Chez les mammifères, deux protéines cytosoliques régulatrices du fer, IRP1 et IRP2 (Iron Regulatory Proteins), sont impliqué dans le métabolisme de ce métal biologiquement essentiel. Ces protéines peuvent fixer des régions spécifiques d'ARNm, appelée Iron Responsive Elements, et ainsi contrôler l'expression des protéines impliquées dans l'importation, le stockage et l'utilisation du fer. IRP1 peut perdre sa capacité de fixer l'ARN en acquièrent un agrégat [4Fe-4S] lui conférant une activité d'aconitase cytosolique catalysant la conversion du citrate en isocitrate. Le rôle physiologique exact d'IRP1 n'est pas encore complètement clair mais des indications laissent à penser qu'elle serait impliquée dans la réponse au stress oxydant.<br />L'IRP1 humaine a une masse moléculaire de 98 kDa. La forme contenant un agrégat [4Fe-4S] a été cristallisée dans des conditions anaérobiques et un jeu de donnée allant jusqu'à 1,85 Å a été collecté. Un remplacement moléculaire a été tenté afin de résoudre la structure de la protéine à partir de la structure de l'aconitase mitochondriale de bœuf qui présente 22% d'identité de séquence avec IRP1 mais sans succès. Par la suite, une seconde forme cristalline diffractant à 2,5 Å de résolution a été obtenue. Le signal anomal de l'agrégat fer-soufre ainsi que celui d'un site zinc inattendu dans l'enzyme native ont été utilisés en plus des données isomorphes d'un dérivés or pour résoudre le problème de la phase. La structure a été affinée et analysée en respectant la fonction aconitase et celle de fixation d'ARN de la protéine et ce en utilisant les résultats des différentes études de mutagenèse dirigée disponibles dans la littérature.

Bakgrund Flera kvantitativa studier redogör för att många elever har svårigheter att tolka likhetstecknet men ger dessvärre inte läsaren en inblick i hur eleverna tänker kring likhetstecknet. Den svenska läroplanen lyfter att eleverna ska ges förutsättningar att utveckla förtrogenhet med grundläggande matematiska begrepp. Resultaten som presenterats i TIMSS-undersökningar visar att svenska elever ligger under genomsnittet och detta kan tolkas bland annat bero på en bristande förtrogenhet med de matematiska begreppen. Av den anledningen ville vi genom en kvalitativ metod undersöka vad eleverna tänker om begreppet likhetstecknet. Den kvalitativa metoden gav oss även möjligheten att undersöka elevernas förmåga att uttrycka sig och föra matematiska resonemang. Syfte Syftet med denna studie är undersöka elevers uppfattningar om och förståelse för likhetstecknet. Vidare vill vi undersöka i vilken form matematiska samtal gestaltar sig i användandet av arbetsmaterialet concept cartoon. Metod För insamlingen av studiens empiriska material genomfördes en matematiklektion med arbetsmaterialet concept cartoon. De deltagande eleverna valdes ut utefter ett kartläggningstest med fokus på uppfattningen av likhetstecknet. Vid lektionen agerade vi båda två som deltagande observatörer. Lektionen spelades in med ljudupptagning. Resultat Studiens resultat synliggör elevernas tankar och uppfattningar om likhetstecknet med utgångspunkt i en concept cartoon. Under lektionen kunde vi även identifiera olika samtalstyper samt hur dessa upplevdes påverka samtalet och elevernas uppfattningar av likhetstecknet.

This essay will treat the Gotlandic textile production during the Viking period (790 AD­–1150 AD) by studying the three grave fields, Barshalder in Grötlingbo parish, Broe in Halla parish and Ire in Hellvi parish. The focus of the study are the textile related objects, spindle whorls, weaving tablets, needles and needle cases found at the grave fields. The study will mainly examine the spindle whorls since differences in the weight and diameter can tell us about what type of yarn or thread that was produced and in extent the textiles that were produced. The results will be catalogued, and a correspondence analysis will be done in hope it will show patterns in the material. The study will also look at if there are differences between the three grave fields and in extent on the island.

