As hialuronidases de peçonhas animais são capazes de clivar o hialuronan presente na matriz extracelular, facilitando a difusão das toxinas no tecido da vítima. Essas enzimas têm sido negligenciadas, devido à instabilidade enzimática e à baixa concentração na peçonha. Assim, a expressão heteróloga das hialuronidases permite a sua obtenção em quantidades que viabilizam seu estudo estrutural e funcional. Associado a isso, o transcriptoma propicia a identificação de novas toxinas e componentes de baixa proporção na peçonha. Portanto, o presente trabalho realizou o transcriptoma da glândula de peçonha do escorpião Tityus serrulatus e a clonagem e expressão heteróloga da hialuronidase. No transcriptoma foram obtidos 558 ESTs, dos quais 61,8% correspondem às toxinas, dentre elas neurotoxinas com ação em canais iônicos, metaloproteinas, hipotensinas, peptídeos antimicrobianos, dentre outros. Foram também identificadas novas toxinas como o neuropeptídeo e Ts16.1, descritos pela primeira vez para o gênero Tityus. Dentre os transcritos obtidos, foi identificado apenas um clone correspondente ao C-terminal incompleto de uma hialuronidase de T. serrulatus. Consequentemente, foi produzido o gene sintético contendo a sequência da TsHyal-1, obtida em bancos de dados, no vetor de expressão pPICZ?A para expressão heteróloga em P. pastoris. A rTsHyal-1 foi expressa em escala laboratorial em meio não suplementado (BMM) em pH 7,0 durante 96 h da indução com alimentação diária de metanol a 0,75%. A rTsHyal-1 foi produzida na sua forma solúvel e ativa (838,31 UTR/mg) e resultou em um rendimento proteico de 250 mg/L de material expresso. O secretoma do meio de expressão mostrou que além da rTsHyal-1, a P. pastoris também secreta proteínas nativas associadas à ligação do ATP, metabolismo de carboidrato e resposta ao estresse oxidativo. A rTsHyal-1 foi parcialmente purificada em troca catiônica fraca e apresentou atividade específica de 1.097,45 UTR/mg. A rTsHyal-1 apresenta massa molecular de 49,5 kDa e o tratamento com PNGase F seguido da análise por espectrometria de massas (MALDI-TOF) indicou que uma possível N-glicosilação de 4,5 kDa. Adicionalmente, o sequenciamento dos digestos trípticos da enzima realizado no MALDI-TOF e pelo Q-Exactive resultaram em 46,8% de cobertura da sequencia da proteína. A rTsHyal-1 apresenta especificidade pelo substrato hialuronan, seguido da condroitina C, A e B e apresentou atividade ótima em pH 6,0 e a 40°C. Adicionalmente, o ensaio do MTT indicou que a enzima recombinante não apresenta citotoxicidade in vitro. Os resultados obtidos determinaram as melhores condições para a expressão heteróloga da rTsHyal-1, que corresponde à primeira hialuronidase recombinante de escorpião expressa em P. pastoris com atividade enzimática preservada / The venom hyaluronidases of animals are able to cleave hyaluronan present in the extracellular matrix, acting as a spreading factor for the toxins into the tissues of the victim. These enzymes have been neglected due to their instability and low concentration in the venom. Thus, heterologous expression of hyaluronidases permits the obtainment of sufficient amount for structural and functional studies. Moreover, the transcriptome allows the identification of new toxins or components with low proportion in the venom. In this way, this study aimed the construction of the transcriptome of Tityus serrulatus venom gland and cloning/heterologous expression of hyaluronidase. In the transcriptome, 558 ESTs were identified and 61.8% corresponded to toxins, such as neurotoxins with action on ion channels, metalloproteins, hypotensins, antimicrobial peptides, among others. In addition, new toxins were identified, comprising one neuropeptide and Ts16.1, described for the first time to the genus Tityus. Among the obtained transcripts, one identified clone corresponded to an incomplete C-terminal of a hyaluronidase. Consequently, a synthetic gene was synthesized containing the sequence of TsHyal-1 (obtained from databases) in the pPICZ?A vector for heterologous expression in P. pastoris. The rTsHyal-1 was expressed at laboratorial scale in unsupplemented medium (BMM) at pH 7.0 for 96 h, after induction time, with daily feeding of 0.75% methanol. The rTsHyal-1 was produced in soluble and active form (838.31 UTR/mg) and resulted in a protein yield of 0,266 mg/mL in final expressed material. Besides, the secretome of the medium showed that P. pastoris also secretes native proteins bound with ATP, proteins related to carbohydrate metabolism and oxidative stress response. The rTsHyal-1 was partially purified in a weak cation exchange and presented specific activity of 1097.45 UTR/mg. The rTsHyal-1 has molecular mass of 49.5 kDa and the treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, the sequencing of tryptic digests performed in the MALDI-TOF and Q-Exactive resulted in 46.8% of protein sequence coverage. The rTsHyal-1 presents substrate specificity for hyaluronan followed by chondroitin C, A and B and showed an optimum activity at pH 6.0 and 40°C. Furthermore, the MTT assay indicated that the recombinant enzyme does not display in vitro cytotoxicity. These results validate the biotechnological process of the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpions expressed in P. pastoris system with enzymatic activity preserved.

