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In living cells, calcium ions (Ca2+) play an important role as an intracellular second messenger. It mediates the regulation of cellular processes such as gene expression, initiation of vesicle fusion in synapses, is used in muscle contraction and is believed to play a fundamental role in synaptic plasticity as a molecular substrate for learning. The Ca2+ signals are created by the fact that the concentration of Ca2+ in the cytosol is four orders of magnitude lower than in the extracellular fluid as well as in cytoplasmic compartments such as the endoplasmic reticulum (ER). This enables fast increments in the cytosol concentration, which is regulated back to normal concentration by different mechanisms. In this project, the connection between Ca2+ signals of different cells was analysed using different correlation techniques: cross-correlation of continuous signals and digitalised signals. Therefore a software tool was developed in MATLAB, which takes Ca2+ recordings from time-lapse fluorescence microscopy as input and calculates the pair wise correlation for all cells. The software was tested by using previous data from experiments with embryonic stem cells from mouse (mES) and human (hES) as well as data from recordings done as part of the project. The study shows that the mathematical method of cross-correlation can successfully be applied to quantitative and qualititative analysis of Ca2+ signals. Furthermore, there exist strongly correlated cells in colonies of mES cells and hES cells. We suggest the synchronisation is achieved by physical coupling implicating a decrease of correlation as the distance increases for strong correlations. In addition, the lag used by the cross-correlation function (an effective phase shift) decreases as the correlation coefficient increases and increases as the intercellular distance increases for high correlation coefficients. Interestingly, the number of cells included in small scale clusters of strongly correlated cells is significantly larger for the differentiating mES cells than for the proliferating mÉS cells. In a broader perspective, the developed software might be usd in for instance analysis of cellular electrical activity and shows the relevance of applying methods from the exact sciences to biology. / QC 20100708

Acinetobacter baumannii is a prevalent nosocomial pathogen where, like many other infectious bacteria, A. baumannii is increasingly considered a multi-drug resistant pathogen. This research study was designed to find a way to affect the persistence of A. baumannii such that it can be applied to a hospital setting to prevent further nosocomial infections. One regulatory mechanism potentially used by A. baumannii to persist on hospital surfaces is through the use of the bacterial second messenger cyclic-di-GMP (c-di-GMP). This nucleotide signal is regulated in response to environmental conditions, and then activates c-di-GMP-binding proteins that induce phenotypic changes. One c-di-GMP-regulated phenotype is bacterial motility, and reducing motility may alter A. baumannii’s ability to colonize and persist on hospital surfaces. I hypothesized that A. baumannii uses c-di-GMP-binding proteins to regulate motility. A. baumannii encodes two potential c-di-GMP-binding proteins of interest, one that contains a sole c-di-GMP-binding PilZ domain and another that pairs a PilZ domain with a hydrolase enzymatic domain. I am also testing two A. baumannii strains: AB5075, a recent multi-drug resistant military hospital isolate and 17978, an established lab strain. A notable difference between these two strains is that AB5075 demonstrates twitching motility where it utilizes type IV pili, while 17978 demonstrates swarming motility that has unknown mechanisms. Both c-di-GMP-binding proteins were tested for their role in motility for the particular A. baumannii strain. While I am still generating the deletion strain for the c-di-GMP-binding hydrolase enzyme in AB5075, the sole PilZ domain protein is required for twitching motility, while both c-di-GMP-binding proteins are required for 17978 swarming motility. [PM1] Future plans include determining the role of the c-di-GMP-binding hydrolase enzyme in twitching motility and identifying the role that these proteins play through binding of c-di-GMP.

