Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Les membranes plasmiques et les membranes du trans-Golgi des cellules eucaryotes présentent une asymétrie des lipides qui les composent, avec les aminophospholipides (APLs : phosphatidylsérine et phosphatidyléthanolamine) enrichis dans le feuillet cytosolique. La dissipation de cette asymétrie est impliquée dans de nombreux processus (patho)physiologiques. Plusieurs études suggèrent que les ATPases P4 sont les candidats les plus probables pour le transport des APLs et le maintien de leur distribution asymétrique ; leur délétion dans la levure inhibe le trafic membranaire. En outre, des études ont montré que les ATPases P4 interagissaient avec les protéines de la famille CDC50 ; cette interaction est essentielle pour l’adressage et peut-être aussi la fonction des ATPases P4. Afin de contribuer à la compréhension du mécanisme de transport des lipides par les ATPases P4, l’objectif de ce travail a été de mettre au point la co-expression fonctionnelle, dans la levure, de l’ATPase P4 Drs2p et de sa protéine partenaire Cdc50p. Nous avons obtenu une fraction membranaire enrichie à 3% avec la protéine Drs2p, majoritairement en complexe avec Cdc50p. L’étude fonctionnelle du complexe nous a permis de mettre en évidence un rôle crucial du phosphatidylinositol-4-phosphate (PI(4)P), un important régulateur du trafic membranaire, au cours d’une étape particulière du cycle catalytique. Nous avons également développé un protocole de purification sur résine streptavidine du complexe Drs2p/Cdc50p. Enfin, comme un site potentiel d’interaction avec le PI(4)P est présent sur l’extrémité C-terminale de Drs2p, nous avons engendré différentes constructions de Drs2p, dans lesquelles une partie de l’extrémité C-terminale a été délétée ; dans une autre construction, l’extrémité N-terminale a également été délétée. Notre travail ouvre la voie à la caractérisation fonctionnelle et structurale détaillée du complexe Drs2p/Cdc50p, et à l’étude du rôle du transport de lipides dans le trafic membranaire. / Trans-Golgi membranes and plasma membranes of eukaryotic cells are asymmetric, with their cytosolic leaflet enriched in aminophospholipids (APLs: phosphatidylserine and phosphatidylethanolamine). Dissipation of this asymmetry is involved in many (patho)physiological processes. P4 ATPases are prime candidates for APL transport and for maintaining asymmetry across membranes. In addition, yeast deleted for P4 ATPases display membrane trafficking defects. Besides, CDC50 proteins have been shown to interact physically with P4 type ATPases, and this interaction is important for addressing the complex to the right destination, and possibly also for its function. To gain insight into the molecular mechanism of lipid transport by P4 ATPases, the goal of my thesis was to develop the co-expression, in yeast, of a functional P4 ATPase, Drs2p, together with its partner, Cdc50p. The strategy we developed allowed us to obtain a membrane fraction enriched in Drs2p (~3%), mainly in complex with Cdc50p. Functional characterization of the complex identified phosphatidylinositol-4-phosphate (PI4P), a major regulator of membrane trafficking, as a crucial component for rapid completion of the Drs2p/Cdc50p catalytic cycle. We also purified the complex in one step on streptavidin beads. Finally, we started investigating the potential auto-inhibitory roles of the C-terminus (as the C-terminus of Drs2p contains a PI4P binding site) and the N-terminus of Drs2p, by expressing various truncated versions of Drs2p. Our work sets the stage for detailed functional and structural characterization of the Drs2p/Cdc50p complex and its role in membrane traffic.