Submitted by Wendell Amoedo (wendell.amoedo@hotmail.com) on 2018-11-26T14:35:47Z No. of bitstreams: 1 dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) / Submitted by Wendell Amoedo (wendell.amoedo@hotmail.com) on 2018-11-28T13:01:00Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-11-28T13:20:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) / Made available in DSpace on 2018-11-28T13:20:12Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertação.kerollen.runa.pinto2017.pdf: 1354749 bytes, checksum: 47321c0e5d660481ffc33d9d3204b2f0 (MD5) Previous issue date: 2017-02-27 / FAPEAM - Fundação de Amparo à Pesquisa do Estado do Amazonas / Enterokinase (EC 3.4.21.9) is a heterodimer serine protease, a natural activator of trypsinogen, capable of cleaving specifically the sequence Asp-Asp-Asp-Asp-Lys. Due to the high specificity of the recognition site, it became a great tool of biotechnological interest. It is usually used to remove affinity tags in vitro, of recombinant proteins. In this work, the molecular cloning strategy resulted in the construction of the pDMK06, capable of programming the regulated expression of a heterologous gene ETK-Trx in E. coli. Through the cleavage process with restriction enzymes NdeI and BamHI, it was possible to obtain the coding sequence of the fusion protein between enterokinase and thioredoxin (ETK-Trx) of approximately 1259 bp from pENTK plasmid. Then, this sequence was subcloned at NdeI and BamHI sites of the expression vector pDM02, originating the recombinant plasmid pDMK06. This vector contains the TH2 promoter, which is efficiently regulated by Lac operator/repressor. E. coli JM110 cells transformed with the recombinant plasmid showed smaller growth in a solid medium when the expression of the heterologous protein was induced by IPTG in comparison with the control; however, this effect was not detected in the liquid medium. Furthermore, the E. coli cells morphology was analyzed through optical microscopy containing the recombinant plasmid pDMK06, when it was observed, all through the growth time, modifications on cell morphology, characterized by the formation of filaments in those induced with IPTG, in comparison with the control. For expression analysis of the recombinant protein ETKTrx, polyacrylamide gel electrophoresis SDS-PAGE was performed with the samples that grew with IPTG induction for eight hours. The results showed that the protein ETK-Trx is about 47 kDa with a high level of expression at the insoluble fraction, probably as an inclusion corpuscle. The high levels of expression of ETK-Trx protein occurred in a perfectly regulated way, showing the functionality of the pDM02 plasmid expression/regulation system. / A enteroquinase (EC 3.4.21.9) é uma serino protease heterodimérica, ativadora natural do tripsinogênio, capaz de clivar especificamente a sequência Asp-Asp-Asp- Asp-Lys. Devido à alta especificidade do sítio de reconhecimento tornou-se uma ferramenta de grande interesse biotecnológico. É comumente utilizada para a remoção in vitro de marcas de afinidade, como etiquetas de fusão (tags) de proteínas recombinantes. No presente trabalho, a estratégia de clonagem molecular resultou na construção do plasmídeo pDMK06, que é capaz de programar a expressão heteróloga regulada do gene ETK-Trx em E.coli. Por meio do processo de clivagem com as enzimas de restrição NdeI e BamHI foi possível obter a sequência codificadora da proteína de fusão entre enteroquinase e tiorredoxina (ETK-Trx) de aproximadamente 1259 pb partir do plasmídeo pENTK, a seguir essa sequência foi subclonada nos sítios de NdeI e BamHI do vetor de expressão pDM02, originando o plasmídeo recombinante pDMK06. Esse vetor contém o promotor TH2 que é regulado eficientemente pelo sistema operador/repressor Lac. Células de E.coliJM110 transformadas com o plasmídeo recombinante mostram menor crescimento em meio sólido quando a expressão da proteína heteróloga era induzida por IPTG em relação ao controle, porém esse efeito não foi detectado em meio líquido. Além disto foi analisado por microscopia ótica a morfologia das células de E.colicom o plasmídeo recombinante pDMK06, onde observou-se no decorrer do tempo de crescimento e indução, alterações na morfologia celular caracterizada de filamentação das células induzidas com IPTG quando comparadas com o controle. Para análise da expressão da proteína recombinante ETK-Trx utilizou-se a eletroforese em gel de poliacrilamida SDS-PAGE das amostras referentes a 8 horas de crescimento e indução com IPTG. Os resultados obtidos apresentaram a proteína ETK-Trx com tamanho aproximado de 47kDa com alto nível de expressão na fração insolúvel, provavelmente em forma de corpúsculo de inclusão. A expressão em altos níveis das proteínas ETK-Trx ocorreu de forma perfeitamente regulada mostrando a funcionalidade do sistema de expressão/regulação do plasmídeo pDM02.

