Organophosphorus compounds (OPs) are used as pesticides, e.g. parathion, which is converted in the body to paraoxon, and chemical warfare nerve agents, such as sarin, soman, cyclosarin, VX, and tabun. Even small amounts of OP exposure can be fatal, depending on the toxicity of the compound. Great stocks of highly toxic chemical warfare nerve agents exit around the world and are considered a serious threat to national security and international stability. OPs exert their toxicity by covalent irreversible inhibition of acetylcholinesterase (AChE) that prevents the enzyme from hydrolyzing acetylcholine (ACh), a neurotransmitter in the central and peripheral nervous systems (CNS and PNS). Therefore, ACh accumulates in the cholinergic synapses throughout the body, which results in overstimulation of the ACh receptors. Removal of the phosphyl moiety from the OP-bound AChE active site has been a promising method to restore AChE’s catalytic activity. However, a secondary process called aging also occurs in the OP-AChE complex. Once aging occurs, currently available oximes are ineffective in removing the phosphyl moiety from the enzyme’s active site, and hence are ineffective as antidotes against the aged enzyme. Several families of alkylating and acylating agents including several classes of agents that combine alkylating moieties with known active site or peripheral cite (PAS) binding motifs were synthesized and evaluated. The general aim of the research was that successful alkylation or acylation of the phosphonate monoanion of aged AChE would produce neutral phosphyl complexes that would either spontaneously reactivate or would be reactivatable in the presence of oxime antidotes. Methoxylamine analogs of the oxime antidote 2-PAM were synthesized with the aim that methyl transfer to the aged AChE adduct would produce a neutral phosphyl AChE adduct simultaneously with 2-PAM in situ, and subsequent 2-PAM nucleophilic attack would reactivate the newly formed neutral phosphyl-AChE adduct. However, none of these 2-PAM analogs resurrected the activity of aged AChE. Another strategy for resurrecting the activity of aged AChE utilizes N-methylpyridiniums that are substituted at the 2-position with a beta-lactam moiety. For these compounds, opening of the electrophilic beta-lactam unmasks a nucleophilic amidine function which could putatively attack at phosphorus to expel the free enzyme. For this class of agents, only the active site directed compound that possessed the 5-CF₃ substituent showed possible resurrection of the activity of aged AChE, though activities in both the control and treated samples were low. Methyl transfers are common in Nature, and the natural transfer agent is S-adenosylmethionine, a sulfonium methyl donor. Consequently, the array of sulfonium compounds were evaluated on the expectation that they would bind to the AChE active site and transfer a methyl group to the phosphonate monoanion of the aged enzyme. Though high-affinity binding was noted for these compounds, none of these resurrected the activity of the aged AChE complex. Finally, several selected agents were evaluated on reactivating the initial OP-AChE complex before aging has occurred. It was observed that degraded samples of selected inhibitors are capable of reactivating initial complexes of sarin and soman inhibited AChE at low concentration that is an important character of efficient reactivators. However, the structure of reactivator is still unknown. Two major challenges still face researchers in the quest to design effective medicinal agents for counteracting poisoning by AChE-inhibiting nerve agents. The first is that there is no universal oxime antidote. Oximes that are effective against certain nerve agents are ineffective against others. The second is that, despite extensive efforts that span two generations, aged phosphyl-AChE adducts have never been reactivated. However, given the powerful tools of modern structural biology, medicinal chemistry and molecular biology, there is still hope that these considerable challenges can be met.

This thesis describes phytochemical studies on Buxus macowanii, a medicinal plant collected from South Africa and the fungal transformation of glycyrrhetinic acid (71), a triterpenoid found in Glycyrrhiza plants. The crude methanolic extract of B. macowanii was active in the acetylcholinesterase inhibition assay (IC50 = 30 μg/mL). Chemical investigation of its alkaline dichloromethane extract afforded five novel triterpenoidal alkaloids, 31-hydroxybuxatrienone (56), macowanioxazine (57), 16α-hydroxyma- cowanitriene (58), macowanitriene (59), and macowamine (60), along with two known Buxus bases, Nb-demethylpapillotrienine (61) and moenjodaramine (62). Compounds 56, 57, and 61 exhibited different level of AChE inhibitory activities with compound 56 being significantly active (IC50 = 8.0 μM). Glycyrrhetinic acid (71) was incubated with Curvularia lunata (ATCC 12017) for ten days to afford one metabolite which was characterized by NMR spectral data as known biotransformed product, 3-oxo-glycyrrhetinic acid (85). This metabolite was previously obtained through biotransformation of 71 by using other fungi.

