Dissertation (PhD)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: To manipulate recombinant microorganisms for industrial processes, controllable genetic systems are needed that can coordinate expression of recombinant metabolic pathways. All components are sensitive to change and thus putative targets for modification and genetic elements and regulatory systems need to be understood and determined. Central in gene regulation is the transcription activators that mediate gene transcription mechanisms by binding to promoters in response to environmental signals. Promoter engineering entails the modification of transcription factors and their target promoters. In this study, a metabolic control system in Saccharomyces cerevisiae was constructed that would allow induction in response to physiological environment, specifically hypoxia and low temperature conditions. Two approaches were undertaken to find such a system. Firstly, a bi-directional reporter gene cloning vector was designed to search for novel hypoxiainducible promoters. Secondly, a transcription regulatory circuit was built, consisting of an inducible transcription regulator and promoter with a reporter gene through which it mediates transcription. Advantage was taken of the modular nature of proteins and functional domains originating from different transcriptional proteins were combined. A search for promoter elements sensitive to hypoxia from a S. cerevisiae genomic DNA (gDNA) library, using a bi-directional cloning vector, did not yield highly inducible promoters. It was concluded that a multitude of signals overlap, rendering genetic induction difficult to control. A synthetic regulatory system would minimize the impact of these multiple interactions. Such a genetic circuit was constructed, consisting of a chimeric transcription activator and a target fusion promoter. The chimeric transcription activator consisted of the GAL4 DNA binding domain, ADR1 TADIII transactivation domain and three domains of the MGA2 regulatory protein. The functional domains of Mga2p responsible for unregulated expression (at high basal levels) under both aerobic and hypoxia conditions were located, as well as a further upregulation under low temperature, and were mapped to the Nterminal and mid-Mga2p regions. A target fusion promoter consisting of a partial GAL10/1 promoter sequence and a Trichoderma reesei core xyn2 promoter were constructed as target for this chimeric transactivator. This synthetic promoter was fused to the T. reesei xyn2 open reading frame encoding for a readily assayable β-xylanase activity. Both the chimeric transactivator and fusion promoter-reporter gene cassettes were expressed from the same episomal plasmid, named pAR. Transformed into S. cerevisiae Y294, this regulatory system induced transcription under aerobic and hypoxia conditions. Furthermore, the reporter gene expression was upregulated by the chimeric transactivator at low temperatures. The chimeric transactivator mediated a seven-fold induction of the reporter gene under aerobic conditions in S. cerevisiae Y294 when transformed with plasmid AR. A two- to three-fold induction at 23ºC was reported under anaerobic conditions, relative to a reference strain expressing a transcription activator without the Mga2p domains. At 30ºC, a two- to three-fold induction under aerobic conditions and similar induction under oxygen-limited conditions were observed. Replacing the reporter gene with your favorite gene (for example a recombinant enzyme) and incorporating such a pAR system into a recombinant yeast should induce expression of the chosen gene under low temperatures, both aerobic and anaerobically (thus creating a controllable system). The system also has wider application in identifying other transcription factors’ signal-sensitive domains. The design of this system provides the ability to add a linker to a transactivator and to either create specific signal sensitivity or relieve the regulator of its signal dependence. It creates an easy system for assessing other transactivators and their domains with unknown functions and thus provides a ”workhorse and prospector in one”. / AFRIKAANSE OPSOMMING: Vir