The apoplastic enzyme, ascorbate oxidase (AO), is a blue copper oxidase that catalyses oxidation of ascorbate (AsA) to monodehydroascorbate (MDHA). In Arabidopsis thaliana, AO is encoded by three genes (At4g39830, At5g21105 and At5g21100) designated AO1, AO2, and AO3 respectively. Since AsA is the most abundant antioxidant in the apoplast and AO is active in this compartment, the regulation of apoplastic AsA redox status by AO and its role in development and environmental perturbations has become a subject of interest. Phylogenetic analysis showed that AO is present in higher plants, pteridophytes, mosses and green algae. Amino acid sequence analysis showed that AO2 and AO3 shared higher sequence identity than AO1. In silico analyses found that AO1 had a distinct expression pattern and subcellular localisation compared to AO2 and AO3, suggesting AO1 might be involved in alternative functions. Consistent with previous studies, AO activity was high in actively growing tissue of wild-type (WT) A. thaliana, supporting a possible role of AO in cell expansion. ao1, ao3 and ao1ao3 T-DNA insertion mutants were characterised. ao1 had similar level of AO activity to WT, while ao3 and ao1ao3 had 10-20% of WT AO activity. Compared with WT, these T-DNA insertion mutants did not show any phenotypic differences under unstressed or stressed (high light and drought) growth conditions. An artificial microRNA construct (amiR-AO) to silence all three AO genes was developed. Also, an overexpression plasmid (35S::AO3) harbouring AO3 gene was constructed. These constructs were used to transform A. thaliana. AO activity was undetectable in the amiR-AO line, while the 35S::AO3 line had 3-fold higher AO activity than the WT. Under unstressed normal growth conditions, the amiR-AO line had bigger rosette size, whereas the 35S::AO3 line exhibited early flowering and smaller number of rosette leaves. The amiR-AO line accumulated more anthocyanin and AsA than WT when acclimated to high light, whereas the 35S::AO3 line accumulated less anthocyanin than WT. In response to drought, the amiR-AO line did not show phenotypic differences compared to WT, while the 35::AO3 line had higher rate of leaf water loss and appeared to have greater sensitivity to drought. These results suggest that AO perturbation could, to some extent, affect the growth and stress response of A. thaliana although the effect is small.

Submitted by Jorge Cativo (jorge.cativo@inpa.gov.br) on 2018-08-23T13:05:02Z No. of bitstreams: 2 TESE_FINAL_.pdf: 4368762 bytes, checksum: 819cc6a5cad9b495a51a1d1b2a102bf7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-23T13:05:02Z (GMT). No. of bitstreams: 2 TESE_FINAL_.pdf: 4368762 bytes, checksum: 819cc6a5cad9b495a51a1d1b2a102bf7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-23 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Frankliniella schultzei (Trybom, 1910) is a highly polyphagous thrips that has a pest status and a tropical and subtropical distribution. It presents plasticity in body coloration and its two morpho-colors (pale and dark) is an open taxonomic question. Although uncertain, it’s likely that they originate in the Neotropics, as most species of that genus. Recent researches in Kenya and Australia (areas where this species was introduced) indicate that morpho-colors are different species, and that the dark form is actually a complex of species. In this study, nuclear (microsatellites - SSRs) and mitochondrial (Cytochrome Oxidase I - COI) molecular markers were used to estimate the genetic diversity, and to evaluate the population structure of F. schultzei in Brazil. The Near Infrared Spectroscopy (NIRS) was used to discriminate morfo-colors. We found that the clear form of F. schultzei is a different species from the dark one, and that it probably corresponds to a single biological unit in the world. We also inferred the existence of a complex of species within the dark form of F. schultzei in Brazil, in which populations (two species) are living in sympatry and sharing the same hosts. These populations are highly structured, with a higher percentage of variation within than between them, and a low or nonexistent gene flow. Adding haplotype network data into this context, we deduced that the southern region of Brazil should be the most likely center of origin and dispersal of F. schultzei, since it presents composition of samples by more stable and genetically diverse localities. These are the first scientific data produced about the population genetics of F. schultzei in their most likely region of origin – South America / southern Brazil, which are in agreement with the reports for for the species analyzed in Kenya and Australia. / Frankliniella schultzei (Trybom, 1910) é um tripes altamente polífogo, com status de praga e distribuição tropical e subtropical. Apresenta plasticidade na coloração corporal e suas duas morfo-cores (clara e escura) é uma questão taxonômica em aberto. Apesar de incerta, tem provável origem nos Neotrópicos como a maioria das espécies do gênero. Recentes pesquisas, no Quênia e na Austrália (áreas de introdução da espécie), indicam que as morfo-cores são espécies diferentes e que a forma escura é um complexo de espécies. Neste estudo, marcadores moleculares nuclear (microssatélites - SSR) e mitocondrial (Citocromo Oxidase I - COI) foram utilizados com o objetivo de estimar a diversidade genética e avaliar a estrutura populacional de F. schultzei no Brasil e a Espectroscopia no Infravermelho Próximo (NIRS) para a discriminação das morfo-cores. Encontramos que a forma clara de F. schultzei é uma espécie diferente da forma escura e que, provavelmente, corresponda a uma única unidade biológica no mundo. Inferimos também a existência de um complexo de espécies dentro da forma escura de F. schultzei no Brasil, com populações (duas espécies) vivendo em simpatria e compartilhando os mesmos hospedeiros. Estas se encontram altamente estruturadas, com maior porcentagem da variação dentro que entre elas, e com baixo ou inexistente fluxo gênico. Acrescentando dados de rede de haplótipos a este contexto, deduzimos que a região sul do Brasil deve ser o provável centro de origem e dispersão de F. schultzei escura, por apresentar composição de amostras por localidade mais estáveis e mais diversas geneticamente. Estes são os primeiros dados científicos produzidos sobre genética populacional de F. schultzei em sua provável região de origem – América do Sul / região sul do Brasil, os quais estão em consonância com os relatos para a espécie analisados no Quênia e Austrália.

