An accurate plasma concentration-response relationship for propafenone (PF), a potent class 1 antiarrhythmic agent, has not yet been defined. A general pharmacological premise suggests that only the free drug is available to contribute to the observed pharmacological response. It has previously been shown that PF is highly bound to α-l-acid glycoprotein (AAG) which results in a low free PF concentration. The correlation of free PF concentration and response failed to adequately describe the dose response relationship. It has subsequently been shown that upon chronic dosing, two active metabolites, namely 5-hydroxypropafenone (5-OH-PF), and n-depropylpropafenone (n-depropyl-PF) accumulate in humans treated with PF. It is highly likely that the free concentration of PF, in addition to those of 5-OH-PF and n-depropyl-PF, contributes to the observed pharmacological effect following administration of PF at steady-state. To date, no accurate estimation of 5-OH-PF binding in serum has been established. This thesis examines the binding characteristics of PF and 5-OH-PF and their interaction in human serum, and in solutions of AAG, human serum albumin (HSA), high density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) using equilibrium dialysis. The binding of PF (2.0 μg/mL) and 5-OH-PF (0.5 μg/mL) was examined in serum when both drug and metabolite were present. The free fraction (FF) of PF and 5-OH-PF in serum was 0.063 ± 0.004 and 0.232 ± 0.020, respectively. Both PF and 5-0H-PF were found to bind to a high affinity, low capacity binding site on AAG, in addition PF showed a second low affinity, high capacity binding site. PF displayed a 10 fold greater affinity for the high affinity binding site on AAG when compared to 5-OH-PF. Both PF and 5-OH-PF showed only one low affinity, high capacity site on HSA of similar affinity. The interaction of PF and 5-OH-PF with HDL, LDL, and VLDL appeared to be due to solubilization, rather than a "true" drug-protein binding interaction, since it correlated well with the concentration of cholesterol within the lipoprotein complex (PF, r²=0.85; 5-OH-PF, r²=0.96). However, PF appeared to show saturable binding to the HDL complex. The uptake of PF and 5-OH-PF was greatest in LDL followed by HDL, and finally VLDL. In serum PF displayed both a high affinity, low capacity, and a low affinity, high capacity binding sites, although a similar observation was expected for 5-OH-PF, only one binding site could be experimentally identified. The uptake of 5-OH-PF by red blood cells (RBC) appeared to be approximately 5 fold greater than that of PF (i.e. The ratio of PF and 5-OH-PF concentration in the red blood cell/plasma was 0.7 ± 0.1 and 3.2 ± 0.5, respectively). When the binding of PF and 5-OH-PF was considered separately, the binding profiles were similar, that is, both drugs showed high affinity binding to AAG, and low affinity binding and/or non-specific binding to other serum proteins such as HSA, HDL, LDL, and VLDL. However, when both drug and metabolite were present, the binding of 5-OH-PF to AAG was found to be reduced. This is thought to occur as a result of the displacement of 5-OH-PF by PF from AAG. Thus, the binding of 5-OH-PF was noted to be more dependent on HSA, and lipoproteins when compared to PF. On the other hand, the binding of PF (2.0 μg/mL), even with the addition of 5-OH-PF, was dependent largely on the concentration of AAG. Although the binding of 5-OH-PF was apparently not altered by the addition of PF in serum, a decrease in the binding of 5-OH-PF by the addition of PF was observed. It is hoped that the understanding gained from this thesis will provide information regarding the relative importance of free PF and 5-OH-PF plasma concentration in future pharmacodynamic studies of PF. / Pharmaceutical Sciences, Faculty of / Graduate

Iron deficiency anaemia is perhaps the most widespread nutritional deficiency disease; as result, the topic of iron absorption has received intensive investigation over a relatively long period of time. Most of the investigative thrust has come from clinical medicine and allied fields, with some associated biochemical investigation. Evidence from the latter has pointed towards the involvement of iron-binding proteins especially ferritin and transferrin in the absorptive process. While the biochemical literature on these two proteins, particularly transferrin, is vast, their roles in iron absorption are obscure. This study was undertaken, therefore, as an investigation into these proteins, their quantitation and role in iron absorption. The physiology of absorption was studied by injection of radiolabelled ferrous ascorbate into isolated intestinal loops and the determination of mucosal, blood and carcass uptake.

