Les œstrogènes sont impliqués dans la prolifération des cellules épithéliales du sein normal et l'exposition prolongée à ces hormones s'accompagne d'une augmentation du risque de développement de cancer du sein. Les œstrogènes exercent leurs effets via les récepteurs des œstrogènes (REs). L'activité de ces récepteurs est finement régulée par un grand nombre de cofacteurs transcriptionnels, mais également par les modifications post-traductionnelles. Mon travail de thèse a eu pour objectif la compréhension de l'impact de ces deux niveaux de régulation sur la signalisation œstrogénique. Il a été récemment décrit que la sumoylation affectait de manière drastique l'activité du RE. La sumoylation est une modification dont le caractère réversible est assuré par des isopeptidases appelé SENPs (SENtrin Proteases). Dans une première étude nous avons montré que SENP2 pouvait fortement réprimer l'activité transcriptionnelle dépendante des œstrogènes ainsi que la prolifération cellulaire. Dans une seconde étude, nous nous sommes attelés à mieux caractériser les mécanismes d'action à l'origine du caractère répresseur du cofacteur transcriptionnel LCoR (Ligand-dependent Corepressor). Nous nous sommes plus précisément intéressés aux relations existant entre LCoR et un autre cofacteur du RE, RIP140 (Receptor Interacting Protein of 140 kDa) répresseur majeur de l'activité œstrogénique. Nous avons pu caractériser, outre les modes de recrutement des deux protéines, les modulations d'expression exercées par les deux cofacteurs. L'ensemble de nos travaux identifie de nouveaux cofacteurs des REs et contribue à une meilleure compréhension de la signalisation œstrogénique. / Estrogens are involved in the proliferation of normal breast epithelial cells. The prolonged exposure to these hormones comes along with an increase of the risk of breast cancer.development. Estrogen receptors (ERs) mediate the effects of estrogens. The activity of these receptors is finely tuned by a large number of transcriptional cofactors, but also by post-translational modifications. This work aimed at understanding the impact of these regulations on estrogenic signalling. It was recently described that sumoylation could strongly affect ER-dependent activity. SUMO conjugation is a dynamic process which is reversed by SUMO specific proteases also known as SENtrin Proteases (SENPs). In a first study, we investigated the role of SENP2, in ER-dependent transcriptional activity. We showed that SENP2 could acts as a transcriptional cofactor independently of its catalytic activity by strongly repressing ER-dependent transcriptional activity. We also provided evidence for a role in in breast cancer cell line proliferation. In a second part of the work we investigated the mechanism of action of the transcriptional cofactor LCoR (Ligand-dependent Corepressor) with a specific emphasis on the relationship between LCoR and another ER cofactor, RIP140 (Receptor Interacting Protein of 140 kDa). We characterized a crossed expression modulation of the two transcription cofactors. We also depicted an interaction between these two corepressors and a regulation of LCoR activity by RIP140. Our work provides new insights in identifying new coregulators of ER and contributes to a better understanding of both LCoR and RIP140 mechanism of action, and therefore of estrogenic signalling.

Cell migration is essential for homeostasis and the development of metastases. We hypothesize that cell migration is regulated by mitochondria and endoplasmic reticulum morphology. Using live cell microscopy, we found that mitochondria specifically migrate into the biochemically dense leading edge of the cell interacting with focal adhesions as well. At the leading edge the mitochondria are visibly shorter and less tubular than the perinuclear area. This is related to the elevated levels of fission events per minute in the leading edge and elevated levels of fusion events per minute in the trailing edge. We observe that mitochondria migrate along microtubules and simultaneously interact with the ER. When the ER is sheet-like the mitochondria are longer and tubular and when the ER is tubular the mitochondria are shorter and punctate. This change in ER and mitochondria morphology changes the cell’s ability to migrate. CLIMP63 cells have more sporadic turns, take longer to make turns, have shorter distances travelled and shorter displacements. To determine whether mitochondria dynamics play a role we examined these cell migration parameters in the presence of OPA1 and Drp1. This allowed us to conclude that the ER morphology is responsible for the distance and displacement the cell travels while the mitochondria is responsible for the angles the cell turns. When the ER is sheet-like the cells will be travel shorter total distances and displacements and when the cell has longer mitochondria it will be sporadic turns and take longer to make these turns.

