背景黃芪是中醫藥中最常用的補益藥之一，現代研究發現其多糖類成分具有抗疲勞、抗氧他和免疫調節等作用，因此研究黃芪多糖的生理活性已成為研究黃芪藥理作用新的主流方向。由於多糖類物質分子量較大，單糖組成及組成方式多樣，所以對多糖的研究除生理活性外還需探討解析其他學特征。目的初步驗證黃芪多糖對免疫系統的生理活性及其自身他學組成，探討展望未來對黃芪多糖研究的新方向。方法本實驗採用水提醇沉法提取分離除黃芪粗多糖，經除蛋白及透析等操作純忙得到黃百多糖。採用高效凝膠色譜分離法及超高效液相色譜法分別求得黃芪多糖相對分子量大小及其單糖組成成分免疫活性探究使用RAW264.7 細胞系巨噬細胞，以脂多糖為陽性對照，採用MTT 法測試細胞毒性，計算加藥后一氧化氮及細胞因子IL-6 和TNF－α 生成量，評價黃芪多糖的免疫調節作用。結果黃芪多糖相對分子量為108.02kDa(±2.73kDa），由阿拉伯糖、葡萄糖、半乳糖、葡萄糖醛酸和半乳糖醛酸組成。MTT 實驗表明黃芪多糖對細胞無明顯毒性﹔ NO 及細胞因子IL-6 和TNF－α 生成量表明其具有免疫調節功能，且作用強度與黃芪多糖濃度在一定範圍內呈正相關。結論黃芪多糖具有免疫調節活性，但其組成成分較多，他學結構複雜，仍需要進行更多研究探討其作用機制及其他學結構與免疫調節機制的關係。【關鍵詞】黃芪多糖﹔化學組成﹔免疫活性

Orientador: Isabel Cristina Cherici Camargo / Banca: Regildo Márcio Gonçalves da Silva / Banca: Eneri Vieira de Souza Leite Mello / Resumo: A Agave sisalana (sisal) é amplamente cultivada em território brasileiro. Foi constatada a presença de cinco saponinas esteroidais nessa planta, responsáveis por várias atividades farmacológicas, destacando-se as atividades antifúngica e anti-inflamatória. Popularmente, o sisal ainda é utilizado para o tratamento de doenças hepáticas, tuberculose e sífilis. Devido à sua ação detergente, as saponinas são dotadas de efeito tóxico em função de sua propriedade de causar ruptura em eritrócitos, liberando hemoglobina, além de outros efeitos ainda relacionados à lise celular como as ações inseticida, anti-helmíntica e ictiotóxica. Considerando-se as diversas ações farmacológicas da A. sisalana e seu amplo uso na medicina popular, torna-se necessário investigar seus possíveis efeitos colaterais na reprodução feminina, os quais ainda não foram relatados na literatura / Abstract: The Agave sisalana (sisal) is largely cultivated in Brazil. It was found the presence of five steroidal saponins that plant, responsible for various pharmacological activities, especially the anti-fungal and anti-inflammatory activities. Popularly, sisal is also used for the treatment of liver diseases, tuberculosis and syphilis. Due to its detergent action, saponins are equipped with toxic effect due to its property to cause disruption in erythrocytes, releasing hemoglobin, and other effects still related to cell lysis as the actions insecticide, anthelmintic and ichthyotoxic. Considering the various pharmacological actions of A. sisalana and its widespread use in folk medicine, it is necessary to investigate their possible side effects on female reproduction, which have not yet been reported in the literature / Mestre