Modification of cellular proteins with molecules of ubiquitin is an important process that regulates the activity of cellular proteins. Cullin RING ligases (CRLs) are multi-subunit complexes that act in concert with E2 enzymes to attach molecules of ubiquitin to protein substrates. There are seven CRLs in mammalian cells (Cul1, Cul2, Cul3, Cul4A, Cul4B, Cul5, and Cul7) that are highly homologous in sequence and structure. CRLs possess a highly conserved C- terminal domain that interacts with E2 enzymes, and a more variable N- terminal domain which recruits substrates through distinct substrate adapter molecules. Despite the structural similarity, these CRLs recognize distinct substrates and carry out unique functions in cells. In order to characterize the functional domains of cullins that are responsible for their unique activity, we generated cullin chimeras for expression and analysis in mammalian cells. These chimeras are Cul3 mutants in which the C- terminal domain or N- terminal domain of Cul3 has been replaced by that of Cul1 or Cul2, respectively. These chimeras were cloned into a mammalian expression vector for the purpose of experimentation in cultured cells. The chimeric cullin constructs provided a valuable tool for investigating how different functional domains of CRLs contribute to their specific functions in cells. In this study, we first investigated if the chimeras that we engineered were able to interact with their respective substrate adapters. We performed co- immunoprecipitation experiments in which we tested the ability of wild type, chimeric, or mutant cullin proteins to bind to three different substrate adapter proteins. We found that the chimera possessing the C- terminus of Cul1 and the N- terminus of Cul3 retains the ability to interact with the BTB substrate adapters Ctb57 and KLHL3. We also found that the chimera that possesses the C- terminus of Cul3 and the N- terminus of Cul1 was unable to interact with BTB proteins. Lastly, we found that the Cul1 adapter Skp1 was able to bind to Cul1, but did not bind to Cul3 or either chimera. We concluded that the chimera possessing the N- terminus of Cul3 likely retains the functional binding abilities of Cul3 at the N- terminus and would therefore be useful for conducting experiments. In this study, we also used the cullin chimeras to investigate the binding interactions between E2 enzymes and cullin RING ligases. We performed co- immunoprecipitation assays to examine the interactions between E2 enzymes and wild type, mutant or chimeric cullin proteins. We found that E2 enzyme UbE2E1 selectively binds to Cul3 and not to Cull. Notably, the BTB binding region at the N- terminus of Cul3 is required for binding to UbE2E1. Furthermore, we found that UbE2E1 also binds to Cul3 substrate adapter protein Ctb57. These experiments revealed a novel interaction between and E2 enzyme and the N- terminus of Cul3, as well as with a Cul3 substrate adapter protein. In conclusion, the chimeras generated in this study have provided valuable information regarding what regions of CRLs are important for interactions with other proteins, and will continue to be a useful tool for investigating CRL structure and function.