This thesis describes phytochemical studies on Buxus macowanii, a medicinal plant collected from South Africa and the fungal transformation of glycyrrhetinic acid (71), a triterpenoid found in Glycyrrhiza plants. The crude methanolic extract of B. macowanii was active in the acetylcholinesterase inhibition assay (IC50 = 30 μg/mL). Chemical investigation of its alkaline dichloromethane extract afforded five novel triterpenoidal alkaloids, 31-hydroxybuxatrienone (56), macowanioxazine (57), 16α-hydroxyma- cowanitriene (58), macowanitriene (59), and macowamine (60), along with two known Buxus bases, Nb-demethylpapillotrienine (61) and moenjodaramine (62). Compounds 56, 57, and 61 exhibited different level of AChE inhibitory activities with compound 56 being significantly active (IC50 = 8.0 μM). Glycyrrhetinic acid (71) was incubated with Curvularia lunata (ATCC 12017) for ten days to afford one metabolite which was characterized by NMR spectral data as known biotransformed product, 3-oxo-glycyrrhetinic acid (85). This metabolite was previously obtained through biotransformation of 71 by using other fungi.

A doença de Alzheimer (DA) é caracterizada pela perda progressiva de memória episódica relacionada à agregação do peptídeo ?-amiloide (A?) e à fosforilação anormal da proteína tau, levando à perda da função colinérgica. É bem conhecido que o comprometimento desta função devido à neurotoxicidade do peptídeo A? contribui significativamente para o declínio cognitivo associado à DA. Os inibidores da enzima acetilcolinesterase (AChE) compõem a principal classe de drogas usadas no tratamento da DA. No entanto, há uma grande necessidade de síntese de novas moléculas, uma vez que os fármacos já em uso pelos pacientes apresentam alta toxicidade hepática, além de vários efeitos colaterais devido à ação nos tecidos periféricos. A hipótese do presente projeto se baseia na atividade de indução de neurodiferenciação e neuritogênese de novos compostos inibidores de AChE, os quais são híbridos sintéticos de donepezila-tacrina, além de atuarem como neuroprotetores em culturas de células neuronais, sob condições experimentais de um estímulo neurotóxico e de estresse oxidativo induzidos pelo peptídeo A?(1-42) e peróxido de hidrogênio (H2O2). Assim, o objetivo deste trabalho foi avaliar a capacidade de neurodiferenciação e neuritogênese, assim como o potencial neuroprotetor de duas moléculas híbridas de donepezila-tacrina na linhagem SH-SY5Y, modelo de diferenciação neuronal. Foram realizados ensaios de citotoxicidade e hepatotoxicidade em células tratadas e coletadas em diferentes tempos (24, 48, 72 e 120 h) nas linhagens celulares SH-SY5Y e HepG2, respectivamente. Ensaios de diferenciação neuronal (análise morfológica), expressão proteica, cinética ciclo celular, proliferação celular, alterações mitocondriais e de estresse oxidativo foram realizados em linhagem neuronal SH-SY5Y. Ainda, foram realizados ensaios de viabilidade e morte celular para avaliar a capacidade de neuroproteção dos novos compostos híbridos na linhagem SH-SY5Y. Estes não se mostraram citotóxicos e não alteraram a viabilidade celular em todas as concentrações avaliadas: TA8Amino 0,0035 a 0,112µM e TAHB3 0,088 a 2,84µM; nos ensaios de hepatotoxicidade, o composto TAHB3 reduziu a viabilidade celular na maior concentração (2,84 µM) e no tempo de 24 h. Ambos os compostos híbridos foram capazes de induzir diferenciação neuronal e neuritogênese, cerca de 55% das células para TA8Amino e 43% para TAHB3. Apenas o composto TA8Amino induziu um aumento de aproximadamente 75% na expressão do marcador ?-III-Tubulina comprovando a formação de neurônios maduros. Nenhum dos tratamentos realizados causaram alterações significativas na distribuição das células nas fases do ciclo celular e no ensaio de proliferação celular. O composto TA8Amino foi capaz de induzir a produção de ROS intracelular (48,27%) e mitocondrial (67,60%) nas células diferenciadas. Ambos os compostos híbridos aumentaram a expressão de SOD1, indicando atuação na atividade antioxidante das células. Entretanto, os compostos não alteraram o potencial de membrana mitocondrial e massa mitocondrial. Esses dados demonstram que o estresse oxidativo induzido pelo TA8Amino não gerou danos por disfunção mitocondrial nas células diferenciadas. Na avaliação de alterações da expressão proteica de PTEN(Ser380/Thr382/383), AKT(Ser473) e COX2 ambos os compostos híbridos promoveram a indução de PTEN e AKT. TA8Amino e TAHB3 induziram um aumento na expressão de COX2 demonstrando umapossível atuação na via de sinalização PI3K/AKT/COX2, compatível com a diferenciação neuronal. Nos ensaios de viabilidade celular, os compostos TA8amino e TAHB3 apresentaram efeito de neuroproteção frente ao dano neurotóxico induzido pelo peptídeo A?