are present in the epiblast, primitive and mesoderm. Antibodies that specifically recognize E-cadherin are not presently available. During EMT, P-cadherin relocalizes from the lateral surfaces of epithelial epiblast cells to a circumferential distribution in emerging mesodermal cells. Cells electroporated with an E-cadherin expression construct undergo EMT and migrate into the mesoderm. An examination of Snail function showed that reduction of Slug (SNAI2) protein levels using a morpholino fails to inhibit EMT, and expression of human or chicken Snail in epiblast cells fails to induce EMT. In contrast, cells expressing the Rho inhibitor peptide C3 rapidly exit the epiblast without activating Slug or the mesoderm marker N-cadherin. Together, these experiments show that epiblast cells undergo EMT while retaining P-cadherin, and raise questions about the mechanisms of EMT regulation during avian gastrulation.

Plant embryogenesis is an intriguing developmental process that is controlled by many genes. AGAMOUS Like 15 (AGL15) is a MADS-domain transcriptional regulator that accumulates preferentially during this stage. However, at the onset of this work it was unknown which genes are regulated by AGL15 or how AGL15 is regulated. This dissertation is part of the ongoing effort to understand the biological roles of AGL15. To decipher how AGL15 functions during plant development, a chromatin immunoprecipitation (ChIP) approach was adapted to obtain DNA fragments that are directly bound by AGL15 in vivo. Putative AGL15 targets were isolated, and binding and regulation was confirmed for one such target gene, ABF3. In addition, microarray experiments were performed to globally assess genes that are differentially expressed between wild type and agl15 young seeds. Among them, a gene, At5g23405, encoding an HMGB domain protein was identified and its response to AGL15 was confirmed. Preliminary results suggest that the loss-of-function of At5g23405 might have an effect on somatic embryogenesis, consistent with AGL15 repression of the expression of this gene. Lastly, to address the question about how the regulator is regulated, the cis elements controlling the expression of AGL15 must be identified. Deletion analysis of the AGL15 promoter indicated the presence of putative positive and negative cis elements contributing to the expression of AGL15. Further analysis suggested that AGL15 regulates the expression of its own gene and this regulation may partially be explained by the direct binding of the protein to the AGL15 promoter. The data presented in this dissertation demonstrate that ChIP can be used to identify previously unsuspected targets of AGL15. Based on ChIP, a ChIP-chip technique is being developed in the lab to allow a more global analysis of in vivo binding sites. The identification of target genes and cis elements in AGL15 promoter is a step towards characterization of the biological roles of AGL15.

Wheat (Triticum aestivum L.) is occasionally exposed to low temperature during flowering and huge economic losses can occur especially in some key production countries such as Australia. Although it is generally predicted that there will be a rise in global temperature there are still predicted to be risks associated with low temperature for temperate crops. Post head emergence frost damage remains a major constraint to increasing wheat production. Five Iraqi varieties (Abu-Ghariab, Fatah, Sham6, IPA95 and IPA99) were screened for their frost hardiness (LT50). Abu-Ghariab was chosen for further investigations along with the European cv. Claire because the acclimated frost hardiness level in these two varieties (LT50 -8.07 and -8.01°C for Claire and Abu-Ghariab respectively) was found to be significantly lower than the other varieties in this study. Several techniques were employed including REC%, IR thermography and molecular analysis of cold acclimation in order to characterise the frost resistance of those two varieties. REC% revealed that both Claire and Abu-Ghariab could tolerate some freezing when the spikes were just visible (ZCK 51-60) with an acclimation shift of the LT50 of -1.6 and -2.11°C respectively but this was not apparent at later growth stages. Based on molecular analysis, cold acclimation was shown to be activated at ZCK 51-60 in both varieties. Cbf14 was expressed after 8 hours exposure to acclimatising temperatures (4°C) and then declined to a low, but still up-regulated level in both varieties