Oncolytic viruses (OVs) have made tremendous strides as a viable cancer therapeutic in recent years; however, variable infectivity rates have since limited clinical efficacy. Residual type 1 interferon (IFN-1) responses are integral to the tumour’s innate antiviral defense and confer resistance to OVs. To combat this, small molecules with viral sensitizing ability can be used in combination to transiently knockdown IFN-1 responses, allowing OVs to gain a foothold for increased infectivity and therapeutic efficacy. Accordingly, we hypothesize that some chemical or genetic manipulations of cellular processes can indirectly antagonize antiviral IFN-1 responses and modulate pro-inflammatory pathways to potentiate oncolytic virotherapy. In this thesis, we identify several avenues to modify cell signalling events to increase OV therapeutic efficacy through IFN-1 inhibition. Firstly, with respect to the demonstrated OV-enhancing effects of vanadium, a pan-phosphatase (PP) inhibitor, we elucidate that its IFN-1 suppressing activity involves activation of the epidermal growth factor receptor (EGFR) pathway via STAT1/2 and NF-κB. Pharmacological inhibition of EGFR abrogated vanadium’s viral sensitizing ability in vivo. Secondly, using high-throughput screening methodology, we identify protein phosphatases that inherently regulate the IFN-1 response as targets for oncolytic vesicular stomatitis virus (VSV∆51) potentiation. Indeed, cloning interfering RNA against one of these PP targets, acid phosphatase 2 (ACP2), into the VSV∆51 platform demonstrated superior infectivity and cancer cell cytotoxicity compared to the non-targeting VSV∆51 control. Thirdly, we characterize pevonedistat, a first in-class neddylation activating enzyme inhibitor, to potentiate OV therapeutic efficacy across several in vitro and in vivo contexts. We demonstrate pevonedistat’s ability to inhibit IFN-1 signalling and pro-inflammatory cytokine production using both neddylation independent and dependent mechanisms. Taken altogether, we dissect multiple signaling mechanisms by which the IFN-1 response can be modulated for the purposes of improving OV therapeutic efficacy. This knowledge can subsequently be directly translated into designing optimized OV strategies for clinical testing.

<p>One of the hallmark features of cancer is altered metabolism, whereby rates of glucose and fatty acid turnover are constitutively elevated to support uncontrolled propagation. The key regulator of energy metabolism is the enzyme AMP-activated protein kinase (AMPK), which suppresses anabolic pathways that increase proliferation and enhanced catabolic pathways that liberate energy, all in an attempt to maintain energy homeostasis in the cell. In addition to regulating metabolism, AMPK has also been implicated as a tumour suppressor and we have suggested that it may be a modulator of radiation responses in cancer cells <em>in vitro</em>. Moreover, we investigated the molecular mechanisms that facilitate ionizing radiation (IR)-induced AMPK activation, as well as demonstrated that certain AMPK activating drugs can work as radiation sensitizers in a variety of cancer cell lines. Stemming from this framework, we also provided experimental evidence that suggests AMPK is centrally involved in pathways that regulate DNA damage and proliferation at the basal level, and in response to IR. One of the targets involved in these pathways that can also influence AMPK regulation is the stress-activated Sestrin 2 protein. We have provided evidence that Sestrin 2 mediates IR-induced activation and expression of AMPK. Taken together, this work has provided novel insight into the ability of IR to modulate the activity and expression of AMPK, which in turn is required to facilitate the appropriate stress-response in cancer cells. Given its emerging interest in the cancer field, AMPK may become an important biomarker for evaluating clinical outcomes in patients undergoing radiation therapy.</p> / Doctor of Philosophy (PhD)

The communication of information between irradiated and non-irradiated bystander cell populations and the subsequent expression of radiation-like responses in the non-irradiated population, formally referred to as the radiation-induced bystander effect, is a very well established phenomenon in the study of radiobiology. Intercellular communication of bystander signals is known to occur via the exchange of soluble factors through biological fluids and via the transfer of molecules between adjacent cells via gap-junctions. Both of these communication methods require some degree of physical contact between biological entities. However, observations made in the literature demonstrating the induction of radiation effects in optically-coupled, yet chemically-separated organisms raises the hypothesis that alternative radiation bystander communication mechanisms may exist that have not yet been explored. Following the detection of significant photon emission from human keratinocyte cells exposed to ionizing beta-radiation by Ahmad in 2013, the involvement of an electromagnetic bystander signal was proposed. While not yet established in the field of radiobiology, intercellular communication