(1-42), promovendo uma proteção de 91,93% para TA8Amino e 54,68% para TAHB3, embora o mesmo efeito não tenha sido observado nas células tratadas com o peróxido de hidrogênio. Entretanto, a indução de morte por apoptose pelas moléculas híbridas nas células tratadas com o peptídeo A?(1-42) foi apenas levemente alterada pelos compostos, havendo uma redução de 5,10 e 4,85%, respectivamente, para TA8Amino e TAHB3. Em geral, os resultados do presente trabalho demonstraram que os compostos híbridos donepezilatacrina (TA8amino e TAHB3), inibidores da AChE, apresentam vantagens como potenciais fármacos, visto não serem citotóxicos nas concentrações de inibição da enzima AChE, além de serem capazes de induzir diferenciação neuronal, neuritogênese e neuroproteção, diferentemente dos fármacos donepezila e tacrina, testados isoladamente. / Alzheimer\'s disease (AD) is characterized by a progressive loss of episodic memory related to aggregation of ?-amyloid peptide (A?) and to abnormal phosphorylation of tau protein, leading to loss of cholinergic function. It is well known that the impairment of this function due to A? peptide neurotoxicity contributes significantly to the cognitive decline associated with AD. Acetylcholinesterase (AChE) inhibitors are the main class of drugs used to treat AD. However, there is a great need for synthesis of new molecules, since current drug treatment presents high hepatic toxicity, besides several side effects due to the action in the peripheral tissues. The hypothesis of this project is based on the activity of AChE inhibitor compounds (synthetic hybrids of donepezil-tacrine) in terms of inducing neurodifferentiation and neuritogenesis, as well as neuroprotective effects in neuronal cells under experimental conditions of a neurotoxic stimulus and oxidative stress induced by the peptide A? (1-42) and hydrogen peroxide (H2O2). Thus, the objective of this work was to evaluate the neurodifferentiation and neuritogenesis abilities, as well as the neuroprotective potential of two hybrid molecules of donepezil-tacrine in the SH-SY5Y model. Cytotoxicity and hepatotoxicity assays were performed on treated cells that were collected at different times (24, 48, 72 and 120 h) in SH-SY5Y and HepG2 cell lines, respectively. Neuronal differentiation assays (morphological analysis), protein expression, cell cycle kinetics, cell proliferation, mitochondrial changes and oxidative stress were performed on SH-SY5Y cell line. Further, viability and cell death assays were performed to evaluate the neuroprotection ability of the novel hybrid compounds in SH-SY5Y cells. The compounds did not present cytotoxic effects and did not alter cell viability at the following concentrations: TA8Amino 0.0035 to 0.112?M and TAHB3 0.088 to 2.84?M; in the hepatotoxicity assays, TAHB3 reduced cell viability only at the highest concentration (2.84 ?M) and at the time of 24 h. Both hybrid compounds were able to induce neuronal differentiation and neuritogenesis, around 55% after TA8Amino and 43% after TAHB3 treatment. Only TA8Amino induced approximately 75% increase in ?-III-Tubulin expression, compatible with the formation of mature neurons. None of the treatments performed caused significant changes in cell cycle kinetics. The synthetic hybrid TA8Amino induced the production of intracellular (48.27%) and mitochondrial ROS (67.60%). Both hybrid compounds increased SOD1 expression, which could be related to the increase of oxidative damage. However, both TA8amino and TAHB3 did not promote changes in mitochondrial membrane and mitochondrial mass potential. This data demonstrate that oxidative stress induced by TA8Amino did not generate damage due to mitochondrial dysfunction in differentiated cells. In the evaluation of changes in PTEN(Ser380/ Thr382/383), AKT(Ser473) and COX2 protein expression, both hybrid compounds were able to induce PTEN and AKT expression. TA8Amino and TAHB3 promoted an increase in COX2expression indicating a possible role in the PI3K/AKT/COX2 pathway, which is related to neuronal differentiation. In the cell viability assays, TA8amino and TAHB3 showed a neuroprotective effect against the neurotoxic damage induced by the A? peptide (1-42), promoting a protection of 91.93% for TA8Amino and 54.68% for TAHB3, although the same effect could not be observed in cells treated with hydrogen peroxide. However, the induction of apoptosis by the hybrid molecules in cells treated with the A?(1-42) peptide was only slightly altered by the compounds, with a reduction of 5.10 and 4.85%, respectively, for TA8Amino and TAHB3, respectively. In general, the results of the present work demonstrate that donepezil-tacrine hybrid compounds (TA8amino e TAHB3), AChE inhibitors, have advantages as potential drugs, since they are not cytotoxic at concentration levels that inhibit AChE enzyme, besides being able to induce neuronal differentiation, neuritogenesis and neuroprotection, unlike the drugs donepezil and tacrine, tested alone.