and this led to expression of the COR15a protein. These molecular changes correlated with the frost tolerance recorded at ZCK 51-60. It was concluded that the possibility existed to up-regulate cold acclimation after spike emergence if there was enough environmental stimulus. Molybdenum (Mo) was demonstrated to work synergistically with low temperature in increasing the expression of Cbf14 and COR15a. The European wheat cv. Claire showed a higher capacity (-8.14°C) to be acclimated than Iraqi wheat (-7.40°C) under the effect of both Mo and acclimation temperatures. Mo alone increased the expression of Cbf14 in both varieties but did not increase the frost tolerance. Observations of ice nucleation using an infrared thermography (IR) revealed that supercooling is highly likely in spikes and some spikes avoided frost damage even when the temperature fell to -12°C. It was observed that the proportion of frozen spikes was 22.8% while the remainder supercooled. Spraying plants with distilled water was not effective in facilitating ice nucleation in wheat spikes. Observations also revealed that spikes that did freeze started freezing at temperatures of -4 to -5 °C close to temperature of the putative constitutive frost hardiness of un-acclimated wheat and it is suggested that this may reflect that many laboratory freezing experiments may not actually freeze until -5°C. The anthesis stage of wheat was found to be the most vulnerable stage and it needs to be given more attention in terms of research to up-regulate cold acclimation. Frost damage to wheat during flowering continues to be a serious problem in certain production areas and therefore continued effort in characterising and finding suitable solutions to this are imperative.

Leptosphaeria maculans ‘brassicae’ (Lmb) est un ascomycète de la classe des Dothideomycètes faisant partie d’un complexe d’espèces présentant différents niveaux d’adaptation au colza. Lmb est responsable d’une des maladies les plus dommageables sur colza : la nécrose du collet. Lmb présente un cycle de vie complexe au cours duquel il alterne différents modes de vie, traduisant l’existence de mécanismes de régulation fine de l’expression des gènes lui permettant de s’adapter rapidement à de nouvelles conditions. Le séquençage de son génome a révélé une structure originale, avec l’alternance de deux types de régions : les isochores GC et les isochores AT. Alors que les isochores GC sont riches en gènes, les isochores AT sont pauvres en gènes et présentent des caractéristiques de l’hétérochromatine (régions génomiques riches en éléments transposables et présentant un faible taux de recombinaison). Bien que pauvres en gènes, les isochores AT représentent une « niche écologique » pour les gènes codant des effecteurs puisque 20 % des gènes des isochores AT codent des effecteurs putatifs contre seulement 4 % des gènes localisés en isochores GC. Les gènes codant des effecteurs situés en isochores AT présentent un comportement transcriptionnel différent de ceux localisés en isochores GC : une faible expression pendant la croissance mycélienne et une forte induction d’expression pendant l’infection primaire du colza. Sur la base de ces observations, l’objectif de ma thèse était de caractériser le déterminisme de la co-expression des effecteurs situés dans les isochores AT et en particulier d’évaluer si la régulation de l’expression de ces gènes se fait par un contrôle épigénétique lié à leur localisation particulière et/ou par l’intervention de régulateurs communs. Afin de déterminer le rôle de la structure des isochores AT, l’analyse fonctionnelle de protéines impliquées dans le remodelage de la chromatine (i.e. HP1, DIM-5 et DMM-1) a été réalisée et leur implication dans la régulation de l’ensemble des gènes prédits dans le génome de L. maculans a été évaluée. Cette étude a permis de démontrer l’implication de la structure hétérochromatinienne des isochores AT dans la répression de l’expression pendant la croissance mycélienne des gènes situés dans cet environnement génomique, en particulier les gènes codant des effecteurs. Parmi les gènes sous contrôle épigénétique, nous avons pu observer qu’en plus des gènes localisés en isochores AT, des zones en isochores GC étaient aussi affectées et pouvaient constituer des « hot-spots » de contrôle épigénétique. Afin d’identifier des régulateurs candidats pouvant être impliqués dans le contrôle de l’expression des effecteurs pendant l’infection, le répertoire