Introduction Heteromeric amino acid transporters (HATs) are composed of a heavy subunit (rBAT or 4F2hc) and a light subunit (b0 + AT, ASC1, LAT1, LAT2, y + LAT1, y + LAT2 and xCT), joined by a disulfide bridge (Chillaron et al. 2001). rBAT and 4F2hc are type II membrane glycoproteins (N-terminal cytoplasmic). Both have a single transmembrane segment, an N-terminal intracellular tail and an extracellular domain (ectodomain). As far as we know, the role of the heavy subunit is facilitating the transit of the light subunit to the plasma membrane. The light subunits are polytopic proteins unglycosylated, with 12 transmembrane segments, and the N-and C-terminal intracellular. The light subunit is the catalytically active subunit which confers specificity to the heterodimer on the transport system (Reig et al. 2002): LAT1 and LAT2 for system L , y+LAT1 and y+LAT2 for system y + L, asc for system ASC1; xCT for system Xc -, and b0+at for system b0, + (Chillaron et al. 2001). Results Overexpression of these human proteins was carried out with the methylotrophic yeast Pichia pastoris (strain KM71H) as expression system (based on Long et al. 2005). The main objective was to generate enough protein in a high level of purity to study the structure and check their function by transport assays. The different subunits, light and heavy, were cloned into the expression vector pPICZ (Invitrogen). To facilitate the purification of the different proteins, a cluster of 10 histidines was introduced by PCR at the N-terminus of the heavy subunits and a StrepTagII (IBA) at the N-terminus of the light subunits. 4F2hc is a glycoprotein with 4 possible targets for glycosylation. The glycosylations confer heterogeneity to protein, thus glycosylation targets were eliminated by directed mutagenesis. From all these human heavy and light subunits and heterodimers, only 4F2hc for the heavy subunits, LAT2 for the light subunits, and the heterodimer 4F2hc/LAT2 were overexpressed and extracted from the yeast membrane in enough amounts to continue with the purification step. The light subunit LAT2 was successfully purified but when the stability was analysed by size exclusion chromatography showed a clear profile of aggregation, concluding further studies. In contrast, the heavy subunit 4F2hc was stable after the exclusion chromatography for two days. The heterodimer 4F2hc/LAT2 proved to be stable after gel filtration analysis during one day. Thus, the heterodimer was significantly more stable than the light subunit alone, which allowed us to make an important statement. The catalytic subunit LAT2 needed their heavy subunit (i.e. 4F2hc) to increase the stability. This statement contrasted with the results for the heterodimer rBAT/b0+AT, in which was the heavy subunit rBAT the one who needed its light subunit b0+AT to a correct folding during its biogenesis (Bartoccioni et al. 2008; Rius et al. 2011). Functional studies with human heterodimer 4F2hc/LAT2 were set up to check the role of 4F2hc in the transport. Firstly the functionality of the heterodimer 4F2hc/LAT2 and the light subunit LAT2 in the living cell was checked successfully, meaning a correct folding at expression level. The apparent KM in both cases was the same, remaining unanswered the role of the heavy subunit 4F2hc in the transport function. Next, reconstitution in liposomes was carried out successfully for 4F2hc/LAT2 but not for LAT2, due to the high aggregation tendency. 4F2hc/LAT2 showed the typical overshoot for an amino acid transporter. To carry out the structural studies and due to the difficulty to maintain a stable soluble heterodimer, it was decided to carry out the technique of Single particle -negative staining (SP-NS) in the laboratory of Prof. Fotiadis in the University of Bern (Switzerland). The 3D model technique based on transmission electron microscopy (TEM) is relatively new and has been imposed for mammalian membrane proteins, allowing structural analysis with relatively small concentration of protein. The pure heterodimer was stained in a grid with uranyl formate at 0.75% (two drops optimized for 1 second, washing with water twice). This sample was analysed by transmission electron microscopy (TEM). Different images of projections in different orientations for 4F2hc/LAT2 were kept in a library of 11,000 picks. The refinement of the whole library allowed the 3D reconstruction of this protein by Mr. Meury. The model showed two asymmetric particles, one smaller, in which the crystal of the human ectodomain 4F2hc (Fort et al. 2007) fitted pretty well, and other bigger, which showed a black hole. Thus, the smaller particle was recognized as the heavy subunit, located on top of the light subunit. The resolution was 19 amstrongs, which was in the normal range for this method (from 16 amstrongs to 25 amstrongs). Discussion It was observed that the heavy subunit was located on top of the light subunit LAT2, and not in contact with the cell membrane as was firstly though. The size for the heavy subunit coincided with the existing 3D crystals of the human ectodomain which can fit quite accurately, always assuming the presence of the transmembrane segment in the 3D model. By contrast, the light subunit did not fit with the crystal structure of the prokaryotic homolog AdiC in the APA family (APC superfamily) (Gao et al. 2009) (Kowalczyk et al. 2011) due to the large amount of detergent surrounding this highly hydrophobic subunit in SP-NS method. In spite of that, when the size was compared with AdiC and Stet (a prokaryotic homolog in the LAT family with 30% of homology) studied in the same SP-NS method (Casagrande et al. 2009) the light subunit LAT2 coincided in size with its homologs, demonstrating that the increased volume was due to the detergent effect. Supporting the 4F2hc/LAT2 model, interaction studies with integrins (Feral et al 2005; Feral et al. 2007) and other membrane proteins involved in cell growth (ICAMI; Liu et al. 2003) and / or overexpressed in tumours (CD147/MCT1; Xu et al. 2005) suggest an effect in the transport function through the heavy subunit 4F2hc, which may be explained with an orientation on top of the light subunit and interaction by the external loops. New Evidences: Recently, the 4F2hc/LAT2 heterodimer model in which the heavy subunit is located on top of the light subunit has been corroborated by cross-linking experiments by Miss Helena Alvarez in our laboratory. This fact, allow us to imagine how interactions between both subunits will carry out also when the disulphide bridge is missing. Analyzing the external loops in AdiC atomic structure (the closest paradigm with LATs at present) is found that the external loop 3 and the external loop 4 are the longest (around 25 residues). These loops are even longer in LAT2, which make possible the interaction between both subunits being the separation of 16 amstrongs in the 3D model. Both loops have important roles in the transport cycle based in LeuT fold. The external loop 3 has an important movement in the transition from outward-open conformation to occluded-outward conformation due to the tilt of 40o of the transmembrane 6. The external loop 4, moves down to lid the substrate pathway during the transition from occluded-outward conformation to the occluded-inward conformation. Our new 3D model of a human heteromeric aminoacid transporter offers the opportunity to study new aspects about the role of the heavy subunit in the holotransporter. If the external loops join 4F2hc and LAT2 modulating the transport function in presence of other transmembrane proteins, or if 4F2hc only acts as a bollard of a multiproteic complex, rest to be studied in the future. / Els transportadors heteromèrics d'aminoàcids (HATS) de metazous estan formats per una subunitat pesada (4F2hc o rBAT) (N-glicoproteïna amb 1 segment transmembrana i un gran ectodomini en el seu extrem C-terminal), i una subunitat lleugera (d'entre 10) unides covalentment per un pont disulfur, fent aquests transportadors únics entre els metazous. En humans, 6 subunitats lleugeres es troben formant heterodímers amb 4F2hc (LAT1, LAT2, y+ LAT1, y + LAT2, XCT i asc1) i una (b0, + AT) amb rBAT. Els HATs tenen incidència en la salut, ja que mutacions en qualsevol de les subunitats ocasionen aminoacidúries (cistinúria, lisinúria amb intolerància a proteïnes), són receptors virals o estan sobre expressats en cèl • lules tumorals. El nostre grup va determinar l'estructura de l'ectodomini de 4F2hc a 2.1 Å (Fort J et al. 2007), i recentment ha resolt l'estructura d'un homòleg procariota (AdiC d' E. coli, amb ~17% d´homologia) de les subunitats lleugeres a 3.0 Å de resolució (Kowalczyk et al. 2011). Per contra no hi ha informació estructural sobre els holo-transportadors HAT. El present treball ens mostra el primer model estructural a 19 Å d'un transportador HAT humà, el transportador 4F2hc/LAT2. La importància de 4F2hc, a part de tenir un paper important en immunologia, es troba en la seva sobreexpressió en cèl•lules tumorals, el que la converteix en una important diana per a tractaments i desenvolupament de vacunes contra el càncer. El model ens mostra com en aquest transportador, l´ectodomini de 4F2hc està situat sobre LAT2, suggerint interacció amb els bucles extracel•lulars del transportador i nos sols interacció a través del pont disulfur del segment transmembrana com es pensava anteriorment. Aquesta nova topologia explica la necessitat i la importància de que l'ectodomini de 4F2hc formi part de l´heterodímer 4F2hc/LAT2 i presenta un escenari estructural per al paper "chaperone-like" de 4F2hc sobre les subunitats lleugeres.