Celulases têm atraído muito interesse nos últimos anos devido a sua habilidade na bioconversão de material lignocelulolítico em glucose, a qual pode, então, ser convertida a etanol por fermentação. O complexo celulolítico capaz de degradar a celulose consiste de várias enzimas (principalmente celulases e β-glucosidases) e proteínas auxiliadoras, que atuam em sinergismo para eficientemente hidrolizar a biomassa. Nesse estudo, investigou-se a hidrólise enzimática do bagaço de cana-de-açúcar pré-tratado utilizando enzimas produzidas por T. harzianum e enzima comerial. O rendimento de hidrólise foi avaliado quanto a diferentes níveis de deslignificação de biomassa, graus de cristalinidade da celulose, composição dos coquetéis enzimáticos e adição de BSA. Estudos de difração de raios-X mostraram que a cristalinidade da lignocelulose não é um fator determinante na recalcitrância ao ataque enzimático. Além disso, a adição de BSA não teve qualquer efeito no rendimento da hidrólise. O mais eficiente coquetel enzimático foi obtido misturando o preparado comercial com o produzido pelo T. harzianum (rendimento acima de 97%). Esse desempenho está, provavelmente, relacionado com níveis adequados de β-glucosidases e xilanases no coquetel. Devido a essa eficiente atividade celulolítica, o fungo T. harzianum tem um grande potencial em aplicação para hidrólise de biomassa. A celobiohidrolase I, uma exoglucanase, é a principal enzima secretada por esse fungo (cerca de 60% do total) e nesse estudo ela foi expressa em bioreator, purificada por cromatografia de troca iônica seguida de gel filtração e caracterizada bioquímica, biofísica e estruturalmente. Conforme confirmado por SAXS, tanto a CBHI inteira quanto seu domínio catalítico, obtido por digestão parcial com papaína, são monoméricos em solução e apresentam distância máxima (DMax) de 110 e 60 Å, e raio de giro (Rg) de 20 e 27 Å, respectivamente. Os resultados indicam que o linker é flexível em solução e confirmam o formato de girino da enzima. A CBHI possui atividade máxima em pH 5.0 e temperatura de 50 °C, com atividade específica contra Avicel &reg e pNPC de 0,28 and 1,53 U/mg, respectivamente. Outras celulases de interesse foram também expressas para caracterização, no entanto, para essas, foi utilizado o sistema de expressão heteróloga em Aspergillus Níger ou Pichia pastoris. O domínio catalítico da endoglucanase I de T. harzianum foi expresso em A. Níger. A proteína tem atividade específica contra CMC de 15,8 U/mg e pH e temperatura ótima de 3 e 50 °C, respectivamente. A proteína é estável nessas condições em até 3 dias de incubação (dados de ensaios de atividade residual). Estudos biofísicos de deslocamento térmico e dicroísmo circular apresentaram alguns parâmetros de estabilidade de estrutura terciária e secundária, respectivamente. A proteína perde estrutura terciária regular, em pH 5, em torno de 30 °C mas sua estrutura secundária é desordenada somente em pH 9 (quando a 25 °C). Experimentos de dicroísmo circular também indicaram a composição de estrutura secundária do domínio catalítico da EGLI de 6% de α-hélice e 42% de folhas- β. / Cellulases have attracted an outstanding interest in the recent years because of its ability in the bioconversion of cellulose-containing raw materials into glucose, which can then be converted into ethanol by fermentation. The cellulase complex able to degrade cellulose consists of several enzymes (mainly cellulases and β-glucosidases) and auxiliary proteins, which act in synergism to efficiently solubilize the biomass. In this study, we investigated the enzymatic hydrolysis of pretreated sugarcane bagasse using crude enzyme extracts produced by Trichoderma harzianum as well as from the extract in combination with a commercial cocktail. The influence of different levels of biomass delignification, degree of crystallinity of lignicellulose, composition of enzymatic activities and BSA on enzymatic hydrolysis yields was evaluated. Our X-ray diffraction studies showed that crystallinity of lignocellulose is not a key determinant of its recalcitrance toward enzymatic hydrolysis. In fact, under the experimental conditions, an increase in crystallinity of lignocellulosic samples resulted in increased glucose release by enzymatic hydrolysis. Furthermore, under the same conditions, the addition of BSA had no significant effect on enzymatic hydrolysis. The most efficient enzyme blends were obtained by mixing a commercial enzymatic cocktail with T. harzianum cellulase preparations (above 97%). Increased hydrolytic efficiencies appeared to correlate with having an adequate level of both β-glucosidase and xylanase activities in the blends. Due to its elevated cellulolytic activity, the filamentous fungus T. harzianum has a considerable potential in biomass hydrolysis applications. The cellobiohydrolase I, an exoglucanase, is the main enzyme secreted by this fungus (about 60% of total) and in this study we have expressed, purified and performed an initial biochemical, biophysical and structural characterization. As confirmed by small angle X-ray scattering (SAXS) both full-length CBHI and its catalytic core domain (CCD), obtained by partial digestion with papain, are monomeric in solution and they have Dmax of 110 and 60 Å, and Rg of 20 and 27 Å, respectively. The results indicate that the linker is flexible in solution and confirmed the tadpole shape of the enzyme. CBHI displays maximum activity at pH 5.0 and temperature of 50 °C, with specific activities against Avicel &reg and pNPC of 0,28 and 1.53 U/mg, respectively. Other celulases were also expressed, however, for them we have used the heterologous expression system in Aspergillus niger and Pichia pastoris. The catalytic domain of endoglucanase I from T. harzianum was expressed in A. niger and partially characterized. The protein has specific activity against CMC of 15.8 U/mg and optimum pH and temperature of 3 and 50 °C, respectively. The protein is stable in these conditions until 3 days of incubation. Biophysical studies of termal shift and circular dichroism (CD) assays have showed some parameters of stability of tertiary and secondary structure of the protein. It loses regulary terciary structure in pH 5 around 30 °C but the secondary structure is desordened only in pH 9 at 25 °C. CD experiments also indicated the secondary structure compsition of the catalytic domain of EGLI: 6% de α-helices and 42% de β-sheet.