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Mediante diferentes técnicas histoquímicas, se analizan los tipos de fibras de la musculatura esquelética (Mm. semitendinoso y flexor carporradial) de perros adultos y cachorros (0-65días). Tomando el músculo semitendinoso como patrón, se establecen los criterios para la correcta identificación de las distintas fibras. En base a la actividad mATPasa se reconocen 4 tipos diferentes, que denominamos: I, IIA, IIp y IIC. Con idéntica metodología y criterios de interpretación, se procede al estudio el M. flexor carporradial. Secciones transversales completas de este músculo obtenidas en criosotato a -20ºC, fueron analizadas desde el punto de vista histoquímico, procediéndose al estudio de los tipos de fibras y distribución de las mismas. Los resultados obtenidos indican que el M. flexor carporradial presenta dos cabezas, radial y cubital, cuya funcionalidad puede ser diferente. La cabeza radial se integra casi de forma exclusiva por fibras tipo I, mientras la cabeza cubital muestra una distribución en mosaico con presencia de fibras I, IIA y IIC. Las fibras IIp no fueron identificadas en ninguna de las dos cabezas. Los resultados parecen sugerir que el M. flexor carporradial del perro no solo participa en la protracción del miembro torácico (flexión del carpo), sino que desarrolla un importante papel en el aplomo del mismo. Los estudios llevados a cabo durante el periodo postnatal (0-65días), confirman la aparición gradual y progresiva de las fibras identificadas en animales adultos, aunque en distintos periodos de tiempo según el músculo y/o cabezas. Las técnicas de m-ATPasa permiten formular una nomenclatura específica para las fibras durante el desarrollo, que puede ser tomada como referencia para futuros estudios en estos periodos. / By jeans of different histochemical techniques, fibre types of skeletal muscle (Mm. semitendinosus and flexor carpi radialis) in adults and young dogs (0-65 days) were analysed. Histochemical criteria to the right identification of the different fibres were established by using semiteninosus muscle as control. According to their m-ATPase activity, we observed four different fibre types designated as I, IIA, IIp and IIC. To study the flexor carpi radialis muscle the same techniques and histochemical criteria were followed too. Cryostat whole cross serial sections were histochemically analysed, and the fibre types as well as their distribution studied. The results showed that flexor carpi radialis muscle has two heads, radial and cubital, probably with different functionality. Radial head was almost exclusively constituted by type I fibres; on the other hand, cubitl head exhibited mosaic pattern distribution, containing I, IIA and IIC fibres. None of the heads showed IIp fibre types. These findings suggest that flexor carpi radialis muscle in dog not only participates in the protraction of the forelimb (flexes carpal joint) but also takes an important part in its own aplomb. This study which was carried out throughout postnatal period (0-65 days) confirmed that occurrence of the different fibre types is gradual and progressive in time depending on muscle and / or head portion. mATPase techniques allow us the formulation of an specific nomenclature for the fibres during their development thus, it is recommended for further studies of muscle development.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Glutaric aciduria type I (GA-I) is an inborn error of metabolism (EIM) characterized biochemically by accumulation of glutaric acid (GA). The clinical manifestations are mainly neurological and develop during childhood. Among these changes, there are the seizures and cognitive deficits, which may be precipitated by infectious processes. Although growing evidence supports that inflammation and oxidative damage are both involved in learning impairment, it is not known whether inflammatory and oxidative stress markers facilitate GA-induced memory impairment. From this, the main objective of this study was to investigate the performance of rat pups chronically injected with GA and lipopolysaccharide (LPS) in spatial memory test on Barnes maze. To evaluate antioxidant defenses, cytokines levels, Na+, K+-ATPase activity, and hippocampal volume. Furthermore, we also evaluated wheter N-acetylcysteine (NAC) could improve these behavioral, biochemical or structural changes induced by GA and LPS administration. For this, the rat pups were injected with GA (5umol g of body weight-1, subcutaneously; twice per day; from 5th to 28th day of life), and were supplemented with NAC (150 mg/kg/day; intragastric gavage; for the same period). In order to mimic a severe infection state, LPS (2 mg/kg; E.coli 055 B5) or vehicle (saline 0.9%) was injected intraperitoneally, once per day, from 25th to 28th day of life.Oxidative stress biomarkers, antioxidant activity and hippocampal volume were assessed. In