Non-claret disjunctional (Ncd) is a kinesin-14 microtubule motor protein involved in the assembly and stability of meiotic and mitotic spindles in Drosophila oocytes and early embryos, respectively. Ncd functions by cross-linking microtubules through the tail and motor domains. It was originally believed that the role of the Ncd tail domain was to only statically bind microtubules. However, the Ncd tail domain has recently been shown to have properties that stabilize and bundle microtubules, and contribute to the overall motility of the Ncd protein. Continued characterization of the Ncd tail domain is essential to understanding the complete role of Ncd in cell division. This work explored the regulatory function and microtubule binding properties of the Ncd tail domain. Ncd activity is regulated during interphase by nuclear sequestration. GFP-Ncd fusion proteins, containing full length Ncd, individual Ncd domains, or combinations of Ncd domains, were used to identify the presence of a nuclear localization signal (NLS) in the Ncd polypeptide. The nuclear localization of only the GFP fusion proteins containing the Ncd tail sequence indicates that the NLS is contained within the tail domain. Subsequent, experiments performed with GFP fusion proteins containing segments of the tail domain indicate that essential NLS amino acid segments may span the length of the tail domain. Attempts to characterize the microtubule binding properties of the Ncd tail domain, using bacterially expressed MBP-Ncd tail-stalk, were unsuccessful. MBP-Ncd tail-stalk proteins aggregated under binding assay conditions, preventing an accurate determination of the stoichiometric binding relationship between Ncd and the tubulin dimer. / Master of Science

El gironí Josep Claret Rubira (1908-1988), arquitecte de professió i artista-dibuixant de vocació, va obtenir el títol l'any 1933, a l'Escola d'Arquitectura de Barcelona. D'ençà d'aquella data i fins a mitjan dels anys 70, va projectar una quantitat enorme d'edificis de múltiples tipologies i nombrosos plans d'urbanització, arreu de les comarques gironines i menorquines. Amb ideologia primerenca propera a les esquerres catalanistes, es va haver d'adaptar al règim franquista, per poder continuar treballant i vivint a Catalunya. Malgrat que inicialment li deuria costar un gran esforç, el procés d'amotllament el va tenir planer (almenys aparentment), gràcies al seu parentiu -per via matrimonial- amb una família que hi estava estretament vinculada. És autor d'obres d'elevat nivell arquitectònic -amb poques que siguin conegudes i reconegudes- i un clar conformador del paisatge de les seves contrades, fet influent en la societat que hi ha estat habitant i que hi viu encara avui dia. La seva arquitectura és un magnífic exemple del que agradava a les generacions de durant cinc dècades. És també autor d'obres que, moltes vegades, no s'adiuen gens amb el que la Història de l'Arquitectura dóna per bones, però que reflecteixen el gust, les preferències i les prioritats d'una societat determinada. / Josep Claret Rubira (Girona, 1908-1988), architect of profession and artist-sketcher of vocation, obtained his university degree from the School of Architecture of Barcelona in 1933. From this year to the middle seventies, he projected an enormous amount of buildings of multiple typologies and numerous plans of urbanization throughout Girona and Menorca. His early political ideology was close to left wing, so he had to adapt to the pro-Franco regime to be able to continue working and living in Catalonia. Although it had probably been hard at the beginning, the connection process was easy (at least apparently), thanks to his relationship by marriage with a family who was closely linked to it. He is the author of works of high architectural level (being a few of them known and recognised) and a clear organiser of the landscape of his territory, an influential fact in the society that has been living there since then. His architecture is a magnificent example of what people from five decades liked. He is also the author of works that do not often fit in with what is considered correct, according to the History of Architecture, but reflect the taste, the preferences and the priorities of a certain society.

Ncd is a member of the kinesin family of motor proteins. Ncd is involved in the processes of meiosis and early mitosis in <i>D. melanogaster</i>. PCR-mediated site-directed mutagenesis was utilized to introduce specific mutations into pET/MC6, a construct containing the motor domain of ncd. Six mutations were generated, two at glutamic acid residue 656, two at proline residue 649, one at arginine residue 623, and one double mutant at arginine residue 623 and threonine residue 632. Mutants proteins were expressed in bacteria and further characterized. Mutagenesis of the proline or glutamic acid residues resulted in insoluble proteins. The one exception is the mutagenesis of glutamic acid residue 656 into a glutamine, which resulted in a partially soluble protein. Mutagenesis of the arginine residue into an alanine (MC6-A623) resulted in a soluble protein while the double mutation of the arginine and threonine was insoluble. MC6-A623 exhibited a similar S-sepharose ion exchange chromatography binding and elution profile as MC6. Peptide antibodies made to conserved ncd motor domain sequences also recognized MC6- A623. The affinity of MC6-A623 (under the conditions tested) for microtubules was less than MC6. Most interestingly, under the conditions tested, MC6-A623 did not exhibit an increased ATPase rate in the presence of microtubules, a hallmark of the kinesin family of microtubule motor proteins. Analysis of the published ncd crystal structure, other motor protein sequences, and the experimental results of the mutagenesis of arginine residue 623, suggest that this residue is involved in the binding of MC6 to microtubules. / Master of Science