A interação da radiação ionizante com moléculas de alanina promove a quebra das ligações químicas entre o átomo de carbono central e os grupos que se ligam a ele, resultando na formação de radicais livres paramagnéticos. A técnica de dosimetria utilizando alanina e a ressonância de spin eletrônico (ESR) baseia-se na determinação da concentração dos radicais livres produzidos pela interação da radiação ionizante com as moléculas da alanina por meio do registro do espectro de ESR da alanina irradiada. Para propósitos de dosimetria, a amplitude da linha central do espectro pode ser correlacionada diretamente com a dose de radiação. Na atualidade, a alanina/ESR é uma técnica de dosimetria amplamente aceita para a dosimetria de altas doses como aquelas usadas nos processos de irradiação de alimentos, esterilização de produtos médicos e radioterapia. A expansão dos tratamentos de câncer por meio de procedimentos de radiocirurgia e radioterapia de intensidade modulada (IMRT) torna cada vez mais relevante a necessidade de realizar a dosimetria em pequenos campos de radiação. Para a dosimetria desses feixes estreitos de radiação, é necessário o uso detectores que também apresentem dimensões reduzidas de forma a proporcionar alta resolução espacial. Uma vez que a sensibilidade dos dosímetros de alanina depende do número de radicais formados pela radiação, a redução do tamanho do detector de alanina implica em redução de seu volume sensível e consequentemente de sua sensibilidade. Assim, pesquisas têm sido realizadas buscando uma melhora de sua sensibilidade. Neste trabalho foram investigados os mecanismos envolvidos na formação da nanoestrutura e a capacidade da alanina em estabilizar e acomodar nanopartículas metálicas de ouro e prata em sua matriz cristalina, para aplicação dos nanocompósitos como detectores de radiação utilizando a técnica de ressonância de spin eletrônico. Foi analisado o tamanho das partículas, sua interação com as moléculas do aminoácido, sua agregação e posicionamento dentro da matriz cristalina da alanina. Além disso, foi investigada a maneira como a morfologia e o estado de agregação e segregação das nanopartículas podem alterar a resposta dosimétrica dos detectores híbridos de alanina. O aumento de sensibilidade dos dosímetros de alanina mostrou estar diretamente associado ao aumento do número de elétrons ejetados por efeito fotoelétrico. No entanto, foi mostrado que aumento da sensibilidade dos dosímetros de alanina é governado por outros fatores além do número de eventos fotoelétricos. A auto-absorção dos elétrons oriundos de uma nanopartícula metálica, por outra nanopartícula vizinha, também influencia na resposta dosimétrica do detector. Assim, sistemas homogêneos onde não houve segregação, apresentaram menor auto-absorção e, consequentemente, os ganhos em sensibilidade foram maiores. Para sistemas onde houve aumento de tamanho e segregação das partículas metálicas, a auto-absorção se tornou mais relevante. Para fótons com energias próximas a energia da camada K do metal presente no nanocompósito, a absorção da radiação foi muito mais relevante do que a organização das nanopartículas na estrutura da alanina. Para energias maiores a probabilidade de eventos fotoelétricos somente diminui e, dessa forma, a agregação e segregação das partículas desempenharam papéis fundamentais na sensibilidade dos dosímetros. Assim, a sensibilidade dosimétrica dos detectores pode ser aumentada e otimizada por meio do uso da nanoestruturação, reduzindo a auto-absorção dos elétrons ejetados. / The interaction of ionizing radiation with alanine molecules produces free radicals. The room temperature stable alanine radical is molecule depleted of the amine group. The Alanine/ESR dosimetry technique is able to measure the amount of free radicals produced by the radiation. For dosimetric purposes, the amplitude of the central ESR spectral line is direct related to the absorbed dose by the alanine molecules. Nowadays, the alanine/ESR technique