des gènes codant des facteurs de transcription (FTs) chez Lmb a été établi et l’analyse de la conservation de ce répertoire parmi les autres espèces du complexe d’espèces Leptosphaeria a permis d’identifier les FTs spécifiques, ou spécifiquement sur-exprimés pendant l’infection du colza, chez Lmb. Des candidats ont été sélectionnés pour réaliser leur analyse fonctionnelle : des gènes codant des FTs sur-exprimés pendant l’infection (9 FTs) ainsi que les orthologues de FTs qui avaient été décrits chez d’autres espèces comme régulateurs majeurs de la pathogénie (StuA, Sge1 et Fox1). L’analyse fonctionnelle de FTs candidats a permis d’établir que StuA, comme chez d’autres champignons phytopathogènes, joue un rôle important dans la mise en place de l’infection et l’expression des effecteurs chez L. maculans. Le « silencing » d’un FT de type AT-Hook, famille de FTs se fixant préférentiellement au niveau de séquences riches en AT, a un fort effet sur la pathogénie du champignon et entraîne une diminution d’expression de 2 effecteurs. Cette thèse a permis d’apporter de nouveaux éléments concernant la régulation des gènes codant des effecteurs chez un champignon phytopathogène impliquant, pour la première fois, un mécanisme épigénétique. / Leptosphaeria maculans is an ascomycete belonging to the Dothideomycete class and is part of a species complex showing different level of adaptation toward oilseed rape. Within this species complex, Lmb is responsible for the most damaging disease of this crop: “stem canker”. Lmb presents a complex life cycle during which it alternates between different life styles and nutritional strategies underlying the involvement of precise regulatory networks for gene expression to rapidly adapt to new conditions. The sequencing of the Lmb genome has revealed an unusual structure, alternating two types of regions, GC- and AT-isochores. While GC-isochores are gene-rich, AT-isochores are gene-poor and have several characteristics of heterochromatin (they are rich in transposable elements and present a lower rate of recombination compared to GC-isochores). Although gene-poor, AT-isochores are “ecological niches” for effector genes as 20% of the genes in these regions encode for putative effectors against only 4% of the genes in GC-isochores. Effector-encoding genes located in AT-isochores present a different transcriptional behavior compared to those located in GC-isochores: a very low expression in axenic culture and a drastic increase in expression during primary leaf infection. On these bases, the aim of my thesis was to characterize the determinism of the concerted effector gene expression. Are AT-isochores targets of reversible epigenetic modifications that affect the regulation of effector genes? and/or are one or several common regulators involved in the control of the concerted expression of effector genes? To assess the role of the structure of AT-isochores, functional analysis of three key players involved in chromatin remodeling (i.e. HP1, DIM-5 and DMM-1) was performed and their role in global gene expression was assessed. This study validated that heterochromatic structure of AT-isochores represses expression of genes located in such a genomic environment, notably effector genes. Among genes under an epigenetic control, we also identified genes located in GC-isochores that were similarly influenced and may represent “hot spots” for epigenetic control. To identify putative regulators of effector gene expression, we established the complete repertoire of transcription factors (TFs) of Lmb and by analyzing the conservation of this repertoire among species of the Leptosphaeria species complex, we identified TFs specific of Lmb, or specifically induced during infection. Functional analysis of 12 TFs was set up: nine TF-encoding genes induced during infection and three orthologs of TFs described as required for pathogenesis in other phytopathogenic fungi (StuA, Sge1, Fox1). This functional analysis showed that StuA, as in other phytopathogenic fungi, plays a major role in infection and expression of effector genes in Lmb. The silencing of an AT-Hook type TF, family of TFs that specifically interact with AT-rich sequences, was associated with a reduction of the expression of two effector genes during infection and with pathogenicity defects. This study brought new insights into the regulation of effector genes in a phytopathogenic fungus involving, for the first time, an epigenetic mechanism.

