The predominant nuclear fuel used globally at present is uranium which is a finite resource. Thorium has been identified as an alternative nuclear fuel source that can be utilized in almost all existing uranium–based reactors and can significantly help in conserving limited uranium reserves. Furthermore, the elimination of proliferation risks associated with thorium–based fuel cycles is a key reason for re–evaluating the possible utilization of thorium in high temperature reactors. In addition to the many advantages that thorium–based fuel has over uranium–based fuel, there are vast thorium resources in the earth's crust that up until the present have not been exploited optimally. This study focuses on determining the amount of uranium ore that can be saved using thorium as a nuclear fuel in HTR's. Four identical 200 MWth high temperature reactors are considered which make use of different fuel cycles. These fuel cycles range from the conventional uranium fuel cycle to a thorium–based fuel cycle in which no U–238 is present and have been simulated using the VSOP–A system of computer codes. This study also considers the effect that protactinium, an isotope that occurs in thorium–based fuel cycles, will have on the decay heat production in the case of a depressurized loss of coolant (DLOFC) accident. / Thesis (M.Ing. (Nuclear Engineering))--North-West University, Potchefstroom Campus, 2011.

No description available.

Recently HER3, member of the epidermal growth factor receptor family (EGFR), has been found to play a crucial role in the development of resistance towards inhibitors that are given to patients with HER1- and HER2-driven cancers. As HER3 is up-regulated or over-activated in several types of human cancers, it is of outmost importance that new innovative drugs target its oncologic activity. The Affibody® Molecule Z08698 inhibits the heregulin induced signalling of HER3 with high affinity (KD~50 pM). As the Affibody® Molecule is small, has high solubility and outstanding folding kinetics, an effective penetration of tumour tissue is suggested together with a rationalized manufacturing process. Further coupling to an albumin binding domain (ABD) expands the plasma half-life of the molecule, hence increasing the molecule's potential of serving as a therapeutic. A process development for production of Z08698-VDGS-ABD094 has been established, where the molecule is efficiently produced in the E. coli host strain BL21(DE3), through a T7 based expression system. Cultivations were performed with a fed-batch fermentation process and the conditions were further optimized in order to obtain highest expression, while avoiding undesirable modifications like gluconoylations. By employing Design of experiments in combination with multivariate data analysis, a production process resulting in ~3.5 g product/ l culture could be verified. Moreover, thermolysis was evaluated as a suitable method for cell disruption, enabling an easy and cost-effective manufacturing process of the ABD fused Affibody® Molecule.

Submitted by Cristiane Shirayama (cristiane.shirayama@fgv.br) on 2011-05-31T15:00:03Z No. of bitstreams: 1 63090100006.pdf: 1094712 bytes, checksum: 977dab8c82f44b506e1fc9eb0c160c62 (MD5) / Approved for entry into archive by Suzinei Teles Garcia Garcia(suzinei.garcia@fgv.br) on 2011-05-31T15:47:01Z (GMT) No. of bitstreams: 1 63090100006.pdf: 1094712 bytes, checksum: 977dab8c82f44b506e1fc9eb0c160c62 (MD5) / Approved for entry into archive by Suzinei Teles Garcia Garcia(suzinei.garcia@fgv.br) on 2011-05-31T15:48:07Z (GMT) No. of bitstreams: 1 63090100006.pdf: 1094712 bytes, checksum: 977dab8c82f44b506e1fc9eb0c160c62 (MD5) / Made available in DSpace on 2011-06-01T16:42:33Z (GMT). No. of bitstreams: 1 63090100006.pdf: 1094712 bytes, checksum: 977dab8c82f44b506e1fc9eb0c160c62 (MD5) Previous issue date: 2011-04-08 / Neste trabalho abordamos alguns "puzzles" da Paridade do Poder de Compra (PPC) ainda não resolvidos; durante esse processo propomos um novo modelo não-linear e estudamos o papel da agregação temporal e de bases de dados abrangendo apenas um pequeno período histórico. A hipótese de que não existe uma força de convergência agindo sobre o câmbio real ajustado (ARER) foi fortemente rejeitada estatisticamente, e a não-linearidade se mostrou um questão importante. As meia-vidas encontradas para o Brasil usando os modelos padrão parecem ser uma das menores já encontradas para um país, e chegamos à conclusão de que a velocidade de convergência em direção a PPC ainda não pode ser considerada um consenso. Pretendemos, em adição, dar contribuições através do levantamento e esclarecimento de alguns resultados e problemas potenciais concernentes ao estudo da PPC. / In this work we address some unresolved purchasing-power parity (PPP) puzzles; during the process we propose a new nonlinear model and check the role of temporal aggregation and of datasets covering only a small period of time. The hypothesis that there is no convergence force acting on ARER has been strongly statistically rejected and the nonlinearity showed itself as an important issue. The half-lives found for Brazil using standard models seem to be one of the smallest ever found for a country. However, we concluded that the speed of converge towards PPP is not a consensus yet. Besides, we expect to give contributions to PPP literature by pointing out important results and potential pitfalls on PPP research.

