Angiogenesis is the growth of new blood vessels from existing vasculature; it requires the breakdown of existing blood vessel walls followed by the migration and proliferation of endothelial cells to form the new vessels. It is a complex process that is regulated by many pro- and anti-angiogenic factors and the roles of some of these factors are still unclear. Angiogenesis is a key feature of many pathological conditions including cancer, polycystic ovary syndrome and endometriosis so is an area of great research interest. There are several methods currently available for the study of angiogenesis, both in vitro and in vivo, and whilst all of these methods have enhanced understanding of angiogenesis, they also have limitations. The ovary is an excellent model for the study of angiogenesis as it undergoes intense vascular morphogenesis in a cyclical manner. The female reproductive system is unique as no other healthy adult tissue undergoes spontaneous angiogenesis. The tissues in the ovary undergo constant remodelling during both folliculogenesis and the formation and regression of the corpus luteum. Blood vessels are recruited from the ovarian stroma at the preantral stage to form vascular sheaths, in the thecal layer, which surround the developing follicle and supply nutrients, hormones and allow gaseous exchange. As follicular development progresses to the antral stage, when gonadotrophin-dependence is established, increased angiogenesis is essential to sustain development of the rapidly expanding follicle. Previous research into ovarian angiogenesis has focussed on the corpus luteum but the mechanisms of the regulation of angiogenesis during folliculogenesis need further elucidation. The work in this thesis aims to develop and utilise an in vitro angiogenesis assay using the culture of intact preantral and early antral follicles to provide a new approach to the study of follicular angiogenesis. During the course of this thesis this assay was utilised to investigate the effect of various factors on follicular angiogenesis and ovarian function. The role of the putative anti-angiogenic factor thrombospondin-1 (TSP-1) in the regulation of physiological angiogenesis was investigated using the in vitro angiogenesis assay developed during the course of this thesis and the role of TSP-1 in normal ovarian function was investigated using the culture of isolated granulosa cells. The results suggest that TSP-1 is able to inhibit angiogenesis and that it has an extravascular role in the ovary, in vitro. These findings were extended to an in vivo angiogenesis model where follicular angiogenesis was assessed by quantitative immunohistochemistry for bromodeoxyuridine and the endothelial cell marker CD31. The extravascular role for TSP-1 was also further investigated in vivo and was assessed by quantitative immunohistochemistry for activated caspase-3. The results confirmed the findings of the in vitro study, indicating that TSP-1 has antiangiogenic action and acts to clear non-dominant follicles from the ovary through the induction of atresia. Vascular endothelial growth factor (VEGF) is the main factor involved in stimulating angiogenesis and many advances have been made into elucidating the role, and the mechanisms of action, of VEGF on angiogenesis. Angiopoietin-1 (Ang-1) is considered to be one of the main factors acting in concert with VEGF to stabilise new blood vessels and its role in angiogenesis has been the subject of much discussion and controversy. This thesis investigates the effects of Ang-1 on follicular angiogenesis and development, using the in vitro angiogenesis assay, granulosa cell culture and RNA knockdown experiments. The results have shown that Ang-1 can induce follicular angiogenesis at high doses and that at low doses stimulates prosurvival pathways and inhibits apoptotic mediators. This thesis describes a novel in vitro culture system for the study of angiogenesis in ovarian follicles. Using this system the effects of various factors on follicular angiogenesis and on follicle development and survival have been investigated. Investigations into the mechanisms of action of these factors have also been performed. These studies have improved understanding of the regulation of follicular angiogenesis and have indicated extravascular roles for angiogenic factors in the ovary. Since angiogenesis is a key feature of many pathological conditions, the ability to manipulate angiogenesis and to investigate and quantify the effects of proor anti-angiogenic compounds may have important clinical implications.

Studien har haft som syfte att undersöka hur lärare i årskurs 1-3 planerar och genomförundervisning i teknik. Frågeställningarna som tagits fram för att för att få svar på studiens syfteär: hur beskriver lärarna att de planerar för teknikämnet, allmänt och i relation till det centralainnehållet för årskurs 1-3?, hur beskriver lärarna att de undervisar relaterat till det centralainnehållet i teknik för årskurs 1-3? samt vilka undervisningsmetoder används, enligt lärarna?Dessa frågor har det sökts svar på genom kvalitativa intervjuer med åtta lärare i årskurs 1-3.I resultatet framkommer att teknik är ett ämne som inte fullt ut har fått fäste i årskurs 1-3, då detvisar sig att flera lärare i studien saknar förtrogenheten med ämnets kursplan och planering ochundervisning beskrivs till stor del bedrivas tillsammans med No-ämnena. Teknikämnet har haften egen kursplan i tjugo år. Trots detta saknas på många skolor lärare med utbildning i tekniksamt material. Det framgår dock också i resultatet att några av de intervjuade lärarna ärintresserade och väl insatt i ämnets kursplan, även material i form av Skellefte-tekniken och NTAanvänds av flertalet av lärarna i studien. Teknikämnet behöver stärkas ytterligare som eget ämneoch i högre utsträckning undervisas separat för att ge eleverna en god uppfattning av vad som är teknik