Paclitaxel is an antitumor diterpene from Taxus spp. that binds tubulin, stabilizes microtubules and induces apoptosis in dividing human cells. It was originally isolated from the bark of Taxus brevifolia and approved for clinic uses by the FDA in 1992. Because of its excellent activity in treatment of various cancers, a significant supply shortage has been created by the enormous demand for this natural product. Thus, researchers have been focusing on the development of effective ways to increase the production of paclitaxel and related bioactive molecules. This shortage was initially solved by over-harvesting of T. brevifolia bark; however, it is not an environment-friendly, effective and sustainable way to supply paclitaxel. A semisynthetic route was then developed to convert the more readily available and renewable 10-deacetylbacatin III into paclitaxel. As an alternative, plant cell cultures have been employed to commercially produce paclitaxel and it is a more environment-friendly and sustainable route to end the supply crisis. However, problems associated with plant cell culturing at an industrial scale, such as cell aggregation and variability in yield, significantly affect paclitaxel production. Therefore, a discovery of a better-performing Taxus cell line might be a solution to overcome these culturing-associated problems. A cambial meristematic cell (CMC) line of Taxus cuspidata has been isolated, cultured and demonstrated to be a cost-effective and environmentally friendly platform for the sustainable production of paclitaxel (Lee et al. 2010). Compared to dedifferentiated cell (DDC) lines, CMC lines are undifferentiated cells and proved to have stem cell-like properties. When cultured at an industrial scale, this cell line contains much smaller cell aggregates with many cells appearing as singletons, the biomass of which is still increasing after 22-month culturing, and has much greater paclitaxel production after elicitation (Lee et al. 2010). In my project, we aimed to identify the transcription factors (TFs) that regulate the expression of paclitaxel biosynthesis genes. We performed Illumina Solexa sequencing on cDNA libraries derived from methyl jasmonate (MeJA)-elicitated CMCs to digitally profile gene expression. Analysis of differentially expressed gene (DEG) abundance led to the discovery of 19 putative TFs and bioinformatic analysis further showed that these 19 TFs belong to 5 different TF families. Further, the DNA binding motifs associated with these TFs can be found in the promoters of the two early, taxadiene synthase (TASY) and taxadiene 5α hydroxylase (T5αH), and three late, 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT), phenylpropanoyltransferase (PAM) and 3’-N-debenzoyl-2-deoxytaxol-Nbenzoyltransferase (DBTNBT), paclitaxel biosynthesis pathway genes. Then, yeast one-hybrid analysis, gel shifting assays and plant transient expression assays (TEA) were employed to assay TFs that interact with these promoters. Although Y1H screening did not show any convincing TF-promoter interactions, the attempted plant transient expression assay in the leaves of Nicotiana benthamiana might be a more suitable system to screen the positive regulators. Finally, the elucidation of a TF regulatory network that controls paclitaxel biosynthesis will guide the rational engineering of CMCs to ultimately increase yields of this important pharmaceutical.

No description available.

Cystic fibrosis (CF) is the most common fatal genetic disorder that afflicts young Canadians. The major cause of morbidity and mortality in patients with CF is chronic pulmonary infection with the opportunistic Gram-negative pathogen Pseudomonas aeruginosa. Once established, P. aeruginosa lung infections cannot be cleared despite sustained and aggressive antimicrobial therapy. Treatment failure of P. aeruginosa lung infections is caused by a combination of antibiotic resistance and tolerance mechanisms. Antibiotic resistance is mainly mediated by multidrug efflux pumps such as MexAB-OprM. Antibiotic tolerance has been attributed to biofilms and to nutrient starvation. In this thesis, I present an analysis of three transcriptional regulators (PA3225, RpoS, and RpoN) and their contributions to resistance and tolerance in P. aeruginosa. PA3225 is a transcriptional regulator that I initially identified as a candidate regulator of a type VI secretion system (T6SS) that had been previously implicated in biofilm tolerance. While a ΔPA3225 deletion mutant did