Cotransin is a cyclodepsipeptide inhibitor of protein translocation and has been demonstrated to inhibit cotranslational translocation of a variety of proteins by targeting the mammalian ER translocation channel Sec61 (Besemer, Harant et al. 2005, Garrison, Kunkel et al. 2005). Genetic screens in cancer cells found out the mutations near the luminal plug domain of Sec61 confer resistance to cotransin inhibition and thus outline the proposed binding site for cotransin (MacKinnon, Paavilainen et al. 2014). However molecular details of cotransin interactions remain unknown. Purpose of my first project was to express the human Sec61 translocation channel in correct stoichiometric ratios. To our knowledge, heterotrimeric expression of Sec61 has not been achieved previously. Baculovirus system was chosen express the Sec61 heterotrimeric complex. To vary expression levels of Sec61α and Sec61γ relative to Sec61β, separate baculovirus constructs were prepared. Proper co-transfection ratios between these viruses to express Sec61subunits in correct stoichiometric ratios were calculated during my pro gradu and insect cell expression was then scaled up using the determined virus ratios. Results from Sec61 expression have returned sufficient quantities of the translocation channel for biochemical analyses. Final expression seems to contain high lipid/protein ratio, which may have been caused due to insect cells not-fully adapted to the media. The expression should be repeated in well-adapted healthy insect cells and then accessed for protein quality. For its isolation for crystallization studies, co-immunoprecipitation may be a preferred way to pull down the Sec61 complex. Detergent based solubilization may be alternatively used to isolate Sec61. The second part of my pro gradu work included expression of SecYEβ translocation channel from Pyrococcus furiosus, along with its various humanized mutants, whose DNA constructs had been provided by Dr. Ville Paavilainen. A major goal of this work was to express the mutants which bind to cotransin. Via photo-crosslinking and click chemistry analyses, a mutant binding to cotransin was identified and scaled-up. Our photo-crosslinking studies were able to demonstrate that the wild-type SecYEβ does not bind to cotransin in vitro. Results from photo-crosslinking assays during this project also demonstrated that other known translocation inhibitors Mycolactone and Apratoxin A can bind to mammalian Sec61 channel. These results are consistent with unpublished work from Paavilainen lab (Paatero et al).

Bacterial proteins DsbB, VKOR, and the mammalian protein VKORC1 share similar functions involving electron transfer processes. While DsbB is not homologous to the bacterial and mammalian VKOR proteins, the three proteins share overall structural features. Based on the similarities between the three proteins and the finding that mtbVKOR can replace DsbB in E. coli, we considered the possibility of rat VKORC1 displaying similar functionality as mtbVKOR. Genetic selection and screening was done on an EMS mutagenized ΔdsbB strain expressing rat VKORC1wt from a plasmid for isolation of E.coli mutants that would facilitate complementation of the lack of DsbB by VKORC1wt. The principle for the genetic selection and screening is the restoration of disulfide bond forming pathway by replacement of DsbB. This phenotype of complementation can be assayed by restoration of motility, resistance to TCEP, and β-galactosidase inactivation on ∆dsbB strain. Results of the selection and screening process revealed mutations in the VKORC1 gene instead of the E.coli chromosome. On the other hand, we used a rational approach other than genetic screening. This approach involved targeting YidC hydrophilic groove, previously identified upon selection of mutants that facilitated functional expression of VKORC1∆AAR; a deletion of amino acids 31–33 (AAR), where other mutations inactivating protease HslV were also identified. For this approach, chromosomal mutations were introduced on selected residues in the YidC hydrophilic groove then functional expression of VKORC1wt or enhanced expression of VKORC1∆AAR in the new strains was assayed. We identified novel YidC mutants enhancing the expression of VKORC1∆AAR. From the analysis of these mutations and the VKORC1 mutations obtained from the screens, we concluded that the charge imbalance by VKORC1wt violates the positive-inside rule impeding its ability to substitute DsbB. The correct assembly of VKORC1∆AAR provided insight on the involvement of E.coli YidC in correct folding and insertion of foreign membrane proteins, with the hydrophilic groove being core for its membrane insertase functions. The improved functional expression of VKORC1∆AAR upon HslV inactivation provided insight on the mis-assembly of foreign membrane proteins as a quality control system. These findings suggested that the native VKORC1wt may not assemble properly in the E.coli inner membrane, and is degraded by proteases as a quality control mechanism.

