Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

Invisible but not forgotten: The Afro-Argentine and Afro-Uruguayan experience from the sixteenth to the twentieth centuries

The problem to be examined in this thesis involves the supposedly overlooked history and contributions of Africans and their descendants in the River Plate countries of Argentina and Uruguay. Therefore, the primary purpose of the study is to narrate the social history of Afro-Argentines and Afro-Uruguayans from the sixteenth to the twentieth century. A secondary purpose, moreover, is to synthesize the academic literature on Blacks in the Rio de la Plata and their many cultural and other contributions to the current nation-states of Argentina and Uruguay. This thesis thereby challenges the regnant historiographical argument that African Argentines and African Uruguayans have been “forgotten” as historical actors by scholars both inside and outside the Rio de la Plata. By synthesizing the large body of historical and social science scholarship on Africans in the River Plate, as well as providing a thorough bibliography on the subject, this study attempts to proffer (to borrow the subtitle of Marvin Lewis' 1996 study of Afro-Argentine literature) “another dimension of the Black Diaspora” to the Americas. ^

The European Parliament, membership expansions, and organizational change

The European Union (EU) is an extraordinary achievement. From a regional economic organization, it grew into a polity within fifty years. The original EU of six members expanded incrementally to 27 over forty years, and it now comprises a population of almost 500 million people. While the five expansions of the European Economic Community/European Community/European Union (EU) have received considerable scholarly attention, surprisingly little attention has been given to their impacts on "Europe's" only legislative body, currently known as the European Parliament (EP). More specifically, little is known about how waves of new members (from widely diverse parties and national backgrounds) affected—and were affected by—the EP's organizational structure and its internal processes. The purpose of this study therefore is to help fill this gap by describing and explaining how the various EEC/EC/EU expansions or "membership shocks" (1973, 1981, 1986, 1995, and 2004) affected the EP's organizational structure and its internal Rules of Procedure (RoP). The central research question of this dissertation is the following: What were the major structural and procedural effects of the five membership expansions of what eventually became the European Union on the European Parliament? This dissertation answers this question by using concepts and measures drawn from organizational theory. While other studies have applied concepts and hypotheses from organizational theory to legislatures, such an approach has never been used to analyze the EP, which is conceptualized here as a "membership organization." This study, through an analysis of the EP, demonstrates that organization theory can help us fully understand the effects of membership expansions on any membership organization. That is, understanding how this particular organization responded to change can inform not only how others in this class (legislatures) do so, but how this process unfolds in a variety of times and places. The principal findings of this study are as follows: (1) EP staff growth revealed an interesting pattern: Staff did not increase concurrently with EP membership. That is, it turned out that the rate of membership growth exceeded the rate of staff increase, suggesting professionalization of EP staff and their relative empowerment vis-à-vis MEPs; (2) The number of rules and the precision within them increased; (3) the largest number of EP rule changes focused on increasing EP efficiency; and (4) The authority was centralized in the hands of EP leadership, that is, the EP President, the Conference of Presidents and also two major political groups.

Trends for Meetings and Expositions Industry

During the last decade, the meetings and expositions industry has flourished, even as it has struggled to cope with difficult challenges. This is a taste of things to me. In the years ahead, the global population will continue to grow and change, science and technology will tighten their hold on business and society and the world will knit itself ever more tightly into a single market. As a result, both opportunities and trials will abound.

Club Medic

For most of us, getting sick is a good way to ruin a vacation. However, for growing numbers of people, needing to see the doctors the whole point of going abroad. When they require surgery or dental work, thy may combine treatment with a trip to the Taj Mahal, A photo safari on the African veldt, or a stay at a luxury hotel-or at a hospital that feels like one – all at bargain-basement prices. This is medical tourism, and it is one of the hottest niche markets in the hospitality industry.