Estudos de consolidação da informação na memória de trabalho visual mostram que, em tarefas de detecção de mudança, a localização do objeto memorizado perde importância após intervalos entre 900ms e 1500ms. No entanto, vários estudos têm mostrado que dicas retroativas baseadas na localização são eficientes para a recuperação da informação memorizada mesmo em intervalos que vão de 100ms a 6000ms. O presente estudo, teve como objetivo investigar o intervalo de decaimento da informação de localização na memória de trabalho visual, quando irrelevante para a tarefa, e verificar se o processo de seleção da informação na representação do binding é afetado pela localização. A apresentação dos itens e o registro das respostas foram realizados por meio do software E-prime 2.0. Os participantes realizaram tarefas de detecção de mudança para estímulos visuais definidos pela conjunção de cor e forma. Nos experimentos 1 (n=18) e 2 (n=18), a tarefa do participante foi memorizar uma cena com figuras coloridas e, depois de um intervalo de retenção (500ms e 1500ms), julgar se a cena teste continha exatamente as mesmas figuras da cena inicial. Nossa suposição nos experimentos 1 e 2, era de que a informação de localização permaneceria atrelada a representação do objeto por intervalo menor que 1500ms. No terceiro experimento (n=18) foi realizada uma tarefa semelhante, acrescida da apresentação de retrodica de cor. Nesse terceiro experimento, nossa suposição era de que o desempenho na seleção da informação na representação memorizada, seria em função da distância entre os itens nessa representação. Os dados obtidos foram analisados através de ANOVA em função do índice de discriminação (d) dos participantes nas condições manipuladas e do tempo de reação. Os dados obtidos nos dois primeiros experimentos indicam que a informação de localização não decaiu em um intervalo de 1500ms, e o desempenho dos sujeitos variou em função da carga de itens apresentados. O terceiro experimento indicou que o espaço continua a interferir na performance dos participantes, em intervalo maior, 2450ms, além disso, as dicas válidas auxiliaram no desempenho dos participantes, e a seleção da informação na representação ocorreu de modo diferente do esperado / Studies on information consolidation in visual work memory shows that, in change detection tasks, the location of the memorized object loses importance after intervals between 900ms and 1500ms. However, several studies have shown that retroactive location-cues are efficient in retrieving stored information, even at intervals ranging from 100ms to 6000ms. The present research aimed to investigate the decay interval of the location information in the visual work memory, when irrelevant to the task, and to verify if the process of selection of the information in the representation of the binding is affected by the location. The presentation of the items and the recording of the answers was performed by the software E-prime 2.0. Participants performed change detection tasks for visual stimuli defined by the conjunction of color and shape. In experiments 1 (n=18) and 2 (n=18), the participant\'s task was to memorize a scene with colored figures and, after a retention interval (500ms and 1500ms), judge whether the test scene contained exactly the same figures as the initial scene. Our assumption for experiments 1 and 2 was that location information would remain tied to the representation of the object by interval less than 1500ms. In the third experiment (n=18), a similar task was carried out, in addition to the presentation of a color cue. Our assumptions about this experiment was that the performance in selecting the information in the memorized representation would be in function of the distance between the items in that representation. The obtained data were analyzed through ANOVA according to the discrimination index (d\') of the participants in the manipulated conditions and the reaction time. The results in the first two experiments indicated that the location information did not decay in a range of 1500 ms, and the performance of the subjects varied according to the load of items presented. The third experiment pointed out that space continues to interfere with the performance of participants, in a larger interval, 2450 ms, in addition, valid cues improved the participants performance, and the selection of information in the representation occurred differently than expected