No description available.

Tissue fibrosis occurs in the advanced stages of various chronic diseases and can account for 45% of all deaths related to chronic diseases worldwide. The extracellular matrix (ECM) components comprising the fibrotic scar are primarily derived from myofibroblasts, which are contractile fibroblasts arising from the trans-differentiation of several cellular progenitors. Disturbances in immune cell infiltration and function could lead to the uncontrolled production of pro/anti-inflammatory mediators, which may alter the phenotype, state, and function of myofibroblasts progenitors, leading to aberrant wound repair and pathological fibrosis. A great deal of knowledge has implicated macrophages in the pathogenesis and exacerbation of the fibrotic process. Nonetheless, much remains to be elucidated on the potential mechanisms regulating macrophage accumulation and pro-fibrotic polarization, and whether these mechanisms can be further investigated to modulate tissue repair. The Endoplasmic reticulum (ER) stress has recently been implicated as a key mechanism that propagates the pathogenesis of the fibrotic process. How ER stress precisely impacts the fibrotic process is still unclear. This thesis partly explored how modulating the outcome of ER stress – the unfolded protein response (UPR), would affect the severity of lung fibrosis and addressed the role of IL-6 signalling in macrophages during fibrosis. The data demonstrated that UPR activation in pro-fibrotic macrophages and partial deficiency of Grp78, the master regulator of the UPR, abrogated pulmonary fibrotic changes and reduced the accumulation of pro-fibrotic (M2-like) macrophages. These findings were later associated with high TUNEL levels, 7AAD positive cells, Chop and cleaved caspase 3 levels, which are suggestive of GRP78 mediated apoptosis in this population. On the contrary, mice lacking a terminal UPR mediator of apoptosis, called Chop, had increased ECM deposition and greater persistence of non-apoptotic macrophages. These findings suggest that UPR-mediated macrophage polarization and apoptosis may alter lung wound repair processes. As IL-6 synergized the effect of IL-4 to promote a hyper M2 macrophage state, it provided a unique and compelling model to study the dynamics of macrophage alternative programming, which has set the stage to investigate whether the UPR was implicated in the generation of a hyper pro-fibrotic macrophage phenotype. This hyper M2 macrophage model led to the identification of ER expansion program and the IRE1-XBP1 arm of the UPR in pro-fibrotic macrophage polarization, and suggested an unprecedented in vivo role of IL-6 in priming macrophages in the injured lungs to possibly potentiate pathological wound repair. Looking forward, many questions remain to be answered in order to precisely identify the vital UPR axis regulating ER expansion in macrophages during pathological wound repair and to get closer to the understanding of whether the UPR modulates the pro-fibrotic/pro-resolving capacity of macrophages. Insights on these mechanisms may facilitate the development of therapeutics that better manage chronic fibrotic diseases which pose fatal consequences and increase public concern. / Thesis / Doctor of Philosophy (PhD)

