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.

A hipóxia-isquemia neonatal (HI) faz parte da etiologia de diversas patologias neurológicas e é causa de graves sequelas. Os mecanismos patofisiológicos dessa lesão começam com o insulto imediato após a HI e se estendem por dias ou semanas, pelo aumento da liberação de espécies reativas de oxigênio associada a redução da defesas anti-oxidantes e reação glial, sendo a lesão secundária parte crucial no processo que culmina no dano final. A acetilcolina (ACh) é um neurotransmissor do sistema nervoso central (SNC) que parece ter uma importante ação neuroprotetora após a HI. A acetilcolinaesterase (AChE) é responsável pela degradação da ACh, inibidores dessa enzima vêm sendo utilizados para o tratamento de danos neurológicos. Sua ação positiva sobre a HI foi demonstrada em estudos realizados em nosso laboratório, onde a administração do extrato de Huperzia quadrifariata (inibidor de AChE) reduziu os déficits cognitivos e histológicos causados por essa lesão Para avaliar os efeitos das administrações pré e pós-hipóxia de galantamina, inibidor da AChE, no modelo de HI perinatal, ratos Wistar no 7º dia de vida pós-natal (DPN7) foram submetidos à combinação da oclusão unilateral da artéria carótida direita e exposição a uma atmosfera hipóxica (8% de O2) durante 60 minutos. Foram aplicadas injeções intraperitoniais de salina para os grupos Sham e HI+Salina (HIS) e de galantamina nos grupos HI+Galantamina 5 mg/kg pré-hipóxia (HIG5-Pré), HI+Galantamina 10 mg/kg pré-hipóxia (HIG10-Pré), HI+Galantamina 5 mg/kg pós-hipóxia (HIG5-Pós) e HI+Galantamina 10 mg/kg pós-hipóxia (HIG10-Pós). Os grupos Pré receberam galantamina imediatamente antes da hipóxia e os grupos Pós nos intervalos de 1, 24, 48 e 72 horas após a cirurgia. No DPN45 foi feita a análise do volume das estruturas encefálicas que demonstrou a redução do volume do hipocampo do grupo HIS em relação ao Sham e uma prevenção desse efeito no grupo HIG10-Pré, mas não nos demais grupos. Análises bioquímicas foram feitas no hipocampo ipsilesional 24 horas após a lesão e revelaram: através da citometria de fluxo uma redução na sobrevivência de neurônios no grupo HIS em relação ao Sham que foi prevenida no grupo HIG10-Pré; através de ELISA uma hipertrofia dos astrócitos no grupo HIS que foi revertida no grupo HIG10-Pré e um aumento na atividade da enzima anti-oxidante catalase. O tratamento pré-hipóxia com galantamina foi capaz de prevenir os déficits histológicos, aumentar a sobrevivência celular, reduzir a reação astrocitária e aumentar a atividade anti-oxidante em ratos submetidos à HI. / Neonatal hypoxia ischemia (HI) has a role in etiology of several neurological pathologies and causes severe sequelae. The pathophysiological mechanisms of this lesion start immediately after HI and last for days or weeks, with the secondary injury being a crucial part the process that culminates in the final damage. Acetylcholine (ACh) is a neurotransmitter of the central nervous system that seems to have an important neuroprotective action after HI. Acetylcholinesterase (AChE) degradates ACh and inhibitors of this enzyme have been used to treat neurological damage. Its positive action on HI has been demonstrated in studies performed in our laboratory, where the administration of the alkaloid extract of Huperzia quadrifariata (An inhibitor of AChE) reduced the cognitive and histological deficits caused by this lesion. To evaluate the effects of the pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in the model oh perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right charotid artery and of exposure to a hypoxic exposure (8% O2) for 60 minutes. Intraperitoneal injections of saline in the groups Sham anf HI+Saline (HIS) and of galantamine in the groups HI+Galantamine 5 mg/kg pre-hypoxia (HIG5-Pre), HI+Galantamine 10 mg/kg pre-hypoxia (HIG10-Pre), HI+Galantamine 5 mg/kg post-hypoxia (HIG5-Post) and HI+Galantamine 10 mg/kg post-hypoxia (HIG5-Post). The Pre groups received galantamine immediately before hypoxia and the Post groups in the intervals of 1, 24, 48 and 72 hours after HI. On PND45 the analysis of the volume of brain structures showed a reduction of the volume of the ipsilesional hippocampus in the HIS group when compared to the sham and a prevention of this effect in the HIG10-Pre, but not in any other group. Biochemical analysis was performed in the ipsilesional hippocampus 24 hours after the lesion and revealed: a reduction of the number of surviving neurons in the HIS group when compared to the Sham that was prevented in the HIG10-Pre; a hypertrophy of the astrocytes in the HIS group that was prevented in the HIG10-Pre group and an increase in the activity of the anti-oxidant enzyme catalase in the HIG10-Pre group. The treatment with galantamine was able to prevent the histological deficits, increase the survival of neurons, reduce astrocytic reaction and increase the anti-oxidant activity in rats submitted to HI.