INTRODUÇÃO: O método padrão de reparo de falhas ósseas é o transplante do osso autólogo. No entanto, novas técnicas de bioengenharia de tecido ósseo poderão substituir o método padrão. A construção de uma técnica em bioengenharia de tecido ósseo é feita pela associação entre fatores ou células indutoras de osso e biomateriais carreadores. O objetivo do presente trabalho foi mensurar a neoformação óssea em falha óssea alveolar de modelo animal após o reparo com fontes diferentes de bioengenharia de tecido ósseo e compará-las com o reparo com o osso autólogo transplantado da região craniana. MÉTODOS: Foi criada uma falha óssea na região alveolar de 28 ratos Wistar medindo 5 mm de diâmetro. Quatro modalidades de reparo foram comparadas ao método padrão: No grupo 1 (método padrão), as falhas ósseas foram reparadas com o transplante de osso autólogo da região parietal da calvária; nos grupos 2 e 3, as falhas ósseas foram reparadas com o biomaterial carreador osso bovino mineral sem e com o emprego de células-tronco mesenquimais humanas indiferenciadas, respectivamente; nos grupos 4 e 5, as falhas ósseas foram reparadas com o biomaterial carreador -tricálcio fosfato sem e com o emprego de células-tronco mesenquimais humanas indiferenciadas, respectivamente. A neoformação óssea na falha alveolar foi aferida por meio de imagens de tomografia computadorizada e avaliação histomorfométrica após 8 semanas da cirurgia. A neoformação óssea obtida por meio da avaliação histomorfométrica possibilitou a comparação dos grupos 2, 3, 4 e 5 com o grupo 1. Foi criado um sistema de pontos para determinar a distribuição do osso na falha óssea alveolar por meio das imagens de tomografia computadorizada em cinco animais por grupo, sendo 1 ponto para ossificação parcial, 2 pontos para ossificação total e heterogênea e 3 pontos para ossificação total e homogênea. O índice de significância estatístico p<0,05 foi determinado pelo teste não paramétrico de Mann-Whitney. RESULTADOS: Na avaliação histomorfométrica, o grupo 1 apresentou 60,27% ± 16,13% de osso na falha (n=7). Os grupos 2 e 3 apresentaram respectivamente, 23,02% ± 8,6% (n=3) Resumo (p=0,01) e 38,35% ± 19,59% (n=5) (p=0,06) de osso na falha. Os grupos 4 e 5 apresentaram respectivamente, 51,48% ± 11,7% (n=3) (p=0,30) e 61,8% ± 2,14% (n=6) (p=0,88) de osso na falha. Na avaliação radiológica, os animais dos grupos 1, 2, 3, 4 e 5 apresentaram média de pontos respectivamente igual a 2; 1,4; 1,5; 1,6, 1,8. CONCLUSÕES: O grupo de animais cujas falhas ósseas alveolares foram reparadas com -tricálcio fosfato e células-tronco mesenquimais apresentou a neoformação óssea mais semelhante a do grupo de animais cujas falhas ósseas foram reparadas com osso autólogo / INTRODUCTION: The current criterion standard to repair bone defects is an autogenous bone transfer. However, bone engineering strategies may become the first choice in the future. Bone bioengineering strategies are created through the association of inductive factors, stem cells and biomaterial matrices. The objective of this study was to measure the bone formation in an alveolar osseous defect animal model using different bone tissue engineering strategies and to compare them with the autogenous bone transfer. METHODS: Alveolar circular bone defects measuring 5 mm of diameter were created in 28 Wistar rats. Four alternative modalities were compared to the traditional modality of autogenous bone transfer: In group 1 (traditional modality), defects were repaired with autogenous bone graft from the calvarial region; in groups 2 and 3, defects were repaired using bovine bone mineral free of cells and loaded with undifferentiated mesenchymal stem cells, respectively; in groups 4 and 5, defects were repaired with - tricalcium phosphate free of cells and loaded with mesenchymal stem cells, respectively. Groups 2, 3, 4 and 5 were compared with group 1. Bone formation was evaluated by computed tomography imaging, and by histomorphometry at 8 weeks after surgery. Radiologically, a score system was developed to determine the bone distribution measured by computed tomography imaging in five animals of each group. Statistical significance was determined as p<0.05 by the non-parametric statistical hypothesis test called the Mann-Whitney test. RESULTS: Histomorphometrically, group 1 showed 60.27% ± 16.13% of bone in the defect (n=7). Groups 2 and 3 showed respectively, 23.02% ± 8.6% (n=3) (p=0.01) and 38.35% ± 19.59% (n=5) (p=0.06) of bone in the defect. Groups 4 and 5 showed respectively, 51.48% ± 11.7% (n=3) (p=0.30) and 61.80% ± 2.14% (n=6) (p=0.88) of bone in the defect. Radiologically, groups 1, 2, 3, 4 and 5 scored on average 2, 1.4, 1.5, 1.6, 1.8, respectively. CONCLUSION: The group of animals whose alveolar osseous defects Summary were repaired with -tricalcium phosphate and mesenchymal stem cells showed the most similar bone formation to the group whose alveolar osseous defects were repaired with autogenous bone

