The initial motivation for crystallization of Bovine Serum Albumin (BSA) is an interest to understand how thiomolybdates interact with BSA and suppress copper intake from the food sources of cattle. The main objective of my research work is to determine the crystal structure of BSA using X-ray crystallography techniques. Once the tertiary structure of BSA is determined, its structural information can help us to study the interactions between BSA, copper, and thiomolybdates, and to understand the way in which thiomolybdates render copper unavailable in cattle. Many trials for the optimal crystallization conditions of BSA were attempted in order to grow high-quality BSA crystals. However, all crystals only diffract to 8 Å resolution limit. Such resolution is not sufficient to solve the tertiary structure of BSA. Another objective of my research was to crystallize Thioredoxin-2 (Trx-2) to obtain larger crystals which may lead to high resolution crystallographic data, better than 2.4 Å, for protein structure refinement. This is because Trx-2 diffraction data that had been collected are split at high resolution. The ambiguous data at high resolution might impede the structure refinement and even can cause the three-dimensional structure of Trx-2 to not be refined successfully. A number of attempts were conducted for crystallizing Trx-2 to grow bigger and higher quality of Trx-2 crystals. However, the improvement of crystal dimensions was not significant, the diffraction resolution limits are similar to previous published data, and the split data at high resolution was still observed.

The initial motivation for crystallization of Bovine Serum Albumin (BSA) is an interest to understand how thiomolybdates interact with BSA and suppress copper intake from the food sources of cattle. The main objective of my research work is to determine the crystal structure of BSA using X-ray crystallography techniques. Once the tertiary structure of BSA is determined, its structural information can help us to study the interactions between BSA, copper, and thiomolybdates, and to understand the way in which thiomolybdates render copper unavailable in cattle. Many trials for the optimal crystallization conditions of BSA were attempted in order to grow high-quality BSA crystals. However, all crystals only diffract to 8 Å resolution limit. Such resolution is not sufficient to solve the tertiary structure of BSA. Another objective of my research was to crystallize Thioredoxin-2 (Trx-2) to obtain larger crystals which may lead to high resolution crystallographic data, better than 2.4 Å, for protein structure refinement. This is because Trx-2 diffraction data that had been collected are split at high resolution. The ambiguous data at high resolution might impede the structure refinement and even can cause the three-dimensional structure of Trx-2 to not be refined successfully. A number of attempts were conducted for crystallizing Trx-2 to grow bigger and higher quality of Trx-2 crystals. However, the improvement of crystal dimensions was not significant, the diffraction resolution limits are similar to previous published data, and the split data at high resolution was still observed.

Determination of whether ion specific potassium halides binding with BSA occurred within the experimental conditions was the aim of the study. Ultrasound velocity measurements using a Resoscan system and density measurements using a DMA 5000M density meter were made to analyse BSA conformation in the presence of potassium salts of the Hofmeister series. It was found that the density and the adiabatic compressibility of BSA-potassium halide mixed aqueous solutions cannot be predicted from the density and the adiabatic compressibility of potassium halide solutions and that of BSA solutions. Interaction occurs between BSA and Br-, I- and F- ions in mixed aqueous solutions of BSA-KBr, BSA-KI and BSA-KF, but there is no interaction between BSA and Cl- ion in BSA-KCl mixed aqueous solution. The interaction of ion to BSA is ion non-specific to the Hofmeister series.

Determination of whether ion specific potassium halides binding with BSA occurred within the experimental conditions was the aim of the study. Ultrasound velocity measurements using a Resoscan system and density measurements using a DMA 5000M density meter were made to analyse BSA conformation in the presence of potassium salts of the Hofmeister series. It was found that the density and the adiabatic compressibility of BSA-potassium halide mixed aqueous solutions cannot be predicted from the density and the adiabatic compressibility of potassium halide solutions and that of BSA solutions. Interaction occurs between BSA and Br-, I- and F- ions in mixed aqueous solutions of BSA-KBr, BSA-KI and BSA-KF, but there is no interaction between BSA and Cl- ion in BSA-KCl mixed aqueous solution. The interaction of ion to BSA is ion non-specific to the Hofmeister series.