Modelling studies were performed on a fermentation system using Saccharomyces cerevisiae NCYC 754. The production of fermentation product and cytochrome P-450 were studied under semi-anaerobic condition in batch cultures. The fermentation was carried out in a 5-litre fermenter and controlled at constant set-points which had been optimized by an earlier worker with respect to enzyme yield. An unstructured model was established to describe the biomass profile which comprised two growth phases; however the system did not demonstrate the classical diauxic growth as expected. Furthermore, against the general belief that glucose is the limiting substrate of the system; the maximum wet biomass seemed to depend on the concentration of peptone and yeast extract in the fermentation broth. Growth kinetics indicated that a second substrate was utilized before glucose metabolism began in spite of the presence of high levels of glucose. Luedeking and Piret type models, combined with ethanol inhibition, were derived to describe the profile of ethanol and cytochrome P-450 concentration. Later, it was demonstrated that a close correlation exists between initial glucose and cytochrome P-450 concentration. Viable count by agar plating techniques was used to test the proposed biomass model. The results were in line with the proposed model, even though the cell viability profile in the system was rather low. The Taguchi method was used to seek out the noise factor in the system, and optimize the operating conditions for a particular performance statistic. Contrary to earlier findings, the stirrer speed was found to have little effect on the yield of cytochrome P-450.

No description available.

Made available in DSpace on 2016-02-05T18:29:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-03-25. Added 1 bitstream(s) on 2016-02-05T18:33:11Z : No. of bitstreams: 1 000856523.pdf: 885289 bytes, checksum: 8ba3eecc7223dda20c63ac810083c4d7 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / As amilases são enzimas pertencentes à classe das hidrolases, atuando na estrutura do amido com a hidrólise das ligações glicosídicas dos tipos α-1,4 e α-1,6, do interior das cadeias de amilose e amilopectina, respectivamente. Atualmente são responsáveis por cerca de 30% do mercado mundial de enzimas e apresentam uma vasta gama de aplicações industriais. Os fungos capazes de produzir enzimas amilolíticas podem ser cultivados mediante insumos de baixo custo empregados na fermentação em estado sólido (FES), o que possibilita a valorização dos subprodutos da agroindústria, bem como fornece suporte semelhante ao encontrado pelo micro-organismo no ambiente natural. O presente estudo visa produzir amilases por Rhizopus microsporus var. oligosporus em FES utilizando o farelo de trigo como substrato, purificar parcialmente o extrato bruto enzimático obtido, caracterizar bioquimicamente o extrato enzimático parcialmente purificado e posteriormente realizar a sacarificação pela hidrólise do amido presente na quirera de arroz e fermentação alcoólica por Saccharomyces cerevisiae para produção de etanol. A atividade enzimática do extrato bruto foi 39,8 U/mL o que equivale a 358 U/g de substrato. O índice de purificação, após as etapas de cromatografia foi parcial, mas suficiente para que a caracterização bioquímica do extrato produzido fosse realizada. Estes testes demonstraram faixa ótima no pH 4,0 e pH 5,5, indicando as condições ácidas como as melhores para este estudo. A estabilidade do pH foi ampla variando do pH 3,5 ao pH 8,5 com cerca de 40 a 60% de atividade relativa. A temperatura ótima de atividade enzimática determinada como ideal foi de 60 a 65 °C, porém a enzima mostrou-se termoestável até 60 °C. Os testes do efeito de íons na reação enzimática amilolítica demonstraram que os íons Cu+2, Zn+2, Al+2 e Na+2 comportaram-se como inibidores da atividade. O íon Mn+2 destacou-se por potencializar em... / Amylases are enzymes belonging to the class of hydrolases acting on starch structure with the hydrolysis of glycosidic bonds of α-1,4 and α-1,6 types, in the interior of the chains of amylose and amylopectin, respectively. Currently they are responsible for about 30% of the world market enzymes and show a wide range of industrial applications. Fungi capable of producing amylases can grow by low cost inputs in solid-state fermentation (SSF), which enables the recovery of the agricultural industry by-products and provides support similar to that found by the microrganism in the natural environment. This study aims to produce amylase by Rhizopus microsporus var. oligosporus in solid-state fermentation using wheat bran as substrate partially to purify the crude enzyme extract obtained biochemically characterize the partially purified enzyme extract and subsequently to carry out the hydrolysis saccharification of starch present in broken rice and fermentation of it by Saccharomyces cerevisiae for ethanol production. Enzymatic activity of the raw extract was 39.8 U/mL equivalent to 358 U/g substrate. The purification ratio after the chromatography step was partial, but sufficient for the biochemical characterization of the produced extract was taken. These tests showed optimal range of pH 4.0 to pH 5.5 indicating the acidic condition as the best for this study. The pH stability was wide range of pH 3.5 to pH 8.5 with 40 to 60% relative activity. The optimum temperature for enzyme activity determined as optimum was 60 to 65 °C, but the enzyme was thermostable up to 60 °C. Ion effect of the amylolytic enzyme tests showed that the reaction Cu+2, Zn+2, Al+2 and Na+2 ions behaved like activity inhibitors. The Mn+2 ions distinguished for enhancing at about 60% relative enzymatic activity to hydrolysis without addition of ions. The enzymatic hydrolysis of broken rice using as substrate allowed the complete conversion of starch to reducing sugars ...