Irreversible electroporation is a minimally invasive technique for the non-thermal destruction of cells in a targeted volume of tissue, using brief electric pulses, (~100 µs long) delivered through electrodes placed into or around the targeted region. These electric pulses destabilize the integrity of the cell membrane, resulting in the creation of nanoscale defects that increase a cell’s permeability to exchange with its environment. When the energy of the pulses is high enough, the cell cannot recover from these effects and dies in a non-thermal manner that does not damage neighboring structures, including the extracellular matrix. IRE has been shown to spare the major vasculature, myelin sheaths, and other supporting tissues, permitting its use in proximity to these vital structures. This technique has been proposed to be harnessed as an advantageous non-thermal focal ablation technique for diseased tissues, including tumors. IRE electric pulses may be delivered through small (ø ≈ 1 mm) needle electrodes, making treatments minimally invasive and easy to apply. There is sub-millimeter demarcation between treated and unaffected cells, which may be correlated with the electric field to which the tissue is exposed, enabling numerical predictions to facilitate treatment planning. Immediate changes in the cellular and tissue structure allow real-time monitoring of affected volumes with imaging techniques such as computed tomography, magnetic resonance imaging, electrical impedance tomography, or ultrasound. The ability to kill tumor cells has been shown to be independent of a functioning immune system, though an immune response seems to be promoted by the ablation. Treatments are unaltered by blood flow and the electric pulses may be administered quickly (~ 5 min). Recently, safety and case studies using IRE for tumor therapy in animal and human patients have shown promising results. Apart from these new studies, previous work with IRE has involved studies in healthy tissues and small cutaneous experimental tumors. As a result, there remain significant differences that must be considered when translating this ablation technique towards a successful and reliable therapeutic option for patients. The dissertation work presented here is designed to develop irreversible electroporation into a robust, clinically viable treatment modality for targeted regions of diseased tissue, with an emphasis on tumors. This includes examining and creating proving the efficacy for IRE therapy when presented with the many complexities that present themselves in real-world clinical patient therapies, including heterogeneous environments, large and irregular tumor geometries, and dynamic tissue properties resulting from treatment. The impact of these factors were theoretically tested using preliminary in vitro work and numerical modeling to determine the feasibility of IRE therapy in heterogeneous systems. The feasibility of use was validated in vivo with the successful treatment of human mammary carcinomas orthotopically implanted in the mammary fat pad of mice using a simple, single needle electrode design easily translatable to clinical environments. Following preliminary theoretical and experimental work, this dissertation considers the most effective and accurate treatment planning strategies for developing optimal therapeutic outcomes. It also experimentally characterizes the dynamic changes in tissue properties that result from the effects of IRE therapy using ex vivo porcine renal cortical tissue and incorporates these into a revised treatment planning model. The ability to use the developments from this earlier work is empirically tested in the treatment of a large sarcoma in a canine patient that was surgically unresectable due to its proximity to critical arteries and the sciatic nerve. The tumor was a large and irregular shape, located in a heterogeneous environment. Treatment planning was performed and the therapy carried out, ultimately resulting in the patient being in complete remission for 14 months at the time of composing this work. The work presented in this dissertation finishes by examining potential supplements to enhance IRE therapy, including the presence of an inherent tumor-specific patient immune response and the addition of adjuvant therapeutic modalities. / Ph. D.

Metazoans have evolved conserved mechanisms to promote cell survival under low oxygen tensions by initiating a transcriptional cascade centered on the action of Hypoxia Inducible transcription Factors (HIFs). In aerobic conditions, HIFs are inactivated by ubiquitin-proteasome-mediated degradation of their a subunit, which is dependent on prolyl hydroxylation by 2-oxoglutarate (2-OG) and Fe(II)-dependent prolyl hydroxylases (PHDs). In hypoxia, HIF-$\alpha$ is no longer hydroxylated and is therefore stabilised, activating a global transcriptional response to ensure cell survival. Interestingly, HIFs can also be activated in aerobic conditions, however the mechanisms of this oxygen-independent regulation are poorly understood. Here, I have explored the role of the vacuolar H+-ATPase (V-ATPase), the major proton pump for acidifying intracellular vesicles and facilitating lysosomal degradation, in regulating HIF-$\alpha$ turnover. Unbiased forward genetic screens in near-haploid human cells identified that disruption of the V-ATPase leads to activation of HIFs in aerobic conditions. Rather than preventing the lysosomal degradation of HIF-$\alpha$, I found that V-ATPase inhibition indirectly affects the canonical proteasome-mediated degradation of HIF-$\alpha$ isoforms by altering the intracellular iron pool and preventing HIF-$\alpha$ prolyl hydroxylation. In parallel, I characterised two putative mammalian V-ATPase assembly proteins, TMEM199 and CCDC115, identified by the forward genetic screen and subsequent mass spectrometry analysis. I confirmed that both TMEM199 and CCDC115 are required for V-ATPase function, and established assays to determine how TMEM199 and CCDC115 associate with components of the core V-ATPase complex. Lastly, to measure how V-ATPase activity leads to changes in the labile iron pool, I developed an endogenous iron reporter using CRISPR-Cas9 knock-in technology. This approach confirmed that iron homeostasis is impaired during V-ATPase inhibition, and demonstrated that exogenous ferric iron can restore the labile iron pool in a transferrin-independent manner. Together my studies highlight a crucial link between V-ATPase activity, iron homeostasis, and the hypoxic response pathway.

