T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

T lymphocytes comprise one arm of the adaptive immune system and are critical for immunity to neoplasia and infection. A full understanding of their development has important implications for the treatment of autoimmunity, immunodeficiency, and leukemias arising from T cell developmental intermediates. The Notch signaling pathway is already known to be absolutely required for T cell commitment and development, but its collaboration with other factors is poorly understood. Unfortunately, deficiency in many of the genes critical to hematopoiesis, including Notch, causes early embryonic lethality by disrupting multiple developmental processes. This complicates the study of such genes by in vivo models or ex vivo hematopoietic progenitors. To circumvent these difficulties, this thesis describes the use of in vitro-differentiated embryonic stem cell-derived T progenitors to examine the roles of two GATA family members during early T cell development. GATA-2, while not required for T cell development, is shown to act downstream of Notch signals to inhibit myelopoiesis. These findings both characterize a novel role for GATA-2, and demonstrate that T progenitor maturation and exclusion of non-T cell fates are distinct and separable events. GATA-3, in contrast to GATA-2, is absolutely required for T lymphopoiesis. However, the current literature does not distinguish between a requirement for GATA-3 in homing to the thymic environment, committing to the T cell fate, or surviving such a commitment event. This thesis demonstrates that GATA-3 is dispensable for commitment itself, but required to permit survival and proliferation after commitment. Taken together, the results presented in this thesis employ a novel model system to characterize the interactions of two important collaborators with Notch signals during T cell development, and further dissect the stages through which early T cell development is enacted.

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

The regulation of cardiac-specific genes such as GATA-4 and its co-factor FOG-2 is paramount for normal heart development and function. Indeed, those mechanisms that regulate GATA-4 and FOG-2 function, such as nuclear transport and the post-translational modification of SUMOylation, are of critical importance for cardiogenesis. Therefore the aims of this study were to: i) elucidate the nuclear transport mechanisms of GATA-4; ii) determine the function of SUMOylation on the biological activity of both GATA-4 and FOG-2; and iii) examine how these mechanisms impact on the role of GATA-4 and FOG-2 in cardiac hypertrophy. Firstly, we characterised a non-classical nuclear localisation signal that mediates active import of GATA-4 in both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial residues within this region that interacted with importin ?? to mediate GATA-4 import via the non-classical import pathway. In addition, a cardiac myocyte-specific CRM1-dependent nuclear export signal, which consists of three essential leucine residues, was identified. We also investigated the residues of GATA-4 that are responsible for its DNAbinding activity and therefore transcriptional control of cardiac-specific genes. Secondly, we demonstrated that SUMOylation of both GATA-4 and FOG-2 is exclusively carried out by SUMO-2/3. Moreover, SUMOylation is involved in the nuclear localisation of both GATA-4 and FOG-2 in cardiac myocytes as well as the transcriptional regulation of cardiac-specific genes, such as cardiac troponin I. Finally, and perhaps most biologically significant, we showed that nuclear transport as well as SUMOylation of GATA-4 is imperative for the ability of GATA-4 to induce cardiac hypertrophy. Moreover, it was determined that FOG-2 SUMOylation is involved in the ability of FOG-2 to protect against cardiac hypertrophy. In conclusion, the current study provides detailed information on the nuclear transport pathways of GATA-4 as well as the SUMOylation of both GATA-4 and FOG-2 and the role these two mechanisms play in gene transcription and cardiac hypertrophy.