this study, GA caused spatial learning deficit in the Barnes maze, and that LPS potentiated the memory impairment induced by GA in rat pups. In addition, GA and LPS increased proinflammatory cytokine levels (TNF- and IL-1), and the co-administration of these compounds potentiated the increase of IL-1 levels but not TNF- levels in the hippocampus of this animals. Although GA and LPS administration increased TBARS (thiobarbituric acid-reactive substance) content, reduced antioxidant defenses and inhibited Na+,K+-ATPase activity (total and subunit α1), GA and LPS co-administration did not have additive effect on oxidative stress markers and Na+, K+ pump. The hippocampal volume did not change after GA or LPS administration. N-acetylcysteine protected against impairment of spatial learning and increase of cytokines levels induced by GA and LPS. The NAC also protected against deleterious effects induced by GA and LPS, as characterized by inhibition of Na+,K+-ATPase activity (total and subunit α1)and increase of TBARS content, as well as the reduction of antioxidant defenses(non protein thiols and glutathione content, superoxide dismutase and catalase activities).These results suggest that inflammatory and oxidative markers may underlie at least in part of the neuropathology of GA-I in this model. Pharmacological protection with NAC during encephalopatic crises could be considered as an adjuvant therapy to prevent hippocampal dysfunction and the progression of disease in children with GA-I. / A acidemia glutrárica do tipo I (AG-I) é um erro inato do metabolismo (EIM) caracterizada bioquimicamente pelo pelo acúmulo de ácido glutárico (AG). As manifestações clínicas são predominantemente neurológicas, e desenvolvem-se principalmente na infância. Entre essas alterações, as quais são precipitadas por processos infecciosos, pode-se citar o déficit cognitivo. Embora estudos recentes sugerem que a inflamação e o estresse oxidativo estão envolvidos no déficit cognitivo, não se sabe se os marcadores inflamatórios e oxidativos facilitam o prejuízo de memória após a administração de AG. A partir disso, o objetivo desta dissertação foi investigar o desempenho de ratos jovens injetados cronicamente com AG e lipopolissacarídeo (LPS) no teste de memória espacial no labirinto de Barnes. Além disso, foi avaliado os níveis das defesas antioxidantes, níveis de citocinas, atividade da enzima Na+, K+-ATPase e volume hipocampal. Como a N-acetilcisteína (NAC) possui propriedades antioxidantes e antiinflamatórias, foi testado se esse composto poderia melhorar as alterações comportamentais, bioquímicas e estruturais induzidas pela administração de AG e LPS. Para isso, os ratos jovens foram injetados com AG (5 μmol/g do peso corporal-1; subcutaneamente; duas vezes por dia; do 5º ao 28º dia de vida), e foram suplementados com NAC (150 mg/kg/dia; por gavagem; pelo mesmo período). A fim de mimetizar um estado infeccioso, LPS (2 mg/Kg: E. coli 055 B5) ou veículo (salina 0.9%) foi injetado intraperitonealmente uma vez por dia, do 25º ao 28º dia de vida. Nesse estudo, AG causou déficit de aprenizagem espacial no labirinto de Barnes, e o LPS potencializou esse prejuízo de memória induzido pelo AG nos ratos jovens. Em adição, a administração de AG e LPS aumentou os níveis de citocinas pró-inflamatórias (TNF- and IL-1), e a associação desses compostos potencializou o aumento dos níveis de IL-1, mas não de TNF-α no hipocampo dos animais. Embora a associação de AG e LPS tenha causado o aumento o conteúdo TBARS (espécies reativas ao ácido tiobarbitúrico), a redução das defesas antioxidantes e inibição da atividade da Na+,K+-ATPase (total e subunidade α1), a associação de AG e LPS não teve efeito aditivo nos marcadores de estresse oxidativo e na atividade da bomba de Na+ e K+. O volume hipocampal não foi alterado após a administração do AG e LPS. A N-acetilcisteína protegeu contra o prejuízo de aprendizagem espacial e aumento de citocinas inflamatórias induzido pelo AG e LPS. A NAC também protegeu contra os efeitos deletérios induzidos pelo AG e LPS, caracterizado pela inibição da atividade da Na+,K+-ATPase (total e subunidade α1) e aumento do conteúdo de TBARS, bem como a redução das defesas antioxidantes (tiós não-proteicos, conteúdo de glutationa, avitidade da superóxido dismutase e catalase). Esses resultados sugerem que marcadores inflamatórios e oxidativos podem estar envolvidos, em parte, na neuropatologia da AG-I neste modelo. Dessa forma, a proteção farmacológica com a NAC durante crises encefalopáticas pode ser considerada como uma terapia adjuvante para prevenir a disfunção hipocampal e a progressão da doença em crianças com AG-I.