not, unfortunately, have dysregulated expression of the T6SS, I fortuitously discovered that the mutant displayed increased resistance to various antibiotics from different functional classes. I linked the increased antibiotic resistance of ΔPA3225 to upregulation of MexAB-OprM and provided evidence that PA3225 may be a direct repressor of mexAB-oprM. Next, I sought to identify a transcriptional regulator of ndvB, which is another gene that plays a role in biofilm tolerance. I found that the stationary phase sigma factor, RpoS, was essential for expression of ndvB in stationary phase and biofilm cells. Moreover, RpoS was important for tolerance of stationary phase cells to tobramycin (TOB), an aminoglycoside antibiotic that is used to treat CF patients. In recent years, several groups have sought to identify novel treatments to combat antibiotic tolerance in P. aeruginosa. A popular strategy is metabolic potentiation, which involves co-administration of an antibiotic with a metabolite to reverse tolerance due to nutrient starvation. For example, one group found that fumarate (FUM) combined with TOB (TOB+FUM) was highly effective at killing tolerant P. aeruginosa. FUM uptake depends on C4-dicarboyxlate transporters, which are transcriptionally regulated by the alternative sigma factor, RpoN. Importantly, rpoN loss-of-function mutations are a recognised mechanism of pathoadaptation in CF clinical isolates. I demonstrated that TOB+FUM was unable to kill ΔrpoN stationary phase and biofilm cells due to loss of FUM uptake and that rpoN alleles from CF clinical isolates were unable to complement the ΔrpoN mutant. These findings could have important implications for TOB+FUM as a treatment modality in CF patients with a high burden of rpoN mutants. Overall, my work has provided interesting and, in the case of RpoN, clinically relevant insights into the regulatory networks that determine antibiotic susceptibility in P. aeruginosa.

The mammary gland is a branched epithelial organ comprised of myoepithelial, ductal and alveolar cells that are derived from resident stem and progenitor cells. The progression from mammary gland stem cell(s) to the differentiated mammary gland cell types is poorly understood. Here, I describe the identification and characterization of two luminal progenitor cell populations in the mouse mammary gland, and investigate the role of the transcription factor C/EBPβ in their development. In Chapter 2, I describe the isolation of two luminal progenitor cell populations (Sca1+ and Sca1- luminal cells) and show that they are differentially primed in their gene expression towards ductal and alveolar cell fates, respectively. Furthermore, I show that in vivo genetic priming affects the in vitro differentiation potential of Sca1+ and Sca1- luminal cells. In Chapter 3, I show that C/EBPβ is required for the appropriate specification of ductal and alveolar lineages, and in its absence, alveolar lineage priming is lost, and ductal lineage priming is up-regulated in both Sca1+ and Sca1- cells. Preliminary data also shows that in addition to severe proliferation defects, the changes in in vivo lineage priming in Cebpb-/- mice also affect the in vitro differentiation potential of Cebpb-/- Sca1+ and Sca1- luminal progenitors. Lastly, in Chapter 4, I describe the genome-wide binding characteristics of C/EBPβ in Sca1+, Sca1- and P16.5 alveolar cells. These experiments reveal that genome-wide C/EBPβ occupancy is correlated with alveolar cells fate, and that C/EBPβ target genes perform distinct cellular functions in alveolar cells (Sca1- cells and P16.5). Furthermore, I show that Elf5 is directly regulated by C/EBPβ, and posit that direct regulation of Elf5 by C/EBPβ may be one mechanism through which C/EBPβ exerts its alveolar cell fate programming.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide. Current treatments are subpar, with late stage diagnosis and poor prognosis contributing to limited treatment options. The evolutionarily conserved, ubiquitously expressed transcription factor LSF is overexpressed in HCC, and its expression is positively correlated with disease severity. Certain small molecules, known as Factor Quinolinone Inhibitors (FQIs), specifically inhibit LSF DNA-binding activity, inhibit HCC cell proliferation in vitro and prevent tumor growth in an endogenous mouse liver cancer model without apparent toxicity. The targeting of transcription factors by small molecule inhibitors has been historically difficult (Dunker and