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-29T12:19:19Z No. of bitstreams: 1 Amanda Camilo Silva.pdf: 1035628 bytes, checksum: 4182f45e5ff155111084077a4179deb4 (MD5) / Made available in DSpace on 2016-07-29T12:19:19Z (GMT). No. of bitstreams: 1 Amanda Camilo Silva.pdf: 1035628 bytes, checksum: 4182f45e5ff155111084077a4179deb4 (MD5) Previous issue date: 2011-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The pregnancies of queens are not always desirable by their owners, who use hormonal contraceptive and surgical methods (ovariohysterectomy - OSH). Another form of contraception used in human medicine is the tubal oclusion, which consists of mechanical occlusion and / or partial resection of this structure (partial salpingectomy). This research had the objetive of a develop a technique for definitive contraception in pregnant queens, that would not compromise the current pregnancy, the birth of kittens, and the concepts, considering that is common in castration campaigns the visualization of gravid uterus during the approach to abdominal cavity. Another objective was evaluate weight gain and changes in behavior of queens submitted to partial salpingectomy, comparing them with those submitted to OSH, and to compare the time between both surgical procedures. Were used 40 queens, divided into two groups of 20 animals, A: Partial salpingectomy, and B: OSH. The group A, was divided in A1, containing 10 queens pregnant and A2 containing 10 non-pregnant. During the surgery were recorded the quantity of embryonic vesicles found, and the execution time of both procedures. All animals were assessed with seven, 60, 180 and 365 days, being observed initially the maintenance of pregnancy and the occurrence of fetal resorption in group A1, and fertility, weight gain and behavior change in all groups. The mean operative time of group A was 353 seconds, with a deviation of 58.476 seconds, and group B corresponded to 448 seconds with diversion of 84.609 seconds, showing statistically significant difference (p = 0.000069) between the groups on the Mann-Whitney test, with significance level of 5%. The gestacional course and birth of kittens were normal in queens of group A, not occurring abortion neither dystocia. A total of 51 embryonic vesicles were visualized during surgery, 49 born live fetuses, no stillbirths, and no fetal withholding, occurring two (3.92%) fetals resorptions. Among the 49 fetuses, seven (14.28%) presented genu recurvatum. With respect to fertility, the animals in group A that cycled and mated, did not get pregnant. Group B showed average weight increase of 20.34%, statistically significant (p = 0.000062) on the Wilcoxon test at a significance level of 5%, while the A2 group showed an increase of 9.57%, considered not significant (p = 0.130570) compared to pre-surgery weight.Regarding the behavioral parameters, group B showed increase in food intake in 60% of the animals, lethargy in 65%, and decreased waking period in 75%, whereas these parameters remained unchanged in most animals of group A. It is concluded that partial salpingectomy in queens is a 100% effective contraceptive method of rapid execution, which can be employed during the transoperative identification of pregnancy, with minimal adverse effects on fetuses, no significant changes of behavior neither weight gain, but with undesirable characteristics on the acceptance of owners. / As gestações de gatas nem sempre são desejáveis por parte dos proprietários, os quais recorrem aos métodos contraceptivos hormonal e cirúrgico (ovarissalpingohisterectomia - OSH). Outra forma de contracepção bastante utilizada na medicina humana é a ligadura ou laqueadura das tubas uterinas, que consiste na oclusão mecânica e/ou ressecção parcial desta estrutura (salpingectomia parcial). Objetivou-se com este trabalho, desenvolver uma técnica de contracepção definitiva em gatas prenhes, que não comprometesse a gestação em curso, o parto, nem os conceptos, tendo em vista que é comum em campanhas de castração a visualização de útero gravídico durante a abordagem à cavidade abdominal; assim como avaliar o ganho de peso e mudanças de comportamento das gatas submetidas à salpingectomia parcial, comparando-as com as submetidas à OSH; e comparar o tempo cirúrgico entre os dois procedimentos. Para tal, foram utilizadas 40 gatas, divididas em dois grupos de 20 animais, A: salpingectomia parcial, e B: OSH; sendo o grupo A subdividido em A1, contendo 10 gatas prenhes, e A2 contendo 10 não prenhes. Transcirurgicamente foram registradas as quantidades de vesículas embrionárias encontradas, e o tempo de execução de ambos os procedimentos. Todos os animais foram reavaliados com sete, 60, 180 e 365 dias, sendo inicialmente observados quanto à manutenção da gestação e ocorrência de reabsorção fetal no grupo