Most pharmaceutical residues in wastewater treatment plants (WWTPs) end up in the hydrosphere where they cause negative effects on the aquatic life and might disrupt ecosystems. By implementing an ozonation step (treatment with ozone) in the wastewater treatment process, these pharmaceutical residues can be reduced.  The purpose of this project was to verify that the ozonation process works in full-scale, thereby verifying a pilot study conducted in 2014 at Tekniska Verken i Linköping AB (TVAB). Additionally, the purpose was to investigate which parameters influence the ozone demand in order to formulate a model for the ozone demand. The initial phases during this thesis were a pre-study and a literature study. This was followed by the multivariate analysis and model construction based on different data from the pilot study. Measurements were performed on the wastewater in the full-scale facility in order to verify the results from the pilot study. Moreover, measurements were performed to find new ozone consuming parameters. The reduction of pharmaceutical residues was similar to the pilot study, although slightly lower. Several parameters and factors that were different between pilot study and new measurements affected the reduction of pharmaceutical residues. For example, DOC and nitrate concentrations have increased since the pilot study in 2014. Also, factors such as the growth in population in Linköping and the differences in design between the pilot plant and the full-scale facility have influenced the reduction of pharmaceutical residues. A control strategy based on a linear relationship between ozone sensitive Ultra Violet Absorption (UVA) left and remaining pharmaceutical residues after ozonation could potentially be used. Moreover, three models were constructed and the Multivariate Analysis 1 (MVA1)-model was deemed as the best, this model includes ozone residual, nitrite, turbidity, simulated Chemical Oxygen Demand (COD(sim)) and ozone dose. The variations in the dose compared to the input parameters for the validation data show that the model predict the ozone dose well. However, in future other interesting parameters can be included in the model to further improve the accuracy in the ozone dose predicted by the model. / Många läkemedelsrester i avloppsreningsverken hamnar i hydrosfären där de kan orsaka negativa effekter på det akvatiska livet och högre ekosystem. Genom att införa ett ozoneringssteg (behandling av ozon) på avloppsreningsverken, kan läkemedelsresterna reduceras. Syftet med det här examensarbetet var att verifiera att ozoneringsprocessen fungerade i fullskala och därmed verifiera en pilotstudie som utfördes år 2014 på Tekniska Verken i Linköping AB (TVAB). Syftet var också att undersöka vilka parametrar som påverkade ozonbehovet för att kunna konstruera en modell för ozonbehovet. De initiala faserna under examensarbetet var en förstudie och en litteraturstudie. Dessa följdes av en multivariat analys och modellkonstruktioner baserat på olika data ifrån pilotstudien. Mätningar på fullskaleanläggningen gjordes också för att hitta nya ozonkonsumerande parametrar. Reduktionen av läkemedelsrester liknande reduktionen under pilotstudien, men var dock något lägre. Flera parametrar och faktorer var värden skiljde sig mellan pilotstudien och fullskala påverkade reduktionen av läkemedelsrester. Till exempel, har DOC och nitratkoncentrationen ökat sedan pilotstudien år 2014. Faktorer så som befolkningsökningen i Linköping och de skillnader som fanns i designen hos de två anläggningarna kan också ha påverkat reduktionen av läkemedelsrester. En kontrollstrategi baserat på ett linjärt samband mellan ozonkänslig ultraviolett absorption (UVA) kvar och kvarvarande läkemedelsrester efter ozonering kan eventuellt användas. Tre modeller konstruerades där Multivariat analys 1 (MVA1)-modellen ansågs vara den bästa. Den här modellen inkluderade ozonresidual, nitrit, turbiditet, simulerad chemical oxygen demand (COD(sim)) och ozondos. Variationerna i dosen jämfört med inputparametrarna för validerade data visade att modellen predikterade ozondosen bra. I framtiden kan andra intressanta parameter inkluderas i modellen för att vidare förbättra trovärdigheten i ozondosen som predikteras av modellen.