is largely used for dosimetry of high doses such as used in food irradiation, sterilization of medical products and radiotherapy. The growing need for cancer treatments using radiosurgery and intensity modulated radiation therapy (IMRT) makes important the dosimetry of small radiation fields. To this end, it is necessary to use small radiation detectors in order to obtain enough special resolution. Once the sensitivity of alanine dosimeters depends on the amount of free radical produced by the radiation, reducing the size of alanine detector also reduces its sensitivity. Thus there is an intense investigation to develop high sensitivity of Alanine/ESR dosimeters. In this work the mechanisms involved in the formation of nanostructures and the ability of alanine to stabilize and accommodate nanoparticles of silver and gold in its crystalline matrix, for applications of the nanocomposite as radiation detectors was investigated using the electron spin resonance technique. The effect of particle size, its interaction with alanine molecules, aggregation and segregation of particles inside the alanine matrix as well as the way the morphology and the state of agglomeration change the dosimetric sensitivity of the alanine detectors were investigated. The increase of sensitivity of the alanine detectors was direct related to the amount of photoelectric events in the nanocomposite. However, the self-absorption of the electrons ejected by one particle, by another nanoparticle in the vicinity, also dictates the sensitivity of the detectors. In this sense, homogeneous systems presented less self-absorption and, consequently, the increase in sensitivity was higher. For systems containing aggregated and segregated particles, self-absorption becomes more relevant. For incident photons with energies near de K-edge of the metal of the nanocomposite, the number of photoelectric events was more relevant than the organization of the particles inside the alanine crystals. For higher energies the probability of a photoelectric interaction decreases, therefore the aggregation and segregation of particles become essential in determining the sensitivity of the dosimeters.

Estudos recentes têm demonstrado que a suplementação de beta-alanina (BA) pode melhorar o desempenho físico. O mecanismo proposto para tal resultado envolve o aumento das concentrações intramusculares de carnosina, um dipeptídeo cuja função mais bem atribuída é a de manutenção do equilíbrio ácido-básico. Apesar do emergente corpo literário acerca dos efeitos ergogênicos da suplementação de BA, a maior parte das evidências provém de estudos conduzidos com indivíduos não treinados ou fisicamente ativos, enquanto os estudos com indivíduos treinados são escassos, e seus resultados, controversos. Tem sido especulado que a diferença na capacidade tamponante muscular entre indivíduos treinados e não treinados é um possível fator mascarando o efeito ergogênico da suplementação de BA em indivíduos treinados, já que têm sido demonstrado que este perfil de indivíduos possui maior capacidade tamponante e conteúdo muscular de carnosina. Assim, o objetivo do presente estudo foi investigar a influência do estado de treinamento sobre o desempenho físico intermitente de membros inferiores em resposta à suplementação de BA. Para tanto, 40 homens jovens e saudáveis foram recrutados para participar do estudo, e divididos em dois grupos de acordo com o seu estado de treinamento [ciclistas treinados (T) ou indivíduos não treinados (NT)]. Os participantes foram aleatoriamente designados a um grupo suplementado com BA ou placebo (dextrose - PL), provendo quatro condições experimentais: NTPL, NTBA, TPL e TBA. A suplementação foi realizada com a ingestão de 6.4 gramas de BA ou PL por dia, durante 4 semanas. Antes e após o período de suplementação, os participantes completaram 4 séries do teste de Wingate para membro inferior, com 30 segundos de duração cada uma e 3 minutos de descanso entre elas. O trabalho total realizado