Malaria is a major threat to the public health worldwide as it is affecting populations in tropical and subtropical areas globally. Among those populations are around 40% of pregnant women and children who are susceptible to this disease. Plasmodium falciparum is the most lethal agent that causes malaria in human. Currently, there is drug resistance against antimalarial drugs in parasite against treatment of malaria infections, it is essential to search for new drug targets in order to find cure and alleviate suffering of human population. There are approximately 100 protein kinases in P. falciparum that are involved in phosphorylation of asexual blood stage. Hence, the phosphorylation plays an important part in the development of different stages of malarial parasites. Due to their significance in the parasite life cycle, one of the protein kinase of P. falciparum belongs to the ABC-1 family of proteins. PfABCK2 can be a therapeutic target due to its higher expression during the late schizont stage of blood stage form. The bioinformatic analysis and preliminary results of PfABCK2 showed the heterologous expression of this protein. Hence, the gene of PfABCk2 was ligated into pET21a+ vector with His-tag at C-terminus and transformed into BL-21 (DE3) competent cells that were verified through Miniprep and DNA sequencing. Furthermore, this gene construct is utilized to heterologous express this protein with IPTG and afterwards purified the recombinant protein kinase using nickel affinity chromatography as shown on 10% SDS-PAGE with the expected 36 kDa protein band. Therefore, the aim of this study is to partially characterize PfABCK2 protein kinase utilizing molecular cloning, heterologous express and protein kinase activity assay.

Natural products have long furnished medical science playing a significant role in drug discovery and development. Their importance notwithstanding, it is estimated that less than 1% of microorganisms can be cultivated from environmental sources using standard laboratory techniques. It is therefore necessary to develop biochemical and genetic techniques to access these uncultivable genomes. Here as a point of departure toward this goal, two cDNA libraries of two microorganisms were constructed along with five fosmid libraries with DNA isolated from marine environmental samples. We describe the construction of cDNA libraries from marine microbial species and detail experiments to exploit these libraries for their natural product biosynthetic pathways and other metabolic enzymes they harbor. However, no useful biosynthetic pathways were detected within the cDNA libraries. Genetic selection by complementation was additionally explored as a method to identify and localize biosynthetic gene clusters within marine microbial DNA libraries. Genetic selection is a fast and economic method which utilizes selection of a part of a pathway to represent the presence of an entire pathway for the complementation of known mutant strains. We describe genetic selection to localize biotin biosynthetic pathways of Hon6 (Chromohalobacter sp.) as a proof of principle experiment for the identification and localization of biosynthetic pathways in general. Instead of developing purification methods or manipulating cultivation conditions, large fragments of non-culturable bacterial genomes can be cloned and expressed using recombinant DNA technology. A strong transcriptional promoter to control high-level gene expression is required in recombinant expression plasmids. We aimed to develop new tools to control gene expression through the use of riboswitches. Riboswitches are metabolite-sensing ribonucleic acid (RNA) elements that possess the remarkable ability to control gene expression. The thiamine pyrophosphate (TPP) riboswitch system was chosen as it will enable use of E. coli as a suitable host strain. This system is particularly attractive because it has one of the simplest structures among the riboswitches elucidated to date. The use of the TPP riboswitch will also enable modulation of pathway gene expression by varying the TPP coccentration as many gene products are toxic. The violacein gene cluster from Chromobacterium violaceum was selected and placed under the control of this riboswitch. We describe modulation of heterologous gene expression by the ThiC/Riboswitch and detail experiments to investigate the expression and manipulation of the gene cluster under various promoters.