Extracellular vesicles (EVs) are small nano-sized particles released constantly by cells into body fluids like blood, saliva, urine, plasma and milk. Depending in the size, EVs are divided into exosomes (30–100 nm), microvesicles (100–1000 nm) and apoptotic bodies (50–5000 nm). During this work, I extracted exosomes from kidney cancer cells using two different centrifugation methods (sequential centrifugation and sucrose gradient ultracentrifugation) under two different conditions (hypoxia and normoxia). The characterization was done by electron microscopy, western blot and nanoparticle tracking analysis (NTA). The effect of exosomes in normal kidney cells was studied in vitro by treating metanephric mesenchyme (MM) cells with exosomes for cell proliferation and motility. The exosomes were injected in chicken embryos in vivo together with renal carcinoma (Renca YFP) cells to see if they have some role in tumor growth. The protein and RNA contents of the exosomes were also analyzed. The cells release more vesicles when they are exposed to hypoxic conditions. Electron microscopy and western blot showed the presence of exosomes expressing CD63 and CD81 markers. Cell proliferation and motility was found to enhance when cells were treated with exosomes. Chicken embryos showed formation of bigger Renca YFP tumor after treatment with exosomes. Different proteins and miRNAs were detected in exosomes which may play active roles in biological processes. In summary, I successfully purified exosomes from kidney cancer cells and characterized them. We concluded that these vesicles play important roles in cell activity and have ability to enhance the tumor growth. The presence of proteins and miRNAs suggest the potential role in cellular communication.

In previous studies we have found that urinary fibrinogen-derived peptides are potential tumor markers for renal cell carcinoma. These peptides occur at low concentrations in urine. Identification of a low-abundant tumor marker requires optimal sample preparation and a highly sensitive analyzer. In this work different chromatographic and mass spectrometric methods were compared and evaluated for tumor marker discovery. We used urine samples from patients with renal cell carcinoma and prostate cancer. Our main targets were peptides derived from fibrinogen beta with unknown sequence that are produced by differential proteolysis. With our optimized workflow we discovered 26 fibrinogen beta derived peptides that have not been identified in urine previously.

Orientador: Sandra Regina Pombeiro Sponchiado / Banca: Iracilda Zeppone Carlos / Banca: Valdecir Farias Ximenes / Banca: Jairo Kenupp Bastos / Dagmar Ruth Stach Machado / Resumo: Do fungo Aspergillus nidulans foi isolado um mutante que apresenta producao aumentada do pigmento melanina. Este pigmento, formado pela polimerizacao oxidativa de compostos fenolicos e/ou indolicos, pode estar presente na parede celular ou no meio de cultura. Embora nao seja essencial para o crescimento dos fungos, a melanizacao parece aumentar a capacidade de sobrevivencia das especies em condicoes desfavoraveis, fornecendo protecao contra radiacao UV, altas temperaturas, enzimas hidroliticas, metais pesados e agentes oxidantes. A obtencao deste pigmento, na forma pura, tem despertado interesse biotecnologico devido a sua utilizacao em formulacoes cosmeticas, principalmente pela sua propriedade fotoprotetora e antioxidante. Tendo em vista o potencial farmacologico do pigmento melanina, este trabalho tem como objetivos fazer a caracterizacao estrutural e funcional do pigmento extraido do fungo A. nidulans. Os resultados de espectrometria de massas indicam que a estrutura da melanina do fungo A. nidulans e composta de duas grandes unidades, representadas pelos fragmentos de massas moleculares igual a 573 e 550, com grupamentos diidroxindol e aneis pirrolicos substituidos. Os ensaios de citotoxicidade mostram que a melanina extraida do fungo A. nidulans nao causa danos significativos aos componentes celulares, tanto antes como pos metabolizacao. Os ensaios de mutagenicidade sugerem que a melanina nao apresenta propriedades mutagenicas para as linhagens TA 97a, TA 98, TA 100 e TA 102 de Salmonella thyphimurium. No teste antimicrobiano, observase que a melanina do fungo nao apresenta potencial como antibacteriano frente a Staphylococcus aureus, Escherichia coli e Enterococcus faecalis, quando comparadas com as substancias de referencia, ampicilina e cloranfenicol. Quanto... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: A mutant that presents increased production of the melanin pigment was isolated from the Aspergillus nidulans fungus. This pigment, formed by the oxidative polymerization of phenolic and/or indolic compounds, can be present in the cellular wall or culture medium. Although it is not essential for the fungal growth, melanization seems to increase the survival capacity of the species in unfavorable conditions, supplying protections against UV radiation, high temperatures, hydrolytic enzymes, heavy metals and oxidants agents. The attainment of this pigment, in its pure form, has taken biotechnological interest due to its use in cosmetic formulations, mainly for its photoprotector and antioxidant property .Considering the pharmacological potential of the melanin pigment, this study aims to make the structural and functional characterization of the pigment extracted from A. nidulans fungus. The results of mass spectrometry indicate that the melanin structure of the A. nidulans fungus is composed of two large units, represented by the fragments of molecular mass equal to 573 and 550, with dihydroxyindole groups and substituted pyrrolic rings. The cytotoxicity assays have shown that the melanin extracted from the A. nidulans fungus does not cause significant damages to the cellular components, before and after metabolization. The mutagenicity assays suggest that the melanin does not present mutagenic properties for the strains TA 97a, TA 98, TA 100 and TA 102 of Salmonella thyphimurium. In the antimicrobial test, it is observed that the fungal melanin does not present antibacterial potential facing Staphylococcus aureus, Enterococcus faecalis and Escherichia coli, when compared to the reference substances, ampicilin and cloranphenicol. Regarding the immunomodulatory activity, it was verified that the melanin does not stimulate NO release... (Complete abstract click electronic access below) / Doutor

