Pin1 is an enzyme central to cell signaling pathways because it catalyzes the cis–trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. This regulatory function makes Pin1 a drug target in the treatment of various diseases. The effects of phosphorylation on Pin1 substrates and the basis for Pin1 recognition are not well understood. The conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 were determined using molecular dynamics simulations. Phosphorylation perturbs the backbone conformational space of Pin1 substrate analogues. It is also shown that Pin1 recognizes specific conformations of its substrate by conformational selection. Dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme–substrate complex when the substrate is in the transition state configuration. This suggests that these motions play a significant role during catalysis. These results provide a detailed mechanistic understanding of Pin1 substrate recognition that can be exploited for drug design purposes and further our understanding of the subtleties of post-translational phosphorylation and cis–trans isomerization. Results from accelerated molecular dynamics simulations indicate that catalysis occurs along a restricted path of the backbone configuration of the substrate, selecting specific subpopulations of the conformational space of the substrate in the active site of Pin1. The simulations show that the enzyme–substrate interactions are coupled to the state of the prolyl peptide bond during catalysis. The transition-state configuration of the substrate binds better than the cis and trans states to the catalytic domain of Pin1. This suggests that Pin1 catalyzes its substrate by noncovalently stabilizing the transition state. These results suggest an atomistic detail understanding of the catalytic mechanism of Pin1 that is necessary for the design of novel inhibitors and the treatment of several diseases. Additionally, a set of constant force biased molecular dynamics simulations are presented to explore the kinetic properties of a Pin1 substrate and its unphosphorylated analogue. The simulations indicate that the phosphorylated Pin1 substrate isomerizes slower than the unphosphorylated analogue. This is due to the lower diffusion constant for the phosphorylated Pin1 substrate.

ABSTRACT Covalent Immobilization of Glucose Isomerase on Poly (2-hydroxyethyl methacrylate) Particles Yildiz, Hakan &Uuml / mit M.S., Department of Chemistry Supervisor: Prof. Dr. Nesrin Hasirci July 2004, 54 pages In this study, poly (2-hydroxyethyl methacrylate), P(HEMA), particles were prepared by suspension polymerization of the monomer 2-hydroxyethyl methacrylate with addition of ethylene glycol dimethyacrylate, EGDMA, as cross linker. Glucose isomerase, GI, enzyme was covalently immobilized on the prepared P(HEMA) particles after activation of the particles with cyanuric chloride. The activities of the free and immobilized enzymes were measured with Ethanol-Carbazole method. The immobilization of GI on P(HEMA) particles promoted enzyme stability and as a result, the enzyme became more stable to temperature, storage, and reuse. For maximum substrate conversion, optimum temperature was determined as 70 oC for free GI and this value shifted to 60 oC for immobilized enzyme. Optimum pH for maximum substrate conversion was found to be 7.0 for free GI and 8.0 for immobilized GI. The change of enzyme activity with substrate concentration were determined to calculate Km and Vmax values of the free and immobilized enzymes. Km values were found to be 1.7x10-2 mol/L and 3.1x10-1 mol/L while Vmax values were 1.01x10-4 mol/L.min, 1.65x10-3 mol/L.min for free and immobilized GI, respectively. Reuse capability of immobilized GI on P(HEMA) particles was measured and compared with commercial GI. Both systems retained 80 % of their original activities after 40th use, within 6 days. The change of enzyme activities upon storage were detected at certain time intervals for the samples stored in buffer solution at 4 oC. Immobilized enzyme was retained 60% of its original activitiy in 60 days of storage at 4 oC. Immobilized GI and commercial GI both retained 90% of their activities under continuous flow after 180 mL of substrate solution passed through the column.