Submitted by SIDNEI CAVASSANA null (sidnei5333@gmail.com) on 2017-09-16T17:06:01Z No. of bitstreams: 1 Mestrado Sidnei versão Final.pdf: 4006618 bytes, checksum: 3e1f03fc6f46516ae312a5bc3c98f9bb (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-19T18:25:30Z (GMT) No. of bitstreams: 1 cavassana_s_me_ilha.pdf: 4006618 bytes, checksum: 3e1f03fc6f46516ae312a5bc3c98f9bb (MD5) / Made available in DSpace on 2017-09-19T18:25:30Z (GMT). No. of bitstreams: 1 cavassana_s_me_ilha.pdf: 4006618 bytes, checksum: 3e1f03fc6f46516ae312a5bc3c98f9bb (MD5) Previous issue date: 2017-08-01 / O medo da dor relacionada à injeção é um empecilho à terapia de injetáveis. As injeções hipodérmicas são motivos de grande ansiedade e de reduzida adesão à aplicação subcutânea de insulina para o controle glicêmico em diabéticos ou no tratamento da esclerose múltipla, aumentando o risco de complicações e mortalidade. Neste trabalho foi analisado como algumas das características da agulha podem influenciar a sensação de dor na injeção. Mediu-se o esforço de penetração de agulhas em um tecido artificial (modelo substituto da pele), para diferentes diâmetros de cânula, rugosidade, profundidade de penetração, lubrificação e ângulos do bisel da ponta perfurante. Este estudo visou encontrar alternativas para facilitar a aplicação e a autoaplicação de injeções hipodérmicas, aumentando a segurança e conforto, diminuindo a intensidade da dor percebida pelo paciente. Para isso, analisou-se no projetor de perfil e no MEV o bisel de agulhas usadas repetidas vezes para verificar a perda do perfil ou a formação de rebarbas que possam dificultar a penetração ou traumatizar o tecido durante o reuso de agulhas. Também foi analisado sob o ponto de vista mecânico, o que pode ser feito para prevenir que as agulhas usadas na aplicação subcutânea não atinjam inadvertidamente o músculo. O maior esforço de penetração observado nas agulhas com maior ângulo do bisel é responsável pela percepção de dor do paciente. / Fear of injection-related pain is a drawback to injectable therapy. Hypodermic injections are a cause for great anxiety and reduced adherence to the subcutaneous application of insulin for glycemic control in diabetics or in the treatment of multiple sclerosis, increasing the risk of complications and mortality. In this work, it was analyzed how some of the characteristics of the needle can influence the sensation of pain in the injection. The needle penetration effort was measured in an artificial tissue (substitute skin model) for different cannula diameters, roughness, depth of penetration, lubrication and angles of the perforating tip bevel. This study aimed to find alternatives to facilitate the application and self-application of hypodermic injections, increasing safety and comfort, reducing the pain intensity perceived by the patient. To do this, the bevel of needles used repeatedly was analyzed in the profile projector and SEM to verify the loss of the profile or the formation of burrs that could hamper the penetration or traumatize the tissue during the reuse of needles. It has also been mechanically analyzed, which can be done to prevent that the needles used in the subcutaneous application do not inadvertently reach the muscle. The greater penetration effort observed in the needles with greater angle of the bevel is responsible for the patient's perception of pain.

Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors that are underutilized for controlling insect pests despite their involvement in various physiological functions. To-date, there are no commercialized insecticides targeting insect mAChRs. In this dissertation, effective target-site synergism was demonstrated in susceptible Drosophila melanogaster where mAChR agonism by pilocarpine enhanced the toxicity of insecticides targeting gamma-aminobutyric acid (GABA)-gated chloride channels, indicating the potential of insect mAChRs as a target for developing novel insecticides/synergists to control resistant pests. A point mutation (A301S) in the GABA-gated chloride channel confers resistance to dieldrin (Rdl), lindane, and fipronil, which I have confirmed using different routes of exposure. However, the same synergistic effect was not achieved in the resistant strain with the presence of this target-site mutation. This difference between two strains is perplexing because there is a change in the efficacy of several compound classes that do not directly act upon GABA-gated chloride channels. Specifically, a point mutation appears to influence how the insect central nervous system (CNS) responds to muscarinic compounds, type I pyrethroids, and acetylcholinesterase (AChE) inhibitors. In the case of acetylcholinesterase, the resistant insect increases the expression of Ace gene encoding this enzyme. Fully understanding how the CNS responds to receptor modifications is not well understood and could have a significant impact to pest management strategies. / Doctor of Philosophy / Insects significantly influence the food production, health, and the economy of the human world. Control of insect pest outbreaks relies on the proper use of insecticides. However, extensive application of insecticides has resulted in pests being able to adapt to these compounds, through insecticide resistance. Ultimately, this will affect currently used pest management strategies. To help alleviate this urgent problem, my dissertation provided an alternative strategy to control pests, which is to use a mixture of two molecules that influence different targets in the insect nervous system that could reduce the use of toxic or deleterious compounds that are the active ingredients. It is important to not solely rely on current insecticides on the market and find new insecticides that work differently. I used the fruit fly to help me understand how insecticide mixtures would work, but also understand how the complex nature of insect adaptations at the level of the nervous system continues to threaten pest management. Based on studies that were performed here, we now have a better understanding on how to investigate the failure of insecticides in the field, which will ultimately help us make new molecules.

Throughout the last five decades, European Honey bee (Apis mellifera) colonies have been heavily damaged by invading Varroa mites (Varroa destructor), and, more recently, small hive beetles (Aethina tumida). These pests infest A. mellifera colonies throughout Virginia, with V. destructor feeding upon the lipids of their hosts and spreading viruses, and A. tumida feeding extensively on hive products and brood. Because V. destructor has historically demonstrated acaricide resistance, this study examined V. destructor resistance to three common acaricides (amitraz, coumaphos, and tau-fluvalinate) throughout the three geographic regions of Virginia using glass vial contact bioassays; the results showed no resistance in the sites tested. To gain better insights into A. tumida pharmacology, several known acetylcholinesterase (AChE) inhibitors and three novel insecticides (previously shown to have low mammalian toxicity) were tested against an A. tumida laboratory colony through in vivo and in vitro bioassays. The results of these bioassays indicated that coumaphos was most selective and topically effective against A. tumida, while only one experimental compound was selective against A. tumida, with 29-fold less potency than coumaphos. These results can help apiculturists in making informed pest management choices and can lead to future studies further examining V. destructor resistance and optimizing A. tumida insecticide treatments. / Master of Science in Life Sciences / Beekeepers throughout the world have experienced great economic loss and observed a troubling decline in European Honey bee colonies over the past fifty years due to Varroa mite infestations. Varroa mites feed on the fat body of bees, depriving them of nutrients and infecting them with various diseases. Attempts made to control Varroa mites with synthetic chemicals throughout the years have led to acaricide resistance. To look at resistance in Virginia’s mite populations, resistance testing was performed on Varroa mite populations throughout the three geographic regions of Virginia, and these studies showed that there was not resistance in these populations. Another significant hive pest that is increasingly prevalent in the United States is the small hive beetle (SHB), which feeds on bee brood and hive products. SHB management tactics are still being explored, and this project tested various known insecticide treatments against small hive beetles and bees, as well as three insecticide treatments that were designed for mosquito control and have low toxicity to mammals. The results of this study showed that, of the insecticides tested, coumaphos was the most selective against SHB. This information can help beekeepers in Virginia make informed choices when deciding how to treat Varroa mite infestations, and can add to the knowledge base of those fighting small hive beetle infestations.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes, in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie, sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro- Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg- Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61 Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang. Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie, informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican; perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante. Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese differensiering en kanker, lê die grondslag vir verdere ondersoeke.

