Development of an affinity pair “Tag-Receptor” for recombinant protein expression and purification

Dissertação apresentada para a obtenção do Grau de Mestre em Biotecnologia, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Novel affinity pairs "tag-receptor" for the purification of fusion proteins

Fundação para a Ciência e Tecnologia - SFRH/BD/48804/2008 and the project PTDC/BI/65383/2006 assigned to Prof. Cecíla Roque and also to Associate Laboratory REQUIMTE (Pest-C/EQB/LA0006/2011)

Update of the Gene Discovery Program in Schistosoma mansoni with the Expressed Sequence Tag Approach

Continuing the Schistosoma mansoni Genome Project 363 new templates were sequenced generating 205 more ESTs corresponding to 91 genes. Seventy four of these genes (81%) had not previously been described in S. mansoni. Among the newly discovered genes there are several of significant biological interest such as synaptophysin, NIFs-like and rho-GDP dissociation inhibitor

Purification of recombinant proteins by chemical removal of the affinity tag.

The efficient removal of a N- or C-terminal purification tag from a fusion protein is necessary to obtain a protein in a pure and active form, ready for use in human or animal medicine. Current techniques based on enzymatic cleavage are expensive and result in the presence of additional amino acids at either end of the proteins, as well as contaminating proteases in the preparation. Here we evaluate an alternative method to the one-step affinity/protease purification process for large-scale purification. It is based upon the cyanogen bromide (CNBr) cleavage at a single methionine placed in between a histidine tag and a Plasmodium falciparum antigen. The C-terminal segment of the circumsporozoite polypeptide was expressed as a fusion protein with a histidine tag in Escherichia coli purified by Ni-NAT agarose column chromatography and subsequently cleaved by CNBr to obtain a polypeptide without any extraneous amino acids derived from the cleavage site or from the affinity purification tag. Thus, a recombinant protein is produced without the need for further purification, demonstrating that CNBr cleavage is a precise, efficient, and low-cost alternative to enzymatic digestion, and can be applied to large-scale preparations of recombinant proteins.

Monitoring of NY-ESO-1 specific CD4+ T cells using molecularly defined MHC class II/His-tag-peptide tetramers.

MHC-peptide tetramers have become essential tools for T-cell analysis, but few MHC class II tetramers incorporating peptides from human tumor and self-antigens have been developed. Among limiting factors are the high polymorphism of class II molecules and the low binding capacity of the peptides. Here, we report the generation of molecularly defined tetramers using His-tagged peptides and isolation of folded MHC/peptide monomers by affinity purification. Using this strategy we generated tetramers of DR52b (DRB3*0202), an allele expressed by approximately half of Caucasians, incorporating an epitope from the tumor antigen NY-ESO-1. Molecularly defined tetramers avidly and stably bound to specific CD4(+) T cells with negligible background on nonspecific cells. Using molecularly defined DR52b/NY-ESO-1 tetramers, we could demonstrate that in DR52b(+) cancer patients immunized with a recombinant NY-ESO-1 vaccine, vaccine-induced tetramer-positive cells represent ex vivo in average 1:5,000 circulating CD4(+) T cells, include central and transitional memory polyfunctional populations, and do not include CD4(+)CD25(+)CD127(-) regulatory T cells. This approach may significantly accelerate the development of reliable MHC class II tetramers to monitor immune responses to tumor and self-antigens.

Protectionist but globalised? Latin American custom duties and trade during the pre-1914 belle époque

While it is true that Latin American republics had no rival on maximising revenues from custom collection during the belle époque, this paper shows that Latin American countries were also generous importers, only behind the larger commercial countries of Western Europe in terms of imports per capita. Latin American citizens were much more linked to international trade than citizens of most regions of the world. Their relation to the world economy was tighter both via their imports and their exports relative to their population and income levels. This paper comes to show that there is no contradiction between the high custom collection by the Latin American republics and their high level of interaction with the global economy in the pre-1914 belle époque, although large country differences can be observed when descending from the regional to the national level.

Three-dimensional computed cementless custom femoral stems in young patients: midterm followup.

We prospectively evaluated the results of our custom cementless femoral stems to ascertain whether this technology produced reasonable clinical function, complication rates, and loosening rates at midterm. Fifty-seven consecutive patients had surgery in 62 hips for primary osteoarthritis at a mean age of 57 years using a three-dimensional computed custom cementless stem. Patients were reviewed at a mean followup of 94.9 months. At review, the mean Harris hip score was 98.8 points (range, 84-100) compared with 61.1 (range, 28-78) points preoperatively. No patient complained of thigh pain. No migration or subsidence was observed. All stems were considered stable according to the radiographic criteria defined by Engh et al. There were no dislocations, no infections, and no reoperations. Our results are comparable with published results from clinical and radiologic points of view. Two problems remain unsolved: the price of a custom stem is twice as expensive as a standard stem; and we need longer term results before definitely recommending this technology as a reasonable alternative to current arthroplasties in younger patients. The data support the continued exploration of this technology with controlled clinical followup. LEVEL OF EVIDENCE: Therapeutic study, Level II-1 (prospective cohort study). See the Guidelines to Authors for a complete description of levels of evidence.