Resumo: O nitrogênio é nutriente absorvido em maiores quantidades na cultura do milho e o que proporciona maiores produtividades de grãos, sendo seu uso no sistema solo-planta alterado pelo sistema de cultivo utilizado. Com o objetivo de avaliar o efeito de sistemas de cultivo e doses de nitrogênio na produção de matéria seca, nitrogênio na planta, nitrogênio foliar, produtividade de grãos e eficiência do nitrogênio no milho, foi implantado experimento de campo nos anos de 2008 e 2009, em um Argissolo Vermelho-Amarelo, distrófico, sob irrigação. Utilizou-se o delineamento experimental em parcelas subdivididas, com oito repetições. As parcelas foram constituídas pela semeadura direta (SD) e plantio convencional (PC). Nas subparcelas, foram aplicadas seis doses de N (0; 40; 80; 120; 160 e 200 kg ha-1) na forma de uréia. Em 2009 a fim de avaliar a velocidade de decomposição e liberação do nitrogênio do feijão utilizado como cobertura morta, esses resíduos foram acondicionados em sacolas de náilon, as quais foram dispostas sobre o solo nas parcelas correspondente a SD e o seu conteúdo analisado em intervalos de 25 dias, até 100 dias após sua instalação. A adubação nitrogenada aumentou significativamente as variáveis relacionadas com a produtividade, sendo que a SD proporcionou a maior produção de matéria seca da parte aérea, matéria seca do grão, nitrogênio na parte aérea, nitrogênio do grão, produtividade de grãos, eficiência de absorção e uso do nitrogênio. A faixa de suficiência de nitrogênio na folha variou de 25,7 a 28,4 g kg-1. As doses máximas econômicas em 2008 e 2009 foram respectivamente de 125 e 160 kg ha-1 N / Abstract: Nitrogen is the nutrient absorbed in largest quantities in maize which provides more grain yields, and its use in soil-plant system is modified by the tillage system utilized. Aiming to evaluate the effect of cropping systems and nitrogen rates on dry matter production, nitrogen in the plant, leaf nitrogen, grain yield and nitrogen efficiency in maize, field experiment was established in 2008 and 2009 in a dystrophic red-yellow Argisoil under irrigation. The split-plot experimental design were used with eight replications. The plots were established by no-tillage (NT) and conventional tillage (CT). Subplots were set in six levels of N(0, 40, 80, 120, 160 and 200 kg ha-1) as urea. In 2009, in order to assess the speed of decomposition and nitrogen release from the bean plant used as cover crop, plant residues were packaged in nylon bags, which were arranged on the ground in the plots corresponding to NT and its contents examined at intervals of 25 days until 100 days after its installation. The nitrogen fertilization increased significantly the variables related to productivity, being that no-tillage provided the highest dry matter yield on shoot, dry matter of grain, nitrogen on shoot, grain nitrogen, grain yield, nitrogen uptake efficiency and nitrogen use efficiency. The sufficiency range of leaf nitrogen ranged from 25.7 to 28.4 g kg-1. The maximum economic rates in 2008 and 2009 season were respectively 125 and 160 kg ha-1N / Orientador: Edson Luiz Mendes Coutinho / Coorientador: Adeodato Ari Cavalcante Salviano / Banca: Luiz Evaldo de Moura Pádua / Banca: Takashi Muraoka / Banca: José Carlos Barbosa / Banca: José Eduardo Corá / Doutor

Radon Study of a Sample of Chosen Municipalities of South Bohemia - Measurment, Results Analysis and Inhabitants´ Perception of Risk The topic of my diploma thesis is radon study of a sample of chosen municipalities of South Bohemia - measurment, results analysis and inhabitants´ perception of risk. Czech inhabitants anxiety about radioactivity is usually focused on artificial sources of radiation, mainly on nuclear radiation. However, most of the irradiation of people is caused by natural radiation, espacially by radon. Within my diploma thesis I distributed 100 of detectors to measure volume activity of radon. After about six months I collected data to be processed. During the time of measuring I got know lots of people and I used the possibility to talk to them. That is why one of the aims of my thesis was to assess reaction of people to radon, to values of radon measured in their houses, people´s fears or indolence. I also tried to make the problem of radon easier to larger amount of inhabitants and I tried to make them understand this problem. I intended to inform people about results of radon radiation on human body. Main goal of my diploma thesis was to process data of volume activity of radon which were measured in the premises, to compare these data with the radon index of subsoil and to set a relation between volume activity of radon and other parameters as the age of the premises, contact with subsoil, tightness of windows, construction material used, and water source.