Protozoários do gênero Trypanosoma infectam milhões de pessoas todo ano e coletivamente contribuem muito para as misérias humanas, pois são causa de muitas das doenças negligenciadas tropicais. Várias vias metabólicas essenciais são encontradas nesses parasitas tornando-os particularmente atrativos para investigações moleculares. Mecanismos de controle pós-transcricional tem sido alvo de estudo por sua peculiaridade nesses organismos. Nesse cenário, proteínas da classe das poli-A-binding proteins (PABP) possuem função no início da tradução, turnover do mRNA e interação com o 5´-CAP. Nesse trabalho foi identificada a homóloga poli-A binding protein 1 (PABP1) de Trypanosoma brucei. O silenciamento do gene pabp1 revelou que a ausência da proteína é letal ao parasita, comprovando sua essencialidade nesse organismo. Da mesma maneira, na ausência da proteína observou-se erro no processamento do mRNA sugerindo possível função nos eventos de cis e trans splicing. Sua localização subcelular foi avaliada indicando localização citoplasmática, bem como o são suas homólogas. No citoplasma, a proteína apresenta-se em estrutura reticulada, co-localizada com proteínas de retículo endoplasmático. Porém, sob estresse induzido a proteína relocaliza para o compartimento nuclear, indicando ser uma proteína com trânsito núcleo-citoplasma ainda não demonstrada na literatura. As funções identificadas sugerem a existência de um sub-complexo a 3´ do mRNA que acopla poliadenilação e splicing. Além disso, a relocalização nuclear parece ocorrer em resposta a estímulo externo, sugerindo que a relocalização do mRNA para o núcleo pode ser uma estratégia da célula para modular sua resposta gênica frente a variações do ambiente. / Protozoa of the genus Trypanosoma infect millions of people every year and collectively contribute to the human misery by causing several neglected tropical diseases. Several intriguing molecular pathways are found in these parasites also, rendering them particularly attractive for biochemical investigation. This unique eukaryotic cells lack mechanisms to control gene expression at the transcriptional level, they mostly control protein synthesis by posttranscriptional regulation process. Several RNAs and proteins are involved in this process, including poly(A) binding proteins. The poly(A)- binding protein of eukaryotes plays a role in polyadenylation, translation initiation and metabolism of mRNA. In this work the poly(A) binding protein 1 (PABP1) was identified in Trypanosoma brucei. Depletion of TbPABP1 showed its essential role in the procyclic form of the parasite. Immunofluorescence assays showed localization in the cytosolic compartment despite of its functions in cis and trans splicing as shown by RNA analysis of cells free from PABP1. As it was shown in the homologs, PABP1 it´s not only a cytosolic protein but it shuttles between the nucleus and the cytoplasm. Together with the literature, these results suggest an active complex in the 3´ end of the mRNA which works in synchrony with the splicing and capping machinery implying PABP1 as possible link between these processes.

Wang, Jia. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 99-115). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cheung, Siu Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 178-200). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General Introduction --- p.1 / Chapter 1.1 --- S100 Proteins --- p.1 / Chapter 1.1.1 --- Structure of S100 proteins --- p.2 / Chapter 1.1.2 --- Possible functions of S100 proteins --- p.4 / Chapter 1.1.3 --- Genomic organization of S100 genes --- p.6 / Chapter 1.1.4 --- Clinical importance of S100 proteins --- p.7 / Chapter 1.2 --- S100A1 Protein --- p.8 / Chapter 1.2.1 --- Possible functions of the S100A1 protein --- p.10 / Chapter 1.2.1.1 --- Regulation of cardiac and skeletal muscle contractility --- p.10 / Chapter 1.2.1.2 --- Functional roles in the central nervous system (CNS) --- p.12 / Chapter 1.2.1.3 --- Other possible functions of the S100A1 protein --- p.13 / Chapter 1.2.2 --- S100A1 knockout mice --- p.14 / Chapter 1.2.3 --- Relationships between S100A1 and S100B proteins --- p.16 / Chapter 1.3 --- S100B Protein --- p.18 / Chapter 1.3.1 --- Possible functions of S100B protein --- p.19 / Chapter 1.3.2 --- S100B knockout mice --- p.20 / Chapter 1.4 --- RNA interference --- p.22 / Chapter 1.4.1 --- Mechanisms of RNA interference --- p.24 / Chapter 1.4.2 --- Efficacy and selectivity of siRNA --- p.25 / Chapter 1.4.3 --- siRNA