Glycolysis is the main metabolic route in Saccharomyces cerevisiae and it is the sequence of enzyme catalyzed reactions that oxidatively convert glucose to pyruvic acid in the yeast cytoplasm. In addition to the basic steps, glycolysis involves branch points providing the intermediary building blocks of the cell (i.e amino acids and nucleotides). One of these pathways is glucose-6-phosphate branch point which is a junction of glycolytic pathway and pentose phosphate pathway. At this point glucose-6-phosphate can be converted to fructose-6-phosphate a metabolite of glycolytic pathway by phosphoglucoisomerase or it can be dehydrogenated to 6-phosphogluconolactone by glucose-6-phosphate dehydrogenase which is the first enzyme of the pentose phosphate pathway. In this study, the influence of different nitrogen sources on the flux distribution through the pentose phosphate pathway and glycolysis in Saccharomyces cerevisiae was examined. For this purpose, four different compositions of nitrogen sources were used in growth media. The growth medium contained one of the following composition of nitrogen sources / only ammonium sulfate, only yeast nitrogen base, ammonium sulfate and histidine, yeast nitrogen base and histidine. Histidine was added because its synthesis branches from pentose phosphate pathway. In order to analyse the effect of the different compositions of nitrogen sources on the physiology of the yeast, specific activities of hexokinase, phosphoglucose isomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase enzymes were measured in the crude extracts of the biomass samples taken in the late exponential phase of the cultures. Addition of histidine caused an increase in the specific activities of all the enzymes analysed in medium containing ammonium sulfate. The specific activity of hexokinase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase in medium containing yeast nitrogen base and histidine were higher than medium containing yeast nitrogen base. However, the specific activity of 6-phosphogluconate dehydrogenase decreased 3.1% in medium containing yeast nitrogen base and histidine medium with respect to medium with only yeast nitrogen base. The OD value and dry weight in the culture containing histidine aminoacid was higher than the cultures contaning only ammonium sulfate and only yeast nitrogen base. Also the period of the exponential phase was shorter in medium containing ammonium sulfate and histidine and yeast nitrogen base and histidine than medium only ammonium sulfate and only yeast nitrogen base.

7,8-Linoleate diol synthase (7,8-LDS) of the take-all pathogen of wheat, Gaeumannomyces graminis, converts linoleic acid to 8R-hydroperoxyoctadecadienoic acid (8-HPODE) by 8-dioxygenase activity (8-DOX), and further isomerizes the hydroperoxide to 7S,8S-dihydroxyoctadecadienoic acid (7,8-DiHODE) by hydroperoxide isomerase activity. Sequence alignment showed homology to prostaglandin H synthase (PGHS), and both enzymes share structural and catalytic properties. The 8-DOX of 7,8-LDS was successfully expressed in Pichia pastoris and in insect cells (Sf21). Site-directed mutagenesis confirmed His379 as the proximal heme ligand and Tyr376 as a residue, which forms a tyrosyl radical and initiates catalysis. Furthermore, mutagenesis suggested His203 could be the proposed distal histidine, and Tyr329 of catalytic relevance for substrate positioning at the active site. Aspergilli are ubiquitous environmental fungi. Some species, in particular Aspergillus fumigatus, are responsible for invasive aspergillosis, which is a life-threatening disease for immunocompromised patients. A. fumigatus and A. nidulans metabolized linoleic acid to 8R-HPODE, 10R-hydroperoxyoctadecadienoic acid (10R-HPODE), 5S,8R-dihydroxyoctadecadienoic acid, and 8R,11S-dihydroxyoctadecadienoic acid. When the genomes of certain Aspergilli strains were published, several species showed at least three homologous genes (ppoA, ppoB, ppoC- psi producing oxygenases) to 7,8-LDS and PGHS. Gene deletion identified PpoA as an enzyme with 8-DOX and 5,8-hydroperoxide isomerase activities, designated 5,8-LDS in homology to 7,8-LDS. In the same way, PpoC was identified as a 10-dioxygenase (10-DOX), which converts linoleic acid to 10R-HPODE. 10-DOX differs from LDS, since it dioxygenates linoleic acid at C-10, after hydrogen abstraction at C-8 and double bond migration. 10-DOX was cloned and expressed in insect cells. Leu384 and Val388 were found to be critical for dioxygenation at C-10. Mutation to the homologous residues of 5,8- and 7,8-LDS (Leu384Val, Val388Leu) increased oxygen insertion at C-8. LDS and 10-DOX are fusion proteins with a dioxygenase and a hydroperoxide isomerase (cytochrome P450) domain with a cysteine heme ligand. The P450 domain of 10-DOX lacked the crucial cysteine heme ligand and was without hydroperoxide isomerase activity. LDSs and 10-DOX are newly characterized heme containing fungal dioxygenases, with homology to PGHS of vertebrates. Their metabolites regulate reproduction, development, and act as signal molecules with the host after pathogen attack.