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The contamination of the aquatic environment occurs as a consequence of industrial, agricultural and anthropogenic activities. Aquatic ecosystems are often contaminated with metals such as copper and zinc, which are essential in low concentrations but toxic at high concentrations. Thus, the aim of this study was to establish the mean lethal concentration (LC50) within 96 hours of zinc for silver catfish (Rhamdia quelen) and to evaluate the possible effects on biochemical parameters of fish exposed to 0.0 (control), 1.0 and 5.0 mg/L zinc for 96h. The parameters analyzed were reactive substances to thiobarbituric acid (TBARS), protein carbonyl, acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), non-protein thiols (NPSH) and Na+/K+-ATPase. The LC50 value found was 8.07 mg/L. The results of sublethal exposure showed that TBARS levels decreased in liver and brain at every tested Zn concentration. In gills, there was an increase on TBARS levels at 1.0 mg/L and a decrease at 5.0 mg/L, while no changes in muscle were observed. Exposure to zinc decreased protein carbonyls in liver of silver catfish exposed to 5.0 mg/L and gills in every tested concentration. In brain there were no changes in protein carbonyl and in muscle there was an increase at both concentrations. In liver, the SOD activity increased at both zinc concentrations. CAT activity in liver did not change at any concentration. The GST activity decreased in liver and brain increased at both concentrations used. In gills, GST increased in fish exposed to 1.0 mg/L and in muscle at 5.0 mg/L. NPSH levels showed no changes in liver either in gills. In brain there was an increase on NPSH levels at 5.0 mg/L, while the muscle declined at both concentrations. The AChE activity in brain decreased at both zinc concentrations, whereas there was no change in muscle activity for this enzyme. The Na+/K+-ATPase activity was inhibited in gill and intestine in every used concentration. We may conclude that zinc altered biochemical parameters of silver catfish and also the inhibition of brain AChE and Na+/K+-ATPase activity in intestine and gills may be used as biomarkers of waters contaminated by zinc. / A contaminação do ambiente aquático ocorre como consequência das atividades industriais, agrícolas e antropogênicas. Ecossistemas aquáticos são frequentemente contaminados com metais como cobre e zinco, que são essenciais em baixas concentrações, mas tóxicos em concentrações elevadas. Sendo assim, o objetivo deste estudo foi estabelecer a concentração letal média (CL50) em 96 horas do zinco para jundiá (Rhamdia quelen) e avaliar os possíveis efeitos sobre parâmetros bioquímicos destes peixes expostos a 0,0 (controle), 1,0 e 5,0 mg/L de zinco por 96h. Os parâmetros analisados foram substâncias reativas ao ácido tiobarbitúrico (TBARS), proteína carbonil, acetilcolinesterase (AChE), superóxido dismutase (SOD), catalase (CAT), glutationa S-transferase (GST), tióis não-proteicos (NPSH) e Na+/K+-ATPase. O valor da CL50 encontrado foi de 8,07 mg/L. Os resultados da exposição subletal mostraram que os níveis de TBARS diminuíram no fígado e no cérebro em todas concentrações de Zn testadas. Nas brânquias, houve um aumento dos níveis de TBARS na concentração de 1,0 mg/L e uma diminuição na 5,0 mg/L, enquanto que no músculo não foram observadas alterações. A exposição ao zinco diminuiu a carbonilação de proteínas no fígado de jundiás expostos a 5,0 mg/L e nas brânquias nas duas concentrações testadas. No cérebro não houve alterações na proteína carbonil e no músculo, houve um aumento nas duas concentrações. No fígado, a atividade da SOD aumentou em ambas as concentrações de zinco testadas. A atividade da CAT no fígado não teve alteração em nenhuma das concentrações. A atividade da GST diminuiu no fígado e aumentou no cérebro para ambas as concentrações utilizadas. Nas brânquias, o aumento da GST ocorreu na concentração