A hipóxia-isquemia neonatal (HI) faz parte da etiologia de diversas patologias neurológicas e é causa de graves sequelas. Os mecanismos patofisiológicos dessa lesão começam com o insulto imediato após a HI e se estendem por dias ou semanas, pelo aumento da liberação de espécies reativas de oxigênio associada a redução da defesas anti-oxidantes e reação glial, sendo a lesão secundária parte crucial no processo que culmina no dano final. A acetilcolina (ACh) é um neurotransmissor do sistema nervoso central (SNC) que parece ter uma importante ação neuroprotetora após a HI. A acetilcolinaesterase (AChE) é responsável pela degradação da ACh, inibidores dessa enzima vêm sendo utilizados para o tratamento de danos neurológicos. Sua ação positiva sobre a HI foi demonstrada em estudos realizados em nosso laboratório, onde a administração do extrato de Huperzia quadrifariata (inibidor de AChE) reduziu os déficits cognitivos e histológicos causados por essa lesão Para avaliar os efeitos das administrações pré e pós-hipóxia de galantamina, inibidor da AChE, no modelo de HI perinatal, ratos Wistar no 7º dia de vida pós-natal (DPN7) foram submetidos à combinação da oclusão unilateral da artéria carótida direita e exposição a uma atmosfera hipóxica (8% de O2) durante 60 minutos. Foram aplicadas injeções intraperitoniais de salina para os grupos Sham e HI+Salina (HIS) e de galantamina nos grupos HI+Galantamina 5 mg/kg pré-hipóxia (HIG5-Pré), HI+Galantamina 10 mg/kg pré-hipóxia (HIG10-Pré), HI+Galantamina 5 mg/kg pós-hipóxia (HIG5-Pós) e HI+Galantamina 10 mg/kg pós-hipóxia (HIG10-Pós). Os grupos Pré receberam galantamina imediatamente antes da hipóxia e os grupos Pós nos intervalos de 1, 24, 48 e 72 horas após a cirurgia. No DPN45 foi feita a análise do volume das estruturas encefálicas que demonstrou a redução do volume do hipocampo do grupo HIS em relação ao Sham e uma prevenção desse efeito no grupo HIG10-Pré, mas não nos demais grupos. Análises bioquímicas foram feitas no hipocampo ipsilesional 24 horas após a lesão e revelaram: através da citometria de fluxo uma redução na sobrevivência de neurônios no grupo HIS em relação ao Sham que foi prevenida no grupo HIG10-Pré; através de ELISA uma hipertrofia dos astrócitos no grupo HIS que foi revertida no grupo HIG10-Pré e um aumento na atividade da enzima anti-oxidante catalase. O tratamento pré-hipóxia com galantamina foi capaz de prevenir os déficits histológicos, aumentar a sobrevivência celular, reduzir a reação astrocitária e aumentar a atividade anti-oxidante em ratos submetidos à HI. / Neonatal hypoxia ischemia (HI) has a role in etiology of several neurological pathologies and causes severe sequelae. The pathophysiological mechanisms of this lesion start immediately after HI and last for days or weeks, with the secondary injury being a crucial part the process that culminates in the final damage. Acetylcholine (ACh) is a neurotransmitter of the central nervous system that seems to have an important neuroprotective action after HI. Acetylcholinesterase (AChE) degradates ACh and inhibitors of this enzyme have been used to treat neurological damage. Its positive action on HI has been demonstrated in studies performed in our laboratory, where the administration of the alkaloid extract of Huperzia quadrifariata (An inhibitor of AChE) reduced the cognitive and histological deficits caused by this lesion. To evaluate the effects of the pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in the model oh perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right charotid artery and of exposure to a hypoxic exposure (8% O2) for 60 minutes. Intraperitoneal injections of saline in the groups Sham anf HI+Saline (HIS) and of galantamine in the groups HI+Galantamine 5 mg/kg pre-hypoxia (HIG5-Pre), HI+Galantamine 10 mg/kg pre-hypoxia (HIG10-Pre), HI+Galantamine 5 mg/kg post-hypoxia (HIG5-Post) and HI+Galantamine 10 mg/kg post-hypoxia (HIG5-Post). The Pre groups received galantamine immediately before hypoxia and the Post groups in the intervals of 1, 24, 48 and 72 hours after HI. On PND45 the analysis of the volume of brain structures showed a reduction of the volume of the ipsilesional hippocampus in the HIS group when compared to the sham and a prevention of this effect in the HIG10-Pre, but not in any other group. Biochemical analysis was performed in the ipsilesional hippocampus 24 hours after the lesion and revealed: a reduction of the number of surviving neurons in the HIS group when compared to the Sham that was prevented in the HIG10-Pre; a hypertrophy of the astrocytes in the HIS group that was prevented in the HIG10-Pre group and an increase in the activity of the anti-oxidant enzyme catalase in the HIG10-Pre group. The treatment with galantamine was able to prevent the histological deficits, increase the survival of neurons, reduce astrocytic reaction and increase the anti-oxidant activity in rats submitted to HI.

Acetylcholinesterase (AChE) belongs to a distinct family of serine hydrolases and is found in both synaptic and non-synaptic locations. At the synapses, it plays a major role in the hydrolysis of the neurotransmitter acetylcholine (ACh) while in non-synaptic tissue its function is unclear. Snake venom, particularly from the Elapidae family, is a common non-synaptic source of AChE. The venom of krait (Bungarus sindanus), an Elapidae snake, contained a high level of AChE activity. As there is no literature study about Bungarus sindanus venom AChE, it was selected as a main source of AChE activity. We characterized the venom AChE following comparative inhibitory studies with human, horse and rat cholinesterase using different ligands (tacrine, malathion, carbofuran, paraquat, antidepressants, TEMED). The enzyme of krait venom showed optimum activity at alkaline pH 8.5 with an optimal temperature of 45oC. We observed a significant reduction in substrate inhibition of krait venom AChE by using a high ionic strength buffer. With a low ionic strength buffer (10 mM PO4 pH 7.5) the enzyme was inhibited by 1.5 mM AcSCh, while with a high ionic strength buffer (62 mM PO4 pH 7.5) the enzyme was inhibited by 1mM AcSCh. Furthermore, we found that krait 5 venom acetylcholinesterase is thermally stable at 45oC. The enzyme lost only 5% of its activity after incubation at 45oC for 40 min. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide was found to be 0.052 mM. We noted that snake venom AChE was also inhibited by ZnCl2, CdCl2 and HgCl2 in a concentration dependent manner. In addition, this enzyme showed high sensitivity to tacrine, which is known to inhibit synaptic AChE. We observed that tacrine caused a mixed type of inhibition in krait venom as well as in human serum BChE. Snake venom AChE presents similar inhibitory behavior toward commonly used pesticides and herbicides as that of human serum BChE. The snake venom AChE exhibited a mixed type of inhibition for the pesticides malathion and carbofuran and the herbicide paraquat while human serum BChE presented a mixed inhibition for malathion and paraquat and an uncompetitive inhibition for carbofuran. The krait venom AChE was also affected by antidepressants such as paroxetine, imipramine, clomipramine and sertraline. Paroxetine and sertraline caused a mixed type of inhibition, while imipramine and clomipramine exhibited a competitive inhibition. Moreover, the well-known chemical N,N,N´,N´-tetramethylethylene diamine (TEMED) caused a mixed type of inhibition in snake (Bungarus) venom as well as in horse serum BChE. Futhermore, the inhibition of TEMED, was also confirmed from in vivo study in different structures of the brain, such as striatum, hippocampus, cortex, hypothalamus and cerebellum. Decrease in AChE activity was observed in all treated groups. The results suggest that TEMED exhibits toxic effect via inhibition of