Protein aggregation is commonly encountered during the manufacture of protein-based bioproducts in processing such as protein expression, purification, refolding, shipping and storage (Volkin and Middaugh, 1992; Brange, 2000). Aggregation may shorten the shelf-life of pharmaceutical proteins (Frokjaer and Otzen, 2005) and induce severe hypersensitivity (Rosenberg, 2006). In addition, several diseases ranging from Alzheimer’s disease to cystic fibrosis are associated with protein aggregation in the form of amyloid fibrils and plaques (Dobson, 1999; Luheshi et al., 2008). Hence, studies on protein aggregation, especially those dealing with high concentrations of proteins, are highly demanded in both academic and industrial laboratories. To address the aforementioned issues, physicochemical factors affecting protein aggregation were investigated systematically in this project. Strategies were developed to inhibit protein aggregation during renaturation and to enhance protein stability against aggregation during and after production, especially when dealing with high protein concentrations. ∆5-3-Ketosteroid isomerase (KSI) was used as a model for aggregation studies during protein renaturation due to its intrinsic aggregation properties. KSI was overexpressed as inclusion bodies (IBs) in Escherichia coli (E. coli). Cost- and time-efficient combination of chemical extraction and one-step affinity purification ensured the production of denatured KSI with high purity at high yield. Several key factors, including protein concentration and ionic strength, were determined to greatly influence KSI aggregation during renaturation. Polymer addition (PEG 3000 and Eudragit S-100) was found to alter KSI aggregation behaviour in a polymer-specific manner, as quantified using reversed phase-high performance liquid chromatography (RP-HPLC) analysis. Light scattering for second virial coefficient (SVC) measurement, surface plasmon resonance (SPR), and microfluidics were applied to study the fundamental mechanism of protein aggregation. Lysozyme was further introduced as a control protein for comparison with KSI. A rapid lumped method was established to measure specific refractive index (∂n/∂c) and SVC values for KSI and lysozyme, which provided quantitative and qualitative information on thermodynamic interactions of molecules in solution. SPR and microfluidics were also used to explore protein aggregation properties. To our best knowledge, it is the first time SPR and microfluidics have been used to investigate protein aggregation behaviour. Both SPR and microfluidics present significant potential for assessing protein aggregation and diagnosis or drug screening of protein aggregation related diseases. The chemical and physical stability of proteins needs to be maintained after successful refolding to ensure an acceptably long shelf life, especially at high protein concentration (Chang and Hermsdorf, 2002). The pharmaceutical effects of lectins on cell growth provided incentive for studies to improve their stability. Human galectin-2 (hGal-2, a homodimeric lectin) was used as a study model in this project. Mutations were introduced at one of the two Cys residues (C57A, C57M, and C57S). Only the C57M variant was highly expressed in bacteria in soluble form. No aggregate of this mutant was detected during 3 weeks of storage. hGal-2 C57M also facilitated site-directed introduction of poly(ethylene glycol) (PEG) into the remaining sulfhydryl group (Cys75). Product analysis revealed rather complete conjugation with one PEG chain per protein subunit in homodimer. Neither secondary structure alteration nor the absence of binding ability to a glycoprotein (asialofetuin) was observed. The results document the feasibility of tailoring a human galectin for enhanced stability against aggregation as well as monoPEGylation, which enables further testing of biological properties including functionality as a growth regulator and the serum clearance rate of hGal-2.

Thesis (M.Sc.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Cell Biology. Title from pdf file main screen (viewed on October 24, 2009). Includes bibliographical references.