die manipulering van rekombinante mikroörganismes vir industriële prosesse word beheerbare genetiese stelsels benodig om gekoördineerde uitdrukking van rekombinante metaboliese weë teweeg te bring. Alle komponente van sulke stelsels is sensitief vir verandering en genetiese elemente en reguleerbare sisteme moet dus deeglik verstaan of bepaal word. Sentraal tot geenregulering is die transkripsie-aktiveerders wat geentranskripsie beheer deur aan promoters te bind in reaksie op eksterne omgewingsfaktore. Promotoringenieurswese behels wysigings van transkripsiefaktore en hul teikenpromotors. In hierdie studie is 'n genetiese beheerstelsel vir Saccaromyces cerevisiae ontwikkel wat induksie in reaksie tot spesifieke fisiologiese omgewingreaksies, naamlik hipoksie- en lae temperatuur, toelaat. Twee benaderings is gevolg: eerstens is ‘n tweerigting verklikker-geen vektor ontwikkel en gebruik om vir unieke induseerbare hipoksie-promoters te soek. Tweedens is ‘n transkripsie reguleringstelsel gebou wat uit ‘n induseerbare transkripsiereguleerder and promotor met ‘n verklikkergeen bestaan, waardeur transkripsie bemiddel kan word. Hierdie benadering benut die modulêre onderbou van proteïene en funksionele domeine afkomstig vanaf verskillende transkripsiefaktore is gekombineer. 'n Soektog na hipoksie-sensitiewe promotors vanuit 'n Saccharomyces cerevisiae-genoom- DNA (gDNA), deur van ‘n tweerigting verklikker-vektor gebruik te maak, het ongelukkig nie hoogs-induseerbare promotors opgelewer nie. Die gevolgtrekking was dat ‘n veelvoud van seine met mekaar oorvleuel en die beheer van genetiese induksie dus bemoeilik. Die ontwikkeling van ‘n sintetiese regulering-sisteem kan die impak van die veelvuldige interaksies verminder. Vir dié doel is ‘n sintetiese reguleringstelsel ontwerp, bestaande uit ‘n chimeriese transkripsie-aktiveerder met ‘n teiken fusie-promotor. Die chimeriese transaktiveerder bestaan uit die GAL4 DNA bindingsdomein, die ADR1 TAD III transaktiveringsdomein en drie domeine van die Mga2 reguleringsproteïen. In die studie is die funksionele domeins van Mga2p betrokke by lae temperatuur-respons en ongereguleerde uitdrukking (teen hoë basale vlakke) onder beide aërobiese en anaërobiese toestande aangedui en is tot die N-terminaal en middel-Mga2p areas gekarteer. ‘n Teiken-fusie-promoter, bestaande uit 'n gedeeltelike GAL1/10 DNA promotoropeenvolging en ‘n Trichoderma reesei kern xyn2-promoter, is as teiken vir hierdie chimeriese transaktiveerder saamgestel. Hierdie sintetiese promotor is aan die T. reesei xyn2 oopleesraam, wat vir ‘n maklik meetbare β-xylanase aktiwiteit kodeer, gekoppel. Beide die chimeriese transaktiveerder and fusie-promoter-verklikker-geenkaset word vanaf dieselfde episomale plasmied, bekend as pAR, uitgedruk. Hierdie reguleringsisteem induseer transkripsie onder aërobiese en hipoksie toestande in S. cerevisiae Y294. Verder word die verklikkergeen se uitdrukking deur die chimeriese transaktiveerder by lae temperature verhoog. Die chimeriese transaktiveerder induseer ‘n sewe-voudige induksie van die verklikkergeen onder aërobiese toestande by 23ºC vanaf die pAR-stelsel in S. cerevisiae Y294. ‘n Twee- tot drie-voudige induksie teen 23ºC is onder hipoksie toestande gevind, relatief tot induksievlakke van ‘n verwysingstam met ‘n transaktiveerder sonder die Mga2 domeine. By 30ºC is ‘n twee- tot drie-voudige induksie onder aërobiese en lae suurstofvlakke waargeneem. Deur die verklikker geen met ‘n jou-gunsteling-geen te vervang (bv. ‘n rekombinante ensiem) en so 'n pAR-sisteem in ‘n rekombinante gis te inkorporeer, word uitdrukking onder lae temperature onder beide aërobiese- en anaërobiese toestande geïnduseer (en sodoende word ‘n reguleerbare sisteem geskep). Die sisteem het wyer toepassing om sein-sensitiewe domeine van ander transkripsiefaktore te identifiseer. Die ontwerp van die stelsel maak dit moontlik om 'n skakel tot die transaktiveerder by te voeg wat óf sensitiwiteit tot 'n spesifieke sein skep, óf die reguleerder vanaf seinafhanklikheid verlos. So word ‘n bruikbare stelsel vir die bestudering van ander transaktivators en hul domeine met onbekende funksie geskep – ‘n “werksesel en prospekteerder in een”.