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) είναι κατιονικοί δίαυλοι, οι οποίοι ενεργοποιούνται κατόπιν πρόσδεσης κατάλληλου νευροδιαβιβαστή και ευθύνονται για την κυτταρική επικοινωνία δια μέσου της ακετυλοχολίνης. Ο κυριάρχος τύπος νικοτινικών υποδοχέων στον εγκέφαλο των θηλαστικών με υψηλή συγγένεια για νικοτίνη, αποτελείται από α4 και β2 υπομονάδες. Ο συγκεκριμένος τύπος υποδοχέων, είναι υπεύθυνος για την εξάρτηση στην νικοτίνη και θεωρείται ότι εμπλέκεται στις ασθένειες Alzheimer και Parkinson. Για το λόγο αυτό οι συγκεκριμένοι δίαυλοι, αποτελούν έναν σημαντικό στόχο σχεδιασμού και ανάπτυξης φαρμάκων. Στην προσπάθειά μας να αποκτήσουμε υδρόφιλες περιοχές του ανθρώπινου υποδοχέα α4β2 για δομικές μελέτες ικανές να προσδένουν αγωνιστές, εκφράσαμε τα εξωκυττάρια τμήματα των συγκεκριμένων υπομονάδων στο ευκαρυωτικό σύστημα έκφρασης ζύμης Pichia pastoris. Στα αγρίου τύπου εξωκυττάρια τμήματα αλλά και στις μεταλλαγμένες μορφές τους, στις οποίες έχει αντικατασταθεί η κυστινική θηλιά με την περισσότερο υδρόφιλη αντίστοιχη περιοχή της πρωτεΐνης η οποία δεσμεύει ακετυλοχολίνη, δεν παρατηρήθηκε καμία ειδική αλληλεπίδραση με γνωστούς προσδέτες. Κρίθηκε λοιπόν αναγκαίο να εκφράσουμε τα εξωκυττάρια τμήματα διασυνδεόμενα με ένα ολιγοπεπτίδιο 24 αμινοξικών καταλοίπων (AGS)8, ως ένα μόριο. Παρατηρήθηκε λοιπόν ότι το συγκαταμερές β2-24-α4-mECD αλλά όχι το α4-24-β2-mECD είναι αρκετά υδατοδιαλυτό μόριο με πολύ καλές ιδιότητες πρόσδεσης αγωνιστών. Η ιωδινιωμένη επιβατιδίνη (125Ι-επιβατιδίνη) και η τριτιωμένη νικοτίνη ([3Η]-νικοτίνη) δεσμεύονται στο συγκαταμερές β2-24-α4-mECD με σταθερές διάσπασης (Kd) 0,38 και 19 nM αντίστοιχα, τιμές οι οποίες προσεγγίζουν τις αντίστοιχες γνωστές από τη βιβλιογραφία, που προσδίδονται σε ολόκληρο τον ανθρώπινο υποδοχέα α4β2. Επιπλέον, το πόσο ειδική είναι η δέσμευση της 125Ι-επιβατιδίνης φαίνεται και από το γεγονός ότι παρεμποδίζεται συναγωνιστικά από τους αγωνιστές νικοτίνη, κυτισίνη, ακετυλοχολίνη και καρβαμυλοχολίνη με τιμές σταθεράς αναστολής (Κi) ίσες με 20,64, 3,24, 242 και 2254 nM αντίστοιχα. Επιπλέον, επιχειρήθηκε η δημιουργία πενταμερών εξωκυττάριων τμημάτων των ανθρώπινων υπομονάδων α4 και β2. Αρχικά, πραγματοποιήθηκε συνέκφραση καθενός από τα δύο συγκαταμερή (α4-24-β2-mECD και β2-24-α4-mECD), τα οποία φέρουν χαρακτηριστική αλληλουχία έξι ιστιδινών (6xHis), με κάθε εξωκυττάριο τμήμα των μεταλλαγμένων υπομονάδων α4 ή β2 τα οποία φέρουν την χαρακτηριστική αλληλουχία του οκταπεπτιδίου FLAG. Στις περιπτώσεις της συνέκφρασης του συγκαταμερούς α4-24-β2-mECD με καθένα από τα εξωκυτταρικά και μεταλλαγμένα τμήματα των υπομονάδων α4 ή β2 (α4-mECD ή β2-mECD) δεν παρατηρήθηκε δέσμευση με 125Ι-επιβατιδίνη και συνεπώς δεν προχωρήσαμε σε περαιτέρω χαρακτηρισμό. Μόνο στην περίπτωση της συνέκφρασης του συγκαταμερούς β2-24-α4-mECD με το β2-mECD παρατηρήθηκε πρόσδεση με 125Ι-επιβατιδίνη. Επίσης, οι μελέτες δυναμικής σκέδασης φωτός εμφάνισαν μία εκτεταμένη ετερογένεια μεγέθους των τελικών προϊόντων όπως απομονώνονται μέσω της χρωματογραφίας μοριακής διήθησης, αποκλείοντας, τουλάχιστον σε αυτό το σημείο οποιαδήποτε προσπάθεια δομικής μελέτης για τα συγκεκριμένα μόρια. Κατασκευάστηκαν επίσης δύο συγκαταμερή τριμερών (β2-24-β2-24-α4-mECD και β2-24-α4-24-β2-mECD). Δυστυχώς, η ανάλυση μέσω SDS-PAGE και η αποτύπωση κατά Western αποκάλυψε την ύπαρξη ενός μείγματος, το οποίο αποτελείται από ολόκληρα τα μόρια (τριμερή) αλλά και από πολυπεπτίδια στα οποία απουσιάζουν ένα ή και δύο από τα μονομερή εξωκυττάρια τμήματα (διμερή και μονομερή αντίστοιχα). Το συγκεκριμένο αποτέλεσμα υποδηλώνει ότι το σύστημα ετερόλογης έκφρασης το οποίο χρησιμοποιούμε (P. pastoris) αν και είναι κατάλληλο για την έκφραση των λειτουργικών διμερών συγκαταμερών (β2-24-α4-mECD), είναι μάλλον ακατάλληλο για την έκφραση πιο περίπλοκων κατασκευών, πιθανότατα εξαιτίας γεγονότων ομόλογου ανασυνδιασμού των πλασμιδίων πριν ή κατά την ενσωμάτωσής τους στο γονιδίωμα της ζύμης. Ακόμα, ενδιαφέρον είναι το γεγονός ότι το απογλυκοζυλιωμένο συγκαταμερές β2-24-α4-mECD εμφανίζει τις ίδιες ιδιότητες πρόσδεσης αγωνιστών. Επιπροσθέτως, το απογλυκοζυλιωμένο συγκαταμερές β2-24-α4-mECD απομονονέται ως μονομερές, γεγονός το οποίο, σε συνδιασμό με την προηγούμενη παρατήρηση, καθιστά την συγκεκριμένη πρωτεΐνη κατάλληλη να χρησιμοποιηθεί σε δομικές μελέτες και πιθανόν για την φαρμακολογική εκτίμηση καινούργιων και ειδικών αγωνιστών για υποδοχείς α4β2. / Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels responsible for cell communication via the neurotransmitter acetylcholine. The predominant nAChR subtype in the mammalian brain with a high affinity for nicotine is composed of α4 and β2 subunits. This nAChR subtype is responsible for addiction to nicotine and is thought to be implicated in Alzheimer and Parkinson diseases