Die EZM bildet ein Netzwerk quervernetzter Proteine, welches alle Zellen im Tumor umgibt. Sie übt direkte Effekte auf die Medikamenteneinbringung und -verteilung aus und somit auch auf die therapeutische Effizienz von Chemotherapeutika. Die LOX(L)-Proteinfamilie katalysiert die oxidative Desaminierung von Lysinresten in Elastin und Kollagenfasern und ermöglicht dadurch eine intra- und intermolekulare Quervernetzung. Diese wird für die Reifung und Stabilisierung der Kollagene in der EZM benötigt. Eine erhöhte LOX(L)-Expression steigert durch eine verstärkte EZM-Quervernetzung die Gewebesteifheit im Tumor und bildet so eine physikalische Diffusionsbarriere. Durch diese Barriere wird die Versorgung mit Sauerstoff und Nährstoffen reduziert. Die resultierende Hypoxie im Tumor kann eine fehlgeleitete Angiogenese triggern und zu einer Aktivierung maligner Signalkaskaden führen. In dieser Arbeit wurden durch eine LOX(L)-Inhibierung mittels βAPN einerseits und eine ektopische LOX-/LOXL2-Überexpression andererseits Auswirkungen solcher Eingriffe auf verschiedene Indikatoren wie Zellproliferation und apoptose, Versorgung mit Sauerstoff und Nährstoffen, Angiogenese, Hypoxie, Makrophageninfiltration und die Expression verschiedener Wachstumsfaktoren analysiert. Die Versuche wurden an fünf verschiedenen Tumoren (4T1-, E0771- und EMT6-Brustkarzinome, LLC-Lungenkarzinome und MT6-Fibrosarkome) durchgeführt. Die Ergebnisse dieser Arbeit demonstrieren eine direkte Verbindung zwischen der EZM und einer Therapieresistenz. Nach βAPN-Behandlung konnte eine verbesserte Versorgung mit Sauerstoff und Nährstoffen beobachtet werden, welches in einer Verringerung maligner Signalkaskaden und folglich auch in einer verbesserten Vaskularisierung resultierte. Als Konsequenz wurde die therapeutische Effizienz von Chemotherapeutika verbessert. Im Gegensatz dazu führte eine LOX-/LOXL2-Überexpression zu einer erhöhten Therapieresistenz. Die vorliegende Studie zeigt, dass die Modifizierung der EZM durch eine Hemmung von LOX(L) das Potenzial birgt, das Ansprechen von Chemotherapeutika in der Behandlung von Krebserkrankungen zu verbessern. / The extracellular matrix (ECM) forms a network of cross-linked proteins, which surrounds all cells in the tumor. The ECM has direct effects on the accumulation and distribution of drugs and therefore also on the therapeutic efficacy of chemotherapeutics. The LOX protein family catalyzes the oxidative deamination of lysin-residues in elastins and collagen fibers and as a consequence allows an intra- and intermolecular crosslinking. This is necessary for the maturation and stabilization of the collagens in the ECM. An elevated LOX(L)-expression increases the stiffness through a higher cross-linked ECM and thus builds a physical diffusion barrier. Because of this barrier the oxygen and nutritional supply is limited. The resulting hypoxia in the tumor could promote errant angiogenesis and lead to an activation of malign signal cascades. In this dissertation, effects on different influences were examined on the one hand through a LOX(L)-inhibition with βAPN and on the other hand through an ectopic LOX /LOXL2-overexpression. These influences were cell proliferation and -apoptosis, supply with oxygen and nutrients, angiogenesis, hypoxia, infiltration of macrophages and the expression of various growth factors. The experiments were performed on five different tumor models (4T1, E0771, EMT6 breast carcinomas, LLC lung carcinomas and MT6 fibrosarcomas). The results of this work demonstrate a direct link between the ECM and a therapeutic resistance of tumors. After βAPN-treatment a better supply with oxygen and nutrients could be determined, which ended up in a reduction of malign signal cascades and thus in a better vascularization. As a consequence the therapeutic efficacy of chemotherapeutics was improved. In contrast, LOX /LOXL2-overexpression lead to a higher therapeutic resistance. This dissertation shows that modification of the ECM through LOX(L)-inhibition has the potential to improve response of chemotherapeutics in the treatment of cancer.