Submitted by Jaqueline Silva (jtas29@gmail.com) on 2016-09-08T19:58:04Z No. of bitstreams: 2 Dissertação - Gabriel Eliseu Silva - 2014.pdf: 1496066 bytes, checksum: 022a464ffaf53a95f5e7c2aba78aeff4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-09-08T19:58:15Z (GMT) No. of bitstreams: 2 Dissertação - Gabriel Eliseu Silva - 2014.pdf: 1496066 bytes, checksum: 022a464ffaf53a95f5e7c2aba78aeff4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-08T19:58:15Z (GMT). No. of bitstreams: 2 Dissertação - Gabriel Eliseu Silva - 2014.pdf: 1496066 bytes, checksum: 022a464ffaf53a95f5e7c2aba78aeff4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-08-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / (Dynamics of the shrub-tree community in three areas of cerrado sensu stricto in the municipality of Jataí, southwest of Goiás). The aim of the study was to assess the changes in shrub-tree community in a range of approximately three years, involving changes in the structure and vegetation composition with soil factors and the fire. The study was conducted in three areas of “cerrado sentido restrito”, using a ten plots of 20 × 50m in each area. All shrub-tree individuals with DAS (diameter at the heigh of the ground) ≥ 5 cm in the first survey (T1) were plaquetados, estimated their heights and identified their species (2009 in BAT and LAJ, and 2010 in FRP). The second survey (T2) was carried out in 2012 in the area of LAJ and in 2013 in the other two areas (BAT and FRP). T2 survivors were re-measured and those who have to enter the inclusion criteria (recruits) were measured and plaquetados. T1 in BAT, it was found 70 species, 853 individuals and basal area of 8.75 m2. T2 is sampled-63 species, 601 individuals and basal area of 9.12 m2. In the interval between the two surveys (4.16 years) nine species disappeared and two joined the community. The diversity index of Shannon and Pielou evenness were 3.26 and 0.77, respectively, for T1. For T2, only the diversity changed, dropping to 3.16. The average annual mortality rate was 11.55% and the recruitment 2.4%. In LAJ met 83 species, 1391 individuals and basal area of 16.82 m2 in T1 and T2 is already sampled-87 species, 1503 individuals and basal area of 18.65 m2. In the period of 3.58 years, eight species entered four disappeared in the community. The diversity index was 3.62 at T1 and T2 3.65. The average annual mortality rate was 2.27% and 4.13% of recruitment. In FARP met 77 species, 1863 individuals and basal area of 10.87 m2 in T1, T2 and already sampled to 75 species, 1395 individuals and basal area of 10.1 m2. In the period of three years, seven species have disappeared and five joined the community. The diversity index was 3,62 at T1 and T2 3,65. For the evenness values were 0,8 for both surveys. The average annual mortality rate was 10,37% and 1,73% of recruitment. The three cerrado narrow sense fragments altered the species richness, the number of individuals and basal area, but no significant changes in the diversity index between the first and the second survey. This may be related to variations in wealth, depending on the species with few individuals. The highest mortality found for BAT and FRP may be related to the presence of fire, while for the LAJ the largest recruitment is associated with absence of disturbance. The change in density and growth of individuals can still be conditioned to the soil characteristics. In LAJ and BAT the clay soil, while allowing the establishment and growth of forest species and individuals susceptible to fire, in BAT, it is believed that the fire has acted as limiting changes in the structure, preventing the density of vegetation; and LAJ, the increased density and increased basal area possibly occurred in the absence of fire. In the FRP, it is suggested that any fire and the sandy soil can contribute to the limitation on structural changes and the wealth of the community. Thus, in the LAJ greater rate of recruitment in relation to the recorded mortality, may possibly be related to the absence of fire and soil characteristics, providing tree compaction stage and a possible succession of the community, indicating the formation of a cerradão. Regarding