Multiple Description Coding (MDC) is a source coding technique which generates several descriptions of a signal such that the reconstruction gradually refines with the number of decoded descriptions. Conventionally, the design of MDC has largely focused on combating the description loss. This thesis considers the construction of robust multiple description lattice vector quantizers (MDLVQ) by addressing the problem of designing an index mapping able to combat bit errors. We consider the scenario when the first description is received correctly at the decoder while the second one may carry bit errors. Our approach is to use a good assignment of the central lattice points to pairs of side lattice points, as developed in previous work, and design the mapping of the second description side lattice points to binary indexes such that the expected channel distortion to be minimized. In this thesis, we propose two methods to tackle this problem. The first method is a binary switching heuristic algorithm, which starts with an initial mapping and iteratively switches two indexes such that the distortion is decreased. This algorithm only guarantees a locally optimal solution whose quality depends on the initial configuration. The second approach attempts to increase the minimum Hamming distance between possible indexes in the second description when the first description is fixed. This is achieved using a structured construction as follows. First the set of second description side lattice points is partitioned into Voronoi regions of a carefully chosen coarse lattice. Next a channel code with high Hamming distance is picked, each Voronoi region is assigned a coset of this channel code and the side lattice points within each Voronoi region are mapped to binary sequences in the corresponding coset. We point out that in order to achieve good performance the mapping of Voronoi regions to cosets of the channel code must assign cosets close in Hamming distance to neighboring Voronoi regions. Two methods to achieve this goal are proposed and bounds on their performance are derived. Finally, simulations are carried out to assess the practical performance of the proposed designs. Our results show significant performance improvement when the proposed index mappings are used versus non-optimized mappings. / Thesis / Master of Applied Science (MASc)

Burkholderia pseudomallei is an intracellular pathogen and the causative agent of melioidosis, a severe disease of humans and animals. One of the virulence factors critical for early stages of infection is the Burkholderia secretion apparatus (Bsa) Type 3 Secretion System (T3SS), a molecular syringe that injects bacterial proteins, called effectors, into eukaryotic cells where they subvert cellular functions to the benefit of the bacteria. Although the Bsa T3SS itself is known to be important for host cell invasion, intracellular replication, and virulence, only a few genuine effector proteins have been identified and the complete repertoire of proteins secreted by the system has not yet been fully characterized. The aims of this study are twofold. The first is to expand the repertoire of known effector proteins using modern proteomics techniques. The second is to explore the function of a subset of effector proteins to better understand their interaction with host cells. Isobaric Tags for Relative and Absolute Quantification (iTRAQ), a gel-free quantitative proteomics technique, was used to compare the secreted protein profiles of the Bsa T3SS hyper-secreting mutants of B. pseudomallei with the isogenic parent strain as well as a mutant incapable of effector protein secretion. This study provides one of the most comprehensive core secretomes of B. pseudomallei described to date and identified 26 putative Bsa-dependent secreted proteins that may be considered candidate effectors. Two of these proteins, BprD and BapA, were validated as novel effector proteins secreted by the Bsa T3SS of B. pseudomallei. To determine the possible function of two effector proteins, BipC and BapA, a yeast two-hybrid system was used to identify host cell proteins the effectors interact with. The proteins were screened against a library of human proteins for interactions. BapA interacted with 2 proteins while BipC interacted with 14. Both BapA and BipC were shown to interact with human C1QBP, a mitochondrial protein involved in inflammation, immunity and autophagy. Finally, the Bsa T3SS protein BipC was characterised in its ability to interact with actin. This study is the first evidence that BipC has the ability to bind to filamentous actin, but not monomeric actin. This binding is direct and no intermediate proteins are required for the interaction. Ectopic expression of BipC in eukaryotic cells caused cytoskeletal rearrangements consistent with an actin-binding protein. The core secretome represents a substantial resource of targets that will be mined for improved diagnostic assays and vaccines. Diagnostics that will detect early stages of disease to allow for more effective antimicrobial intervention are currently lacking. Furthermore, there is scope to design diagnostic assays with dual use such as to detect both melioidosis and infection of cystic fibrosis patients with the closely related opportunistic pathogen B. cepacia. The description of novel T3SS effector proteins is also of considerable value since T3SS proteins are often potent B- and T- cell antigens representing promising components of sub-unit vaccines. Such effector proteins commonly modulate cellular processes such as phagocytosis, inflammasome activation and cell cycle progression, hence the function of the predicted T3SS effectors will provide a series of future research opportunities.