This thesis reports the synthesis and copper coordination chemistry of covalently-linked aryl-imidazole derivatives designed as models for the crosslinked imidazole-phenol sidechains of the His-Tyr cofactor in the CcO. Three new imidazole- (HL1 - HL3) and three new indole- (HL4 - H2L6) containing tripodal ligands were synthesised. The conjugate addition of an imidazole to activated quinone derivatives was developed as a new route to organic models for the Tyr His cofactor. Two monodentate imidazole-aryl, Im-hq(OH)2 and Im-ArOH, and an imidazole-quinone, Im bq were obtained using this route. The X-ray crystal structure of Im-hq(OH)2.EtOH was determined. The route was also used to give new chelating ligands, H2L10 and HL12, containing a cross-linked imidazole-phenol surrogate for the Tyr244-His240 cofactor. Copper complexes of Im-hq(OH)2, Im-bq, Im-ArOH, H2L10-HL12, and HL1-H2L6 were prepared, and the X-ray crystal structures of [Cu(terpy)(Im-bq)][BF4]2 and five other copper complexes were determined. The physiochemical properties of the copper complexes were characterized by FT-IR, UV-Vis-NIR, EPR and (spectro)electrochemical studies. Key results include: the oxidation of Im-ArO- anion affords the semiquinone radical, Im-sq(4OH)(1O??????), in a hydrous solvent. However, the oxidations of neutral Im-ArOH and [Cu(tpa)(Im-ArOH)]2+ produce the corresponding phenoxy radical species that rapidly and reversibly dimerise to give quinol cyclohexadienone, QCHD, dimers. Significantly [Cu(tpa)(Im-sq(4OH)(1O??????))]2+ was EPR silent, perhaps due to antiferromagnetic coupling between the Cu(II) (S=1/2) and semiquinonyl radical (S=1/2) centres. Deprotonation of the hydroquinone in [Cu(tpa)(Im-hq(OH)2]2+ produces the hydroquinone dianion which reduces the Cu(II) centre. The semiquinone radical is coordinatively labile and dissociates from the Cu(I) centre. The biological implications of these results are mentioned.

Kaposi's sarcoma (KS), caused by the oncogenic Kaposi's sarcoma herpesvirus (KSHV), is an angiogenic tumor characterized by intense angiogenesis, inflammation and proliferation of KSHV-infected spindle cells. We describe the characterization of a mouse model of KS by transfection of a KSHV bacterial artificial chromosome (KSHVBac36) into mouse bone marrow endothelial-lineage cells which generated a cell (mECK36) that forms KS-like tumors in mice. Our results define mECK36 as a biologically sensitive animal model of KSHV-dependent KS with the following characteristics: (1) the pathological phenotype is a consequence of KSHV gene expression in normal progenitor cells subjected to in vivo growth conditions, (2) the histopathologic phenotype of the tumors resembles KS lesions, and (3) the model is suitable for analysis of vGPCR-driven tumorigenesis in the context of the whole KSHV genome. The mechanism by which vGPCR promotes tumorigenesis is not fully understood. The characterization of a Rac1 transgenic mouse model that produces KS-like lesions that highly resemble human KS has helped us to identify the potential role of Rac1, which is activated by vGPCR, in the pathogenesis of KS. The results from the RacCA transgenic mouse suggest that viral and host genes triggering Rac1 and ROS production may play an important role in KS tumorigenesis. We set out to determine how vGPCR physiologically activates Rac1 in KSHV-infected cells in the KS model mECK36. We found that KSHV oncogenesis in mECK36 is promoted by vGPCR activation of a paracrine oncogenic mechanism through PDGF-BB, which requires a Rac1- and ROS-mediated loop, leading to STAT3 transcriptional activation of c-Myc, VEGF and KSHV latent viral gene expression. We also found that the latency-associated nuclear antigen (LANA) upregulates the PDGFR in vivo, priming latently-infected cells to the PDGF signaling pathway. This oncogenic mechanism can be targeted with the antioxidant N-acetylcysteine (NAC) and FDA-approved PDGF receptor inhibitors to control KSHV-induced tumorigenesis. Our results highlight a ROS-dependent axis whereby Rac1 activating oncogenes and inflammatory signaling drive paracrine stimulation of neoplastic growth and angiogenesis in neighboring cells, defining this axis and its components as attractive anti-tumor targets in KS pathogenesis.