The development of recombinant proteins for therapeutic applications has revolutionized the pharmaceutical industry. In particular, monoclonal antibodies are the safest class of all therapeutic molecules and account for the majority of recombinant proteins currently undergoing clinical trials. A variety of technologies exist to engineer antibodies with a desired binding specificity and affinity, both of which are a prerequisite for therapeutic applications. This dissertation describes the implementation of a novel combinatorial library screening technology for the discovery and engineering of antibodies with unique binding properties. Combinatorial library screening technologies are used for the in vitro isolation of antibodies from large ensembles of proteins (libraries) typically produced by microorganisms using molecular biology techniques. Our lab has developed a powerful antibody discovery technology that relies on E. coli display by anchored periplasmic expression, otherwise known as APEx. First, I compared the effects of using combinatorial libraries comprising either smaller, monovalent single-chain antibody fragments (scFv), or the much larger, bifunctional full-length IgG antibodies. These technologies were used to isolate a small panel of antigen specific antibodies from the same library of antibody variable domains amplified from a mouse immunized with the Protective Antigen (PA) component from Bacillus anthracis, the causative agent of anthrax. Overall, IgG display resulted in the isolation of a broader panel of variable domain sequences. Most of these variable domains exhibited substantially reduced affinity when expressed as scFvs, which is consistent with the finding that none of these could be isolated from the equivalent scFv library. These results indicate that the antibody format used during in vitro selection affects which antibody variable domains will be discovered. Second, I developed several modifications of the APEx methodology to allow for more efficient recovery of antibodies with desired properties. Specifically, the system was reengineered to simultaneously account for antibody binding and expression levels in order to isolate the highest affinity antibodies with favorable expression characteristics. Third, the new approach, coupled with optimized fluorescence activated cell sorting (FACS) settings, was used to increase the affinity of an antibody by 35-fold resulting in a K[subscript D] of 100 pM. It was demonstrated that genetic transfer of this high affinity antibody specific for the V antigen of Yersinia pestis, the etiologic agent of the plague, conferred increased protection against intranasal challenge with a 363 LD₅₀ of Y. pestis in mice. / text

In this dissertation the investigation of potential applications of hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) harboring CTL epitopes for anticancer vaccine development is presented. The objective of this study was to investigate the potential of recombinant HaPyV VP1 based VLPs for anticancer vaccine generation in model systems, including investigation of VP1 applicability for heterologous CTL epitopes insertions, VLPs assembly and ability to induce insert specific immune response in vivo. HaPyV VP1 VLPs carrying CLT epitopes derived from different proteins were generated, most suitable positions for insertion into VP1 protein were selected, the ways to improve assembly and yield of the chimeric VLPs were determined and new VLPs purification procedure was created allowing to purify VLPs cheaper, faster and more efficiently. HaPyV VP1 based VLPs ability to induce CTL immune response in vivo was evaluated for the first time. It was demonstrated that model chimeric VLPs were able to stimulate antigen specific CTL cells in vitro and in vivo, induced insert specific humoral and CTL immune response in vivo and protected mice from insert specific virus infection and antigen-specific tumor growth. Presented data confirmed that HaPyV protein VP1 is universal carrier for CTL epitopes, capable to tolerate insertions, to form VLPs and to induce effective, long lasting immune response against inserted antigens in vivo. / Disertacijoje yra aprašomas perspektyvų panaudoti žiurkėno poliomos viruso (HaPyV) pagrindinio struktūrinio baltymo VP1 formuojamas į virusus panašias daleles priešvėžinių vakcinų kūrimui tyrimas. Pagrindinis disertacijos darbo tikslas buvo modelinėse sistemose parodyti rekombinantinių HaPyV VP1 baltymų formuojamų į virusus panašių dalelių panaudojimo priešvėžinių vakcinų kūrimui galimybes, įvertinant svetimų CTL epitopų įterpimo į VP1 baltymą toleravimą, VPD formavimosi efektyvumą bei sukeltą įterptam antigenui specifinį imuninį atsaką. Disertacijoje atlikta tyrimo srities literatūros apžvalga, smulkiai aprašomi darbe naudoti metodai, atlikti eksperimentai, pateikiami bei analizuojami gauti rezultatai. Darbe pirmą kartą buvo nuodugniai ištirtos HaPyV viruso VP1 baltymo formuojamų VPD savybės, parodytas jų tinkamumas būti CTL epitopų nešikliais, ištirtos įterpimui palankiausios VP1 baltymo vietos, išbandyti nauji VPD gavimo ir gryninimo būdai, pagerinantys chimerinių VPD formavimąsi bei išeigas. Panaudojant modelines chimerines VPD in vivo buvo ištirtas chimerinių HaPyV VP1 pagrindu sukonstruotų VPD sukeliamas humoralinis ir ląstelinis imuninis atsakas. Gauti rezultatai parodė, kad HaPyV VP1 baltymas yra vienas iš nedaugelio virusų struktūrinių baltymų, kurie ne tik formuoja VPD, bet pasižymi ir universaliomis baltymo – nešiklio savybėmis, o in vivo sukelia efektyvų, ilgalaikį, įterptam epitopui specifinį imuninį atsaką.