via electromagnetic signalling is widely studied in the field of biophotonics. The emission of electromagnetic radiation from biological material, called biophoton emission, and the subsequent communication of effects using those signals has been characterized both spontaneously and as a result of perturbation by various stressors. This thesis therefore aimed to investigate intercellular communication via electromagnetic signalling stimulated by low-dose ionizing radiation to identify a possible convergence between the fields of biophoton communication and radiation-induced bystander effects. The characterization of biophoton emission from human cell cultures was accomplished using a single photon counting photomultiplier tube. The results revealed that biophoton emission is exacerbated by external stimulation (beta-radiation), it possesses a dependence upon the activity of radiation delivered, the density of the irradiated cell culture, and cell viability. These results suggest that biophoton emission is governed by physical transitions between excited and ground states and may further be modulated by metabolic processes. An effect of beta-radiation-induced biophoton emission upon non-irradiated bystander cells was identified and manifested as a reduction in cell survival. The modulatory effects observed following the application of photomodulating agents to the bystander cultures support ultraviolet electromagnetic radiation as a responsible factor in the communication of bystander signals. Observation of photon emission across the entire ultraviolet, visible and infrared spectra lead to the suggestion that ultraviolet wavelengths are only a portion of the signal responsible for eliciting bystander responses and that coherent interaction of multiple wavelengths is probable in the intercellular exchange of information. The possibility of a link between biophoton bystander signalling and soluble factor mediated bystander effects was investigated next by isolating exosomes from biophoton-exposed bystander cultures. Positive bystander responses were exhibited by secondary reporter cells incubated with the exosomes isolated from the biophoton-exposed bystander cultures, thereby suggesting that biophoton signalling is a possible form of biological redundancy where it acts as an intermediary to trigger soluble factor release and further reinforce intercellular communication. Finally, the effect of beta-radiation-induced biophoton signals upon mitochondrial activity was assessed and revealed the capacity for biophotons to downregulate Complex I and ATP synthase activity. The demonstrated effect of biophotons upon mitochondria elucidates a candidate mechanism worthy of further exploration to determine how biophotons may trigger responses in bystander cells. Overall, this thesis elucidates an additional mechanism for intercellular communication between biological systems perturbed by low doses of ionizing radiation, in the form of an electromagnetic signal. This work contributes to the current perspective on biophoton bystander signalling as a potential source of biological redundancy, facilitating a means of intercellular communication when optical coupling but not chemical contact is available in a given system. / Thesis / Doctor of Philosophy (PhD)

Yes / Interferon lambda (IFNλ) is important for epidermal defence against viruses. It is produced by, and acts on, keratinocytes, whereas fibroblasts were previously considered to be unresponsive to this type III IFN. Herein we report findings revealing cell type-specific differences in IFNλ signalling and function in skin resident cells. In dermal fibroblasts, IFNλ induced the expression of MxA, a potent antiviral factor, but not other IFN signature genes as it does in primary keratinocytes. In contrast to its effect on keratinocytes, IFNλ did not phosphorylate STAT1 in fibroblasts, but instead activated MAPKs. Accordingly, inhibition of MAPK activation (p38 and p42/44) blocked the expression of MxA protein in fibroblasts but not in keratinocytes. Functionally, IFNλ inhibited proliferation in keratinocytes but not in fibroblasts. Moreover, IFNλ upregulated the expression of TGFβ1-induced collagens in fibroblasts. Taken together, our findings identify primary human dermal fibroblasts as responder cells to IFNλ. Our study shows cutaneous cell type-specific IFN signalling and suggests that IFNλ, whilst important for epidermal anti-viral competence, may also have a regulatory role in the dermal compartment balancing type I IFN-induced inhibition of tissue repair processes.

Yes / The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/ 2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.