The amino acid proline is unique – within a polypeptide chain, proline adopts either a cis or trans peptide bond conformation while all other amino acids are sterically bound primarily in the trans configuration. In proteins, the isomeric state of a single proline can have dramatic consequences on structure and function. Consequently, cis-trans interconversion confers both barrier and opportunity – on one hand, isomerization is a rate limiting step in de novo protein folding and on the other can be utilized as a post-translational regulatory switch. Peptidyl-prolyl isomerases (PPIs) are a ubiquitous superfamily that catalyzes the interconversion between conformers. Although pervasive, the functions and substrates of most PPIs are unknown. The two largest subfamilies, FKBPs and cyclophilins, are the intracellular receptors of clinically relevant immunosuppressant drugs that also show promise in the treatment of neurodegenerative disorders and cancer. Therefore, narrowing the knowledge gap has significant potential to benefit human health. FKBP25 is a high-affinity binder of the PPI inhibitor rapamycin and is one of few nuclear-localized isomerases. While it has been shown to bind DNA and associate with chromatin, its function has remained largely uncharacterized. I hypothesized that FKBP25 targets prolines in nuclear proteins to regulate chromatin-templated processes. To explore this, I performed high-throughput transcriptomic and proteomic studies followed by detailed molecular characterizations of FKBP25’s function. Here, I discover that FKBP25 is a multifunctional protein required for the maintenance of genomic stability. In Chapter 2, I characterize the unique N-terminal Basic Tilted Helical Bundle (BTHB) domain of FKBP25 as a novel dsRNA binding module that recruits FKBP25’s prolyl isomerase activity to pre-ribosomal particles in the nucleolus. In Chapter 3, I show for the first time that FKBP25 associates with the mitotic spindle apparatus and acts to stabilize the microtubule cytoskeleton. In this chapter, I also present evidence that this function influences the stress response, cell cycle, and chromosomal stability. Additionally, I characterize the regulation of FKBP25’s localization and nucleic acid binding activity throughout the cell cycle. Finally, in Chapter 4, I uncover a role for FKBP25 in the repair of DNA double-stranded breaks. Importantly, this function requires FKBP25’s catalytic activity, identifying for the first time a functional requirement for cis-trans prolyl isomerization by FKBP25. Collectively, this work identifies FBKP25 as a multifunctional protein that is required for the maintenance of genomic stability. The knowledge gained contributes to the exploration of PPIs as important drug targets. / Graduate

Cysteine rich with EGF-like domains 2 (Creld2) is a novel endoplasmic reticulum (ER) resident molecular chaperone that has been recently implicated in the ER stress signalling response (ERSS) and the unfolded protein response (UPR). Global transcriptomic data derived from in vivo mouse models of rare chondrodysplasias; Multiple Epiphyseal Dysplasia (MED Matn3 p.V194D) and Metaphyseal chondrodysplasia type Schmid (MCDS Col10a1 p.N617K), identified a significant upregulation in Creld2 expression in mutant chondrocytes. These chondrodysplasias share a common disease signature consisting of aberrant folding of a matrix component often as a result of inappropriate alignment of intramolecular disulphide bonds. This in turn culminates in toxic protein aggregation, intracellular retention mutant polypeptides and a classical ER stress response. The aim of this study was to further analyse the function of Creld2 in cartilage development and chondrodysplasias in which endochondral bone growth is perturbed. Protein disulphide isomerases (PDIAs) were amongst the most up-regulated genes in the MED and MCDS mouse models, consistent with the prolonged exposure of normally 'buried' cysteine residues. This led to the hypothesis that Creld2 was functioning as a novel PDI-like oxidoreductase to assist in the correct folding and maturation of aggregated misfolded polypeptide chains through REDOX regulated thiol disulphide exchange. A series of Creld2-CXXA substrate trapping mutants were generated in order to determine whether Creld2 possessed inherent isomerase activity. Here potential substrates interacting with Creld2 were 'trapped' as mixed disulphide intermediates, then isolated by immunoprecipitation and identified by mass spectrometry analysis. It was demonstrated that Creld2 possessed a