foi significantemente aumentado após o período de suplementação em ambos os grupos NTBA (+1349 ± 1411 kJ; P = 0.03) e TBA (+1978 ± 1508 kJ; P = 0.002), foi significantemente reduzido no grupo NTPL (-1385 ± 2815 kJ; P = 0.03), e não se alterou no grupo TPL (-219 ± 1507 kJ; P = 0.73). Comparada ao período pré-suplementação, a potência média no período pós-suplementação foi significantemente maior na série 4 para o grupo NTBA (P = 0.0004), enquanto a mesma foi maior nas séries 1, 2 e 4 (P <= 0.05) para o grupo TBA. Não foram observadas diferenças na potência média entre o período pré- e pós-suplementação para os grupos NTPL e TPL. Em conclusão, quatro semanas de suplementação de BA foram efetivas em melhorar o desempenho físico intermitente de membros inferiores em ambos os participantes treinados e não treinados. Estes dados ressaltam a eficácia ergogênica da suplementação de BA para exercícios de alta-intensidade, independentemente do estado de treinamento do indivíduo / Recent studies have demonstrated that beta-alanine (BA) supplementation can improve performance. The proposed mechanisms for this result involve an increased muscle carnosine content, a dipeptide whose function is attributed to the maintenance of acid-base balance. Even though the body of evidence surrounding the ergogenic effects of BA supplementation is increasing, most of the evidences come from studies conducted with physically active or untrained individuals, while studies with trained participants are scarce, and their results, controversial. It has been speculated that the difference in muscle buffering capacity between trained and untrained individuals is a possible factor masking the ergogenic effect of BA supplementation in trained individuals, who have already been demonstrated to have greater buffering capacity and muscle carnosine content. Therefore, the aim of this study was to investigate the influence of training status on intermittent lower-body performance in response to BA supplementation. For this purpose, forty young males were divided into two groups according to their training status (trained - T, and untrained - NT cyclists). Participants were further randomly allocated to BA or placebo (dextrose - PL) groups, providing four experimental conditions: NTPL, NTBA, TPL, TBA. BA or PL was ingested by 6.4 g·d-1, during for 4 weeks. Before and after the supplementation period, participants completed four 30-seconds lower-body Wingate bouts, separated by 3 minutes. Total work done was significantly increased following supplementation in both NTBA (+1349 ± 1411 kJ; P = 0.03) and TBA (+1978 ± 1508 kJ; P = 0.002), and it was significantly reduced in NTPL (-1385 ± 2815 kJ; P = 0.03) with no difference for TPL (-219 ± 1507 kJ; P = 0.73). Compared to pre-supplementation, post-supplementation mean power output was significantly higher in bout 4 for NTBA (P = 0.0004), and higher in bouts 1, 2 and 4 (P <= 0.05) for TBA. No differences were observed in mean power output for NTPL and TPL from pre- to post-supplementation period. In conclusion, four weeks of BA supplementation was effective at improving intermittent lower-body performance in both untrained and trained individuals. These data highlight the efficacy of BA as an ergogenic aid for high-intensity exercise regardless of the training status of the individual

Objetivo: Evaluar la asociación entre los niveles elevados de transaminasemia y resistencia a la insulina en una población de individuos sin alteraciones laboratoriales previas de glicemia, insulinemia, ni tiroideos. Métodos: Realizamos un modelo lineal generalizado crudo y ajustado de la familia Poisson con una varianza robusta, para evaluar la asociación entre los niveles elevados de transaminasemia y resistencia a la insulina. Las asociaciones se presentaron como razón de prevalencia (RP) con sus respectivos intervalos de confianza al 95%. Resultados: Se incluyeron 261 participantes. La mediana de edad fue de 39 años (31-45) y el 23,7% de los