La ténascine-X (TNX) est une glycoprotéine de la matrice extracellulaire. Son expression est fortement réprimée dans de nombreux cancers et l’invasion tumorale est accrue chez des souris TNX-/-. La TNX apparaît donc comme un répresseur potentiel du développement des tumeurs. L’objectif de notre travail est d’étudier cet effet présumé et d’en comprendre les mécanismes, en analysant in vitro le rôle de la TNX sur la croissance et la migration de cellules de fibrosarcome HT-1080 dans des modèles de culture bi- et surtout tridimensionnels, plus représentatifs de l’environnement cellulaire in vivo. Nos résultats montrent que la TNX inhibe la croissance des cellules tumorales, sans induire de mort apoptotique ou nécrotique. Des observations par microscopie confocale ont montré que la présence de TNX réduit l’étalement des cellules ainsi que leur efficacité de migration. Nous avons pu mettre en évidence que la TNX provoque un ralentissement de la migration des cellules tumorales ainsi qu’une diminution de la directionnalité de leurs trajectoires. L’observation de la protéolyse du collagène de type I par les cellules en migration montre qu’elle est inhibée en présence de TNX. Par ailleurs, la TNX réduit l’expression et l’activation des MMP 2, MMP-9, et MT1 MMP. Certaines voies de signalisation associées ont été étudiées : la TNX inhibe la phosphorylation de FAK sur sa tyrosine 397, ainsi que l’activation des GTPases RhoA et Rac, sans affecter celle de Cdc42. Par une régulation fine de ces molécules, qui sont impliquées dans le contrôle de la croissance et de la migration cellulaire, la TNX se caractérise comme un inhibiteur extracellulaire de l’invasion tumorale / Tenascin-X (TNX) is involved not only in the organisation of the extracellular matrix architecture but also in the regulation of cell behaviour. This matrix glycoprotein is down-regulated in many tumor types, while tumor invasion is promoted in TNX-deficient mice. In order to decipher the mechanisms by which TNX modulates tumor cell growth and migration, we compared the behaviour of HT1080 fibrosarcoma cells in conventionnal 2D culture model or embedded in 3D collagen gels, both containing or not recombinant TNX. Some experimentations have permit us to demonstrate that TNX inhibits tumor cells growth, without inducing apoptotic or necrotic cell death. Laser confocal microscopy observations demonstrated that the presence of TNX reduces cell spreading and migration efficency. Moreover, video time-lapse analysis showed that TNX reduces both velocity and directionnality of cell migration. This result is partly due to a decrease of pericellular proteolysis, as observed in situ using FITC-collagen-containing gels. Besides, we showed that TNX led to a decrease of MMP 2, MMP-9, MT1 MMP expression and activity. Then, we determined that both FAK phosphorylation on tyrosine 397 and activation of Rac1/2/3 and RhoA small GTPases were inhibited in TNX conditions. An inactivation of these small GTPases of the Rho family is known to deregulate cell cycle and highly decrease tumor cell spreading and migration efficiency in 3D environment. Taken together, these results indicate that TNX is an extracellular inhibitor of cell invasion, which acts by downregulating the main signalling pathways responsible for cell growth and motility in 3D-collagen gels

L’adénomyose est une pathologie chronique bénigne de l’utérus caractérisée par une infiltration du myomètre par du tissu endométrial composé de glandes et de stroma avec une hypertrophie et une hyperplasie des cellules musculaires lisses adjacentes. Cette maladie fréquente de la femme en âge de procréer cause des symptômes invalidants comme des dysménorrhées, des saignements utérins anormaux et une infertilité. L’adénomyose utérine est souvent associée à d’autres pathologies gynécologiques bénignes œstrogéno-dépendantes comme les léiomyomes utérins et l’endométriose. Les options thérapeutiques médicamenteuses sont purement symptomatiques et non-curatives et l’adénomyose reste une cause majeure d’hystérectomie. Les mécanismes physiopathologiques qui aboutissent au développement de l’adénomyose sont probablement multifactoriels et ne sont que partiellement compris actuellement. Selon la théorie la plus communément admise, l’adénomyose trouve son origine dans la couche basale de l’endomètre avec une invagination de cellules entre les faisceaux musculaires et/ou le long de vaisseaux lymphatiques. De multiples facteurs pourraient être impliqués dans l’initiation de cette invasion, notamment une résistance à l’action de la progestérone, une production intra-lésionnelle d’œstrogènes par activation de l’aromatase, des anomalies myométriales prédisposant à l’invasion, des lésions tissulaires induites par la grossesse, l’accouchement, le dyspéristaltisme utérin ou iatrogènes et des anomalies de l’endomètre le prédisposant à l’invasion. Dans un premier temps nous détaillons, dans un article de revue, les traitements médicamenteux actuellement utilisés pour traiter les symptômes causés par l’adénomyose et discutons les mécanismes physiopathologiques qui pourraient être la cible de nouveaux traitements médicamenteux. Ensuite, nous exposons les résultats de l’étude in vitro des voies de signalisation cellulaires des mitogen-activated protein kinases (MAPKs) et phosphatidylinositol 3 kinase/Akt/mammalian target of rapamycin (PI3K/mTOR/Akt) dans les cellules musculaires lisses utérines issues de femmes avec de l’adénomyose et de témoins sans adénomyose. Nous montrons une augmentation de la prolifération des cellules myométriales avec une activation in vitro de la voie MAPK/ERK chez les femmes avec de l’adénomyose en comparaison avec les