Els transportadors heteromèrics d'aminoàcids (HATs) estan formats per dues subunitats: una subunitat pesada (HSHAT) i una subunitat lleugera (LSHAT) unides per un pont disulfur. Mutacions en algun dels seus membres són responsables d'aminoacidúries hereditàries, com la lisinúria amb intolerància a proteïnes (LPI) o la cistinúria. Es coneixen dues HSHATs: 4F2hc i rBAT que són glicoproteïnes de membrana de tipus II amb un domini intracel·lular N-terminal, un domini transmembrana i un gran ectodomini C-terminal.Aquesta tesi recull els resultats obtinguts a partir de l'expressió en E. coli i la posterior purificació de l'ectodomini de 4F2hc (4F2hc-ED) humà. El principal resultat és la resolució de l'estructura tridimensional d'aquest domini de la proteïna mitjançant cristal·lització i difracció de raigs X. S'han resolt dos tipus de cristalls, un de monoclínic a 2,1 Å (amb una molècula en la unitat asimètrica) i un d'ortoròmbic a 2,8 Å (amb dues molècules en la unitat asimètrica coordinant un àtom de zinc). 4F2hc-ED té una estructura molt semblant a les α-glucosidases de bacteris i consta d'un domini A, que és un barril (β/α)8 o barril TIM i un domini C, format per 8 fulles beta antiparal·leles. El treball presenta l'estructura de 4F2hc-ED i la compara amb les estructures conegudes de glucosidases de bacteris. També es presenta un model estructural per a rBAT, l'altra subunitat pesada dels transportadores heteromèrics d'aminoàcids. Però la producció i purificació d'aquesta proteïna ens ha permès realitzar, també, estudis de funció i interacció amb altres molècules. L'homologia amb les α-glucosidases de bacteris ens han portat a provar activitats relacionades amb aquests enzims. En aquest sentit s'han usat substrats per determinar l'activitat α-glucosidasa i s'han fet estudis d'interacció amb glúcids, sense èxit. L'estudi de l'estructura revela una superfície de càrregues polaritzada, amb una superfície carregada positivament al voltant de l'N-terminal (al voltant del coll que uneix l'ectodomini amb el domini transmembrana) que s'hipotetitza que interaccionaria amb els caps polars de la membrana cel·lular i ens dóna una idea de com podria aquesta proteïna interaccionar amb altres proteïnes com les LSHATs.Aquesta tesi aporta informació per a l'estudi estructura-funció d'aquests transportadors, però també per a l'estudi de les diferents funcions i processos en els quals 4F2hc ha estat vinculat en la literatura, com activació d'integrines i tumorigènesis. Finalment es discuteix sobre l'existència fisiològica del homodimer de l'ectodomini descobert en el cristall ortoròmbic i el paper de la coordinació de zinc. / The heteromeric amino acid transporters (HATs) are composed by two subunits: a heavy subunit (HSHAT) and a light subunit (LSHAT) linked by a disulfur bridge. Mutations in some of their members are responsible for two aminoaciduries: lisinuric protein intolerance (LPI) and cystinuria. There are two described HSHATs, 4F2hc and rBAT and they are a type II membrane glycoprotein, with an intracellular domain N-terminal, a transmembrane domain and a bulky ectodomain C-terminal.This thesis gathers the results obtained from the expression in E. coli and subsequent purification of human 4F2hc ectodomain (4F2hc-ED). The main result is the resolution of the three-dimensional structure of this domain using crystallization and x-ray diffraction. We solved two structures from two different crystal forms: a monoclinical at 2.1 Å (with one molecule in the asymmetric unit) and an orthorhombic at 2.8 Å (with a homodimer in the asymmetric unit coordinating a zinc atom). The 4F2hc-ED structure is very similar to bacterial α-glucosidases and is composed by two domains: a A domain with is a (β/α)8 barrel or TIM barrel architecture, and a C domain formed for eight antiparallel beta sheets. This work presents the structure of the 4F2hc ectodomain and compares it with known structures of bacterial α-glucosidases. Furthermore, a 3D model for rBAT is shown and compared with 4F2hc-ED and α-glucosidase structures. We tested the α-glucosidase activity of the purified domain using α-amylase substrates. Unfortunately, the results were negative in all cases. This thesis contributes in a better understanding of the structure-function relationships of the HATs, but also for the study of the different physiological implications of 4F2hc described in the literature, like integrin activation and tumorigenesis. Finally, the physiological existence of the homodimer of the ectodomain and the role of the zinc coordination, found in the orthorhombic crystal, is discussed. We suggest a model for interaction of 4F2hc-ED with cell membrane.