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The maternal consumption of alcohol during pregnancy produces a wide range of abnormalities in the offspring. The major objectives of this thesis were to investigate (1) the correspondence between prenatal ethanol-induced alterations in behavior and in hypothalamicpituitary- adrenal (HPA) activity, (2) the ability of early postnatal handling as an environmental manipulation to attenuate at least some of the adverse behavioral and physiological consequences of prenatal ethanol exposure, and (3) possible mechanisms mediating the HP A hyperresponsiveness to stressors observed in animals prenatally exposed to ethanol and the possible influence of postnatal handling on those mechanisms. Sprague-Dawley rats from prenatal ethanol (E), pair-fed (PF) and ad libitum fed control (C) treatment groups were tested as young adults (-35-120 d of age) or mid-aged adults (13-14 months of age). The first study investigated the effects of prenatal ethanol exposure (E) and postnatal handling (H) on behavior and HPA activity during a conditioned taste aversion (CTA) task. We tested the hypothesis that E animals which underwent postnatal handling would show improved conditioned aversion learning and reduced HPA activity compared to E animals that did not experience handling (nonhandled, NH). We found that prenatal ethanol exposure and postnatal handling independently resulted in an increased rate of consumption of a saccharin solution over five preexposure days. In addition, we found that handling differentially affected posttoxicosis consumption of the conditioned solution as well as corticosterone (CORT) levels in E, PF and C animals. H-E animals showed increased posttoxicosis intake compared to H-PF and H-C animals during reexposure under non-deprived conditions; CORT levels were lower in PF and C than E males compared to their N H counterparts during reexposure under food- and waterdeprived conditions. Thus, E animals were less able to utilize environmental cues in the present study, displaying a more rapid reduction in neophobia compared to PF and C animals and, following postnatal handling, showing a decreased acquisition of conditioned aversion and an increased CORT response during reexposure to the conditioned solution. The second study examined spatial learning and memory in young adult (2 months) and mid-aged (13-14 months of age) H and N H E and control animals utilizing a Morris water maze. We investigated the hypothesis that postnatal handling would improve spatial navigation in E animals compared to E animals that did not experience handling and/or attenuate differences among E and control animals, and that this effect might be age-dependent. We also examined whether performance deficits in mid-aged animals would correspond to increases in CORT levels on the last day of testing. Young E males showed impairments in spatial navigation compared to young PF and C animals, taking longer to find the hidden platform over the course of testing and displaying an alteration in search pattern when the platform was removed. Interestingly, differences in young E, PF and C animals in escape latency and in distance traveled prior to finding the platform were apparent in H but not in N H animals. There were no differences in performance on the Morris water maze in mid-aged E, PF and C animals, but CORT levels were elevated in mid-aged E compared to C animals, supporting previous data indicating that E animals demonstrate HPA hyperresponsiveness to stressors. Lastly, although mid-aged animals had longer escape latencies and an altered search pattern compared to young animals, handling did not appear to attenuate impairments associated with aging. The third study investigated the hypothesis that postnatal handling might attenuate stress-induced ACTH and/or CORT differences among E, PF and C animals. Furthermore, the ability pf postnatal handling to modulate HPA feedback deficits in E animals was examined during exposure to a restraint stressor following dexamethasone (DEX) administration. Both E females and males showed increased ACTH and CORT compared to PF and/or C animals following saline administration. Administration of DEX to block HPA activity significantly suppressed both plasma ACTH and CORT in all animals. However, E females exhibited increased and/or prolonged elevations in ACTH and CORT compared to PF and C animals following DEX blockade. These data suggest that the insult of prenatal ethanol exposure affects both male and female offspring, but that there may be a sex-specific difference in sensitivity of the mechanism(s) underlying HPA hyperresponsiveness. Postnatal handling reduced ACTH levels in both females and males following saline administration. Following DEX administration, H males had lower CORT than NH males. Postnatal handling resulted in a more rapid decrease in stress-associated CORT elevations in C females, and attenuated differences in CORT between PF and C females. However, postnatal handling did not attenuate deficits in negative feedback inhibition in E females; E females in both the H and N H treatments showed elevated CORT compared to their C counterparts, and H-E females also showed elevated CORT compared to H-PF females. Thus, postnatal handling did not attenuate the typical HPA hyperresponsiveness to stressors observed in E animals (saline condition), nor did it eliminate deficits in HPA feedback inhibition in E females (DEX condition). The fourth study examined whether the mechanisms resulting in HPA hyperresponsiveness in E animals are similar to those underlying the effects of postnatal handling. Differences in HPA responsiveness between H and NH animals appear to be dependent upon basal CORT activity and not stress-induced elevations in CORT. Therefore, we tested the hypothesis that differences in HPA activity among E and control animals would not occur following adrenalectomy (ADX) but could be reestablished following replacement with basal levels of exogenous CORT. In the absence of a CORT feedback signal or in the presence of a constant, basal CORT feedback signal, E, PF and C animals did not significantly differ in their abilities to regulate ACTH secretion, indicating that during the trough of the circadian rhythm, E, PF and C animals are equally capable of regulating HPA activity utilizing either CORT-independent feedback or feedback mediated through basal CORT activity. Thus, the effects of prenatal ethanol exposure on HPA function do not appear to be dependent upon the feedback signal provided by basal CORT levels. In conclusion, handling did not attenuate the effects of prenatal ethanol exposure examined in the present experiments. This may be because the effects of postnatal handling and prenatal ethanol exposure on HPA function are mediated through different mechanisms as well as the finding that handling is, at least partly, mediated through mother-pup interactions. Therefore, postnatal handling might exert differential effects on litters in which pup behavior has already been altered by prenatal treatments, underscoring the enduring effects of prenatal ethanol exposure. / Arts, Faculty of / Psychology, Department of / Graduate