catalytic active CXXC motif in its N-terminus that enabled the molecular chaperone to participate in REDOX regulated thiol disulphide exchange with at least 20 potential substrates including; laminin (alpha3,β3,γ2), thrombospondin 1, integrin alpha3 and type VI collagen. There was also numerous co-chaperones and foldases thought to be part of a specialised protein-protein interactome (PPI) for folding nascent polypeptides translocating the ER lumen. Moreover, co-immunoprecipitation experiments supported a protein-protein interaction between Creld2 and mutant matrilin-3, thereby inferring a potential chondro-protective role in resolving non-native disulphide bonded aggregates in MED. An established biochemical approach was employed to test the hypothesis that all MATN3-MED disease causing mutations have a generic cellular response to the β-sheet V194D mutation, consisting of intracellular retention, protein aggregation and ER stress induction. Several missense mutations were selected for analyses which encompassed a spectrum of disease severity and included examples of both β-sheet and alpha helical mutations. It was possible to define a reliable and reproducible assay for categorising MATN3 missense mutations into pathological or benign based on these basic parameters. This study was extended further to determine whether there were common pathological mechanisms behind MED and Bethlem myopathy (BM) caused by missense mutations in von Willebrand Factor A domain (vWF-A) containing proteins (matrilin-3 and type VI collagen respectively). We chose to compare and contrast the effects of an archetypal MATN3-MED causing mutation (R121W) with the equivalent COL6A2-BM causing mutation (R876H). These mutations compromised protein folding and maturation, resulting in the familiar disease profile of intracellular retention, protein aggregation and an ER stress response in an artificial overexpression system. However, the mutant C2 domain was efficiently targeted for degradation whilst mutant matrilin-3 vWF-A domain appeared to be resistant to these molecular processes.Molecular genetics was employed to study the role of Creld2 in vivo. Creld2-/- null mice (both global and conditional) were generated to directly examine the role of Creld2 in endochondral bone growth. Global knock-out mice were viable with no overt phenotype at birth. However, female Creld2-/- null mice showed a significant reduction in body weight and tibia bone length at 3 weeks of age. A cartilage specific knock-out was generated to determine whether these skeletal abnormalities were attributed to a systemic or a direct effect on cartilage development. [Creld2Flox/Flox Col2Cre (+)] demonstrated a severe chondrodysplasia with significantly reduced body weight and long bone growth compared to control littermates. Morphological and histochemical analysis of mutant growth plates revealed gross disorganisation of the chondrocyte columns with extensive regions of hypocellularity. These pathological features were confirmed to be the result of reduced chondrocyte proliferation and increased/spatially dysregulated apoptosis throughout all zones of differentiation. Taken together, these data provide evidence that Creld2 possesses isomerase activity and exhibits distinct substrate specificity. Furthermore, Creld2 has a fundamental role in post-natal cartilage development and chondrocyte differentiation in the growth plate.

Made available in DSpace on 2016-06-02T19:56:52Z (GMT). No. of bitstreams: 1 5338.pdf: 2812584 bytes, checksum: c4ea2f65f591bec0e6ad7105c6ec591e (MD5) Previous issue date: 2013-06-20 / Universidade Federal de Minas Gerais / In this work, it was studied the simultaneous isomerization and fermentation of xylose to ethanol (SIF) using xylose isomerase (XI) and S. cerevisiae co-immobilized in calcium alginate gel. XI was immobilized on chitosan gel activated with glutaraldehyde (IXI-Ch). The influence of the concentration of enzyme/yeast in the reactor, the pH, temperature and yeast strain on yield and selectivity in ethanol was studied. The concentrations of enzyme and yeast in the reactor were varied by changing the mass of IXI-Ch and yeast per gram of alginate solution, maintaining fixed the ratios of biocatalyst weight: volume of medium in the reactor (1:1). The SIFs were carried out in batch with xylose (65g.L-1), antibiotics and other salts. The first experiment, with 16% Itaiquara® yeast and 5% enzyme biocatalyst (% wenzyme or yeast/wbiocatalyst) showed that pH drop occurred during the test, preventing full conversion of xylose, due to