Resumo: Cepas bacterianas resistentes aos antibióticos convencionais representam um problema de saúde global com forte impacto social e econômico. Desta maneira, há uma urgente necessidade de desenvolver antibióticos com novos mecanismos de ação. O grupo da Profª Drª Mariana S. Castro isolou e determinou a seqüência do peptídeo GWLDVAKKIGKAAFNVAKNFI-NH2, secretado na pele da rã Hypsiboas albopunctatus e qual mostrou atividade antimicrobiana. O objetivo do presente trabalho foi avaliar 3 análogos para obter informações a respeito da relação estrutura-atividade biológica. Os peptídeos foram sintetizados por SPFS utilizando a estratégia química Fmoc. As propriedades conformacionais dos peptídeos foram investigadas por CD em água, em TFE (agente indutor de estrutura secundária) e em micelas zwitteriônicas (LPC). As atividades antimicrobianas foram analisadas pela medida da concentração inibitória mínima em bactérias (uma Gram-positiva e duas Gram- negativa) e em fungos. O aminoácido triptofano foi usado como sonda para analisar a profundidade da inserção em miméticos de membrana. Os resultados mostraram que a SPFS foi útil, obtendo um material com alto índice de pureza. Os estudos de CD demonstraram que os peptídeos em água apresentam uma estrutura ao acaso, adquirindo um alto teor de a-hélice na presença de TFE e LPC. As interações peptídeo/miméticos de membranas indicaram que todos interagiram fortemente com as micelas, mas não com vesículas, com exceção do peptídeo "wild type", que mostrou forte interação com ambos os modelos. Os resultados biológicos demonstraram que todos os peptídeos tem atividade (antibacteriana e antifúngica), porém, o "wild type" mostrou valores mais baixos de CIM. Além disso, nossos resultados sugerem que os peptídeos Hy- D-V 16, Hy-D-V 5,16 e Hy-K9 podem vir a ser potenciais modelos para o desenvolvimento de novas... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. Castro's group isolated and determined the sequence of the peptide GWLDVAKKIGKAAFNVAKNFI-NH2 of skin secretion from the frog Hypsiboas albopunctatus which showed antimicrobial activity. The aim of the present work was evaluated 3 analogues to supply information about the relationship structurebiological activity. The peptides were synthesized by SPPS using the Fmoc chemical approach. The conformational properties of the peptides were investigated by CD techniques in water, TFE (secondary structure-inducing agent) and in zwitterionic micelles (LPC). The antimicrobial activity was assayed by measuring growth inhibition of bacteria (one Gram-positive and two Gram-negative) and fungus. The tryptophan amino acid was used as probe to analyze the deeply of insertion in membrane mimetic. The result showed that the SPFS proved to be useful, and material with high purity was obtained. The CD studies demonstrated that peptides in water have random coil structure, acquiring high amount of a-helix in the presence of TFE and LPC. The interactions peptide/mimetic of membranes indicated that all strongly interacted with micelles, but not with vesicles, with the exception of "wild type" peptide, that showed strong interaction with the both models. The biological results demonstrated that all peptides have activity (antibacterial and antifungal), nevertheless, the "wild type" showed lower MIC values. In addition, our findings suggest that peptides Hy-D-V 16, Hy-D-V 5,16 and Hy-K9 as potential templates for the development of new drugs, therefore these analogous have the higher relationshipantimicrobial/hemolytic activity. These findings highlight the importance of single a-amino acid... (Complete abstract click electronic access below) / Orientador: Eduardo Maffud Cilli / Coorientador: Reinaldo Marchetto / Banca: Dulce Helena Siqueira Silva / Banca: Mariana de Souza Castro / Mestre