A1, e posteriormente quanto à fertilidade, ganho de peso e mudança de comportamento em todos os grupos. O tempo cirúrgico médio do grupo A foi de 353 segundos com desvio de 58,476 segundos, e o grupo B correspondeu a 448 segundos com desvio de 84,609 segundos, apresentando diferença estatisticamente significativa (p= 0,000069) entre os grupos diante do teste de Mann-Withney com nível de significância de 5%. Nas gatas do grupo A1 o curso gestacional e o parto foram normais, não ocorrendo abortos, nem distocias. No total foram visualizadas 51 vesículas embrionárias transcirurgicamente, nasceram 49 fetos vivos, nenhum natimorto, e nenhuma retenção fetal, logo, ocorreram duas (3,92%) reabsorções fetais. Dentre os 49 fetos, sete (14,28%) apresentaram a anormalidade genu recurvatum. Quanto à fertilidade, os animais do grupo A que ciclaram e copularam, não engravidaram. O grupo B apresentou aumento de peso médio de 20,34%,estatisticamente significativo (p= 0,000062) diante do teste de Wilcoxon com nível de significância de 5%, enquanto que o grupo A2 apresentou aumento de 9,57% considerado não significativo (p= 0,130570) em relação ao peso pré-cirúrgico. Com relação aos parâmetros comportamentais, no grupo B se observou aumento na ingestão de alimentos em 60% dos animais, da letargia em 65%, e diminuição do período de vigília em 75%; enquanto que estes parâmetros permaneceram inalterados na maioria dos animais do grupo A. Conclui-se que a salpingectomia parcial em gatas é um método contraceptivo 100% eficaz, de rápida execução, que pode ser empregado durante a identificação transcirúrgica do estado de prenhez, com mínimo efeito prejudicial aos conceptos; e sem alterações significativas de conduta e ganho de peso, mas com características indesejáveis quanto à aceitação dos proprietários.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Congenital heart disease (CHD) is a risk factor for premature cardiovascular complications. Great advances have occurred in the past years leading to the identification of several genes essential for proper cardiac formation such as GATA4/5/6 mutated in some individuals with CHD. GATA6 is a zinc finger transcription factor whose presence is crucial for early embryonic development. GATA6 is expressed in many cell types of the heart including myocardial, endocardial, neural crest, and vascular smooth muscle. In human, mutations in GATA6 result in variable cardiac phenotypes. The objective of this thesis was to determine the roles that GATA6 play in the different cell types of the heart and to elucidate the molecular basis of the cardiac defects associated with Gata6 haploinsufficiency. For this, a combination of cell and molecular techniques were used in vitro and in vivo. First, we show that Gata6 heterozygozity leads to RL-type bicuspid aortic valve (BAV)- the most common CHD affecting 2% of the population. GATA6-dependent BAV is the result of disruption of valve remodeling and extracellular matrix composition in Gata6 haploinsufficient mice. Cell-specific inactivation of one Gata6 allele from Isl-1 positive cells, but not from endothelial or neural crest cells, recapitulates the phenotype of Gata6 heterozygous mice revealing an essential role for GATA6 in secondary heart field myocytes during valvulogenesis. We further uncovered a role for GATA6 as an important regulator of the cardiac conduction system and revealed that GATA6 expression regulates the activity of the cardiac pacemaker. GATA6 exerts its role via regulation of the cross-talk among the different cell types of the SAN. Lastly, some CHDs are characterized by abnormalities of both the limbs and the heart such as the Holt Oram syndrome (caused by mutation in TBX5 transcription factor). The molecular basis for limb-heart defects remain poorly understood. In the course of this work, we discovered that Gata6 haploinsufficiency resulted in a partially penetrant polysyndactyly (extra digits fused together) phenotype. Together, the data provide novel molecular and cellular insight into GATA6 role in normal and pathologic heat development. Our results also suggest that GATA6 should be added to the list of genes whose mutations are potentially associated with heart and limb abnormalities. Better knowledge of the molecular basis of CHD is a prerequisite for the development of diagnostic and therapeutic strategies to improve care of individuals with congenital heart disease.

The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.