Abstract : The chymase-dependant pathway responsible for converting Big ET-1 to ET-1 was established in vitro. It has only been recently, in 2009, that our group demonstrated that the conversion of Big ET-1 to ET-1 (1-31) can occur in vivo in mice (Simard et al., 2009), knowing that ET-1 (1-31) is converted to ET-1 via NEP in vivo (Fecteau et al., 2005). In addition, our laboratory demonstrated in 2013 that mMCP-4, the murine analogue of human chymase, produces ET-1 (1-31) from the Big ET-1 precursor (Houde et al. 2013). Thus far, in the literature, there are no specific characterizations of recombinant chymases (human or murine). In fact, the group of Murakami published in 1995 a study characterizing the CMA1 (human chymase) in a chymostatin-dependent fashion, using Angiotensin I as a substrate (Murakami et al., 1995). However, chymostatin is a non-specific inhibitor of chymase. It has been shown that chymostatin can inhibit elastase, an enzyme that can convert Angiotensin I to Angiotensin II (Becari et al., 2005). Based on these observations, the proposed hypothesis in the present study suggests that recombinant as well as extracted CMA1 from LUVA (human mast cell line), in addition to soluble fractions of human aortas, convert Big ET-1 into ET-1 (1-31 ) in a TY-51469 (a chymase-specific inhibitor) sensitive manner. In a second component, we studied the enzyme kinetics of CMA1 with regard to the Big ET-1 and Ang I substrate. The affinity of CMA1 against Big ET-1 was greater compared to Ang I (KM Big ET- 1: 12.55 μM and Ang I: 37.53 μM). However, CMA1 was more effective in cleaving Ang I compared to Big ET-1 (Kcat / KM Big ET-1: 6.57 x 10-5 μM-1.s-1 and Ang I: 1.8 x 10-4 ΜM-1.s- 1). In a third component involving in vivo experiments, the pressor effects of Big ET-1, ET-1 and Ang I were tested in conscious mMCP-4 KO mice compared to wild-type mice. The increase in mean arterial pressure after administration of Big ET-1 was greater in wild-type mice compared to mMCP- 4 KO mice. This effect was not observed after administration of ET-1 and / or Ang I. / Résumé : La voie de conversion de Big ET-1 en ET-1, chymase dépendante a été établie in vitro. Ce n'est que récemment, en 2009 que notre groupe a démontré que la conversion de Big ET-1 en ET-1 (1-31) peut avoir lieu in vivo chez la souris (Simard et al., 2009), sachant que ET-1 (1-31) est convertie en ET-1 via NEP in vivo (Fecteau et al., 2005). En plus, en 2013, notre laboratoire a démontré que la mMCP- 4, l'analogue murin de la chymase humaine, produit l'ET-1 (1-31) à partir du précurseur Big ET-1 (Houde et al., 2013). Jusqu'a présent, dans la littérature, on ne trouve pas de caractérisations spécifiques de chymases (humaine ou murine) recombinantes. En fait, le groupe de Murakami, en 1995, a publié une étude caractérisant, d'une façon chymostatin dépendante, la CMA1 (chymase humaine) en utilisant l'Angiotensine I comme substrat (Murakami et al., 1995). Cependant, le chymostatin est un inhibiteur non-spécifique de la chymase. Il a été démontré que le chymostatin peut inhiber l'élastase, une enzyme pouvant convertir l'Angiotensine I en Angiotensine II (Becari et al., 2005). Basé sur ces observations, l'hypothèse formulée dans la présente étude est que la CMA1 recombinante ou extraite des cellules LUVA (lignée humaine de mastocytes) ou des fractions solubles des aortes humaines convertit la Big ET-1 en ET-1 (1-31) d'une façon TY-51469 (un inhibiteur spécifique