delivery --- p.27 / Chapter 1.5 --- Objective --- p.31 / Figures and legends --- p.34 / Chapter Chapter Two --- S100A1 expression in normal mouse embryos and characterization of S100A1 knockout mouse embryos --- p.40 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.2 --- Materials and Methods --- p.44 / Chapter 2.2.1 --- Mouse strains --- p.44 / Chapter 2.2.2 --- RNA extraction --- p.46 / Chapter 2.2.3 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 2.2.4 --- Protein extraction --- p.48 / Chapter 2.2.5 --- Western blotting --- p.49 / Chapter 2.2.6 --- Immunohistochemical staining --- p.50 / Chapter 2.3 --- Results --- p.53 / Chapter 2.3.1 --- S100A1 mRNA expression in normal mouse embryo --- p.53 / Chapter 2.3.2 --- S100A1 protein expression in normal mouse embryos --- p.55 / Chapter 2.3.2.1 --- Temporal expression of the S100A1 protein --- p.55 / Chapter 2.3.2.2 --- Spatial expression of the S100A1 protein --- p.57 / Chapter 2.3.3 --- Morphological and histological characterization of SI00A1 knockout mouse embryos --- p.60 / Chapter 2.3.4 --- S100B protein expression pattern in Wt and S100A1 KO mouse embryos --- p.62 / Chapter 2.4 --- Discussion --- p.64 / Tables --- p.73 / Figures and legends --- p.76 / Chapter Chapter Three --- Knockdown of S100A1 in S100B in knockout mouse embryos --- p.118 / Chapter 3.1 --- Introduction --- p.118 / Chapter 3.2 --- Materials and Methods --- p.128 / Chapter 3.2.1 --- Mouse strains --- p.128 / Chapter 3.2.2 --- Short-interfering RNA (siRNA) --- p.129 / Chapter 3.2.3 --- In-uterus surgery --- p.130 / Chapter 3.2.4 --- RNA extraction and RT-PCR --- p.132 / Chapter 3.2.5 --- Immunohistochemical staining of S100A1 and S100B --- p.132 / Chapter 3.3 --- Results --- p.133 / Chapter 3.3.1 --- Characterization of S100B knockout mouse embryos --- p.133 / Chapter 3.3.2 --- S100A1 knockdown in S100B wild-type (Wt) mouse embryos --- p.133 / Chapter 3.3.3 --- S100A1 knockdown in S100B knockout (KO) mouse embryos --- p.139 / Chapter 3.4 --- Discussion --- p.146 / Tables --- p.153 / Figures and legends --- p.154 / Chapter Chapter Four --- General Discussion and Conclusions --- p.175 / Reference --- p.178

Thesis (Ph. D.)--Ohio State University, 2006. / Full text release at OhioLINK's ETD Center delayed at author's request

From opportunistic protist Cryptosporidium parvum we identified and functionally assayed a fatty acyl-CoA-binding protein (ACBP) gene. The CpACBP1 gene encodes a protein of 268 aa that is three times larger than typical ~10 KD ACBPs of humans and animals. Sequence analysis indicated that the CpACBP1 protein consists of an N-terminal ACBP domain (approximately 90 aa) and a C-terminal ankyrin repeat sequence (approximately 170 aa). The entire CpACBP1 open reading fragment (ORF) was engineered into a maltose-binding protein fusion system and expressed as a recombinant protein for functional analysis. Acyl-CoA-binding assays clearly revealed that the preferred binding substrate for CpACBP1 is palmitoyl-CoA. RT-PCR, Western blotting and immunolabelling analyses clearly showed that the CpACBP1 gene is mainly expressed during the intracellular developmental stages and that the level increases during parasite development. Immunofluorescence microscopy showed that CpACBP1 is associated with the parasitophorous vacuole membrane (PVM), which implies that this protein may be involved in lipid remodelling in the PVM, or in the transport of fatty acids across the membrane. We also identified two distinct oxysterol binding protein (OSBP)-related proteins (ORPs) from this parasite (CpORP1 and CpORP2). The short-type CpOPR1 contains only a ligand binding (LB) domain, while the long-type CpORP2 contains Pleckstrin homology (PH) and LB domains. Lipid-protein overlay assays using recombinant proteins revealed that CpORP1 and CpORP2 could specifically bind to phosphatidic acid (PA), various phosphatidylinositol phosphates (PIPs), and sulfatide, but not to other types of lipids with simple heads. Cholesterol was not a ligand for these two proteins. CpOPR1 was found mainly on the parasitophorous vacuole membrane (PVM), suggesting that CpORP1 is probably involved in the lipid transport across this unique membrane barrier between parasites and host intestinal lumen. Although Cryptosporidium has two ORPs, other apicomplexans, including Plasmodium, Toxoplasma, and Eimeria, possess only a single long-type ORP, suggesting that this family of proteins may play different roles among apicomplexans.