Viruses have evolved a multitude of mechanisms, which allow immune evasion in both initial and persistent infection. Understanding the intricacies of these pathways is essential to our future ability to combat primary and reactive viral infections. The murine gamma-2 herpesvirus 68 (γHV68) encodes a protein mK3, which targets Major Histocompatibility Complex (MHC) class I heavy chains for ubiquitin-dependent proteasome degradation. MK3 is able to target and ubiquitinate MHC class I by binding to Endoplasmic Reticulum (ER) resident proteins tapasin, Transporter associated with antigen processing (TAP) 1 and TAP2 that are subunits in the complex known as the peptide-loading complex (PLC). The aforementioned characteristics of mK3 make this novel protein an excellent vehicle to study MHC class I assembly, immune evasion, and ER associated degradation (ERAD). Deepening our understanding of class I assembly and viral immune evasion will impact both the fields of immunology and virology. The homology between γHV68 and many of the human γ-herpesviruses makes this an indispensable model to clarify mechanisms that can then be applied to a broader spectrum of viruses. ERAD, an emerging field of study, is known to play a key role in numerous cellular housekeeping pathways as well as a number of disease states. Illuminating the mechanisms implicated in the mK3-mediated ubiquitination of MHC class I, specifically requirements for substrate recognition and degradation, will yield an increased understanding of cellular pathways involved in ERAD. The studies in this dissertation aim to expand our understanding of the relationship between mK3 and adapter proteins TAP/tapasin as well as mK3 and mK3-targeted substrates. The results show that TAP/tapasin act as adapter proteins by recruiting substrates for mK3. Further, mK3 ubiquitinates TAP/tapasin-associated substrates as long as the substrates have a tail greater than 6aa in length and the tail possesses an ubiquitin acceptor residue (lysine, serine or threonine). These studies also confirm that location of a protein within the PLC will determine the substrate’s susceptibility to mK3-mediated degradation. In the field of ubiquitin ligases and ERAD, these studies lend support to the concept of adapter mediated substrate recruitment.

Databases are a critical part of Information Technology. Following a rigorous methodology in the database lifecycle ensures the development of an effective and efficient database. Conceptual data modeling is a critical stage in the database lifecycle. However, modeling is hard and error prone. An error could be caused by multiple reasons. Finding the reasons behind errors helps explain why the error was made and thus facilitates corrective action to prevent recurrence of that type of error in the future. We examine what errors are made during conceptual data modeling and why. In particular, this research looks at expertise-related reasons behind errors. We use a theoretical approach, grounded in work from educational psychology, followed up by a survey study to validate the model. Our research approach includes the following steps: (1) measure expertise level, (2) classify kinds of errors made, (3) evaluate significance of errors, (4) predict types of errors that will be made based on expertise level, and (5) evaluate significance of each expertise level. Hypotheses testing revealed what aspects of expertise influence different types of errors. Once we better understand why expertise related errors are made, future research can design tailored training to eliminate the errors.

Studien behandlar kompetenser och kunskapsnivåer hos gymnasieelever inom geometriavsnittet, matematikkurs 2b. Syftet är att undersöka i vilken utsträckning elever behärskar de matematiska kompetenserna och vilken kunskapsnivå inom avsnittet geometri elever uppnår på ett kunskapstest som konstruerats av uppgifter från nationella prov och elevernas läromedelsbok. Datainsamlingen genomfördes på en svensk gymnasieskola och omfattar 47 elever från två olika klasser i årskurs 2 på det samhällsvetenskapliga programmet. Samtliga elever har besvarat kunskapstest skriftligt. En innehållsanalys med utgångspunkt från kompetenser och kunskapsnivåer har sedan utförts på elevernas skriftliga svar och resultatet visar att eleverna behärskar algoritmkompetens och att en del elever behärskar begreppskompetens. Resultatet visar också att de flesta av eleverna befinner sig på kunskapsnivå 2 och 3. Många elever saknar således en djupare förståelse för geometrin enligt analysen med kunskapsnivåer. Rekommendationen efter studien är att eleverna behöver mera undervisning om begreppens definitioner för att förbättra de andra kompetenserna såsom resonemangskompetens, modelleringskompetens och kommunikationskompetens.