Proton transfer is one of the most common reactions in biological systems. During energy conversion inside a cell, proton transfer is crucial to maintain an electrochemical proton gradient across the cell membrane. This gradient is in turn used to e.g. produce ATP, the energy currency of the cell. One of the key components of the build-up of this gradient is cytochrome c oxidase. This membrane-bound enzyme catalyzes the reduction of molecular oxygen to water, using protons and electrons, and in the process protons are pumped across the membrane. All protons used during oxygen reduction and those that are pumped, are transferred via hydrophilic pathways inside the hydrophobic interior of the enzyme. One of these pathways, called the D pathway, is used to transfer protons both to the catalytic site and towards a pump site. It is yet not fully understood how these proton-transfer reactions are timed, coupled and controlled.   This thesis is focused on studies of proton-transfer reactions through the D pathway in variants of cytochrome c oxidase that lack the ability to pump protons. The results suggest that changes in pKa values of key residues, as well as structural changes inside the pathway, can explain the non-pumping phenotypes. The results have led us to propose that an internal proton shuttle (Glu286I) can adopt two different conformations that are in equilibrium with each other, and that this equilibrium is altered in non-pumping variants of cytochrome c oxidase. We also observed that proton transfer through the D pathway could occur with the same rate as in the wild-type enzyme even when one of the key residues (Asp132I) is absent. This result contradicts previous assumptions that acidic residues must be present at an orifice of proton pathways. We therefore suggest that this specific residue could have an additional role, e.g. as a selectivity filter that excludes all ions except protons from entering the pathway. / <p>At the time of doctoral defence the following papers were unpublished and had a status as follows: Paper 2: Accepted; Paper 3: Manuscript</p>

Thesis (Ph. D.)--OGI School of Science and Engineering at OHSU, 2001.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

No description available.

Amyloid fibrils are a misfolded state formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them an excellent target for enzyme immobilisation and their strength, stability and nanometre size are attractive features for exploitation in the creation of new bionanomaterials. The aim of this thesis was to functionalise amyloid fibrils by conjugation to glucose oxidase (GOD). GOD is a relatively stable glycoprotein that catalyses the oxidation of glucose and the release of hydrogen peroxide. The consumption of glucose can be measured to assess glucose levels, and the release of hydrogen peroxide is cytotoxic to cells and is thus an effective antibacterial agent. Three methods of attachment were used: cross-linking using glutaraldehyde, periodate oxidation of the glycoprotein shell, and cross-linking using glutaraldehyde following deglycosylation. GOD retained activity upon attachment by all three methods. These attachment methods were assessed using electrophoresis, centrifugation, sucrose gradient centrifugation and TEM. Gel electrophoresis indicated a high degree of cross-linking and TEM showed no significant change of fibril morphology upon cross-linking. Centrifugation experiments suggested a non-covalent interaction was occurring between amyloid fibrils and GOD, and a covalent attachment was occurring upon addition of glutaraldehyde. Sucrose gradient centrifugation provided increased separation of cross-linked material compared to other separation methods, and showed greater cross-linking to crystallin amyloid fibrils than insulin fibrils. Cross-linking native GOD using glutaraldehyde was chosen for further experiments, as it was found to be most effective for GOD attachment to amyloid fibrils. The resulting functionalised enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The distribution of the functionalised fibrils through the film was characterised using SEM and confocal microscopy, where film components were found to be unevenly dispersed. The antibacterial effect of the functionalised film was then tested on E. coli and the antifungal effect of the film was tested on Fusarium, Rhizopus and Penicillium. Growth of E. coli was inhibited around functionalised film circles, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. However, no growth inhibition of fungal species was observed. This work is of significance as it demonstrates the ability to convert a waste material, bovine lens crystallin, to high value protein nanofibres and incorporate functionality via GOD attachment. The incorporation of the GOD-functionalised amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalised amyloid fibril scaffold. Future development of this model system has the potential to lead to the production of a novel biomaterial for use in food packaging due to the antimicrobial properties of GOD.