the areas of BAT and FARP the highest mortality rates in relation to recruitment may ester associated with the presence of fire, preventing the thickening in the community and keeping the cerrado vegetation type narrow sense. / (Dinâmica da comunidade arbustivo-arbórea de três áreas de cerrado sentido restrito no município de Jataí, sudoeste de Goiás). O objetivo do trabalho foi verificar as mudanças ocorridas na comunidade lenhosa em um intervalo aproximado de três anos, associando as alterações na estrutura e na composição vegetacionais com os fatores edáficos e o fogo. O estudo foi realizado em três áreas de cerrado sentido restrito, utilizando dez parcelas de 20 × 50m em cada área, onde foram amostrados todos os indivíduos lenhosos com DAS (diâmetro a altura do solo) ≥ 5 cm no primeiro levantamento (T1), plaquetados, estimados suas alturas e identificados suas espécies (2009 no BAT e na LAJ, e 2010 na FRP). O segundo levantamento (T2) foi realizado em 2012 na área da LAJ e em 2013 nas outras duas áreas (BAT e FRP). Em T2 os sobreviventes foram remedidos e aqueles que passaram a entrar no critério de inclusão (recrutas) foram mensurados e plaquetados. Em T1 no BAT, encontrou-se 70 espécies, 853 indivíduos e área basal de 8,75 m2. Em T2 amostrou-se 63 espécies, 601 indivíduos e área basal de 9,12 m2. No intervalo entre os dois levantamentos (4,16 anos) nove espécies desapareceram e duas ingressaram na comunidade. O índice de diversidade de Shannon e a equabilidade de Pielou foram 3,26 e 0,77, respectivamente, para T1. Para T2, apenas a diversidade alterou, caindo para 3,16. A taxa média anual de mortalidade foi 11,55% e a de recrutamento 2,4%. Na LAJ encontrou-se 83 espécies, 1391 indivíduos e área basal de 16,82 m2 em T1, e já em T2 amostrou-se 87 espécies, 1503 indivíduos e área basal de 18,65 m2. No período de 3,58 anos, oito espécies ingressaram e quatro desapareceram na comunidade. O índice de diversidade em T1 foi de 3,62 e em T2 3,65. A taxa média anual de mortalidade foi de 2,27% e a de recrutamento 4,13%. Na FARP encontrou-se 77 espécies, 1863 indivíduos e área basal de 10,87 m2 em T1, e já em T2 amostrou-se 75 espécies, 1395 indivíduos e área basal de 10,1 m2. No período de 3,0 anos, sete espécies desapareceram e cinco ingressaram na comunidade. O índice de diversidade em T1 foi de 3,62 e em T2 3,65. Para a equabilidade os valores foram 0,8 para ambos os levantamentos. A taxa média anual de mortalidade foi de 10,37% e a de recrutamento 1,73%. Os três fragmentos de cerrado sentido restrito alteraram a riqueza de espécies, o número de indivíduos e área basal, porém não houve mudanças consideráveis nos índices de diversidade entre o primeiro e o segundo levantamento. Isso pode estar relacionado à variação da riqueza, em função das espécies que apresentaram poucos indivíduos. As maiores mortalidades encontradas para a BAT e FRP podem estar relacionadas à presença do fogo, enquanto para a LAJ o maior recrutamento esteja associado à ausência da perturbação. A alteração na densidade e o crescimento dos indivíduos podem estar condicionados ainda às características do solo. Na LAJ e no BAT o solo argiloso, apesar de possibilitar o estabelecimento e crescimento dos indivíduos de espécies florestais e sensíveis ao fogo, no BAT, acredita-se que o fogo tem atuado como limitante nas alterações da estrutura, impedindo o adensamento da vegetação; e na LAJ, o aumento da densidade e incremento em área basal, possivelmente ocorreu devido à ausência de queimadas. Já na FRP, sugere-se que o fogo eventual e o solo arenoso possam contribuir com a limitação nas alterações estruturais e na riqueza da comunidade. Assim, na LAJ a maior taxa de recrutamento registrada em relação à de mortalidade, possivelmente pode estar relacionada à ausência do fogo e às características do solo, proporcionando o adensamento arbóreo e um possível estágio sucessional da comunidade, indicando a formação de um cerradão. Em relação às áreas do BAT e da FARP as maiores taxas de mortalidade em relação às de recrutamento podem estar associadas à presença de queimadas, impedindo o adensamento na comunidade e mantendo a fitofisionomia de cerrado sentido restrito.