Le cancer de la vessie représente un vrai problème de santé publique, avec une surveillance et suivi clinique à long terme en raison de l’importance des fréquences de récidives. La chimiothérapie reste souvent inefficace. L’objectif de cette thèse est donc la recherche de marqueurs sérologiques et moléculaires à valeur pronostique dans le cancer de la vessie qui peuvent servir à prédire la maladie. D’abord, nous avons étudié trois facteurs de transcriptions des lymphocytes T activées qui sont T-bet, GATA-3 et Bcl-6. Nous avons montré une surexpression de T-bet chez les malades à stade invasif et de haut grade, cependant, la surexpression de GATA-3 et Bcl-6 a été corrélée au stade superficiel et de bas grade. La survie a été corrélée avec le groupe des malades sans histoires de récidive ou progression et avec la surexpression de Bcl-6 et GATA-3. Cependant les malades qui expriment fortement T-bet répondent mieux au BCG. Ensuite, nous avons visé la détection de FasL et TRAIL solubles dans le sérum des malades atteints du cancer vésicale. Nous avons montré une surexpression de sFasL et sTRAIL chez les malades à stade superficiel et de bas grade. Le rôle anti-tumoral de ces cytokines a été confirmé sur deux lignées du cancer de la vessie montrant que le traitement avec le sérum riche en sFasL ou en sTRAIL diminue la viabilité cellulaire in vitro. A la fin de cette thèse, nous avons testé l’activation p-Akt dans la tumeur vésicale. Nous avons montré une surexpression de p-Akt au sein des tumeurs comparées au tissu sain adjacent, et au sein des malades à stade invasif et de haut grade. Akt semble être un marqueur de progression tumorale dans le cancer de la vessie. / Bladder cancer is the first most common urogenital cancer in men in Tunisia, with a high recurrence rate. Patients with muscle-invasive disease develop metastasis. The need for expensive continuous surveillance. In this thesis we try to search some candidate biomarkers. Their use for cancer staging and personalization of therapy at the time of diagnosis in order to identify a better treatment could improve patient care. The aim of this first part of our study was to investigate the clinical significance of three immune cell-related transcription factors, T-bet, GATA-3 and Bcl-6 in Tunisian patients with bladder cancer. We found that T-bet level was significantly higher in invasive carcinoma with high- grade. However, T-bet is predictive of response to BCG. On the contrary, the expression of GATA-3 and Bcl-6 was significantly higher in non-invasive carcinoma with low grade. We furthermore studied the effect of activation of soluble FasL and TRAIL molecule in bladder cancer. We demonstrate that the mean serum level of sFasL was higher in patients than in normal donors. sFasL was only higher than in sera of healthy donors where patients had superficial stage and low- and medium-grade cancer. sTRAIL was significantly lower in sera from patients with invasive and high-grade bladder carcinoma than in controls. Finally, we demonstrate that p-Akt levels in patients with invasive carcinoma and high-grade bladder cancer were significantly elevated compared to patients with non-invasive and low grade bladder cancer. Altogether, our results suggest that Akt activation can provide useful prognostic information.

Hintergrund: Der Transkriptionsfaktor GATA-4 ist für die normale Entwicklung des Endoderms essentiell. Mausmutanten mit einer homozygoten Deletion des gata-4 Gens versterben zwischen den embryonalen Tagen 8.5 - 10.5 aufgrund einer Störung der ventralen Morphogenese und der Ausbildung des Herzschlauches. Zielsetzung und experimentelle Strategie: Um die Bedeutung von GATA-4 auch nach der embryonalen Entwicklung untersuchen zu können, wurden doppelt-transgene Mäuse generiert. Diese Mausmutanten exprimieren einen Tetrazyklin-Repressor und eine gegen GATA-4 gerichtete short hairpin RNA (shGATA-4). Die Expression der shGATA-4 steht