de 1,0 mg/L e no músculo dos peixes expostos a 5,0 mg/L. Os níveis de NPSH não mostraram alterações no fígado e nem nas brânquias. No cérebro houve um aumento nos níveis de NPSH na concentração de 5,0 mg/L, enquanto que no músculo diminuiu nas duas concentrações. A atividade da AChE em cérebro diminuiu nas duas concentrações de zinco, enquanto que no músculo não houve alteração na atividade desta enzima. A atividade da Na+/K+-ATPase foi inibida em brânquias e intestino em todas as concentrações usadas. Com estes resultados, podemos concluir que o zinco alterou os parâmetros bioquímicos dos jundiás e que a inibição da AChE cerebral e da Na+/K+-ATPase no intestino e brânquias podem ser utilizadas como biomarcadores de águas contaminadas por zinco.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Die peroxisomalen ABC-Transporter Pxa1p und Pxa2p sind Halbtransporter. Genetische Studien ergaben Hinweise, dass sie zur Bildung aktiver Transporter heterodimerisieren und am Import von langkettigen Fettsäuren in die Peroxisomen von S. cerevisiae beteiligt sind. Es wurden epitopmarkierte Varianten der Proteine als Komplex isoliert. Damit wurde gezeigt, dass Pxa1p und Pxa2p ein stabiles Heterodimer bilden. Zur Charakterisierung der ATP Bindeeigenschaften wurden die Transporter mit 8-azido-[alpha-32P]-ATP inkubiert und kovalent verknüpft. Dabei konnte gezeigt werden, dass Pxa1p und Pxa2p eine unsymmetrische Bindung des ATP Analogons aufweisen. Pxa2p bindet deutlich mehr azido-ATP als Pxa1p, bei sehr ähnlichen Dissoziationskonstanten. Die reduzierte ATP Bindung von Pxa1p spiegelt sich durch degenerierte Sequenzmotive der an der ATP Bindung beteiligten Sequenzen wieder. Die isolierten ABC-Transporter wurden für ATPase Messungen eingesetzt. Sie zeigten eine basale ATPase Aktivität, die durch Zugabe langkettiger Coenzym A aktivierter Fettsäuren, wie Oleoyl-CoA und Palmitoyl-CoA stimulierbar war. Eine Lysin Mutation im Walker A Motiv von Pxa1p hatte keine Funktionalitätseinbuße zur Folge. Dieselbe Mutation bei Pxa2p führte im Wachstumstest auf Festmedium mit Ölsäure als Kohlenstoffquelle zu einem deutlich verlangsamten Wachstum. Diese Ergebnisse korrespondieren mit der beobachteten unsymmetrischen ATP Bindung von Pxa1p und Pxa2p, da bei dem schwächer bindenden Pxa1p die Mutation wirkungslos blieb. Keine Übereinstimmung war bei den ATPase Aktivitätsmessungen der aufgereinigten Mutanten zu verzeichnen. Beide Mutanten zeigten eine unbeeinträchtigte ATPase Aktivität. Die ABC-Transporter wurden in Proteoliposomen eingebaut und für Transportmessungen mit einem Spin-Label markierten Oleoyl-CoA verwendet. Die Transportmessungen zeigten einen ATP abhängigen Transport, woraus geschlossen wurde, dass Pxa1p-Pxa2p tatsächlich Coenzym A Ester langkettiger Fettsäuren transportiert. / The peroxisomal ABC-transporters Pxa1p and Pxa2p are half transporters. Previous genetic investigations have demonstrated that Pxa1p and Pxa2p have to dimerise in order to build a functional transporter, which is very likely involved in the import of long chain fatty acids into peroxisomes of S. cerevisiae. In this work, tagged versions of the proteins were purified as a complex. This proved for the building of a stable hetero dimer. For characterisation of the ATP binding properties, the transporters were incubated and cross linked with 8-azido-[alpha-32P]-ATP. This revealed an asymmetric binding of the ATP analogue. Pxa2p binds much more azido-ATP, than Pxa1p, while the dissociation constants are rather similar. The poorer ATP binding of Pxa1p is reflected by degenerated sequence motifs in the nucleotide binding fold. The purified ABC-transporters have been used for ATPase assays. They showed a basal ATPase activity, which could be stimulated by addition of long chain fatty acid CoAs, like oleoyl-CoA and palmitoyl-CoA. Mutants with a lysine mutation in the walker A motive of Pxa1p led to no functional impairment, while the corresponding lysine mutation in Pxa2p led to reduced growth on agar plates with oleic acid as sole carbon source. The result corresponds with the ATP binding properties of Pxa1p. Because of the poorer ATP binding, even in the wild type protein, the mutation was not supposed to have a big influence. No accordance was found in respect to the ATPase measurements of the isolated mutant proteins. Both mutants revealed unaffected ATPase activity. The purified ABC-transporters were reconstituted in proteoliposomes and used for translocation assays of a spin-labelled oleoyl-CoA derivative. The measurements revealed an ATP dependent transport of the oleoyl-CoA analogue. This led to the conclusion, that Pxa1p-Pxa2p is indeed the transporter of long chain acetyl CoA esters, which were transported in an ATP dependent manner.