cholinesterase. Taken together the krait venom AChE showed similar behavior towards different ligands (tacrine, malathion, carbofuran, paraquat, antidepressants, TEMED) like other sources of cholinestease. Furthermore, krait venom contains large amount of acetylcholinesterase having highest catalytic activity and comparatively more stable than any other sources, making it more valuable for biochemical analysis. / A enzima acetilcolinesterase (AChE, E.C. 3.1.1.7) faz parte de uma família distinta de serina hidrolases, sendo uma importante enzima regulatória, encontrada principalmente no encéfalo, músculos, eritrócitos, neurônios colinérgicos e também em veneno de cobra. Nas sinapses, a sua principal função é a hidrólise no neutrotransmissor acetilcolina (ACh), enquanto que em tecidos não-sinápticos a sua função é desconhecida. O veneno de cobra, principalmente da família Elapidae, é uma fonte não sináptica abundante de AChE. Por exemplo, a espécie Bungarus sindanus, possui um alto conteúdo de AChE. No entanto, na literatura não existem muitos relatos sobre o estudo desta enzima neste veneno. Sendo assim, o veneno de cobra da espécie Bungarus sindanus, foi selecionado comno a principal fonte de AChE neste estudo. A partir dele fez-se estudos de caracterização, comparação com outras fontes de colinesterases (soro eqüino, encéfalo de ratos e sangue humano) e estudos de inibição da enzima por diversos agentes (tacrina, malation, carbofuran, paraquat e 2 antidepressivos). Foi observado que a enzima de veneno de cobra possui um pH ótimo alcalino de pH 8.5 e uma temperatura ótima de ensaio de 45oC. A inibição por substrato, característica cinética da AChE, foi reduzida significativamente ao se usar um tampão com alta força iônica (10 mM PO4 pH 7.5). A enzima possui estabilidade térmica a 45oC. A enzima perdeu somente 5% de sua atividade após 45 min de incubação à 45oC. A constante de Michaelis-Menten (Km) para a hidrólise de ACh foi de 0.052 mM com uma Vmax de 530 μmoles/min/mg de proteína. Também, foi observado que a AChE de veneno da cobra Bungarus sindanus é inibida por ZnCl2, CdCl2 e HgCl2. A enzima apresentou alta sensibilidade a tacrina, a aqual é sabidamente um inibidor da AChE sináptica. A inibição observada foi de tipo mista, tanto em veneno de cobra, tanto quanto em cholinesterase de soro humano (BChE). Também, a enzima apresentou padrão inibitório similar com a colinesterase de soro humano quando foram testados agentes pesticidas e herbicidas. A AChE de veneno de cobra exibiu uma inibição de tipo mista para os pesticidas malation e carbofuran e para o herbicida paraquat. A única diferença foi o padrão de inibição da BChE em relação ao carbofuran, que foi do tipo incompetitivo. A atividade da enzima AChE de veneno de cobra foi inibida, também, pelos antidepressivos paroxetina, imipramina, clomipramina e sertralina. A paroxetina e a sertralina causaram uma inibição de tipo mista, enquanto que a imipramina e a clomipramina exibiram um padrão de inibição competitivo. Também, o bem conhecido composto químico N,N,N´,N´-Tetramethylethylene diamine (TEMED), o qual é rotineiramente utilizado para iniciar processos de polimerização de géis de poliacrilamida causou uma inibição de tipo mista na Ache de veneno de cobra bem como em BChE de soro eqüino. Além disto, a inibição da AChE por TEMED foi observada em diferentes estruturas cerebrais de ratos, tais como estriato, hipocampo, cortex, hipotálamo e cerebelo. Estes resultados confirmam a hipótese de que o TEMED é colinotóxico. Analisando, em conjunto, os resultados podemos concluir que a AChE do veneno da cobra Bungarus Sindanus apresentou padrão cinético inibitório quase similar a outras colinesterases para os compostos tacrina, malation, carbofuran, paraquat, antidepressivos e TEMED. Além disto, o veneno desta cobra possui altas quantidades de AChE o que o torna uma fonte valiosa para estudos bioquímicos e cinéticos desta enzima.