Conceptualizing American power and security in a post -9/11 security context: Conflict, resistance, and global security, 2001–present

In a post-Cold War, post-9/11 world, the advent of US global supremacy resulted in the installation, perpetuation, and dissemination of an Absolutist Security Agenda (hereinafter, ASA). The US ASA explicitly and aggressively articulates and equates US national security interests with the security of all states in the international system, and replaced the bipolar, Cold War framework that defined international affairs from 1945-1992. Since the collapse of the USSR and the 11 September 2001 terrorist attacks, the US has unilaterally defined, implemented, and managed systemic security policy. The US ASA is indicative of a systemic category of knowledge (security) anchored in variegated conceptual and material components, such as morality, philosophy, and political rubrics. The US ASA is based on a logic that involves the following security components: (1) hyper militarization, (2) intimidation,(3) coercion, (4) criminalization, (5) panoptic surveillance, (6) plenary security measures, and (7) unabashed US interference in the domestic affairs of select states. Such interference has produced destabilizing tensions and conflicts that have, in turn, produced resistance, revolutions, proliferation, cults of personality, and militarization. This is the case because the US ASA rests on the notion that the international system of states is an extension, instrument of US power, rather than a system and/or society of states comprised of functionally sovereign entities. To analyze the US ASA, this study utilizes: (1) official government statements, legal doctrines, treaties, and policies pertaining to US foreign policy; (2) militarization rationales, budgets, and expenditures; and (3) case studies of rogue states. The data used in this study are drawn from information that is publicly available (academic journals, think-tank publications, government publications, and information provided by international organizations). The data supports the contention that global security is effectuated via a discrete set of hegemonic/imperialistic US values and interests, finding empirical expression in legal acts (USA Patriot ACT 2001) and the concept of rogue states. Rogue states, therefore, provide test cases to clarify the breadth, depth, and consequentialness of the US ASA in world affairs vis-à-vis the relationship between US security and global security.

Conceptualizing American power and security in a post-9/11 security context : conflict, resistance, and global security, 2001-present

In a post-Cold War, post-9/11 world, the advent of US global supremacy resulted in the installation, perpetuation, and dissemination of an Absolutist Security Agenda (hereinafter, ASA). The US ASA explicitly and aggressively articulates and equates US national security interests with the security of all states in the international system, and replaced the bipolar, Cold War framework that defined international affairs from 1945-1992. Since the collapse of the USSR and the 11 September 2001 terrorist attacks, the US has unilaterally defined, implemented, and managed systemic security policy. The US ASA is indicative of a systemic category of knowledge (security) anchored in variegated conceptual and material components, such as morality, philosophy, and political rubrics. The US ASA is based on a logic that involves the following security components: 1., hyper militarization, 2., intimidation, 3., coercion, 4., criminalization, 5., panoptic surveillance, 6., plenary security measures, and 7., unabashed US interference in the domestic affairs of select states. Such interference has produced destabilizing tensions and conflicts that have, in turn, produced resistance, revolutions, proliferation, cults of personality, and militarization. This is the case because the US ASA rests on the notion that the international system of states is an extension, instrument of US power, rather than a system and/or society of states comprised of functionally sovereign entities. To analyze the US ASA, this study utilizes: 1., official government statements, legal doctrines, treaties, and policies pertaining to US foreign policy; 2., militarization rationales, budgets, and expenditures; and 3., case studies of rogue states. The data used in this study are drawn from information that is publicly available (academic journals, think-tank publications, government publications, and information provided by international organizations). The data supports the contention that global security is effectuated via a discrete set of hegemonic/imperialistic US values and interests, finding empirical expression in legal acts (USA Patriot ACT 2001) and the concept of rogue states. Rogue states, therefore, provide test cases to clarify the breadth, depth, and consequentialness of the US ASA in world affairs vis-a-vis the relationship between US security and global security.