The main goal of this thesis is to design control board for the experimental electroporation device and to develop control firmware. The first chapter of this work focuses on the electroporation phenomenon itself. Behaviour of the cell model in external electrical field is described there as well as simulation and overview of how electroporation affects living tissue. It also explains the main requirements for parameters of the electroporation pulses as well as need for ECG synchronization. Furthermore, some remarks are given about novel high frequency electroporation method, which involves use of nanosecond bipolar high voltage pulse bursts. The second chapter briefly introduces commercial electroporation device called Nanoknife, including control part, power part, and it's limits. The third chapter consists of introduction of the novel experimental electroporation device developed at BUT. Power part of this device is discussed as well. Next chapter focuses on design of the control board for this device and also on description of the particular schematic parts. There is a control algorithm explanation in the fifth chapter of this thesis followed by the brief manual to machine operation.

L'obésité est un facteur de risque majeur de cancer. Il est connu qu’une adiposité élevée prédispose à un stress inflammatoire accru et potentialise la croissance tumorale. Néanmoins, les mécanismes restent mal définis. De façon intéressante, la sénescence cellulaire, ou le programme moléculaire causant l’arrêt du cycle cellulaire suite à un stress insurmontable, favorise l'inflammation chronique et délétère pendant l'obésité. Nous avons donc émis l’hypothèse que l'obésité puisse être un inducteur de sénescence protumoral qu’il est possible d’exploiter, via une stratégie sénolytique, pour ralentir ou même bloquer le développement de tumeurs. Grâce à des marquages de coupes histologiques de tumeurs métastatiques, nous avons montré que les masses malignes de patients ayant un indice de masse corporelle (IMC)>35 sont associées à des marqueurs de sénescence. Cette découverte suggère une charge élevée de cellules sénescentes chez ses patients. Alors que la sénolyse, ou l’élimination thérapeutique des cellules sénescentes, s'est révélée très prometteuse dans le traitement de plusieurs maladies liées à l'âge, son efficacité en tant que traitement du cancer est souvent mitigé et dépend des antécédents du patient. Dans notre étude, nous avons utilisé un modèle murin d'obésité induit par la diète combinée avec un modèle d’injections syngéniques de différentes lignées cancéreuses occasionnant des réponses immunogéniques faibles, légères ou hautes. Chez les souris sur une diète riche en gras, nous avons identifié des cellules cancéreuses sénescentes spécifiquement dans les tumeurs faiblement immunogènes, soit faiblement reconnue par le système immunitaire et donc difficile à traiter. Un traitement sénolytique avec l'inhibiteur de la famille BCL-2 ABT-263 abolit la réponse protumorale observée via l'ablation des cellules cancéreuses sénescentes. Ainsi, nous proposons que les thérapies combinatoires avec des agents sénolytiques devraient être envisagées pour traiter les patients cancéreux présentant une adiposité accrue. De plus, dans la même cohorte de patients où nous avons rapporté des marqueurs de sénescence dans les tissus malins, les patients obèses ont aussi montré une expression importante de Toll-like receptor 4 (TLR4). Nous avons donc émis l’hypothèse que le récepteur TLR4 joue un rôle important dans l’établissement d’un microenvironnement tumoral qui favorise la sénescence cellulaire et la croissance tumorale de souris en surplus de poids. Dans notre étude, nous rapportons que l'expression systémique de TLR4 est importante pour la croissance tumorale induite par l'obésité. Nous montrons également que l’induction d’un stress du réticulum endoplasmique médié par Inositol requiring enzyme 1a (IRE1ɑ) dans les cellules myéloïdes associées à une tumeur, favorise la sénescence des cellules cancéreuses, dans un contexte de faible immunogénicité, via TLR4. Ce travail établit les fondements d’une compréhension moléculaire du lien entre les régimes à forte teneur calorique et l'immunité protumorale. / Obesity is a major risk factor for cancer. High adiposity predisposes to increased inflammatory stress, which potentiates tumor growth. However, the mechanisms remain poorly defined. Interestingly, cellular senescence, or the molecular program causing cell cycle arrest following insurmountable stress, is known to promote chronic and deleterious inflammation during obesity. We therefore hypothesized that obesity could be an inducer of a protumoral senescence that can be exploited, via a senolytic strategy, to slow down or even block tumor development. Through histological sections of metastatic tumor, we show that malignant masses from patients with a body mass index (BMI)>35 are associated with markers of senescence, suggesting a high burden of senescent cells in these patients. While senolysis, or the therapeutic elimination of senescent cells, has shown great promises in the treatment of several age-related diseases, its efficacy as a treatment for cancer is often elusive and depends on patients’ history. In our study, we used a mouse model of diet-induced obesity (DIO) combined with a model of syngeneic injections of different cancer cell lines causing low, mild, or high immunogenic responses. In mice under a DIO, we have identified senescent cancer cells specifically in weakly immunogenic tumors, or tumors poorly recognized by the immune system, and therefore difficult to treat. Moreover, a senolytic treatment with the BCL-2 family inhibitor ABT-263 abolishes the protumor response seen in these mice via the ablation of senescent cancer cells. Thus, combination therapies using senolytic agents should fall into consideration to treat cancer patients with increased adiposity. In addition, in the same cohort of patients where we reported markers of senescence in malignant tissues, obese patients also showed significant expression of TLR4. We therefore hypothesized that the TLR4 receptor plays an important role in establishing a tumor microenvironment that promotes cellular senescence and tumor growth in mice subjected to experimental obesity. In our second study, we report that systemic expression of TLR4 is important for obesity-induced tumor growth. Moreover, we show that the induction of an IRE1ɑ-mediated endoplasmic reticulum stress, in tumor-associated myeloid cells, promotes the senescence of cancer cells, in a context of low immunogenicity, via TLR4. This work lays the foundation for a molecular understanding of the link between high-calorie diets and protumoral immunity.