and therefore presents an important target for drug design. In an effort to obtain water-soluble, ligand-binding domains of the human α4β2 nAChR appropriate for structural studies, we expressed the extracellular domains (ECDs) of these subunits in the eukaryotic expression system Pichia pastoris. The wild-type ECDs and their mutants containing the more hydrophilic Cys-loop from the snail acetylcholine-binding protein (individually expressed or coexpressed) did not demonstrate any specific interaction with ligands. We then linked the mutated ECDs with the 24 amino acid peptide (AGS)8 and observed that the β2-24-α4-mECD concatamer, but not the α4-24-β2-mECD one, exhibited very satisfactory water solubility and ligand binding properties. The 125I-epibatidine and [3H]nicotine bound to β2-24-α4-mECD with dissociation constants (Kd) of 0.38 and 19 nM, respectively, close to the published values for the intact α4β2 nAChR. In addition, 125I-epibatidine binding was blocked by nicotine, cytisine, acetylcholine, and carbamylcholine with inhibition constants (Ki) of 20.64, 3.24, 242, and 2,254 nM, respectively. In addition, we attempted to create pentameric domains of the extracellular human subunits α4 and β2. Initially, we accomplished the coexpression of the particular concatamers, α4-24-β2-mECD and β2-24-α4-mECD, bearing the six histidine tag (6xHis), with each of the mutated extracellular domains of the subunits α4 and β2 (α4-mECD and β2-mECD), bearing the characteristic octapeptide FLAG tag. The coexpression of the concatamer α4-24-β2-mECD with each of α4 or β2-mECD did not lead to binding to 125I - epivatidine and therefore we did not proceed to any further characterization. Only in the case of the coexpression of the concatamer β2-24-α4-mECD with the β2-mECD we observed binding to 125I - epivatidine. Aditionally, the dynamic light scattering studies demonstrated a widespread size heterogeneity of the final product as isolated by gel filtration chromatography, blocking, at least at this point, any further attempt for structural studies of the specific molecules. We then constructed two trimeric concatamers (β2-24-β2-24-α4-mECD and β2-24-α4-24-β2-mECD) . Unfortunately, the analysis by SDS-PAGE and Western blot revealed the presence of a mixture which consists of entire molecules ( trimers ), but also from polypeptides which are missing one or two of the monomer extracellular domains (dimers and monomers, respectively). This result indicates that the heterologous expression system which is used (P. pastoris), is suitable for the expression of functional bilateral concatamers (β2-24-α4-mECD), but is rather unsuitable for the expression of more complex structures, probably due to events of homologous recombination of the plasmids before or during integration into the yeast genome . Interestingly, deglycosylation of the concatamer did not affect its ligand binding properties. Furthermore, the deglycosylated β2-24-α4 ECD existed mainly in monomeric form, thus forming an appropriate material for structural studies and possibly for pharmacological evaluation of novel α4β2 nAChR-specific agonists.

Antibody-mediated rejection is central to ABO incompatible transplantation as well as to xenotransplantation. The xenoantigen alpha-Gal has a highly analogous carbohydrate structure to the human blood group antigens, and both require memory B cell activation for antibody production. We hypothesize that B cells, reactive to the alpha-Gal xenoantigen and B blood group antigen, require the presence of fully activated T cells in order to survive and proliferate in vitro, contrary to the traditional theory that humoral response to carbohydrate antigens is a T cell-independent process. When we compared the capacity of B cells to proliferate, we observed that activated T cells were necessary for B cell proliferation even in the presence of carbohydrate-derived antigens. A relevant question was also to investigate the role of a specific class of T cells: the CD1d-restricted iNKT cells, in the activation of alpha-Gal and B blood group-reactive B cells. The iNKT cells have the specificity of being reactive to glycolipids and are capable of producing both T helper 1 and T helper 2 cytokine responses. We therefore wanted to determine the role of the iNKT cells as mediators of a T helper 2-type response when B cells were exposed to a glycolipid antigen expressing the alpha-Gal epitope or the human B blood group antigen. We observed that, if the interaction between B cells and iNKT cells is blocked, neither B cell proliferation nor antibody production occurs. These results suggest therefore the importance of the iNKT cell category of T helper cells in the response to alpha-Gal and ABO-blood group glycolipids.