En este trabajo se ha estudiado cuál es el punto común que regula el metabolismo del C y del N y como afectan ambos, sobre la regulación del influjo del ión NH4+ y la expresión del gen CitAMT1 en plantas de cítricos (Citrus sinensis L. Osbeck x Poncirus trifoliata blanco). Se ha aislado y caracterizado, por primera vez, los genes CitAMT1 y CitNRT2, los cuales están implicados mayoritariamente en el transporte, mediado por el HATS, de los iones NH4+ y NO3-, respectivamente. Se ha encontrado que el nexo de unión entre el metabolismo del N y el metabolismo del C, en plantas de cítricos, es la variación en la síntesis de sacarosa y su transporte a las raíces a través de la regulación de la fotosíntesis. La sacarosa regula de manera específica la expresión del gen CitAMT1 y el influjo del ión NH4+ en las raíces. Por otra parte, las plantas de cítricos prefieren absorben NH4+ frente a NO3- independientemente de la concentración y relación de N disponible. El influjo del ión NH4+ y la expresión del gen CitAMT1 se de-reprimen y de-inducen, por diferentes mecanismos que implican tanto regulación a nivel transcripcional como postranscripcional, en función del tiempo de carencia de N y se inducen y reprimen en función de la fuente de N disponible y de su concentración. Por último, parece ser que la regulación del influjo del ión NH4+, mediado por el LATS, no está sometida a los cambios en el metabolismo del C y del N.