dabei unter der Kontrolle eines H1-Promotors, welcher durch ein Tetrazyklin-Operator Element modifiziert wurde. Dadurch ist das System durch Doxyzyklin induzierbar. Ergebnisse: Die Integration der Transgene in dem Genom der Maus wurde durch Southern-Blot Analyse nachgewiesen. Die Expression der shGATA-4 wurde durch die Applikation von Doxyzyklin über das Trinkwasser (20 mg/ml) induziert. Langzeitstudien am Herzen haben dabei eine signifikante Suppression von GATA-4 nach 38 Tagen ergeben (80 %). Diese Reduktion konnte durch Western-Blot Analyse bestätigt werden. Obwohl die Expression verschiedener Zielgene von GATA-4 (ANF, BMP-4) ebenfalls herunterreguliert war, fiel bei den transgenen Mäusen kein kardialer Phänotyp auf. Jedoch wurde die GATA-4 Expression in den Hoden und Ovarien transgener Mäuse supprimiert, nachdem shGATA-4 durch die Applikation von Doxyzyklin induziert wurde. Weiterführende Untersuchungen an adulten Mäusen zeigten eine GATA-4 Reduktion von 20 % auch in nicht mit Doxyzyklin-induzierten Mausmutanten. Diese Reduktion könnte durch einen sog. leaky-Effekt des shGATA-4 Transgens hervorgerufen worden sein, wodurch die stark eingeschränkte Fertilität dieser Mauslinie erklärt werden könnte. Interessanterweise haben ca. 10 % der mit Doxyzyklin behandelten transgenen Weibchen Ovarial-Teratome ausgebildet. Histologisch wiesen diese Teratome überwiegend (neuro-) ektodermale, vereinzelt mesodermale und nahezu keine endodermalen Strukturen auf. Schlussfolgerung: In diesem Modell hat die Suppression von GATA-4 keinen Einfluss auf die Funktion des Herzens der adulten Maus. Jedoch scheint GATA-4 für die Fertilität der Maus von großer Bedeutung zu sein. Weiterhin scheint die Suppression von GATA-4 mit der Ausbildung von Ovarial-Teratomen assoziiert zu sein. / Background: The transcription factor GATA-4 is crucial for the normal endodermal development. In mice, homozygous deficiency of GATA-4 causes defects in ventral morphogenesis and heart tube formation, resulting in embryonic death between day e8.5 and e10.5. Aim and experimental strategy: To analyze the implication of GATA-4 beyond embryonic development a double transgenic mouse expressing the tetracycline repressor (TetR) and an inducible small interfering RNA directed against GATA-4 was generated. This expression construct contains a H1 promoter modified with a tetracycline operator upstream of the coding region for the GATA-4 short hairpin RNA (shGATA-4). Results: The integration of the transgenes in FvB mice (H1:G4/TetR) was confirmed by Southern blot. To induce the expression of the shGATA-4 construct, transgenic mice were treated with doxycycline (20 mg/ml drinking water). In longitudinal analysis, most efficient GATA-4 suppression was detected after 38 days. Quantitative PCR revealed a GATA-4 reduction of about 80 % in the heart, if normalized against the wildtype. Reduction of GATA-4 was confirmed by Western Blot. Although GATA-4 target genes (ANP, BMP-4) were down regulated, the animals showed no clinical phenotype. In opposite to wildtype mice, GATA-4 expression was undetectable in the ovaries and testis of transgenic mice with induced shGATA-4. Additional analysis in adult transgenic mice, which were not treated with doxycycline, also showed a reduction of GATA-4 expression of about 20 %, probably caused by a leaky-effect of the transgene. This may explain the significantly reduced fertility of the colony. Importantly, 10 % of transgenic females treated with doxycycline developed ovarian teratomas. Histological examination of teratomas showed predominantly (neuro-) ectodermal and to a lower degree mesodermal, but almost no endodermal compounds. Conclusions: GATA-4 reduction in the adult murine heart is – at least to a certain degree – clinically redundant. GATA-4 seems to be required for normal fertility. In our model GATA-4 deficiency seems to be associated with an increased risk for developing ovarian teratoma.