témoins. L’activation de la voie PI3K/mTOR/Akt n’est pas significativement différente. La production de dérivés réactifs de l’oxygène et leurs voies de détoxification ne sont pas différentes dans les cellules myométriales de femmes avec de l’adénomyose et celles de témoins, ce qui suggère une activation de la voie des MAPK/ERK indépendante des dérivés réactifs de l’oxygène. Nos résultats montrent que des inhibiteurs des protéines kinases et le rapanalogue temsirolimus contrôlent la prolifération des cellules myométriales in vitro, ce qui suggère une implication des voies de signalisation MAPK/ERK et PI3K/mTOR/Akt dans la prolifération des cellules musculaires lisses dans l’adénomyose et les léiomyomes. Finalement, nous avons étudié l’ostéopontine comme biomarqueur sérique dans une cohorte de femmes en âge de procréer opérées pour des pathologies gynécologiques bénignes. La présence d’endométriose a été déterminée chirurgicalement et les lésions endométriosiques ont été confirmées histologiquement et classées en lésions superficielles, endométriomes ou lésions invasives profondes. La présence d’adénomyose a été déterminée par imagerie par résonance magnétique préopératoire et deux types d’adénomyose ont été caractérisés : l’adénomyose diffuse, l’adénomyose focale avec ou sans lésions diffuses associées. L’ostéopontine sérique est diminuée en cas d’adénomyose focale et de lésions d’endométriose profonde en comparaison avec des témoins sains et augmentée dans l’endométriose superficielle en comparaison avec l’endométriose profonde. (...) / Adenomyosis is chronic benign uterine disease characterized by myometrial infiltration by endometrial tissue – both glands and stroma – with hypertrophy and hyperplasia of surrounding smooth muscle cells. This frequent disease occurring in reproductive age women causes invalidating symptoms such as dysmenorrhoea, abnormal uterine bleeding and infertility. Adenomyosis is frequently associated with other estrogen-dependant gynaecologic diseases such as uterine leiomyomas and endometriosis. Medical treatments are non-curative and act purely by alleviating symptoms and adenomyosis remains a major cause of hysterectomy. Physiopathological mechanisms underlying the disease are probably multifactorial and currently not fully elucidated. According to the most widely accepted theory adenomyosis originates from the basal layer of the endometrium which invaginates between smooth muscle cell bundles and/or along lymphatic vessels. Multiple factors could be implicated in triggering this invasion, amongst others resistance to progesterone, intra-lesional production of estrogens through aromatase activation, myometrial anomalies predisposing to invasion, tissue lesions induced by pregnancy, labour, uterine dysperistaltism or iatrogenic and endometrial anomalies predisposing to invasion. First, in a clinical review article, we detail current medical therapies used to alleviate adenomyosis-associated symptoms and discuss physiopathological mechanisms that could be targets for novel medical treatments. We then describe an in vitro study on the activation of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol three kinase/mammalian target of rapamycin/Akt (PI3K/mTOR/Akt) signalling pathways in uterine smooth muscle cells derived from women with adenomyosis and from adenomyosis-free controls. We show an increased proliferation of uterine smooth muscle cells related to the in vitro activation of the MAPK/ERK pathway in women with adenomyosis compared to controls. The activation of PI3K/mTOR/Akt was not significantly different. The production of reactive oxygen species and their detoxification enzymes were not different in uterine smooth muscle cells of women with adenomyosis compared to controls suggesting a reactive oxygen species independent activation of the MAPK/ERK pathway. Our results also show that inhibitors of protein kinases and the rapanalogue temsirolimus control the in vitro proliferation of uterine smooth muscle cells suggesting an implication of both MAPK/ERK and PI3K/mTOR/Akt in the proliferation of uterine smooth muscle cells in adenomyosis and leiomyomas. Finally, we studied osteopontin as a serum biomarker in a cohort of reproductive-age women undergoing surgery for benign gynaecological conditions. The presence of endometriosis was determined surgically and endometriosis lesions were confirmed histologically and classified into superficial lesions, endometriomas and deep infiltrating lesions. The presence of adenomyosis was determined by magnetic resonance imaging before surgery and women were classified according to two types of adenomyosis: diffuse adenomyosis, focal adenomyosis with or without associated diffuse lesions. Osteopontin levels were decreased in case of focal adenomyosis and deep infiltrating endometriosis compared to disease-free women and increased in superficial endometriosis compared to deep infiltrating endometriosis. Osteopontin, a secreted glycoprotein implicated in inflammation and in tumor-metastasis, is not a biomarker of disease severity in endometriosis and adenomyosis but could reflect events implicated in peritoneal dissemination of endometriosis lesions.