Tekniken expanderar på olika sätt inom skola och utbildning i den digitala tidsåldern. Ett uttryck för detta är så kallade 1:1-lösningar, det vill säga tekniklösningar där varje elev har sin egen dator. Det anstår forskningen att klargöra hur tekniken bör integreras i undervisningen för att på bästa sätt främja elevernas lärande, vilket torde vara särskilt angeläget nu då det ännu inte är vederhäftigt belagt att digitala verktyg faktiskt har en potential att främja elevers lärande. Denna fallstudie utgör formen av ett förslag på hur tekniken kan integreras i undervisningen för att främja elevers lärande i 1:1-lösningar. Genom en fenomenologiskt inspirerad ansats fokuseras gymnasieelevers upplevelser av ett digitalt verktyg, lärplattformen Socrative. Centrala frågeställningar är hur Socrative kan fungera som ett stöd för elever i deras lärande och hur detta verktyg kan påverka elevers motivation. Studien har därtill ett deliberativt perspektiv i det att fokus ägnas Socratives potential att främja deliberativa samtal. Studien visar att Socrative har ett flertal effekter som torde framstå som önskvärda i undervisningssammanhang. Arbetsron i lärosalarna tycks bli bättre än vad som tidigare framgått i 1:1-studier. Socrative kan synliggöra lärande, både för lärare och elev, vilket verkar stimulera formativa processer och utveckling av fördjupade kunskaper. Lärandet upplevs som mer lustfyllt när Socrative används, vilket verkar ha positiv betydelse för elevers studiemotivation. Därtill visar studien hur Socrative kan främja deliberativa samtal som utvecklar elevers demokratiska kompetenser och fördjupar deras kunskaper. Det återstår emellertid mycket att undersöka inom detta forskningsområde. Särskilt gäller detta kopplingen mellan lärares praktik och forskning om digitala verktyg i undervisning. Dessutom förefaller det vanskligt att mäta effekterna som Socrative har på elevers lärande. Testkulturer har blivit allt starkare med fokus på mätbar kunskap samtidigt som samtal har fått en central plats i skolans styrdokument. Diskussionen om deliberativa samtal och Socrative tangerar därför diskussionen om svårigheten att mäta kunskap som utvecklas i samspel mellan elever.

The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.

No description available.

The transcription factor Interferon Regulatory Factor-1 (IRF-1) has been demonstrated to suppress tumour growth through the regulation of many anti-oncogenic genes. Pro- and anti-apoptotic factors, cell cycle control genes, DNA damage response genes and prometastatic factors are all under the control of IRF-1, which effects both transcriptional activation and repression. In addition to these cell autonomous tumour suppressor activities, IRF-1 is also a key regulator of the immune system and, as such, mediates immune surveillance of tumours. Numerous studies have confirmed that loss or mis-regulation of IRF-1 is a key factor in several different types of cancer. Despite strong evidence for the crucial role of IRF-1 in cancer, and frequent assertions that this protein warrants further investigation as a drug target, very little is known about its regulation. Furthermore, since recent studies have linked upregulation of IRF-1 to the development of autoimmune diseases, it is particularly important that drugs be able to decouple autoimmune and anti-cancer functions of IRF-1 to avoid harmful side effects. This thesis describes how phosphorylation of IRF-1 in its regulatory C-terminal Mf1 domain modulates transactivatory and tumour suppressor activity. Phosphospecific antibodies were developed as tools to study the C-terminal phosphorylation. Using these, it was shown that treatment of cells with Interferon-γ(IFN-γ) not only causes accumulation of IRF-1 protein, but also results in phosphorylation of IRF-1 at two sites in the C-terminal Mf1 domain. Phosphomimetic mutants demonstrated that these phosphorylations enhanced the transactivatory activity of IRF-1 at various promoters, but did not affect repressor activity. Gel shift assays revealed that dual phosphorylation of IRF-1 (IRF-1 D/D) promoted DNAbinding and suggested this was through increased interaction with the cofactor/histone acetylase p300 which induces a conformational change in IRF-1, favouring DNA-binding. Acetylation by p300 appears to be important although it is not yet clear whether this directly or indirectly affects IRF-1 activity. Since the tumour suppressor activity of IRF-1 is of particular interest, the effect of phosphorylation was examined in clonogenic and invasion assays. IRF-1 D/D more efficiently suppressed colony formation in both anchorage dependent and independent assays, and may improve inhibition of invasion in Transwell assays. Thus, cell treatment with the therapeutic agent IFN-γ nduces phosphorylation of IRF-1, resulting in enhanced DNA binding of IRF-1 through improved p300 binding. In cells the outcome is more effective tumour suppression and inhibition of metastasis.