Excitatory glutamatergic inputs from limbic regions such as the hippocampus and the basolateral amygdala (BLA), and dopaminergic inputs from the ventral tegmental area converge in the nucleus accumbens (NAc). It has been proposed that interactions between these glutamatergic and dopaminergic pathways play an important role in adaptive behaviors. The present thesis employed a multidisciplinary approach to study these interactions, with a specific emphasis on the importance of mesoaccumbens dopamine (DA) transmission, in order to obtain a better understanding of the neural mechanisms by which the NAc transforms signals from the temporal lobes into behavior. The experiments of Chapter 2 utilized extracellular single-unit recordings of individual NAc neurons in combination with electrochemical measures of DA efflux in the NAc. Recordings from NAc neurons which received input from the hippocampus but not the BLA revealed that increased efflux of mesoaccumbens DA, evoked by tetanic stimulation of the fimbria, potentiated hippocampal-evoked neural activity in these cells. These effects were mediated by both DA and NMDA receptors. Similar recordings from neurons which received converging input from both the hippocampus and the BLA revealed tetanic stimulation of the fimbria again potentiated hippocampal evoked spiking activity, while concurrently suppressing BLA-evoked spiking activity in the same neurons. The suppression of BLA-evoked spiking activity was activity-dependent, and was mediated by both D, and adenosine A, receptors. Chapter 3 showed that random foraging on a radial-arm maze, which is dependent on a neural circuit linking the hippocampus to the NAc, was correlated with an increase in mesoaccumbens DA extracellular levels, as measured with microdialysis. In Chapter 4, pharmacological blockade of DA or NMDA receptors in the NAc, or selective disruption of dopaminergic modulation of ventral subicular inputs to the NAc (using an asymmetrical infusion procedure) significantly disrupted random foraging. These effects were mediated by the Dl receptor. In Chapter 5, the present data are integrated with previous research to formulate a model of ventral striatal function. It is proposed that the NAc mediates behavior through distinct patterns of activity and inactivity driven by excitatory limbic input projecting to different groups of neural ensembles. Mesoaccumbens DA transmission plays an essential role in regulating the synchrony ensemble activity, augmenting activity in one ensemble while suppressing activity in another. It is argued that the modulatory effects of DA appears to be essential when an organism must switch from one form of adaptive behavior to another in response to a constantly changing environment. / Arts, Faculty of / Psychology, Department of / Graduate

Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.

Cytotoxic natural products represent a class of cancer drug candidates that have remained largely untapped as payloads in the antibody-drug conjugate (ADC) therapeutic modality. The epothilones, a class of exquisitely cytotoxic natural products and their synthetic analogs, are a prime example, and our work has focused on the development of epothilones as ADC payloads. Strategies toward this goal have included total synthetic efforts toward four structurally distinct epothilone analogs equipped with linker functionality and structural modifications designed to improve metabolic and chemical stability. In addition, we have pursued the synthesis of octreotide- epothilone conjugates, structures designed to target epothilones into cells that overexpress somatostatin receptors 2 and 5. Biological evaluation via in vitro cellular assays revealed one of our epothilone analogs as a promising epothilone-inspired ADC payload. Synthetic efforts toward these goals will be discussed.

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Master of Science