Made available in DSpace on 2017-07-21T19:25:54Z (GMT). No. of bitstreams: 1 Wellington Claiton Leite.pdf: 3789073 bytes, checksum: f4c16b4260fbd54f4eada652038ae5bc (MD5) Previous issue date: 2016-09-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacterial RecA protein plays a role in the complex system of DNA damage repair. In the presence of ATP, RecA proteins polymerize onto single-strand DNA (ssDNA) as righthanded helical nucleoprotein laments, and catalyze strand exchange reaction between the ssDNA and homologous double-strand DNA (dsDNA) molecules. These activities are supported or stimulated by accessory proteins, as the single-stranded binding protein (SSB).Here, we report a functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA).We report the crystal structure of HsRecA-ADP/ATP complex to 1.7 Å of atomic resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, similarly to homologous RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. The bacterial polymerization motif contains the sequence SV/IMR/KLG which interacts with the core ATPase domain residues DNLLLV/CS. In the inactive RecA, it is a loop - strand interaction, respectively, while in the active RecA it becomes a dyad strand. In both RecA forms, the polymerization motif seems to stabilize the subunitsubunit interface by hydrophobic interactions. The methionine of this motif may play an important role in the stability and formation of a right-handed helical nucleoprotein lament. The ATPase activity and the structure of the nucleoprotein lament of HsRecA and Escherichia coli RecA (EcRecA) were analyzed in the presence and absence of SSB. When SSB was added after RecA+ssDNA, HsRecA and EcRecA showed similar ATPase activity and nucleolament structure. However, when SSB was either not included or it was added before RecA+ssDNA, the HsRecA showed higher ATPase activity and formed longer nucleoprotein laments than EcRecA. Thus, HsRecA protein is more ecient at displacing SSB from ssDNA than EcRecA protein. HsRecA promoted DNA exchange more eficiently: a greater yield of nicked circular products were obtained in a shorter time. Reconstruction of electrostatic potential from the hexameric structure of HsRecAADP/ ATP revealed a high positive charge along the inner side, which is consistent with the fact that ssDNA binds inside the filament. It may explain the enhance capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote eficiently the DNA strand exchange reaction. Keywords: RecA, Crystallography, RecA nucleoprotein filament, ATPase activity, DNA strand exchange, crystal structure, structural analysis. / A proteína RecA bacteriana desempenha um importante papel no complexo sistema de reparo de danos ao DNA. Na presença de ATP, a proteína RecA se auto-polimeriza sobre o DNA simples ta (ssDNA) (do inglês single-strand DNA (ssDNA)) como um lamento de nucleoproteína helicoidal, cataliza a reação de troca de fitas entre as moléculas ssDNA e a ta de DNA dupla fita homóloga (dsDNA) (do inglês double-strand DNA (dsDNA)). Estas atividades são suportadas ou estimuladas por proteínas acessórias, como a proteína ligadora de ssDNA SSB (do inglês single-stranded binding protein (SSB)). Neste trabalho é apresentado a caracterização estrutural e funcional da proteína RecA da bactéria Herbaspirillum seropedicae. A estrutura tridimensional do complexo HsRecA-ADP/ATP foi resolvida numa resolução 1,7 Å. A estrutura monomérica da proteína HsRecA consiste em um pequeno domínio N-terminal, um domínio central contendo um sitío ATPásico e e um grande domínio C-terminal, similar com proteínas RecAs homólogas. Análises estruturais comparativas mostraram que o motivo de polimerização da região N-terminal de proteí- nas da familia RecA que incluem archaea e eucariotos, também está presente na proteína RecA bacteriana. O motivo de polimerização da região N-terminal