reduced enzyme activity. calcium carbonate (0.5-1.0%) was then included in the biocatalyst, which maintained the pH between 5,2 to 5,6, allowing complete conversion of the sugar at all concentrations tested (%Yeast -Enzyme in biocatalyst): 5-20, 17-5, and 10 yeast (Itaiquara ®) with 5, 10 and 20-. The maximum ethanol productivity, 2,44 ± 0,26g.L-1.h- 1 was obtained for the highest cell concentration and the highest selectivity ethanol/xylitol, 2,57 ± 0.4 and 2,42 ± 0,01 for the highest enzyme concentrations (10 and 20% with 10% yeast). These results indicated that the highest concentration of xylulose favored more selectivity to ethanol. Fermentation was then performed using no enzyme in biocatalyst with a prior isomerized syrup concentrated in xylulose containing 58g.L-1 xylulose and 9g.L-1 xylose and another with xylose only. At first, xylulose was completely assimilated in 5 hours, xylose was barely consumed in both assays, and ethanol selectivity was lower than that obtained in the SIF tests. Xylitol show thus to be produced mainly from xylulose and selectivity contrary to expectations did not directly increase with increasing xylulose concentration, indicating that the formation of ethanol/xylitol depends not only on external xylulose, and it is probably finely regulated in yeast. The concentrations of enzyme and yeast 20 and 10% (equivalent to 100gderived.L-1 reactor and 50gwd.L-1 reactor) were selected as the best, which were used to study the influence of pH and temperature, and also different strains. The increase of initial pH from 5.6 to 6.5 did not improve the productivity, yield, neither selectivity in ethanol. Temperatures tested for Itaiquara ® were 32, 35 and 37 ° C, and for industrial strains CAT-1 and BG-1: 32, 37 and 40 ° C. Viability remained above 90% for all assays at 24 hours. All three strains showed increased selectivity in ethanol with temperature reduction, obtaining the maximum selectivity for industrial strains (3,06 ± 0,24 - CAT-1 and 3,19 ± 0,11 BG-1) with yield and productivity equal or greater than those obtained in higher temperatures. At 32 ° C and pH 5.6, Itaiquara ® showed lower conversion time, but lower selectivity, while the BG-1, demonstrated the highest selectivity, but low conversion and productivity. The strain CAT-1 combines high productivity, 2,17 ± 0,17 g.L-1.h-1, and selectivity, 3,06 ± 0,24, with 90% conversion in 9 hours, 32 ° C, which is apparently the best performance among the tested yeasts. The results were very promising, indicating the technical feasibility of producing ethanol from xylose with the biocatalyst developed. / Neste trabalho foi estudada a simultânea isomerização e fermentação de xilose a etanol (SIF) usando xilose isomerase (XI) e S. cerevisiae coimobilizadas em gel de alginato de cálcio. XI foi imobilizada em gel de quitosana ativado com glutaraldeido (IXI-Ch).. Foram estudadas as influências das concentrações de enzima/levedura no reator, do pH, da temperatura e da linhagem de levedura na produtividade e na seletividade em etanol. As concentrações de enzima e levedura no reator foram variadas mudando-se a massa de IXI-Ch e levedura por grama de solução de alginato, mantendo-se fixas as proporções 1:1 massa de biocatalisador:volume de meio no reator. As SIFs foram realizadas em batelada com xilose (~65g.L-1), antibiótico e outros sais. O primeiro experimento realizado, biocatalisador com 16% levedura Itaiquara® e 5% enzima (% menzima ou levedura/mbiocatalisador), mostrou que ocorria queda de pH durante o ensaio, impedindo conversão total da xilose, devido à redução da atividade enzimática. Foi incluído carbonato de cálcio 0,5-1,0% no biocatalisador, o que manteve o pH entre 5,2-5,6, permitindo total conversão do açúcar, em todas as concentrações testadas (%Levedura-Enzima no biocatalisador): 5-20, 17-5 e 10 levedura (Itaiquara®)com 5, 10 e 20% enzima. A máxima produtividade em etanol, 2,44 ± 0,26g.L-1.h-1, foi obtida para a mais alta concentração celular e a mais alta seletividade etanol/xilitol, 2,57± 0,4 e 2,42± 0,01, para as mais altas concentrações de enzima (10 e 20% com 10% levedura). Esses resultados indicavam que quanto mais alta a concentração de xilulose, mais favorecida a seletividade em etanol. Foi então realizada uma fermentação usando o biocatalisador sem enzima, com um xarope previamente isomerizado e concentrado em xilulose contendo 58g/L de xilulose e 9g/L xilose e outro apenas com xilose. No primeiro, xilulose foi totalmente assimilada em 5 horas, xilose foi pouco consumida nos dois ensaios, e a seletividade em