Bovine herpesvirus-1 (BoHV-1) is a pathogen of cattle, which most commonly affects the upper respiratory tract to cause infectious bovine rhinotracheitis (IBR). It can also spread systemically to cause fatalities in calves and abortion in pregnant cattle. The virally encoded mechanisms of this systemic spread are poorly understood and therefore have been addressed by comparing isolates from the respiratory form of disease with isolates that have previously demonstrated systemic spread. A survey of 400 bovine abortions in Scotland from 2007-2009 demonstrated a BoHV-1 prevalence of 2.5%. It also demonstrated the importance of real-time PCR as a diagnostic technique when analysing samples from natural cases. The study of BoHV-1 distribution in the placenta and foetal tissue provided support for a haematogenous route of viral spread. Whole genome sequencing of 11 BoHV-1 isolates using Illumina Solexa technology was completed and added significantly to the sequencing data of BoHV-1. In terms of identifying genetic variation between isolates causing respiratory infection and those causing systemic infection, no differences were observed by SNP or phylogenetic analysis. However, there were significant differences in the extent of variation between essential and non-essential genes, which may reflect the evolution of BoHV-1. An in vivo challenge of the natural host to compare two isolates representing the respiratory and systemic forms of infection showed differences in clinical presentation, histopathological analysis, viral distribution and viral transcript expression, measured throughout the infection period. In particular, it was noted that a more severe ocular infection, rather than respiratory based infection was caused by infection with the ‘systemic’ isolate. Differences in the tropism of the virus were observed early in the infection with the ‘systemic’ isolate showing more association with the nasal mucosa than the trachea. The tonsils demonstrated different responses to the virus and differences in viral transcript expression. However, this may simply represent different stages of virus infection. Both isolates demonstrated spread to the brain at day 10 post infection. In vitro methods were used to study the differences in transcript expression in more detail. In a bovine turbinate cell infection faster replication of the respiratory isolate was observed by a significantly faster development of cytopathic effect. This was also reflected in the higher gene expression levels of the respiratory isolate in the first 12 hours of infection. More isolates were studied to investigate whether these differences were consistent, or as suggested by the sequencing, random differences between isolates. Six isolates were used to infect bovine lung slices. Differences in transcript expression were minimal between the two isolate groups. Immunofluorescence did not provide the sensitivity to detect virus in all samples where PCR showed replication. This compromised the study of co-localization but did show promise as a model to study the tropism of respiratory viruses. Overall, this work has showed that systemic spread of BoHV-1 does not appear to be controlled by virally encoded mechanisms. The in vivo experimental infection suggested host factors may play a large part. Further work is also needed to consider any differences that may exist between reactivated virus and the original infecting isolate.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