de bactérias contêm a sequência de resíduos (Serina, Valina ou Isoleucina, Metionina, Arginina ou Lisina, Leucina, Glicina) que interage com a sequência de resíduos do core ATPásico (Aspartato, Asparagina, Leucina, Leucina, Leucina, Valina, Cisteína, Serina). Na proteína RecA inativa esta interação é do tipo loop - strand, respectivamente, enquanto na proteína RecA ativa essa interação se torna uma dupla -strand. Em ambas formas da RecA, o motivo de polimerização parece estabilizar a interface subunidade-subunidade por interações hidrofóbicas. No motivo N-terminal a presença de uma Metionina altamente conservada talvez desempenha um importante papel na estabilidade e formação do lamento de nucleoproteína. A atividade ATPásica e a estrutura do lamento de nucleoproteína da proteína HsRecA e da Escherichia coli RecA (EcRecA) foram analisadas na presença e ausência da proteína SSB. Quando a SSB foi adicionada após RecA+ssDNA, as proteínas HsRecA e EcRecA mostraram similar atividade ATPásica e estrutura de nucleo lamento. Entretanto, quando a SSB não estava incluída ou quando adicionada anteriormente a adição RecA+ssDNA, a proteína HsRecA mostrou maior atividade ATPásica e formou maiores lamentos de nucleoproteína que a proteína EcRecA. Ainda, a proteína HsRecA é mais eficiente em deslocar a SSB do ssDNA que a proteína EcRecA. A proteína HsRecA também promove a reação de troca de fitas mais eficientemente: uma maior quantidade de produtos duplex substrato convertido em duplex circular foram obtidos em um curto intervalo de tempo. A reconstrução do potencial eletrostático da estrutura hexamérica da proteína HsRecA revelou uma maior densidade de cargas positivas no seu interior, que é consistente com o fato que o ssDNA ligar-se internamente ao filamento hexamérico. Isto talvez possa explicar capacidade melhorada da proteína HsRecA ligar-se ao ssDNA, formando um continuo filamento de nucleoproteína, deslocando a SSB e ainda promovendo de forma eficiente a reação de trocas de fitas.

Three novel mutations (G159D, L29Q and E59D/D75Y) in cardiac troponin C (CTnC) associate their clinical outcomes with a given cardiomyopathy. Current paradigms propose that sarcomeric mutations associated with dilated cardiomyopathy (DCM) decrease the myofilament calcium sensitivity while those associated with hypertrophic (HCM) cardiomyopathy increase it. Therefore, we incorporated the mutant CTnCs into skinned cardiac muscle in order to determine if their effects on the calcium regulation of tension and ATPase activity coincide with the current paradigms and phenotypic outcomes. This required the development of new calculator programs to solve complex ionic equilibria to more accurately buffer and expand the free calcium range of our test solutions. In accordance with the DCM paradigms, our result show that G159D and E59D/D75Y CTnC decrease the myofilament calcium sensitivity and force generating capabilities which would likely increase the rate of muscle relaxation and weaken the contractile force of the myocardium. Alternatively, the lack of myofilament change from L29Q CTnC (associated with HCM) may explain why the only proband is seemingly unaffected. Notably, the changes in the calcium sensitivity of tension (in fibers) do not necessarily occur in the isolated CTnC and vice versa. These counter-intuitive findings are justified through a transition in calcium affinity occurring at the level of cardiac troponin (CTn) and higher, implying that the true effects of these mutations become apparent as the hierarchal level of the myofilament increases. Despite these limitations, the regulated thin filament (RTF) retains its role as the calcium regulatory unit and best indicates a mutation's ability to sensitize (+) or desensitize (-) the muscle to calcium. Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse (+ or -) these aberrant changes in myofilament sensitivity. Therefore, we have developed an RTF mediated High Throughput Screening assay to identify compounds in libraries of molecules that can specifically modulate the calcium sensitivity of cardiac contraction. The knowledge gained from these studies will help us and others to uncover new pharmacological agents for the investigation and treatments of cardiomyopathies, hypertension and other forms of cardiovascular diseases.