Der RAF-MEK-ERK-Signalweg steuert grundlegende, oftmals entgegengesetzte zelluläre Prozesse wie die Proliferation und Apoptose von Zellen. Die Dauer des vermittelten Signals wurde als entscheidener Faktor für die Steuerung dieser Prozesse identifiziert. Es ist jedoch nicht eindeutig geklärt, wie die verschiedenen früh und spät reagierenden Genexpressionsmodule kurze und lange Signale unterscheiden können und durch welche kinetischen Merkmale ihre Antwortzeit bestimmt wird. In der vorliegenden Arbeit wurden sowohl Proteinphosphorylierungsdaten als auch Genexpressionsdaten aus HEK293-Zellen gewonnen, die ein induzierbares Konstrukt des Proto-Onkogens RAF tragen. Hierbei wurde ein neues Genexpressionsmodul identifiziert, dass sich aus sofort induzierten aber spät antwortenden Genen zusammensetzt. Es unterscheidet sich in der Genexpressionsdynamik und Genfunktion von anderen Modulen, und wurde mit Hilfe mathematischer Modellierung experimenteller Daten identifiziert. Es wurde festgestellt, dass diese Gene aufgrund von langen Halbwertszeiten der vermitteltenden mRNA in der Lage sind spät auf das eingehende Signal zu reagieren und die Dauer des Signals in die Amplitude der Genantwort zu übersetzen. Trotz der langsamen Akkumulation und damit späten Antwortzeit, konnte aufgrund einer GC-reichen Promoterstruktur zunächst vermutet und mit Hilfe eines Markerverfahrens bestätigt werden, dass die Transkription dieser Gene instantan mit Beginn der ERK-Aktivierung startet. Eine vergleichende Analyse zeigte, dass das Prinzip der Signaldauer-Entschlüsselung in PC12-Zellen und MCF7-Zellen, zwei paradigmatischen Zellsystemen für die ERK-Signaldauer, konserviert ist. Insgesamt deuten die Ergebnisse der Untersuchung darauf hin, dass das neu identifizierte Genexpressionsmodul der Entschlüsselung der ERK-Signaldauer dient und das mRNA Halbwertszeiten sowohl hierfür, als auch für die zeitliche Abfolge der Genantwort eine entscheidende Rolle spielen. / The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals and what features govern their timing. Both protein phosphorylation and gene expression time course data was obtained from HEK293 cells carrying an inducible construct of the proto-oncogene RAF. A new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function was identified by mathematical modelling. It was found that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis showed that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, the findings of this study indicate that ILGs decode ERK signal duration and that both decoding capacity and gene expression timing are governed by mRNA half-life.