Master of Science / Department of Physics / Brian R. Washburn / Rare-earth doped optical fibers can be used to make optical amplifiers in the near infrared with large optical gain in an all fiber based system. Indeed, erbium doped fibers made gain possible within the 1532 to 1560 nm band which makes long span fiber optical communication systems a possibility. Erbium doped fibers have also been used to make narrow linewidth or mode-locked lasers. Other rare-earth doped fibers can be used for amplifiers in other near-infrared spectral regions. Recently, fiber amplifier technology has been pushed to produce watt level outputs for high power applications such as laser machining. These high power amplifiers make new experiments in ultrafast fiber optics a possibility. This report reviews the current literature on Watt-class continuous wave erbium doped amplifiers and discussed our attempt to develop a high power Yb/Er amplifier. After the design of the cladding pump in 1999, the world’s first single mode fiber laser with a power greater than 100 Watts of the continuous wave light was introduced. After 2002 there was a huge spike in the output powers (up to 2 kW) of lasers based on rare-earth doped fibers. Our own work involved developing a 10 W amplifier at 1532 nm and 1560 nm. A high power amplifier was made by seeding a dual-clad Yb/Er co-doped fiber pumped at 925 nm using a lower power erbium doped fiber amplifier. We will discuss the design and construction of the amplifier, including the technical difficulties for making such an amplifier.

Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.

Autophagy is a major degradation mechanism, responsible for clearing damaged and dysfunctional organelles, including the endoplasmic reticulum, a structure essential for protein synthesis and myocellular hypertrophy. Alterations in autophagy throughout various tissues of the body have been linked to various negative side effects such as decreased myocellular hypertrophy and insulin resistance. High fat diets lead to changes (both increases and decreases) in autophagy in various tissues throughout the body in a tissue-specific manner. Skeletal muscle autophagy is decreased in myotubes cultured from obese women, however the mechanism by which this occurs is unknown. As the largest organ system in the human body, skeletal muscle serves an important role in overall metabolic health. Therefore, sufficient skeletal muscle autophagy is important for proper metabolic function. Moreover, a decrease in liver and pancreas autophagy has been found to lead to endoplasmic reticulum (ER) stress and the development of insulin resistance. Understanding the relationship between autophagy and ER stress in the skeletal muscle following a high fat diet may help elucidate a novel target for decreasing negative side effects. Interestingly, both acute and chronic exercise have been shown to increase skeletal muscle autophagy. This points to a potential therapeutic treatment for those suffering with decreased skeletal muscle autophagy and may help improve ER stress. The purpose of this study was to compare the in vivo and in vitro effects of high fat exposure on skeletal muscle autophagy. Additionally, the relationship of autophagy and ER stress in skeletal muscle was explored. Lastly, this project identified changes in skeletal muscle autophagy and ER stress following cyclic stretch, an in vitro model of exercise in C2C12 myotubes. Eight-week-old C57BL/6J were fed a high fat diet for 16 weeks and tibialis anterior muscle examined for changes in autophagy markers. Gene expression (mRNA content) of autophagy markers Atg3 (p=0.011, fold change 1.37), Atg12 (p=0.026, 1.38), and Atg16L (p=0.004, 1.49) were increased in skeletal muscle of obese mice. Protein content was also measured, where increases in Atg3 (p = 0.04, 1.22), Atg12 (p = 0.027, 1.21), and Atg16L1(p = 0.021, 1.59) were found. However, there was no difference in LC3 II:I ration. No changes were seen in Atg5 or LC3. Additionally, C2C12 myotubes were treated with equimolar palmitate and oleate for 24h then assessed for mRNA content of genes involved in autophagy and ER stress. Autophagy genes Atg5 (p = 0.007, fold change 1.78), Atg12 (p = 0.001, fold change 1.99), and LC3 (p = 0.01, fold change 2.02) were decreased with high fat treatment. Paradoxically, there was an increase in Atg16L (p = 0.005, fold change 1.90). There were no changes in protein content. ER stress was increased indicated by an increase of sXBP1 (p = 0.005, fold change 1.33). Furthermore, inhibition of autophagy lead to changes in ER morphology and ER stress. To identify the impact of cyclic stretch on skeletal muscle autophagy and ER stress, C2C12 myotubes were subjected to 30 minutes of equibaxial stretch and examined for changes in autophagy and ER stress. Autophagy flux, measured by tyrosine release, increased by 34% (p = 0.04) following exercise and ER stress was decreased. In conclusion, this study provides the novel finding that decreased skeletal muscle autophagy is sufficient for inducing ER stress. Additionally, cyclic stretch increases autophagy and improves ER homeostasis.