Kort beskrivelse av prosjektet: Oppgaven har vært å tegne et samlingshus for et antall omgivende videregående skoler. Ved å sentralisere ulike funksjoner som bibliotek, sosiale arealer og matsal fungerer huset som et felles læresenter for elevene.  I en tidlig fase fant jeg ut at jeg ville fokusere på å gjøre huset tilgjengelig for offentligheten slik at det ikke blir stående tomt utenom skoletidene. Dette ville jeg uttrykke både i form og i tolking av programmet. Så hvordan viser man at en bygning er åpen for alle i form? Jeg valgte å ta inspirasjon fra det mest offentlige jeg kjenner til, nemlig det offentlige rommet. Det jeg anser som spesielt med dette er at rommet bare fortsetter og fortsetter og består ikke av stengte vegger som man ofte finner i konvensjonelle bygninger. Ved å la gulvet være sammenhengende gjennom hele huset oppnås et interiør som jeg tror kan oppfattes som et offentlig rom. Jeg forsterket denne opplevelsen ved å koble gulvet til fjellet som grenser til tomten og huset ble dermed en offentlig vei per definisjon.  Hvordan fungerer interiøret? En utfordring har vært å definere rom når konseptet er at det skal være helt åpent. Derfor har jeg valgt ut fire parametere for å definere funksjonene: Møbler, et flatt gulv, dobbel takhøyde og til slutt det stengte rommet (se diagram, romdefineringer, s. 5). Hvilken rolle spiller programmet i konseptet? Flere av funksjonene i programmet var hva man ofte tenker på som soner kun for ansatte, som for eksempel administrasjon, rengjøring og kjøkken Jeg valgte å utfordre denne ideen og gjøre det slik at disse funksjonene ble tilgjengelige for folk flest. Gjennom et internt bookingsystem kan selv de som ikke er elever anvende huset og dets muligheter. For eksempel kan man booke, utenom skoletider, kjøkkenet for å lage mat i større volumer, black boxen for å holde en forestilling, et singelrom som kontor, et konferanserom for lesesirkelen på søndager, det multifunksjonelle torget for en bryllupsfest, osv. Hva oppnår man ved å koble huset med Enskede Gård? I planprogrammet står det: ”Stockholms ytterstad har till stor del vuxit fram etappvis och resulterat i stadsdelar som sinsemellan skiljer sig åt, men som i sig själva har ett homogent bostadsbestånd med likartade typologier och prisnivåer. (...) Slakthusområdet, som är tänkt att omvandlas under drygt ett decennium, är en del av denna utveckling. (...) En mer differentierad prisbild och lägre hyresnivåer finns att hitta i det befintliga beståndet, vilket gäller både bostäder och lokaler. Därför spelar programmets ambition att koppla samman Slakthusområdet med omkringliggande stadsdelar en viktig roll i att minska den geografiska segregationen mellan de olika områdena och skapa en funktionell helhet av dagens separata delar”. Utover dette ligger Lindeparken gymnasiesärskola i området og kan med grepet ta del av læresenterets tilbud.  Materialer? Ved å bruke materialer som ofte forekommer i et byrom kan følelsen av et offentlig og åpent rom forsterkes. I huset forekommer derfor stein, asfalt, betong, glass og stål. I tillegg har jeg fokusert på å la grensen mellom ute og inne defineres kun med glass samtidig som den dras inn ved den offentlige veien. Utover dette har jeg latt de rommene som er stengte få en varmere karakter, slik at de blir som små hus man går inn i, fra gaten. / Project description: Three planned high schools in the area were to have a common building with different social functions, such as a library, social areas and a dining room.  Early on in the process, I figured that the program had many functions that potensially could be interesting for the general public, such as a library, social areas and an exhibition. This led to the concept of opening up the building and making it accessible to everyone, creating potensial for an active building, even after school hours.  So how can openness be communicated in form? My concept was to extend the public space and let it continue uninterrupted through the whole interior., in short: a ramp. By connecting the ramp to Enskede, lying ten metres above the plot, the building became a public street. Stockholm municipality had an ambition for opening up Slakthusomradet, and the building became a part of this project. The program in the assignment excisted of many zones, usually associated with staff areas. I wanted to change that conception and make these rooms open to the public. These rooms included for example administration, cleaning and kitchen.  The materials used were all taken from typical public spaces, such as concrete, stone, asphalt, steel and glass. Furthermore, the character of the materials were to change when you moved from “the street” and into the closed spaces. Here you would find wooden walls and furniture, as well as curtains of cotton.