Os youtubers e a representação do certo, errado, adequado e inadequado no trabalho com a variação linguística em sala de aula

Some of the current discussions in the teaching of Portuguese Language (LP) pertain to how the school should deal with the phenomenon of language variation in the classroom. In 2010, for example, an explosion of talk took over the academic corridors: a book, entitled "Por uma vida melhor", the collection "Viver, Aprender", published by the MEC (Ministry of Education and Culture) to students EJA (Youth and Adults) brought notions regarding linguistic variation, even in their first chapter. In it is clear the notion that it is possible to make use of structures as "pretty boy", instead of "pretty boys", depending on the context in which such use is insert. Therefore, the discussions focused around the notions of variety cultivated, standard and popular measuring them to the possibilities of linguistic appropriateness. The community was surprised by the defense of the "power" to use, since it would be the school space to teach a standard "default", and not the possibility of legitimate use of grammatical patterns that clashed with those recommended in traditional grammars. The television media has been responsible for a major blaze that MEC had endorsed the use in schools of a book that legitimized such linguistic patterns. The quarrel was released on Youtube and in that space, netizens expressed themselves for or against the proposal of LD often directing the discussion to questions of a purely political. We observed that, on one side, loomed arguments related to Sociolinguistics (BAGNO , 2002, 2003, 2007, 2009; BAGNO, M.; STUBBS, M., Gagne, G., 2006; Bortoni - RICARDO, S.M., 2008; Tarallo, F., 1982; U. Weinreich, MARVIN I. HERZOG, Labov, W., 1968, Labov 1972, etc.); another, arguments concentrated on defending the school is the area of language teaching standard, and not fit to bring certain discussions within an LD. It was from these words, that this research was born. Interested in the particular way that the community media, which seemed to have no training in linguistics, understand the concepts of right, wrong, appropriate and inappropriate, so intimate in academic circles. Our thoughts take as reference the theoretical studies on the question of sociolinguistic variation and education, official documents that guide the "work" with the Portuguese language in the classroom, like the NCP (National Curriculum) and Curriculum Proposal for Education Youth and Adult (PCEJA). In our analysis, we found that LD" For a better life "makes no apology for teaching the "error", but it raises discussions about the possibility of "change", linked to factors and different order. We realize how significant it is to observe how speakers of a language are positioned in relation to language teaching which they are not speakers and scholars. Our study showed that certain issues regarding the teaching of the Portuguese language, as is the case of linguistic variation, points are far from being resolved, either for linguists and/or grammarians, whether for language speakers.

Downhole logging of the AND-2A borehole, Victoria Land Basin, McMurdo Sound, Antarctica

Under the framework of the ANDRILL Southern McMurdo Sound (SMS) Project successful downhole experiments were conducted in the 1138.54 metre (m)-deep AND-2A borehole. Wireline logs successfully recorded were: magnetic susceptibility, spectral gamma ray, sonic velocity, borehole televiewer, neutron porosity, density, calliper, geochemistry, temperature and dipmeter. A resistivity tool and its backup both failed to operate, thus resistivity data were not collected. Due to hole conditions, logs were collected in several passes from the total depth at ~1138 metres below sea floor (mbsf) to ~230 mbsf, except for some intervals that were either inaccessible due to bridging or were shielded by the drill string. Furthermore, a Vertical Seismic Profile (VSP) was created from ~1000 mbsf up to the sea floor. The first hydraulic fracturing stress measurements in Antarctica were conducted in the interval 1000-1138 mbsf. This extensive data set will allow the SMS Science Team to reach some of the ambitious objectives of the SMS Project. Valuable contributions can be expected for the following topics: cyclicity and climate change, heat flux and fluid flow, seismic stratigraphy in the Victoria Land Basin, and structure and state of the modern crustal stress field.

Sea surface temperature and primary productivity reconstruction of sediment core SO130-275KL from the northeastern Arabian Sea during the late Holocene

Variability in the oceanic environment of the Arabian Sea region is strongly influenced by the seasonal monsoon cycle of alternating wind directions. Prominent and well studied is the summer monsoon, but much less is known about late Holocene changes in winter monsoon strength with winds from the northeast that drive convective mixing and high surface ocean productivity in the northeastern Arabian Sea. To establish a high-resolution record of winter monsoon variability for the late Holocene, we analyzed alkenone-derived sea surface temperature (SST) variations and proxies of primary productivity (organic carbon and d15N) in a well-laminated sediment core from the Pakistan continental margin. Weak winter monsoon intensities off Pakistan are indicated from 400 B.C. to 250 A.D. by reduced productivity and relatively high SST. At about 250 A.D., the intensity of the winter monsoon increased off Pakistan as indicated by a trend to lower SST. We infer that monsoon conditions were relatively unstable from ~500 to 1300 A.D., because primary production and SST were highly variable. Declining SST and elevated biological production from 1400 to 1900 A.D. suggest invigorated convective winter mixing by strengthening winter monsoon circulation, most likely a regional expression of colder climate conditions during the Little Ice Age on the Northern Hemisphere. The comparison of winter monsoon intensity with records of summer monsoon intensity suggests that an inverse relationship between summer and winter monsoon strength exists in the Asian monsoon system during the late Holocene, effected by shifts in the Intertropical Convergence Zone.