L'ARN d'interférence (ARNi) est un mécanisme de régulation du gène qui permet un ciblage spécifique d'ARNmessager (ARNm) par reconnaissance de séquence. Les effecteurs de l'ARNi sont de petites molécules d'ARN non codants (siARN, microARN et piARN). Evoluant dans le contexte de l'ARNi, les virus de différentes familles ont adopté des stratégies afin utiliser l'ARNipour leur propre bénéfice. Le principal objectif de ma thèse a été d'étudier la fonction de l' ARNmiRTAR, un miARN viral dérivé de l'extrémité 3 'de l'ARN VIH-1 TAR dans la réplication du VIH-1. Nous avons constaté que miRTAR réguleà la fois l'activité basale et la transactivation induite par Tat du promoteur du VIH-1. L'effet de miRTAR ne nécessite pas de sa liaison à l'ARN TAR. miRTAR agit en augmentant l'activité de NF-kB, un facteur important pour la transcription du promoteur du VIH-1. En effet, la mutation des sites NF-kB, mais pas des sites Sp1 dans le LTR, abroge l'amélioration miRTARmédiée par la transcription. De plus, l'inhibition de l'expression de NF-kBpardes siARNspécifiquesdes les sous-unités p50 et p65, entraîne une perte d'activité dumiRTAR. Bien que nous n'avons pas pu identifier le(s) gène(s) cellulaire(s) ciblé par miRTAR, sa surexpression conduit à l'activation de la voie NF-kB. Mutation de l'extrémité 3 'du VIH-1 dans les résultats TAR réduction spectaculaire de la réplication virale Tat sans affecter médiée par la transcription, en raison de la perte de production de miRTARsauvage. Enfin, la surexpression de miRTARrestaure la réplication de ce virus contenant une mutation au niveau de la séquence TAR. En conclusion, nos résultats démontrent clairement que lemiRTARencodé par le VIH-1 joue un rôle clé dans la réplication du virus. Sur la base de ces résultats, nous proposons le modèle suivant pour la fonction de miRTAR. La transcription du LTR du VIH-1 conduit à la production de courts ARN dénommés TAR contenant une structure en épingle à cheveux. TAR est « processé » pour générer le miARN miRTAR. L'ARN miRTAR est ensuite chargé dans le complexe RISC et régule l'expression de plusieurs gènes cellulaires impliqués dans la régulation négative de NF-kB. miRTARmédiée par l'activation de NF-kB résultats dans la régulation de gènes viraux et par conséquent augmente la production de virus. L'ensemble de nos résultats montrent que le VIH-1 utilise la voie de l'ARNiinterférence pour optimiser l'environnement intracellulaire requis pour une réplication optimale. / RNA interference (RNAi) is a gene regulatory mechanism that offers a sequence specific targeting of mRNA. Evolving in the context of RNAi, viruses of different families adopted strategies to use RNAi for their benefit. The main objective of my thesis was to understand the function of miRTAR, a viral miRNA derived from 3' end of the HIV-1 TAR RNA, in HIV-1 replication. We found that miRTAR regulates both basal and Tat-mediated transactivation of HIV-1 promoter. The effect of miRTAR does not require its binding to TAR RNA. miRTAR acts by inducing NF-κB transcription factor important for the LTR activity. Indeed, mutation of NF-κB sites but not Sp1 sites within the LTR abrogate miRTAR-mediated enhancement of transcription from the LTR. Additionally, Inhibition of NF-κB using specific siRNA directed against p50 and p65 subunits results in loss of miRTAR activity. Although, we were unable to identify the cellular gene(s) targeted by miRTAR, its overexpression lead to the activation of NF-κB pathway. Mutation of the 3' end of HIV-1 TAR results in dramatic reduction of viral replication without affecting Tat-mediated transcription. Importantly, overexpression of miRTAR rescued the replication of miRTAR HIV-1 mutant virus.In conclusion, our results strongly demonstrate that the HIV-1 encoded miRTAR plays a key role in virus replication. On the basis of these findings, we propose the following model for the function of miRTAR. Transcription of the HIV-1 LTR leads to production of short, TAR containing, RNA hairpin sequences. TAR is processed to generate miRNA, miRTAR. miRTAR is then loaded into the RISC complex and regulates the expression of several cellular genes involved in the negative regulation of NF-κB. miRTAR-mediated activation of NF-κB results in up regulation of viral genes and consequently enhances virus production. Taken together, our results demonstrate that HIV-1 uses RNAi pathway to optimize the intracellular environment for optimal replication.

Celiac disease is an autoimmune disease which occurs in susceptible individuals after ingestion of food containing gluten. Gluten and its monomeric fraction gliadin induce inflammatory damage of the small intestine by activating the immune cells that react strongly to gluten peptides. Gluten peptides have the ability to activate cells of adaptive as well as innate immune system. This work is focused on the production of interleukin (IL)-1 in antigen presenting cells stimulated with peptic gliadin digest. We found that monocytes and peripheral blood mononuclear cells (PBMC) isolated from blood of celiac patients secrete significantly more IL-1α and IL-1β than cells of healthy donors after stimulation with gliadin digest. The gliadin-induced IL-1β expression is controlled by a signaling cascade that includes MAPK kinase family molecules and transcription factor NF-κB. Moreover, we found that the adaptor proteins MyD88 and TRIF as well as Toll-like receptor (TLR) 2 and 4 play a role in the signaling cascade underlying gliadin-induced IL-1β expression by using murine bone marrow derived dendritic cells (BMDC). The precursor form of IL-1β in gliadin- stimulated PBMC and murine BMDC is maturated by caspase-1. In celiac PBMC the gliadin- induced maturation and secretion of IL-1β depends on the potassium...