There is scarce information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Furthermore, the role of acetylcholinesterase (AChE) during heart development is unknown, despite record of its presence in the myocardium. Although three molecular variants of AChE (R, H and T) exist due to alternate splicing, temporal and spatial distribution of these splice variants during cardiac ontogeny is not well characterized. We hypothesized that the AChE “R” splice variant (AChE-R) is involved in directing lineage commitment of mouse ventricular CPCs to the conduction cell phenotype. It is possible that AChE may promote the breakdown of ACh and block the effects of ligand-binding via M2 receptors present on the surface of CPCs. Our study has also provided a platform to suggest that AChE may play a role in the molecular mechanisms underlying functional diversification of myocardial cells into conduction system cells during ontogenesis.

Orientador: Maria Filomena Spatti Sandalo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Estudos da Linguagem / Made available in DSpace on 2018-08-12T09:39:51Z (GMT). No. of bitstreams: 1 Roessler_Eva-Maria_M.pdf: 84135419 bytes, checksum: 0dfdaa306092baf9a06759531f7755fb (MD5) Previous issue date: 2008 / Resumo: Este trabalho é um estudo piloto de alguns aspectos fonológicos e morfológicos da língua achê, uma língua indígena falada por aproximadamente 300 falantes fluentes na região oriental do Paraguai. O trabalho apresenta um estudo fonológico, que inclui a descrição dos segmentos vocálicos e consoantais, como também de aspectos suprassegmentais como o espraiamento de nasalidade. Além disso, discute-se a estrutura silábica, o acento lexical e a estrutura da palavra mínima do achê. A segunda parte apresenta uma descrição da marcacão de pessoa/número, de tempo-aspecto-modo, da construção passiva e também das categorias lexicais da língua achê. É importante destacar que o trabalho tem um segundo objetivo: contextualizar estas descrições gramaticais em um debate sociolingüístico. Durante os últimos dois séculos, e até hoje, a literatura histórica, antropológica e lingüística apontam para a hipotése de o achê ser uma língua de contato baseada no léxico da língua guarani. Como o meu trabalho aborda a questão do contato lingüístico a partir de uma visão interdisciplinar, informações etnográficas e históricas relevantes para o entendimento da possível gênese da língua achê são incluídas. Apresento dados lingüísticos, resultados da minha pesquisa de campo, que podem contribuir para esta discussão. Observo que. há mudanças, essencialmente no subsistema morfossintático, que podem ser consideradas abruptas, e, portanto, podem indicar uma transmissão lingüística nãolinear. O subsistema morfossintático conta com recursos morfológicos reduzidos quando comparado com a possível língua base - o guarani do Paraguai. Estes recursos, além disso, passaram por mudanças, tanto na sua forma fonológica, quanto na sua função. As alterações na função, que em muitos casos são caracterizadas por uma ampliação de funções morfossintáticas e semânticas, podem ser entendidas como resultado de processos de analogia e reinterpretação. / Abstract: This work Is a pilot study of some aspects of phonology and morphology of Aché, an indigenous language spoken by approximately 300 fluent speakers in the oriental region of Paraguay. It presents a phonological study of the phonemic inventory as well as a study of suprasegmental phenomena such as nasalization, stress, syllable structure, and some considerations about minimal word constraints. In the second section of the descriptive part, I outline aspects of person and number agreement, tense-aspect-mood marking, followed by a description of passive constructions and a discussion on lexical categories of the Achá language. The preceding grammatical description is particularly relevant for a wider sociolinguistic discussion of language genesis and evolution. The historical, anthropological and linguistic literature of the past two centuries suggests the hypothesis that the Aché language might be a contact language based on the lexicon of Paraguayan Guarani. My work approaches this hypothesis from an interdisciplinary standpoint, and includes some ethnographic and historic information crucial for the understanding of a possible genesis of the Aché language. In the final section I review linguistic data - the results of my field research - that can contribute to that discussion. Specifically, inside the morphosyntactic subsystem of the Aché grammar, I observe changes that can be considered abrubt and therefore the result of possible non-linear transmission. The morphosyntactic subsystem contains reduced flexional morphemes if compared to the hypothesized lexifier language - Paraguayan Guarani. The remaining flexional morphemes changed both their phonological structure and their grammatical functions. In grammatical terms, the changes can be characterized as amplifications of morphosyntactic and semantic function and might be possibly understood as outcome of processes such as analogy and reinterpretation. / Mestrado / Mestre em Linguística