participantes eran hombres. La prevalencia de transaminasas séricas elevadas para TGO y TGP fue de 13.8% y 26.1%, respectivamente. La prevalencia de resistencia a la insulina fue del 34,1%. En el análisis en bruto encontramos significancia estadística entre TGP y TGO elevados y resistencia a la insulina (RP = 3,18; IC del 95%: 2,33-4,34 y RP = 2,44; IC del 95%: 1,88 a 3,30; respectivamente). Sin embargo, en el análisis multivariado ajustado, la asociación entre el nivel elevado de transaminasas séricas y la resistencia a la insulina permaneció estadísticamente significativa con TGP, pero se perdió con TGO; un PR = 1.90; CI95%: 1.31-2.77 y un PR = 1.23; CI95%: 0,93-1,61; respectivamente. Conclusión: niveles séricos elevados de TGP se asociaron con resistencia a la insulina. TGP podría usarse en la práctica clínica como una herramienta adicional para evaluar la resistencia a la insulina en personas sin alteraciones laboratoriales previas de glicemia, insulinemia, ni tiroideos. / Aim: To evaluate the association between elevated serum transaminase levels and insulin resistance in a population of individuals without alterations in their laboratorial values of glycemia, insulinemia and thyroid panel. Methods: We performed a crude and adjusted generalized linear model of the Poisson family with robust variance, in order to evaluate the association between elevated serum transaminase levels and insulin resistance. The associations were presented as prevalence ratio (PR) with their respective 95% confidence intervals (95% CI). Results: We included 261 participants in the study. The median age was 39 years (31-45) and 23,7% of the participants were men. The prevalence of elevated serum transaminase for TGO and TGP were, 13.8% and 26.1%, respectively. The prevalence of insulin resistance was 34,1%. In the crude analysis we found statistical significance between elevated TGP and TGO and insulin resistance (PR=3,18; 95% CI: 2,33-4,34 and PR=2.44; 95% CI: 1.88-3.30; respectively). However, in the multivariate analysis adjusted for age, sex, body mass index and thyroid hormones, the association between the elevated serum transaminase level and insulin resistance remained statistically significance with TGP, but lost its significance with TGO; a PR = 1.90; CI95%: 1.31-2.77 and a PR = 1.23; CI95%: 0.93-1.61; respectively. Conclusion: Elevated serum levels of TGP were associated with insulin resistance. TGP could be used in clinical practice as an additional tool to assess insulin resistance in people without laboratorial alterations in glycemia, insulinemia and thyroid panel. / Tesis

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (Vm), intracellular K+, Cl-, and Na+ activities (aik, aCliand aNai), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 μml-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 m m tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control Vm was -33±1 mV. l-alanine uptake first depolarized Vm by 2±0.2 mV and then hyperpolarized Vm by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, aNaiincreased by 30% from 19±2 to 25±3 m m (P < 0.01), and aKidid not change significantly from 83±3 m m. However, with added ouabain (1 m m) l-alanine caused only a 2-mV increase in Vm, but now aKidecreased from 61±3 to 54±5 m m (P < 0.05). Hyperpolarization of Vm by l-alanine uptake also resulted in a 38% decrease of aClifrom 20±2 to 12±3 m m (P < 0.001). Changes in Vm and VCl - Vm voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl- with the Vm in hepatocytes. Added Ba2+ abolished the l-alanineinduced hyperpolarization, and aCliremained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte aKiis regulated by increased Na+-K+ pump activity during l-alanine uptake in spite of cell swelling and increased Vm due to increased K+ conductance. The hyperpolarization of Vm during l-alanine uptake provides electromotive force to decrease aCli. The latter may contribute to hepatocyte volume regulation during organic solute transport.