de la chymase) sensible. Dans un deuxième volet, on a étudié la cinétique enzymatique de CMA1 en vers le substrat Big ET-1 et Ang I. L’affinité de CMA1 contre la Big ET-1 était plus grande comparé à l’Ang I (KM Big ET-1 : 12.55 μM et Ang I : 37.53 μM). Cependant CMA1 était plus efficace dans le clivage de l’Ang I comparé à la Big ET-1 (Kcat/KM Big ET-1 : 6.57 x 10-5 μM-1 .s-1 et Ang I : 1.8 x 10-4 μM-1 .s-1 ). Dans un troisième volet impliquant des expériences in vivo, l’effet presseur de la Big ET-1, l’ET-1 et l’Ang I a été testé chez des souris conscientes mMCP- 4 KO comparé à des souris de type sauvage. L’augmentation de la pression artérielle moyenne a été plus importante chez les souris de type sauvage après l’administration de Big ET-1 que chez les souris mMCP-4 KO. Cet effet n’a pas été observé après l’administration d’ET-1 et/ou d’Ang I ce qui explique le rôle de la chymase dans l’effet de la conversion de Big ET-1 en ET-1 (1-31).

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat.

The gonococcal transferrin binding proteins (Tbps) are two surface-exposed outer membrane proteins, TbpA and TbpB, which together function to remove and internalized iron from human transferrin. Iron is an essential nutrient to the gonococcus, without which it cannot survive. The Tbps have been established as virulence factors, demonstrating their importance in establishing infection. Both TbpA and TbpB are well conserved among gonococcal isolates, and have been considered potential vaccine targets. Vaccine studies with the closely related species Neisseria meningitidis, have demonstrated these proteins to be protective in murine challenge studies. Though the meningococcal Tbps have demonstrated promise, no similar gonococcal vaccine experiments have been conducted prior to the current studies. Here we demonstrate purification of recombinant TbpA and TbpB. These recombinant proteins were utilized to evaluate the human immune response to these proteins during natural infections, and their immunogenicity in murine vaccine studies. Our results demonstrate a paucity of antibodies elicited to these proteins during natural infections in serum and mucosal secretions from infected individuals. From this study we hypothesized the induction of both serum and genital antibodies to these proteins could serve to protect an individual from infection. To begin testing this hypothesis, we immunized mice both intranasally (IN) and subcutaneously (s.c.) with full-length Tbps in conjunction with the B subunit of cholera toxin (Ctb) as an adjuvant. We also performed another vaccine study using domains from both proteins in genetic fusions with Ctb and E. coli heat labile toxin IIb (LtbIIb). Both studies demonstrated that these antigens were immunogenic, as Tbp-specific antibodies were elicited in the serum and vaginal washes of female Balb/C mice. Intranasal immunization however was the only route with which we were able to elicit vaginal Tbp-specific IgA, and IgG, whereas subcutaneous immunization only elicited vaginal IgG. Furthermore, we found the full-length Tbps and the Ctb/LtbIIb chimeras were able to elicit bactericidal antibodies, which were also effective in killing heterologous gonococcal strains. This body of work comprises the first published study using the gonococcal transferrin binding proteins as vaccine antigens, and highlights their potential as vaccine antigens in the development of an efficacious gonococcal vaccine.