Uversky, 2010), warranting further molecular investigation into the requirement for LSF in HCC to confirm that the anti-tumor effects of FQIs are the consequence of LSF inhibition. This body of work investigates a dual approach for inhibiting LSF function in order to determine the molecular consequences for HCC cells. To identify the specific point of the cell cycle where LSF is required for HCC proliferation, synchronous HCC cells were treated with FQI or with short interfering RNA to reduce levels of LSF. The results indicate that LSF is required for proper mitotic progression in HCC cells. Specifically, these data show a reduction of key mitotic regulators Aurora Kinase B and Cdc20, at the level of mRNA and protein expression. Time-lapse microscopy also demonstrated an increase in the time for progression through mitosis, with a prometaphase/metaphase delay. Immunofluorescence analysis revealed a prometaphase delay plus aberrant cell division and generation of multi-nucleated cells. These findings were consistent with both FQI1 treatment and RNA interference. Additionally, shorter incubation with FQI1 surprisingly revealed a distinct, non-transcriptional regulation of mitosis in HCC cells, suggesting that mitotic regulation by LSF is multi-faceted. As a targeted therapy for use in the clinic, the in vivo toxicity of FQIs is critical to investigate. Whole blood provides populations of rapidly dividing normal cells that can test susceptibility to anti-mitotic compounds. When mice were treated with FQI1, the blood analysis showed no toxicity. Taken together, these findings indicate that LSF is a mitotic regulator in HCC, further supporting the therapeutic promise of molecular therapies targeting LSF. / 2019-03-04T00:00:00Z

The regulated differentiation and polarisation of macrophages are essential for successful wound healing process. During wound repair, macrophages are involved in the early inflammatory process of healing, as well in later regenerative phases by producing cytokines and growth factors relevant for each stage. Their plasticity made macrophages able to change their phenotype from M1 inflammatory during the inflammatory phase of healing to M2 reparative during regenerative phases of healing. Diabetes affects the ability of macrophages to mature from the bone marrow and on their ability to polarise to different phenotypic subsets. Whereas the non-diabetic macrophages can mature normally to M2 macrophages during mid-stages of healing, diabetic wound continues o display immature proinflammatory macrophages resulting in mixed M1/M2 macrophages in the wound that remain until late stages of healing. We previously showed that sustained expression of Hoxa3 reduced the-the excessive number of leukocytes recruited to the wound, suggesting an anti-inflammatory effect of Hoxa3 upon all leukocytes population. Hoxa3 protein transduction also promoted the differentiation of HSC/P into pro-angiogenic Gr1+CD11b+ myeloid cells. Here we showed that Hoxa3 promoted the differentiation of macrophages and upregulated the transcriptional machinery controlling macrophage differentiation, in THP-1 monocytes and primary macrophages from non-diabetic and diabetic mice. Using qRT-PCR and protein analysis of bone marrow derived macrophages from diabetic mice, we showed that Hoxa3 upregulated the master regulator of macrophages differentiation, Pu.1 transcriptionally and post- transcriptionally and that Hoxa3 protein interacted with Pu.1 protein in vitro and in vivo within macrophages proposing a mechanism of their regulation. Hoxa3 also inhibited proinflammatory markers in classically activated macrophages and augmented pro-healing markers in alternatively activated macrophages. Investigating the IL-4/Stat6 pathway of M2 macrophage activation revealed that Hoxa3 upregulated Stat6 and increased Stat6 phosphorylation, a novel effect of Hoxa3 on the signaling pathway of alternative macrophage activation. In vivo analysis of Hoxa3's effect on wound derived macrophages in diabetic mice, confirmed that Hoxa3 promoted the generation of pro-healing macrophages and showed reduced Nos2+ (M1) cells and increased Arg1+ (M2) cells suggesting that Hoxa3 can rescue the phenotype of diabetic macrophages in the wound. Altogether, this work has delineated the specific role of Hoxa3 in rescuing maturation and phenotype of diabetic macrophages thereby providing a better understanding of the therapeutic role of this transcription factor for myeloid cells dysregulation in diabetes.