Changes in physiological function occurring during a body water deficit may result in significant decrements in performance, cognitive function and fine motor control during exercise. This may be due to the magnitude of the body water deficit. Rehydration strategies are important to prevent these deleterious effects in performance. The purpose of this study was to examine the changes before and after prolonged exercise of an alanine-glutamine dipeptide (AG) on cognitive function and reaction time. Twelve male endurance-trained runners (age: 23.5 [plus or minus] 3.7 y; height: 175.5 [plus or minus] 5.4 cm; weight: 70.7 [plus or minus] 7.6 kg) participated in this study. Participants were asked to run on a treadmill at 70% of their predetermined VO2max for 1 h and then run at 90% of VO2max until volitional exhaustion on four separate days (T1-T4). T1 was a dehydration trial and T2-T4 were all different hydration modalities (electrolyte drink, electrolyte drink with a low dose of AG, electrolyte drink with a high dose of AG, respectively) where the participants drank 250 mL every 15 min. Before and after each hour run, cognitive function and reaction tests were administered. Hopkins Magnitude Based Inferences were used to analyze cognitive function and reaction time data. Results showed that physical reaction time was likely faster for the low dose trial than the high dose trial. Dehydration had a possible negative effect on the number of hits in 60-sec compared to both the low and high dose trials. Comparisons between only the electrolyte drink and the high dose ingestion appeared to be possibly negative. Analysis of lower body quickness indicates that performance in both the low and high dose trials were likely improved (decreased) in comparison to the dehydration trial. Multiple object tracking analysis indicated a possible greater performance for dehydration and low dose compared to only the electrolyte drink, while there was a likely greater performance in multiple object tracking for the high dose trial compared to consumption of the electrolyte drink only. The serial subtraction test was possibly greater in the electrolyte drink trial compared to dehydration. Rehydration with the alanine-glutamine dipeptide during an hour run at a submaximal intensity appears to maintain or enhance subsequent visual reaction time in both upper and lower body activities compared to a no hydration trial. The combination of the alanine-glutamine dipeptide may have enhanced fluid and electrolyte absorption from the gut and possibly into skeletal tissue to maintain neuromuscular performance.

No description available.

<p>Peptidoglycan (PG) remodelling and cell division are two important cellular processes that are the major target of antibiotics. Due to rising resistance, the need for new antibiotics today has never been greater. Therefore it is important to fill the gaps in our understanding of these two important processes in order to discover new and promising antibiotic targets. Peptidoglycan synthesis and remodelling is a highly coordinated event that involves a wide number of enzymes and processes which are not well understood. N-acetylmuramoyl-L-alanine amidases, whose function is to cleave the amide linkage between the stem peptides and the lactyl moiety of N-acetylmuramic acid, is a major class of PG-active proteins. Their role in daughter cell separation during cell division is well established in <em>Escherichia coli</em> however little is known about it in other systems. Using enzymatic assays we characterize AmiC as a novel amidase in <em>Pseudomonas aeruginosa. </em>Through mutational analysis and microscopy we show that AmiB and AmiC are required for daughter cell separation. A deletion of both enzymes results in a cell chaining phenotype with abnormal cell morphology. Transmission electron microscopy reveals that the double mutant is arrested at the septal peptidoglycan separation step. In addition to cell chaining, the ∆<em>amiB/amiC</em> mutant exhibits a significant increase in susceptibility to antibiotics. We also demonstrate that the LysM motif of AmiB is not required for its role in cell separation. Furthermore, the <em>amiB</em> mutant has significantly shorter cells than the wildtype indicating an additional role for the enzyme in the cell. Lastly, through a novel bioinformatics strategy we identify PA5047 as a potential PG amidase.</p> / Bachelor of Science (BSc)