Erschienen ist der herrliche Tag

Soitinnus: Urut.

SaaS and Public Cloud from Custom Application Provider’s Perspective

The main objective of this master’s thesis is to provide a comprehensive view to cloud computing and SaaS, and analyze how well CADM, a unit of Capgemini Finland Ltd., would fit to the cloud-based SaaS business. Another objective for this thesis is to investigate how public clouds would fit for CADM as a delivery model, if they would provide SaaS applications to their customers. This master’s thesis is executed by investigating characteristics of cloud computing and SaaS especially from application provider point of view. This is done by exploring what kinds of researches and analysis there have been done regarding these two phenomena during past few years. Then CADM’s current business model and operations are analyzed from SaaS’s and public cloud’s perspective. This analyzing part is conducted by using SWOT analysis which is widely used analytical tool when observing company’s strategic position and when figuring out possibilities how to improve company’s operations. The conducted analysis and observations reveals that CADM should pursue SaaS business as it could provide remarkable advantages and strengthen their position in current markets. However, pure SaaS model would not be the optimal solution for CADM because they do not have own product which could be transformed to SaaS model, and they lack of Infrastructure Management ability. Also public cloud would not be the most suitable delivery model for them if providing SaaS services. The main observation of this thesis is that CADM should adopt the SaaS model via Capgemini Immediate offering.

Candace Bushnellin Trading Up Edith Whartonin romaanin The Custom of the Country uudelleenkirjoituksena

Candace Bushnellin (1958–) vuonna 2003 julkaistu romaani Trading Up kuvaa nuoren naispäähenkilön määrätietoista pyrkimystä rikkauteen, maineeseen ja New Yorkin suihkuseurapiireihin. Se on uudelleenkirjoitus Edith Whartonin romaanista The Custom of the Country (1913). Wharton (1862–1937) käsitteli tuotannossaan New Yorkin vanhaa yläluokkaa ja kuvasi usein liikettä yhteiskuntaluokassa ylös tai alas. 1990-luvulta alkaen Bushnell on käsitellyt teoksissaan samankaltaisia aiheita, joskin oman aikansa kontekstissa. Hän on itse todennut Whartonin teosten olevan merkittäviä esikuvia omalle tuotannolleen. Pro gradu -tutkielmassani tarkastelen Bushnellin romaania Trading Up Whartonin The Custom of the Country -teoksen uudelleenkirjoituksena. Analysoimalla teoksia rinnakkain selvitän, millaisia keinoja Bushnell käyttää uudelleenkirjoituksessaan ja mikä merkitys Whartonin romaanin uudelleenkirjoituksella on. Tutkimusnäkökulmani on feministinen, ja keskityn valtasuhteisiin liittyviin kysymyksiin. Tarkastelen etenkin yhteiskuntaluokkaan, sukupuoleen ja seksuaalisuuteen liittyviä valtasuhteita ja sitä, miten valtasuhteet Bushnellin uudelleenkirjoituksessa eroavat Whartonin romaanissa esiintyvistä. Tarkastelen tutkimiani romaaneja myös genren näkökulmasta, Trading Up -romaania osana chick lit -genreä ja The Custom of the Country -teosta taparomaanina. Chick lit on 1990- luvulla syntynyt viihteellinen naisten kirjallisuuden genre, joka kuvaa nuoria naisia ja käsittelee esimerkiksi ihmissuhteita ja perheeseen, työelämään tai ulkonäköön liittyviä kysymyksiä. Kulutuskulttuuri ja uusliberalistinen yksilöllisyys kuuluvat chick lit -genreen. Se on kytketty myös postfeminismiin: chick litissä feminismistä joko vaietaan, tai se esitetään pikemminkin yksittäisten naisten elämäntapavalintoina kuin poliittisena liikkeenä. Bushnellin lisäksi myös muut chick lit -kirjailijat ovat teoksissaan viitanneet taparomaaneihin. Tutkielmassani totean Bushnellin uudelleenkirjoittavan The Custom of the Country -romaania paikoitellen hyvin suoraan. Vaikka teosten ajallisen etäisyyden ja yhteiskunnallisten muutosten takia esimerkiksi yhteiskuntaluokkaan liittyvissä valtasuhteissa on tapahtunut muutoksia, esitetään monet asiat muuttumattomina. Etenkin sukupuolen kuvaus ja sukupuoltenväliset valtasuhteet näyttäytyvät samankaltaisina kummassakin teoksessa. Postfeminismissä ja uusliberalismissa yksilön mahdollisuutta tehdä valintoja korostetaan. Bushnellin romaanin naishenkilöhahmoilla pitäisi olla enemmän valinnan mahdollisuuksia kuin Whartonin sankarittarilla, mutta näiden tekemät ratkaisut ovat lopulta hyvin samankaltaisia. Vaikka Trading Up toistaa Whartonin teoksen tarinaa melko tarkasti, ei siitä välity Whartonin romaanissa merkittävää yhteiskuntakriittisyyttä.

Tag vara på biprodukterna vid slakten!

14 x 21 cm

Tag mig med!

10 x 16 cm

1939 uudet Chrysler mallit Royal Imperial Custom Imperial

7 x 16 cm