Conventional and 40Ar/39Ar K-Ar ages of volcanic rocks at DSDP Leg 55 Holes

Conventional K-Ar, 40Ar/39Ar total fusion, and 40Ar/39Ar incremental heating data on hawaiite and tholeiitic basalt samples from Ojin (Site 430), alkalic basalt samples from Nintoku (Site 432), and alkalic and tholeiitic basalt samples from Suiko (Site 433) seamounts in the Emperor Seamount chain give the following best ages for these volcanoes: Ojin = 55.2 ± 0.7 m.y., Nintoku = 56.2 ± 0.6 m.y., and Suiko = 64.7 ± 1.1 m.y. These new data bring to 27 the number of dated volcanoes in the Hawaiian-Emperor volcanic chain. The new dates prove that the age progression from Kilauea Volcano on Hawaii (0 m.y.) through the Hawaiian-Emperor bend (- 43 m.y.) to Koko Seamount (48.1 m.y.) in the southernmost Emperor Seamounts continues more than halfway up the Emperor chain to Suiko Seamount. The age versus distance data for the Hawaiian-Emperor chain are consistent with the kinematic hot-spot hypothesis, which predicts that the volcanoes are progressively older west and north away from the active volcanoes of Kilauea and Mauna Loa. The data are consistent with an average volcanic propagation velocity of either 8 cm/year from Suiko to Kilauea or of 6 cm/year from Suiko to Midway followed by a velocity of 9 cm/year from Midway to Kilauea, but it appears that the change in direction that formed the Hawaiian- Emperor bend probably was not accompanied by a major change in velocity.

(Table 1, page 1169) Chemical composition of land (fossil) manganese deposits rich in todorokite

Chemical, x-ray and other data are given for todorokite, (Mn, Mg, Ca, Ba, Na, K)2.Mn5O12.3H2O, from Charco Redondo, Cuba, Farragudo, Portugal, and Hüttenberg, Austria. Additional localities at Romanèche, France, Saipan Island, Bahia, Brazil and Sterling Hill, New Jersey, are noted. Delatorreite of Simon and Straczek (1958) is identical with todorokite.

Nanotechnology in the food industry I: applications

Nanotechnology is a multidisciplinary science that is having a boom today, providing new products with attractive physicochemical properties for many applications. In agri/feed/food sector, nanotechnology offers great opportunities for obtaining products and innovative applications for agriculture and livestock, water treatment and the production, processing, storage and packaging of food. To this end, a wide variety of nanomaterials, ranging from metals and inorganic metal oxides to organic nanomaterials carrying bioactive ingredients are applied. This review shows an overview of current and future applications of nanotechnology in the food industry. Food additives and materials in contact with food are now the main applications, while it is expected that in the future are in the field of nano-encapsulated and nanocomposites in applications as novel foods, additives, biocides, pesticides and materials food contact.

Nanotechnology in food industry II: risk assessment and legislation

Currently, there is increasing use of nanomaterials in the food industry thanks to the many advantages offered and make the products that contain them more competitive in the market. Their physicochemical properties often differ from those of bulk materials, which require specialized risk assessment. This should cover the risks to the health of workers and consumers as well as possible environmental risks. The risk assessment methods must go updating due to more widespread use of nanomaterials, especially now that are making their way down to consumer products. Today there is no specific legislation for nanomaterials, but there are several european dispositions and regulations that include them. This review gives an overview of the risk assessment and the existing current legislation regarding the use of nanotechnology in the food industry.