The work focused on addressing four main objectives. The first objective was to quantify protein and amino acid substitution reactions. Michael addition reactions were used to modify the amino acids and protein. Amino acids alanine, cysteine, and lysine, and protein ovalbumin (OA) were substituted with different concentrations of ethyl vinyl sulfone (EVS). The substituted products were analyzed using Raman spectroscopy and UV-spectroscopy based ninhydrin assay. In case of alanine, Raman and UV results correlated with each other. With cysteine at lower EVS substitutions amine on the main chain was the preferred site while the substitution shifted to thiols at higher substitutions. This could only be discerned using Raman spectroscopy. Lysine has amines on the main chain and side chain while main chain amine was the most reactive site at lower concentrations of EVS while at higher concentrations side chain amines were also substituted. This information could be discerned using Raman spectroscopy only and not UV spectroscopy. In case of protein as observed by Raman and UV spectroscopy the reaction continued at higher concentrations of EVS indicating the participation of glutamine and asparagines at higher substitutions. However, the reaction considerably slowed down at higher EVS substitutions. The second objective of the study was to decrease the glass transition temperature (Tg) of OA through internal plasticization and also study the effects of the substituents on the thermal stability of OA. The hypothesis was by covalently attaching substituents to OA, number of hydrogen bonds can be reduced while increasing the free volume and this would reduce Tg. EVS, acrylic acid (AA), butadiene sulfone (BS) and maleimide (MA) were the four groups used. EVS was the most efficient plasticizer of all the four substituents. The Tg decreased with the increasing concentration of EVS until all of the reactive of groups on OA were used up. Tg decreased slightly with AA and BS while no change was observed with MA. However, the substituents showed exact opposite trend in thermal stability as measured using thermogravimetric analysis (TGA). The thermal stability of MA substituted OA was the highest and that of EVS substituted OA was least. FT-IR spectroscopy results indicated that all four substituents caused structural changes in OA. This implied that there were intermolecular interactions between substituted protein chains in case of AA, BS, and MA. This caused an increase in the thermal stability. EVS on the other hand is a linear chain monomer with a hydrophobic end group and hence could not participate in the intermolecular interactions and hence caused a decrease in Tg. As mentioned above the limitation to this technique is the number of available reactive groups on the protein. However, we successfully demonstrated the feasibility of this method in decreasing Tg of protein. The third objective was to create hydrogels by crosslinking OA with divinyl sulfone (DVS). Protein hydrogels due to their biocompatible nature find applications in drug delivery and tissue engineering. For tissue engineering applications the hydrogels need to be mechanically stable. In this study the protein was substituted with EVS or AA and then crosslinked with DVS. The swelling ratio was measured as a function of pH. All the hydrogels showed the same trend and swelled the least at pH 4.5 which is the isoelectric point of the protein. At basic pH conditions EVS substituted hydrogels swelled the most while AA substituted hydrogels showed least swelling. The static and dynamic moduli of the hydrogels were determined using tensile tester and rheometer respectively. The static modulus values were three times the dynamic modulus. The modulus of the control which is crosslinked OA was least and that of AA substituted OA was highest. The stress relaxation test also showed similar results in which AA substituted OA relaxed the most and the control relaxed the least. FT-IR of the dry hydrogels showed that the amount of hydrogen bonding increased with AA substitution. The hydrophilic AA end groups interacted with each other forming hydrogen bonds. These hydrogen bonds served as additional crosslinks there by increasing the modulus of the hydrogels. EVS on the other hand was incapable of interactions due to the lack of hydrophilic end groups. We were successfully able to create protein hydrogels and control the swelling and mechanical properties by varying the amount of substituted group. The final objective of the study was to create and characterize microstructures from substituted alanine and lysine. Alanine and lysine were substituted with different concentrations of EVS. Bars and fibers were observed for alanine at moderate substitutions while at higher concentrations random structures were observed using scanning electron microscopy (SEM). Lysine formed tubes at moderate EVS substitutions and rosettes at high concentrations of EVS as evidenced by SEM. FT-IR results suggested that instead of carbonyl one of sulfonyl bonded to the available amine in modified amino acids. And only in this case fibers, tubes and rosettes were observed. X-ray diffraction (XRD) results supported this observation. Using these results we hypothesized that the self assembled structures very much depended on the amount of EVS present in the substituted product and sulfonyl forming β-sheet analogs with amine. / Ph. D.