A hipóxia-isquemia neonatal (HI) faz parte da etiologia de diversas patologias neurológicas e é causa de graves sequelas. Os mecanismos patofisiológicos dessa lesão começam com o insulto imediato após a HI e se estendem por dias ou semanas, pelo aumento da liberação de espécies reativas de oxigênio associada a redução da defesas anti-oxidantes e reação glial, sendo a lesão secundária parte crucial no processo que culmina no dano final. A acetilcolina (ACh) é um neurotransmissor do sistema nervoso central (SNC) que parece ter uma importante ação neuroprotetora após a HI. A acetilcolinaesterase (AChE) é responsável pela degradação da ACh, inibidores dessa enzima vêm sendo utilizados para o tratamento de danos neurológicos. Sua ação positiva sobre a HI foi demonstrada em estudos realizados em nosso laboratório, onde a administração do extrato de Huperzia quadrifariata (inibidor de AChE) reduziu os déficits cognitivos e histológicos causados por essa lesão Para avaliar os efeitos das administrações pré e pós-hipóxia de galantamina, inibidor da AChE, no modelo de HI perinatal, ratos Wistar no 7º dia de vida pós-natal (DPN7) foram submetidos à combinação da oclusão unilateral da artéria carótida direita e exposição a uma atmosfera hipóxica (8% de O2) durante 60 minutos. Foram aplicadas injeções intraperitoniais de salina para os grupos Sham e HI+Salina (HIS) e de galantamina nos grupos HI+Galantamina 5 mg/kg pré-hipóxia (HIG5-Pré), HI+Galantamina 10 mg/kg pré-hipóxia (HIG10-Pré), HI+Galantamina 5 mg/kg pós-hipóxia (HIG5-Pós) e HI+Galantamina 10 mg/kg pós-hipóxia (HIG10-Pós). Os grupos Pré receberam galantamina imediatamente antes da hipóxia e os grupos Pós nos intervalos de 1, 24, 48 e 72 horas após a cirurgia. No DPN45 foi feita a análise do volume das estruturas encefálicas que demonstrou a redução do volume do hipocampo do grupo HIS em relação ao Sham e uma prevenção desse efeito no grupo HIG10-Pré, mas não nos demais grupos. Análises bioquímicas foram feitas no hipocampo ipsilesional 24 horas após a lesão e revelaram: através da citometria de fluxo uma redução na sobrevivência de neurônios no grupo HIS em relação ao Sham que foi prevenida no grupo HIG10-Pré; através de ELISA uma hipertrofia dos astrócitos no grupo HIS que foi revertida no grupo HIG10-Pré e um aumento na atividade da enzima anti-oxidante catalase. O tratamento pré-hipóxia com galantamina foi capaz de prevenir os déficits histológicos, aumentar a sobrevivência celular, reduzir a reação astrocitária e aumentar a atividade anti-oxidante em ratos submetidos à HI. / Neonatal hypoxia ischemia (HI) has a role in etiology of several neurological pathologies and causes severe sequelae. The pathophysiological mechanisms of this lesion start immediately after HI and last for days or weeks, with the secondary injury being a crucial part the process that culminates in the final damage. Acetylcholine (ACh) is a neurotransmitter of the central nervous system that seems to have an important neuroprotective action after HI. Acetylcholinesterase (AChE) degradates ACh and inhibitors of this enzyme have been used to treat neurological damage. Its positive action on HI has been demonstrated in studies performed in our laboratory, where the administration of the alkaloid extract of Huperzia quadrifariata (An inhibitor of AChE) reduced the cognitive and histological deficits caused by this lesion. To evaluate the effects of the pre and post-hypoxia administrations of galantamine, a cholinesterase inhibitor, in the model oh perinatal HI, Wistar rats in the post-natal day 7 (PND7) were subjected to a combination of unilateral occlusion of the right charotid artery and of exposure to a hypoxic exposure (8% O2) for 60 minutes. Intraperitoneal injections of saline in the groups Sham anf HI+Saline (HIS) and of galantamine in the