The aim of this master thesis is to expand on the understanding of the resource utilisation on the 19 Gotlandic Pitted Ware Culture sites: Ajvide, Alvena, Fridtorp, Grausne, Gullrum,Gumbalde, Hau, Hemmor, Hoburgen, Ire, Kinner/Tjauls, Rangvide, Barshalder, Stenstugu,Stora Förvar, Sudergårds II, Visby, Västerbjers and Västerbys. The study utilises theoretical frameworks such as Site Catchment Analysis, Site Territorial Analysis and Optimal ForagingTheory and is based on two main questions: Which animals did the diet on each site consist of? And are there any apparent connections between diet and topography/environment? To answer these questions, osteological records have been studied to get an idea of the animal food resources utilised on each site. ArcGIS has also been used to create height- and soil maps with contemporary shorelines which show how the sites were located in the middle Neolithic Gotlandic landscape. The study has shown that most sites appear to have included a variety of animals like pig/boar, cattle, sheep/goat, fish, seal, porpoise and birds in their diet. The sites with the lowest number of confirmed animals also tend to have undergone the least archaeological investigation, suggesting that further excavations on these sites might unearth more animal species. Additional discoveries show a small albeit noticeable emphasis on marine animal resources, especially porpoise, on southern sites. Sites located in areas mostly consisting of sandy, meager soils also show an increased marine resource utilisation. This might suggest that the area around these sites were somewhat barren and lacking in terrestrial prey animals.

Ce mémoire porte sur la politique étrangère d’Élisabeth 1ère, reine d’Angleterre (1558-1603), face à la France, durant le règne de Henri III, roi de France de la dynastie des Valois. Le premier objectif fut de déterminer comment Élisabeth 1ère, reine protestante, et ses ambassadeurs ont réagi face aux guerres civiles entre catholiques et protestants en France. Nous avons aussi étudié leurs réactions face à la montée en puissance d’une famille princière ultra catholique, les Guise, et le groupe qu’ils dirigent, la Sainte Ligue. Si Élisabeth a préconisé les menées secrètes avec les rebelles protestants dans un premier temps (1574-1577), elle a adopté une diplomatie sincère avec la couronne française dans un second temps (1584-1589), pour éviter que la France ne tombe aux mains des Guise et de leur allié, le roi d’Espagne Philippe II. / The subject of this thesis is the foreign policy of Elizabeth I, Queen of England (1558-1603), toward France during the reign of Henry III, King of France of the dynasty of Valois. The first purpose was to determinate how Elizabeth I, protestant queen, and her ambassadors, reacted toward the wars of Religion between French Catholics and Protestants. We also study their reactions toward the princely ultra catholic family of Guise and the group they leaded, the Holy League. Elizabeth chose to act secretly with the protestants rebels first (1574-1577) but, she adopted a sincere diplomacy with the crown subsequently (1584-1589) to obviate that France fall into Guise’s and their ally’s hands, Phillip II, King of Spain.