In insects, the cuticle provides protection against physical injury and water loss, rigidness for muscle attachment and mechanical support, and flexibility in inter-segmental and joint areas for mobility. As most insects undergo metamorphosis, they need to shred off old cuticle and synthesize new cuticle to fit the body shape and size throughout their life cycles. The newly formed cuticle, mainly composed of cuticular proteins, chitin, and sclerotizing reagents, needs to be hardened through the crosslinks between cuticular proteins and sclerotizing reagents. This dissertation concerns the biochemical activities of several pyridoxal 5-phosphate (PLP)-dependent decarboxylases with most of them involved in insect cuticle hardening. Herein, we first present a detailed overview of topics in reactions and enzymes involved in insect cuticle hardening. Aspartate 1-decarboxylase (ADC) is at the center of this dissertation. beta-alanine, the product of ADC-catalyzed reaction from aspartate, is the component of an important sclerotizing reagent, N-beta-alanyldopamine; the levels of beta-alanine in insects regulate the concentrations of dopamine, therefore affecting insect sclerotization and tanning (collectively referred as cuticle hardening in this dissertation). Biochemical characterization of insect ADC has revealed that this enzyme has typical mammalian cysteine sulfinic acid decarboxylase (CSADC) activity, able to generate hypotaurine and taurine. The result throws lights on research in the physiological roles of insect ADC and the pathway of insect taurine biosynthesis. Cysteine was found to be  an inactivator of several PLP-dependent decarboxylases, such as ADC, glutamate decarboxylase (GAD) and CSADC. This study helps to understand symptoms associated with the abnormal cysteine concentrations in several neurodegenerative diseases. A mammalian enzyme, glutamate decarboxylase like-1 (GADL1), has been shown to have the same substrate usage as insect ADC does, potentially contributing to the biosynthesis of taurine and/or beta-alanine in mammalian species. Finally, the metabolic engineering work of L-3, 4-dihydroxyphenylalanine decarboxylase (DDC) and 3, 4-dihydroxylphenylacetaldehyde (DHPAA) synthase has revealed that the reactions of these enzymes could be determined by a few conserved residues at their active site. As both enzymes have been implicated in the biosynthesis of sclerotizing reagents, it is of great scientific and practical importance to understand the similarity and difference in their reaction mechanisms. The results of this dissertation provide valuable biochemical information of ADC, DDC, DHPAA synthase, and GADL1, all of which are PLP-dependent decarboxylases. ADC, DDC, DHPAA synthase are important enzymes in insect cuticle hardening by contributing to the biosynthesis of sclerotizing reagents. Knowledge towards understanding of these enzymes will promote the comprehension of insect cuticle hardening and help scientists to search for ideal insecticide targets. The characterization of GADL1 lays groundwork for future research of its potential role in taurine and beta-alanine metabolism. / Ph. D.

The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA), a non-protein amino acid, first attracted attention in correlation to reports of high incidence of the unusual neurological disease amyotrophic lateral sclerosis/Parkinsonism-dementia (ALS/PDC) among the people of Guam in the South Pacific Ocean. Experimental studies have revealed that BMAA causes neuronal cell death. The neurotoxin is suggested to act via excitotoxicity through interaction with glutamatergic receptors. More importantly, BMAA is suggested to misincorporate in the synthesis of proteins, and contribute to protein misfolding and/or deleterious aggregation, which are hallmarks of several neurodegenerative disorders. A selective uptake of BMAA in the rat neonatal hippocampus can interfere with brain development, causing learning and memory impairments in adult rats. The aim of the present study was to investigate the effects of BMAA in human neuroblastoma SH-SY5Y cells. These cells were exposed to BMAA (10 μM, 50 μM, 100 μM or 500 μM) for 72 hours, and the expression of five selected proteins, including heat shock protein-27 (HSP-27), lysosomal associated membrane protein-1 (LAMP-1), CCAAT-enhancer-binding protein homologous protein (CHOP), Golgi associated plant pathogenesis related protein-2 (GLIPR-2), and glucose regulated protein-78 (GRP-78). They were carried out with immunohistochemistry (IHC). Results revealed an increased expression of all selected proteins, which indicates an uptake and shows the effects of BMAA in the cell cultures. Taken together, BMAA caused cellular stress, including endoplasmic reticulum (ER) stress that is correlated with HSP-27, LAMP-1, CHOP, GLIPR-2, and GRP-78. Further studies are needed in order to support the results. The experiments require being repeated using the same biomarkers as well as a combination of them with other biomarkers to elucidate the effects of BMAA.