Der Mensch besitzt ~25 funktionelle Bitterrezeptoren (TAS2R), die für die Wahrnehmung potenziell toxischer Substanzen in der Nahrung verantwortlich sind. Aufgrund der großen genetischen Variabilität der TAS2R-Gene könnte es eine Vielzahl funktionell unterschiedlicher TAS2R-Haplotypen geben, die zu Unterschieden der Bitterwahrnehmung führen. Dies konnte bereits in funktionellen Analysen und sensorischen Studien für einzelne Bitterrezeptoren gezeigt werden. In dieser Arbeit wurden die häufigsten Haplotypen aller 25 Bitterrezeptoren verschiedener Ethnien funktionell charakterisiert. Das Ziel war eine umfassende Aussage über die funktionelle Diversität der TAS2Rs, die die molekulare Grundlage für individuelle Bitterwahrnehmung bildet, treffen zu können. Fehlende Varianten wurden aus genomischer DNA kloniert oder durch gezielte Mutagenese bereits vorhandener TAS2R-Konstrukte generiert. Die funktionelle Analyse erfolgte mittels Expression der TAS2R-Haplotypen in HEK293TG16gust44 Zellen und anschließenden Calcium-Imaging-Experimenten mit zwei bekannten Agonisten. Die Haplotypen der fünf orphanen TAS2Rs wurden mit über hundert Bitterstoffen stimuliert. Durch die gelungene Deorphanisierung des TAS2R41 in dieser Arbeit, wurden für die 21 aktivierbaren TAS2Rs 36 funktionell-unterschiedliche Haplotypen identifiziert. Die tatsächliche funktionelle Vielfalt blieb jedoch deutlich hinter der genetischen Variabilität der TAS2Rs zurück. Neun Bitterrezeptoren wiesen funktionell homogene Haplotypen auf oder besaßen nur eine weltweit vorherrschende Variante. Funktionell heterogene Haplotypen wurden für zwölf TAS2Rs identifiziert. Inaktive Varianten der Rezeptoren TAS2R9, TAS2R38 und TAS2R46 sollten die Wahrnehmung von Bitterstoffen wie Ofloxacin, Cnicin, Hydrocortison, Limonin, Parthenolid oder Strychnin beeinflussen. Unterschiedlich sensitive Varianten, besonders der Rezeptoren TAS2R47 und TAS2R49, sollten für Agonisten wie Absinthin, Amarogentin oder Cromolyn ebenfalls zu phänotypischen Unterschieden führen. Wie für den TAS2R16 bereits gezeigt, traten Haplotypen des funktionell heterogenen TAS2R7 und TAS2R41 ethnien-spezifisch auf, was auf lokale Anpassung und verschiedene Phänotypen hinweisen könnte. Weiterführend muss nun eine Analyse der funktionell-variablen TAS2Rs in sensorischen Tests erfolgen, um ihre phänotypische Relevanz zu prüfen. Die Analyse der funktionsmodulierenden Aminosäurepositionen, z.Bsp. des TAS2R44, TAS2R47 oder TAS2R49, könnte weiterführend zum besseren Verständnis der Rezeptor-Ligand- und Rezeptor-G-Protein-Interaktion beitragen. / Bitter taste perception varies markedly from person to person, due to a high number of polymorphisms present in the 25 known functional bitter receptors (TAS2Rs). These polymorphisms lead to a number of haplotypes for each receptor, which are common in different populations, but vary in frequency. The individual combination of receptor variants seems to determine the person’s sensitivity of bitter perception, as could already be shown for single TAS2Rs. Bitter is an aversive taste quality, indicating the ingestion of harmful substances. Different sensitivity could have an impact on food choice. In order to characterize functional consequences of the genetic diversity, we performed calcium imaging experiments with all main haplotypes for the 25 bitter receptors. The obtained information about receptor properties enables us on the one hand to analyze structure-function relationships and on the other hand gives us the functional diverse candidates to focus on in psychophysical studies. The overall aim is to show genotype-phenotype correlation for bitter taste perception and their impact on food choice and therefore diet and health. Our first aim was to identify agonists for the 5 receptors, which could not be deorphaned in previous screens. We challenged all main haplotypes of these TAS2Rs with 106 bitter compounds and could identify the antibiotic chloramphenicol as agonist for bitter receptor TAS2R41. In total we identified 36 functionally different receptor variants of the 21 deorphaned TAS2Rs. Main haplotypes of nine TAS2Rs were functionally homogeneous while twelve TAS2Rs possessed between two and three functionally heterogeneous receptor variants. In summary the observed functional diversity is not as big as expected. Based on our in vitro findings the shown functional diversity of these twelve bitter receptors might be the molecular basis for individual differences in bitter taste perception and will be further analyzed in psychophysical studies.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.