etanol foi menor que a obtida nos ensaios SIF. Xilitol mostrou, assim, ser produzido majoritariamente a partir de xilulose e contrariamente ao esperado a seletividade não aumenta diretamente com o aumento da concentração de xilulose, indicando que o metabolismo etanol/xilitol não depende apenas da concentração externa de xilulose, devendo ser finamente regulado dentro da levedura. Selecionaram-se as concentrações de enzima e levedura de 20 e 10% (equivalente a 100gderivado.L-1 reator e 50gms.L-1 reator) como as melhores, as quais foram utilizadas para estudo da influência do pH e da temperatura e ainda de diferentes linhagens. O aumento do pH inicial do meio de 5,6 para 6,5 não favoreceu a produtividade, rendimento e nem a seletividade em etanol. As temperaturas testadas para Itaiquara® foram: 32, 35 e 37°C; e para as linhagens industriais CAT-1 e BG-1: 32, 37 e 40°C. A viabilidade manteve-se acima de 90% para todos os ensaios em 24 horas. As três linhagens mostraram aumento da seletividade em etanol com a redução da temperatura, obtendo-se a máxima seletividade para as linhagens industriais (3,06± 0,24- CAT-1 e 3,19± 0,11 BG-1), com rendimento e produtividade iguais ou maiores que os obtidos nas temperaturas maiores. A 32°C e pH 5,6, Itaiquara® apresentou menor tempo de conversão, mas a menor seletividade, já a BG-1, obteve maior seletividade, mas baixa conversão, rendimento e produtividade. A linhagem CAT-1 alia alta produtividade, 2,17 ± 0,17 (g.L-1.h-1), e seletividade, 3,06 ± 0,24, com 90% de conversão em 9 horas, 32°C, sendo aparentemente a de melhor desempenho dentre as testadas. Os resultados foram muito promissores, indicando viabilidade técnica de produção de etanol a partir de xilose com o biocatalisador desenvolvido.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Anna Zolkiewska / Cancer cells exploit key signaling pathways in order to survive, proliferate, and metastasize. Understanding the intricacies of the aberrant signaling in cancer may provide new insight into how to therapeutically target tumor cells. The goal of my research was to explore the role of two modulators of transmembrane signaling, the secretory pathway and cell surface proteolysis, in the aggressiveness of breast cancer cells. To study the role of the secretory pathway, I focused on the family of endoplasmic reticulum (ER) chaperones. I found that several ER chaperones were upregulated in breast cancer cells grown under anchorage-independent conditions as mammospheres versus those grown under adherent conditions. Furthermore, certain members of the protein disulfide isomerase (PDI) family were consistently upregulated in two different cell lines at both the mRNA and protein levels. Knocking down these PDIs decreased the ability of the cells to form mammospheres. I demonstrated that the requirement for PDI chaperones in mammosphere growth is likely due to an increased flux of extracellular matrix (ECM) components through the ER. Next, I examined the role of cell surface proteolysis in modulating the aggressiveness of breast cancer cells. Cell-surface metalloproteases release soluble growth factors from cells and activate the corresponding growth factor receptors. I determined that specific metalloproteases (ADAM9 or ADAM12), modulate the activation of Epidermal Growth Factor Receptor (EGFR). I demonstrated that EGFR activation enhances the CD44⁺/CD24⁻ cell surface marker profile, which is a measure of cancer cell aggressiveness. I found that the MEK/ERK pathway, which is a downstream effector of EGFR activation, modulates the CD44⁺/CD24⁻ phenotype. When DUSP4, a negative regulator of the MEK/ERK pathway, is lost, activation of EGFR by metalloproteases no longer plays a significant role in cancer cell aggressiveness. This indicates that the ligand dependent activation of the EGFR/MEK/ERK pathway is a critical step in DUSP4-positive aggressive breast cancer. Finally, I examined the importance of metalloproteases in the regulation of Programmed-death ligand 1 (PD-L1), a transmembrane protein expressed by some cancer cells that plays a major role in suppressing the immune system. I demonstrated that cell-surface metalloproteases have the ability to cleave PD-L1 and release its receptor-binding domain to the extracellular environment. Collectively, these data indicate that (a) ER chaperones support anchorage-independent cell growth, (b) metalloproteases are important in regulation of an aggressive phenotype through the EGFR/MEK/ERK pathway, and (c) metalloproteases cleave PD-L1, a key component of immunosuppression in cancer.