Prostate cancer is one of the most common types of cancer worldwide and claims hundreds of thousands of lives annually. Currently the most common treatment for prostate cancer is external beam radiotherapy, however, this treatment comes with serious side effects since it lacks selectivity for the cancer cells. Therefore, less harmful treatments are needed and sought for, such as targeted treatments that are intended to only affect cancer cells and thereby reduce the side effects. Targeted treatments require a target that differentiates the cancer cells from healthy cells. A promising target candidate that has gained attention in recent years is gastrin releasing peptide receptor (GRPR), a protein commonly overexpressed in prostate cancer cells. Furthermore, a targeting molecule intended to bind to the target is also required. For this purpose, the bombesin analogue RM26, a high affinity GRPR binder, shows promise. Previous studies have led to the development of RM26-conjugates for the purpose of targeted prostate cancer radiotherapy. In these conjugates RM26 has been linked to a DOTA-chelator for radiolabeling, and an albumin binding domain (ABD) to prolong the conjugate’s half-life in vivo by binding to human serum albumin (HSA). The idea is that the RM26-conjugate will bind to both HSA in the blood and to GRPR on the prostate cancer cells and eliminate the cancer cells with the radiation from the radionuclide attached to the DOTA-chelator. Although these earlier studied conjugates have been very promising some improvements of certain aspects need to be achieved, mainly to improve the biodistribution with retained GRPR binding affinity. Therefor the purpose of this project was to produce three new versions of previous RM26- conjugates and evaluate if they are suitable for further prostate cancer therapy studies. The three RM26-conjugates were developed with primarily recombinant expression in E. coli cells and solid phase peptide synthesis (SPPS). The characterization phase in this project was carried out with mainly five different methods: matrix-assisted laser desorption ionization time- of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization- mass spectrometry (ESI-MS), circular dichroism (CD), surface plasmon resonance (SPR) and flow cytometry. The results showed that all three new RM26-conjugates were possible to produce and yielded final products corresponding to the expected molecular weights. Furthermore, the results indicate that all three RM26-conjuagtes are stable and maintain their structural properties under in vivo- temperatures and that they have high binding affinity for HSA. Further studies need to be conducted before drawing any certain conclusions regarding GRPR binding affinity. / Prostatacancer är en av de mest vanligt förekommande cancertyperna världen över och skördar hundratusentals liv årligen. I nuläget är extern strålbehandling det vanligaste terapialternativet mot prostatacancer, men denna behandling kommer med allvarliga biverkningar på grund av att den saknar selektivitet för cancerceller. Därför finns ett stort behov av mindre skadliga behandlingsformer, såsom riktade behandlingar som endast är avsedda att påverka cancerceller och därigenom minska biverkningarna. Riktade behandlingar kräver ett mål som skiljer cancercellerna från friska celler. En lovande målkandidat som har uppmärksammats de senaste åren är gastrinfrisättande peptidreceptor (GRPR), ett protein som vanligtvis överuttrycks i prostatacancerceller. I tillägg så krävs också en målsökande molekyl avsedd att binda till målet. För detta ändamål visar bombesinanalogen RM26, en GRPR-bindare med hög affinitet, sig vara lovande. Tidigare studier har utvecklat RM26-konjugat för målinriktad strålbehandling av prostatacancer. Dessa konjugat består av en RM26-peptid bunden till en DOTA-kelator för radioinmärkning och en albuminbindande domän (ABD) för att förlänga konjugatens halveringstid in vivo genom att binda till humant serumalbumin (HSA). Syftet med RM26- konjugaten är att de ska binda till både HSA i blodet och GRPR på prostatacancercellerna, och därmed eliminera cancercellerna med strålning från den radioinmärkta DOTA-kelatorn. Även om de tidigare RM26-konjugaten har varit mycket lovande krävs det att vissa förbättringar av några aspekter uppnås, främst affiniteten för GRPR. Syftet med detta projekt var därför att producera tre nya versioner av tidigare RM26-konjugat och utvärdera ifall de uppvisar tillfredsställande egenskaper. De tre RM26-konjugaten utvecklades primärt rekombinant i E. coli-celler och fastfas- peptidsyntes (SPPS). Karaktäriseringsfasen i detta projekt genomfördes med huvudsakligen fem olika metoder: MALDI-TOF-MS, elektrosprejjonisering-masspektrometri (ESI-MS), cirkulär dikroism (CD), ytplasmonresonans (SPR) och flödescytometri. Resultaten visade att alla tre nya RM26-konjugat var möjliga att producera och gav slutprodukter motsvarande de förväntade molekylvikterna. Vidare indikerar resultaten att alla tre RM26-konjugat är stabila och bibehåller sina strukturella egenskaper under in vivo-temperaturer och att de har hög affinitet för HSA. Ytterligare studier bör utföras innan säkrare slutsatser kan dras angående GRPR-bindningsaffinitet.