ATP-dependent phosphoenolpyruvate carboxykinase (E. C. 4.1.1.49; PCK) is an enzyme that catalyses the reversible conversion of oxaloacetate and ATP into phosphoenolpyruvate, ADP and CO2. PCK is made up of about 500 to 600 amino acid residues and is divided into two roughly equal domains. Upon binding of substrates, the two domains of PCK move towards each other. PCK is well known for its role in gluconeogenesis but in some species, it can have an anaplerotic role. In other species, PCK is important for metabolic steps involved in fermentation.<p>Presented in this thesis are five solved crystal structures of the ATP-dependent form of PCK. Three of the PCK crystal structures determined were from <i>Escherichia coli</i>; one was a complex of ATP, Mg2+ and CO2, the second structure was an ATP, Mg2+, Mn2+, CO2 and oxaloacetate complex and, the third <i>E. coli</i> structure was a Lys213Ser mutant complexed with ATP, Mg2+and Mn2+. Two <i>Anaerobiospirillum succiniciproducens</i> PCK crystal structures were also solved; one was in the native form and the other was an ATP-Mg2+-Mn2+-oxalate complex. <p>In the <i>E. coli</i>-PCK-ATP-Mg2+-CO2 crystal complex structure, the observed location of CO2 was in agreement with a previously determined <i>E. coli</i> PCK-CO2 crystal structure, which incorporated CO2 into the structure by a different technique. The findings from the <i>E. coli</i> PCK-ATP-Mg2+-CO2 crystal structure allowed the reaction mechanism presented in this work to be proposed.<p>The PCK-ATP-Mg2+-Mn2+-CO2-oxaloacetate structure is the first structure where oxaloacetate is observed bound to PCK. Surprisingly, the observed location of oxaloacetate in this structure is 5 Angstroms away from its expected position near Mn2+. Oxaloacetate is weakly bound to a non-catalytic region of the enzyme. It is proposed that when the domains of PCK move towards each other upon binding nucleotide, oxaloacetate experiences steric crowding which results in it being pushed towards the active site to react. <p>Previous kinetic studies on the <i>E. coli</i> PCK mutant Lys213Ser have determined that Mn2+ is unexpectedly inhibitory. A crystal structure of K213S-PCK-ATP-Mg2+-Mn2+ demonstrates that Mn2+ is tetrahedrally coordinated in the active site, not octahedrally as occurred in other structures. By having Mn2+ in the tetrahedral coordination state, substrate binding in the active site of PCK is altered in a way that does not allow catalysis to occur.<p>The two crystal structures of <i>A. succiniciproducens</i> PCK were useful in quantifying the substrate-induced domain movement. A surface active site lid made up of residues 385 to 405 that had never been observed in any of the previous PCK crystal structures was observed in the <i>A. succiniciproducens</i> PCK-ATP-Mg2+-Mn2+-oxalate crystal structure. Mutational studies of this lid have shown it to be essential for the function of PCK; however, its exact function is not certain. It has been proposed that the lid has multiple functions. One is to sequester the substrates from bulk solvent. Another function may be to assist in domain closure. The third function may be to assist in the proper positioning of substrates in the active site.