Chloride channel family has been shown to play a significant role in physiological homeostasis processes. The function mechanism of these proteins has not yet been clearly understood. Their deficiency or mutation causes serious human illnesses. Our understanding of the chloride channels' transporting mechanisms can lead to better treatment of these illnesses. As mammalian chloride channels are difficult to prepare in laboratory, the experiments are usually done on homologous chloride channels from prokaryotic organisms. The structures of prokaryotic chloride channels have been solved and moreover they are produced with high yields. Most experiments currently use protein crystallography and provide a static picture of the system. This thesis is focused on the study of structural changes of an E. coli chloride channel using hydrogen/deuterium exchange. This method enables us to monitor dynamic conformation changes dependent on pH and exchanged ions. The measurements were done for the protonated (pH 4.5) and deprotonated state (pH 7.5) and/or in the presence of various anions: Cl− , SCN− , I− , F− , TAR. (tartaric anion). The obtained results justified the theories explaining the function of chloride channel as Cl− /H+ antiporter and provided new findings. Subject words biochemistry, protein...

The goal of this study was to map the glycosylation pattern and antigenic properties of the salivary proteins of two closely related sand fly species, Phlebotomus perniciosus and P. orientalis. Affinity blotting with commercially available lectins revealed that many salivary proteins of these species are N-glycosylated, while the presence of O-glycosylation could not be confirmed. The level of N-glycosylation of most of these proteins is quite low, a larger number of potential N-glycosylation sites were found only in the amino acid sequences of P. orientalis hyaluronidase and endonucleases of both species tested. Four antigens from P. perniciosus salivary glands were selected for expression in a bacterial expression system; two of these proteins (PpeSP01 and PpeSP01B) were not glycosylated and the glycosylation level of the remaining two (PpeSP03B and PpeSP07) was low. The antigenic properties of the four chosen recombinant proteins were subsequently tested using immunoblot and ELISA. During the initial experiments with the sera of dogs experimentally bitten by P. perniciosus, two proteins (rSP07 and rSP01B) were proven unsuitable and they were excluded from further experiments. Recombinant proteins rSP03B and rSP01 were recognized by the same IgG antibodies as the native forms of these proteins...

Calculating the price of an option commonly uses numerical methods and can becomputationally heavy. In general, longer computations result in a more precisresult. As such, improving existing models or creating new models have been thefocus in the research field. More recently the focus has instead shifted towardcreating neural networks that can predict the price of a given option directly.This thesis instead studied how the number of time-steps parameter can beoptimized, with regard to precision of the resulting price, and then predict theoptimal number of time-steps for other options. The number of time-stepsparameter determines the computation time of one of the most common models inoption pricing, the Cox-Ross-Rubinstein model (CRR). Two different methodsfor determining the optimal number of time-steps were created and tested. Bothmodels use neural networks to learn the relationship between the input variablesand the output. The first method tried to predict the optimal number oftime-steps directly. The other method instead tried to predict the parameters ofan envelope around the oscillations of the option pricing method. It wasdiscovered that the second method improved the performance of the neuralnetworks tasked with predicting the optimal number of time-steps. It was furtherdiscovered that even though the best neural network that was found significantlyoutperformed the benchmark method, there was no significant difference incalculation times, most likely because the range of log moneyness and pricesthat were used. It was also noted that the neural network tended tounderestimate the parameter and that might not be a desirable property of asystem in charge of estimating a price in the financial sector.