Marknaden för proteinbaserade läkemedel, de så kallade biologiska läkemedlen, är idag en industri som omsätter miljarder. Ett vanligt sätt att utveckla dessa läkemedel på är med hjälp av monoklonala antikroppar då de kan binda till sitt mål med hög specificitet. Däremot begränsas denna teknik av en lång och dyr produktion som dessutom kräver däggdjursbaserade uttrycksystem. En alternativ teknik till de monoklonala antikropparna är att använda små proteiner som enkelt kan produceras i bakterier till en låg kostnad. Dock begränsas denna metod av de små proteinernas korta cirkuleringstid i blodet. I ett tidigare projekt, har ett litet protein vid namnet ABDderived affinity ProTein (ADAPT) på cirka 7 kDa, utvecklats för att kunna binda till både humant serumalbumin (HSA) för att förlänga cirkulationstiden i blodet och Interleukin 17c (IL17c) som är ett pro-inflammatorisk cytokin. Studien visade dock att ADAPT proteinet inte samtidigt kunde binda till de båda molekylerna tillräckligt effektivt. Syftet med denna uppsats är därför att undersöka om det nämnda proteinet kan optimeras genom så kallad multimering och/eller manipulering av bindningssätet för HSA i syfte att åstadkomma en effektiv och mer långvarig cirkulationstid i blodet samtidigt som det binder sig till sitt mål, IL17c. Tio nya versioner av ADAPT proteinet har utvecklats genom att klona och transformera proteiner till en högt producerande Escherichia coli (E. coli) stam. Proteinerna har sedan producerats och renats fram. Det kunde observeras att proteinerna hade den önskade renheten för att kunna karaktäriseras. Vidare var det möjligt att se att proteinerna hade sin önskade molekylvikt och erhöll sin förväntade struktur som en alfahelix. Proteinernas smältpunkter hade förbättrats eller var liknande jämfört med det ursprungliga proteinet. Dessutom kunde alla proteiner återgå till sin ursprungliga struktur efter upphettning. Utvärderingen av proteinernas bindningskapacitet, med original proteinet som referens, visade på en ökad affinitet till sitt mål, IL17c, för två dimerer och trimeren samt en jämförbar affinitet för två av monomererna med ett manipulerat bindingssäte till HSA. Interaktion till HSA var jämförbar med den ursprungliga ADAPT molekylen för alla nya varianter förutom monomererna med ett manipulerat bindingssäte och dimeren med två manipulerat bindingssäten till HSA. Evaluering av de nya proteinernas kapacitet att binda samtidigt till HSA och IL17c visade att det var gynnsamt med en dimereiserad molekyl då det skapade en distans mellan molekylerna och dess bindningssäten. Vidare kunde det också visas att ordningen som molekylerna interagerade med varandra påverkade proteinernas simultana bindning. / The market for protein-based drugs, or the so-called biopharmaceuticals, is a multibillion-dollar industry today. In the development of protein-based drugs it is common to use monoclonal antibodies (mAbs) due to their ability to bind to its target with high specificity. However, therapeutical development of mAbs is limited by its long and expensive production in mammalian expression system. An alternative to mAbs are the so-called alternative scaffolds which are small proteins that can be produced in bacteria at lower costs. Although a drawback with the latter proteins is their short serum half-life. A small scaffold protein, ABD-Derived Affinity ProTein (ADAPT) of approximate 7 kDa was earlier engineered to obtain bispecific affinity, to Human Serum Albumin (HSA), to extend its half-life, as well as to the pro-inflammatory cytokine, Interleukin 17c (IL17c). Unfortunately, it was shown that the simultaneous binding was not efficient enough for its desired purpose. The aim with this project was therefore to investigate if the previous mentioned binder could be optimized by multimerization and/or manipulation of the HSA binding site for an efficient half-life extension. By generating ten new designs of the ADAPT variants, it was observed that the new variants had stable alpha helical structures and an improved or similar melting temperature as the original variant. The evaluation of the target binding displayed an improved affinity to the target, IL17c, for two of the dimeric versions as well as for the trimer and a comparable affinity for two of the monomers with a manipulated HAS binding site. The interaction to HSA was comparable to the original ADAPT for all binders except from the monomers with impaired HSA binding and the dimer with two impaired HSA binding sites. The evaluation of the simultaneous binding showed that it was favored by dimerization when a distance between the two molecule and their binding surfaces was added. Moreover, it could also be seen that the order of binding events had an impact on the simultaneous binding.