groups HI+Galantamine 5 mg/kg pre-hypoxia (HIG5-Pre), HI+Galantamine 10 mg/kg pre-hypoxia (HIG10-Pre), HI+Galantamine 5 mg/kg post-hypoxia (HIG5-Post) and HI+Galantamine 10 mg/kg post-hypoxia (HIG5-Post). The Pre groups received galantamine immediately before hypoxia and the Post groups in the intervals of 1, 24, 48 and 72 hours after HI. On PND45 the analysis of the volume of brain structures showed a reduction of the volume of the ipsilesional hippocampus in the HIS group when compared to the sham and a prevention of this effect in the HIG10-Pre, but not in any other group. Biochemical analysis was performed in the ipsilesional hippocampus 24 hours after the lesion and revealed: a reduction of the number of surviving neurons in the HIS group when compared to the Sham that was prevented in the HIG10-Pre; a hypertrophy of the astrocytes in the HIS group that was prevented in the HIG10-Pre group and an increase in the activity of the anti-oxidant enzyme catalase in the HIG10-Pre group. The treatment with galantamine was able to prevent the histological deficits, increase the survival of neurons, reduce astrocytic reaction and increase the anti-oxidant activity in rats submitted to HI.

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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / In this study, the effects of Aluminium (Al) and of different diets, both individually and in association, were investigated through the determination of NTPDase, 5 -nucleotidase and acetylcholinesterase (AChE) activities in rat brain. In addition, we investigated said effects on oxidative stress by determining activity of the antioxidant enzyme catalase as well as lipid peroxidation by measuring TBARS levels. Male rats were exposed to Al (50 mg/kg/day) by gavage during three months. NTPDase and 5 -nucleotidase activities were then determined in synaptosomes of cerebral cortex and hippocampus, as well as in platelets. ATP, ADP and AMP hydrolysis was increased in both synaptosomes of cerebral cortex and hippocampus, as well as in platelets. AChE activity and TBARS levels were determined in homogenate of different brain structures in mice exposed to Al (2.7 mg/kg/day) by gavage during three months. The group that received Al+sodium citrate presented an increase in AChE activity in hippocampus, striatum, cortex and hypothalamus. On the other hand, the group that received only Al presented a decrease in AChE activity in hypothalamus and an increase in striatum. Moreover, AChE was determined in S1 of different brain structures, synaptosomes of cerebral cortex and erythrocytes of male rats exposed to Al (50 mg/kg/day) by gavage during three months. There was an increase in AChE activity in S1 of striatum and hypothalamus and in synaptosomes of cerebral cortex and erythrocytes. However, in S1 of cerebellum, hippocampus and cortex there was a decrease. In addition, the effect of diets rich in saturated fat and refined sugar on AChE activity in homogenate of different brain structures, on catalase activity in liver and on TBARS levels in plasma and liver were determined in female and male rats. There was a decrease in AChE activity in hippocampus, cortex and hypothalamus of male and female rats given both a diet rich in saturated fat and a diet rich in refined sugar. There was no alteration of AChE activity in cerebellum and striatum. For both diets, catalase activity was increased in liver of male and female rats. In addition, considering the alterations brought about by the individual exposure to both environmental factors, Al and diets, the effect of the association of both factors was evaluated. Thus, after a period of three months of exposure to both Al (50 mg/kg/day) by gavage and diets rich in saturated fat and saturated/polyunsaturated fat ad libitum, NTPDase and 5 -nucleotidase