Digital Craft is a project that explores the interface between three-dimensional (3-D) computer technology and costume technology. I combine seasoned millinery techniques with modern methods of object construction and design to diversify the costume artisan’s toolbox and encourage practical and useful ways of moving between the virtual and physical world. Through a series of theoretical projects the dichotomy of modern artistic process is explored. The task of this thesis project is to explore the impact of 3-D imaging software on design and construction methods by applying them to the sculptural process of hat making. I collaborate with designers and technicians to develop methods of hat making for performance culminating in an exhibition presented at the University Co-op Cohen New Works Festival, April 2011. This research is applied to cultivate new methods of hat making, by exploring new media and expanding creative possibilities. Craft objects are created directly through the hand of the maker; it is thorough technique that the hand informs the craft object (Risatti, 108). Furthermore, the traditional notion of tools and craft objects is that they are conditioned, controlled and limited by the hands. In order to update and improve methods of object construction, this project will expand the traditional concept of craft, combining hands-on methods with machining. I hope to improve efficiency and decrease the cost of realizing authentic and original hat designs by exploring alternative digital spaces that can be used to communicate, develop and actualize ideas. / text

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Thesis (Ph. D.)--University of Minnesota, 1982. / Illustrations not filmed. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 285-289).

Det her studie undersøger hvordan man finder frem til den bedst mulige løsning, når man udfordrer sine egne kompetencer i et kunstnerisk projekt. Gennem en redegørelse af begrebet Innovation opstilles en række modeller, der illustrere den innovative proces og kreative tænkning. Undersøgelsen belyser Schumpeter’s definition af innovation, Wegge’s senere definition af samme, Edward de Bono’s Six Thinking Hats og Dobbeltdiamantmodellen. I resultatet gennemgås processen af gennemførelsen af et innovativt interaktivt videomapping projekt. Helt lavpraktisk vises der, hvordan de forskellige metoder tages i brug, og hvordan de er med til at skubbe arbejdsprocessen videre. Konkrete eksempler på de tekniske overvejelser og prototyper udstilles, så det er tydligt at se, hvordan de er blevet eksekveret. Til sidst diskuteres der hvorvidt metoderne er en hjælp eller hinder til at gennemføre et innovativt projekt og om de samme metoder og modeller ville fungere, hvis implementeret i et projekt, som skulle udføres af en større gruppe.

"The primary purpose of this study was to identify health education strategies which are effective in increasing the use of protective devices which can prevent injury. The specific protective device studied was the helmet worn by off-road vehicle [including All-Terrain Vehicles [ATVs] and snowmachines] riders in rural Alaska"--Leaf 3. / Thesis (Ed. D.)--Boston University, 1996.

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