O câncer é um problema de saúde pública, especialmente entre os países em desenvolvimento. Está entre as doenças de maior taxa de mortalidade e é responsável por mais de 12% de todas as causas de óbito no mundo. Uma proteína denominda Amblyomin-X foi obtida a partir de uma biblioteca de cDNA das glândulas salivares do carrapato Amblyomma Cajennense e expressa na bactéria Escherichia coli BL21DE3. Essa proteína é capaz de induzir a morte de células tumorais, além de ter pouca ou nenhuma atividade contra células normais, diminuindo assim os efeitos colaterais nos pacientes em tratamentos contra o câncer. Através deste trabalho foi possível elaborar um protocolo para obtenção de elevadas concentrações celulares e produção da proteína recombinante AmblyominX em biorreatores com capacidade total de 15 L. Uma vez que a proteína é intracelular, foram estudados alguns parâmetros e estratégias de cultivo do microrganismo para obtenção de alta concentração celular, tais como: composição do meio de cultura, identificação dos substratos limitantes e inibitórios e vazão de alimentação da fonte de carbono. Foi definido um meio de cultura adequado para atingir elevadas concentrações celulares na etapa de crescimento da E. coli, visando garantir elevadas concentrações de produto recombinante. Ensaios conduzidos em biorreatores com esse meio resultaram em concentrações celulares e densidade ótica (DO600nm) iguais a 40,0 g/L e 76,6, respectivamente, para a etapa de crescimento celular e 59,8 g/L e 161, respectivamente, no final da etapa de produção da proteína de interesse. Os ensaios conduzidos em reatores mostraram que concentrações de ácido acético acima de 0,4 g/L causam inibição do crescimento celular, portanto, foi elaborada uma estratégia eficaz de alimentação exponencial dos substratos limitantes (fontes de carbono e magnésio) na etapa de crescimento dessa bactéria para atingir elevadas concentrações celulares, mantendo-se a velocidade específica de crescimento (µx) menor que 0,2 h-1, abaixo do valor máximo, com a finalidade de evitar a formação de ácido acético. Este trabalho permitiu também conhecer os parâmetros cinéticos para a produção da proteína Amblyomin-X, tais como, a velocidade específica máxima de crescimento (max), a produtividade celular global (Pxglobal), a produtividade celular máxima (Pxmáx) e o fator de conversão de substrato em célula (Yx/s) na etapa de batelada alimentada, cujos valores foram, respectivamente, de 0,30 h-1, 13,8 g de célula/h, 14,1 g de célula/h e 0,37 g de célula/g de substrato. Para a avaliação da biossíntese da proteína recombinante foram realizadas análises de gel de poliacrilamida por eletroforese e a quantificação foi feita pela análise de Bradford. A concentração de proteína Amblyomin-X no final da etapa de síntese alcançou 8,57 g/L. / Cancer is a public health problem, especially among developing countries. It is among the diseases with the highest mortality rate and accounts for more than 12% of all causes of death in the world. A protein called Amblyomin-X was obtained from a cDNA library of Amblyomma Cajennense tick salivary glands and it was expressed in the bacterium Escherichia coli BL21DE3. This protein is able to induce death of tumor cells, as well as having little or no activity against normal cells, thus reducing the side effects in patients on cancer treatments. In this work it was possible to elaborate a protocol for obtaining high cellular concentrations and production of the recombinant Amblyomin-X protein in bioreactors with a total capacity of 15 L. Since the protein is intracellular, some parameters and strategies of culture of the microorganism for obtaining a high cell concentration, were: composition of the culture medium, identification of limiting and inhibitory substrates and feed rate of the carbon source. A culture medium suitable for achieving high cell concentrations in the growth stage of E. coli was defined in order to guarantee high concentrations of recombinant product. Experiments conducted in bioreactors with this medium resulted in cellular concentrations and optical density (DO600nm) of 40.0 g / L and 76.6, respectively, for the cell growth step and 59.8 g / L and 161, respectively, at the end of the production stage of the protein of interest. Experiments conducted in reactors showed that acetic acid concentrations higher than 0.4 g/L cause inhibition of cell growth, therefore, an effective strategy of exponential feeding of the limiting substrates (carbon and magnesium sources) was elaborated in the growth stage of this bacteria to reach high cellular concentrations, maintaining the specific growth rate (µx) less than 0.2 h-1, below the maximum value, in order to avoid the formation of acetic acid. This work also allowed to know the kinetic parameters for Amblyomin-X protein production, such as the maximum specific growth rate (max), the overall cellular productivity (Pxglobal), the maximum cellular productivity (Pxmax) and the conversion factor of substrate in cell (Yx/s) in the fed batch stage, whose values were respectively 0.30 h-1, 13.8 g cell/h, 14.1 g cell/h and 0.37 g of cell/g of substrate. For the evaluation of recombinant protein biosynthesis, polyacrylamide gel analyzes were performed by electrophoresis and quantification was done by the Bradford analysis. The Amblyomin-X protein concentration at the end of the synthesis step reached 8.57 g/L.