activities were determined in synaptosomes of cerebral cortex and platelets of rats. Animals receiving both diets in association with Al and Al/Ci presented an increase in ATP, ADP and AMP hydrolysis in synaptosomes of cerebral cortex and platelets. The results obtained in the present study demonstrate that exposure to both environmental factors, Al and diets rich in saturated fat and refined sugar, either individually or in association, affected the purinergic and cholinergic systems and caused oxidative stress in rats. / Neste estudo, investigamos os efeitos do alumínio (Al) e de diferentes dietas, individualmente e em associação, através da determinação da atividade das enzimas NTPDase, 5 -nucleotidase e acetilcolinesterase (AChE) no encéfalo de roedores. Adicionalmente, investigamos o estresse oxidativo, através da atividade da enzima antioxidante catalase, e a peroxidação lipídica pela medida dos níveis de TBARS. Ratos machos foram expostos ao Al (50 mg/kg/dia) através de gavagem, por um período de 3 meses. Após o tratamento, foi determinada a atividade das enzimas NTPDase e 5 -nucleotidase em sinaptossoma de córtex cerebral, hipocampo e plaquetas. A hidrólise dos nucleotídeos ATP, ADP e AMP foi aumentada, nas frações sinaptossomais de córtex cerebral e hipocampo bem como nas plaquetas. A atividade da AChE e os níveis de TBARS foram determinados em homogeneizado de diferentes estruturas cerebrais de camundongos expostos ao Al (2,7 mg/kg/dia), através de gavagem, por um período de 3 meses. Quanto à atividade da AChE, o grupo que recebeu Al+citrato de sódio apresentou um aumento da atividade desta enzima em hipocampo, estriado, córtex e hipotálamo. Já o grupo que recebeu só Al apresentou uma diminuição da atividade em hipotálamo e um aumento em estriado. Além disto, a atividade da AChE foi determinada em S1 de diferentes estruturas cerebrais, sinaptossoma de córtex cerebral, e em eritrócitos de ratos machos expostos à Al (50 mg/kg/dia), através de gavagem, por 3 meses. A atividade da AChE apresentou um aumento em S1 de estriado e hipotálamo, e em sinaptossoma de córtex cerebral e eritrócitos. Porém, em sobrenadante (S1) de cerebelo, hipocampo e córtex houve uma diminuição. Adicionalmente, foi determinado o efeito de dietas ricas em gordura saturada e açúcar refinado sobre a atividade da enzima AChE em homogeneizado de diferentes estruturas encefálicas, e atividade da enzima catalase em fígado, e os níveis de TBARS em plasma e fígado de ratos machos e fêmeas. A atividade da AChE em hipocampo, córtex e hipotálamo de ratos machos e fêmeas foi diminuída, após exposição a ambas as dietas, rica em gordura e rica em açúcar. Nas estruturas cerebelo e estriado não houve alteração na atividade da AChE. Após o consumo de ambas as dietas, a atividade da enzima catalase foi aumentada em fígado de ratos machos e fêmeas. Adicionalmente, considerando todas as alterações ocasionadas pela exposição individual aos fatores ambientais, Al e dietas, foi avaliado o efeito da associação entre esses dois fatores. Dessa forma, após um período de 3 meses de exposição conjunta ao Al (50 mg/kg/dia) através de gavagem e o consumo ad libitum de dietas ricas em gordura saturada e gordura saturada/poliinsaturada, foram determinadas a atividade das enzimas NTPDase e 5 - nucleotidase em sinaptossomas de córtex cerebral e plaquetas de ratos. Os animais que receberam ambas as dietas administradas em conjunto com Al e Al/Ci apresentaram um aumento na hidrólise dos nucleotídeos ATP, ADP e AMP, em sinaptossoma de córtex cerebral e plaquetas. Os resultados obtidos no presente estudo relatam que a exposição de roedores a ambos os fatores ambientais Al e dietas ricas em gordura saturada e açúcar refinado, individualmente e em conjunto, afetam os sistemas purinérico e colinérgico, e causam estresse oxidativo.