The mass of five proteins (Bovine serum albumin (BSA), casein, lysozyme, ovalbumin and pepsin) adsorbed to five different membrane materials (of various hydrophobicities) was quantified using a static system and analysed to establish any trends. Comparing the results from the five membranes it seems that there were no obvious trends between the protein masses adsorbed indicating that it may not be just one aspect of protein structure that is important in the adsorption process. Many investigations have indicated that the protein may undergo a conformational change during the adsorption process. Disulphide bridges contribute readily to the stability of the protein molecule and it was hypothesised that if such a structural change occurred, it would result in the breakage of these covalent bonds. To this end, the free thiol group content of the proteins was quantified before and after adsorption.

Neste trabalho estudamos por espalhamento de raios-X a baixos ângulos (SAXS) quatro diferentes sistemas de interesse biológico. Visamos investigar a auto-agregação de proteínas e de complexos proteicos que darão origem a fibras amilóides, interação proteína-proteína, simulando ambientes altamente concentrados, interação proteína-membrana simulando vesículas de matriz extracelular (MVs) de sistemas de biomineralização e interações proteína-agentes desnaturantes. No caso de formação de amilóides, investigamos a agregação do domínio GTPase da septina 6 (SEPT6G) e do complexo formado com o domínio GTPase da septina 2 (SEPT2G-SEPT6G). A temperaturas de até 15°C, tanto SEPT6G quanto SEPT2G-SEPT6G apresentam-se predominantemente diméricas em solução. Já a 25°C, o heterodímero SEPT2G-SEPT6G permanece estável enquanto agregados maiores de SEPT6G evoluem e coexistem em solução com SEPT6G-SEPT6G dimérica, sendo que a proporção de dímeros diminui com a temperatura. No estudo das MVs, mostramos que miméticos lipossomais de DPPC e DPPC:DPPS (9:1) possuem as mesmas características estruturais na ausência e presença de cálcio na solução. A interação da proteína anexina V humana (A5), envolvida em processos de biomineralização, impacta na membrana modelo induzindo a formação de nanoporos. A adição da fosfatase alcalina tecido não-específico (TNAP) não altera as propriedades estruturais do proteolipossomo na presença de A5. A ação do surfactante dodecil sulfato de sódio (SDS) a 30 mM não altera a conformação da albumina soro bovina (BSA), de maneira que é observada a formação de micelas de SDS coexistindo com a proteína livre em solução. Já a adição de 50 mM de SDS induz um desenovelamento parcial da proteína, identificado pela análise das curvas de SAXS via modelo de \"colar de pérolas\". A ação de uréia a 3 M e 8 M promove um desenovelamento parcial e total da BSA, respectivamente, com subsequente agregação de proteína dependente da temperatura (T > 30°C). A adição de 6 mM de SDS em proteínas parcialmente desenoveladas pela ação da uréia promove um desenovelamento mais acentuado. O potencial efetivo resultante da interação entre duas proteínas distintas, BSA e lisozima a concentração total de 100 mg/mL em solução, pH 7.0, foi obtido da análise de curvas de SAXS. Para isto, utilizou-se uma análise simplificada (em primeira aproximação) considerando um potencial efetivo de interação entre BSA-BSA, lisozima-lisozima e lisozima-BSA. Variamos a razão molar BSA:LISO até 1:42. No pH estudado, BSA tem uma carga residual superficial de -11e, enquanto a lisozima possui +9e. Conforme variamos a razão molar BSA:LISO, observamos dois regimes para o potencial efetivo resultante: i) até BSA:LISO 1:2, a carga efetiva do sistema é praticamente nula com um potencial resultante de caráter atrativo e ii) para razões entre BSA:LISO 1:3 a 1:42, a carga efetiva aumenta e o potencial resultante tem caráter repulsivo. Assim, lisozima e BSA coexistem sem agregar, através de um delicado balanço de forças atrativas e repulsivas no sistema. / In this work we have used small-angle x-ray scattering (SAXS) to study four systems of biological interest. We aim to investigate the self aggregation of proteins and protein complexes that would form amyloid fibers; protein/protein interaction, simulating high concentrations; protein/cell-membrane interaction, simulating extracellular matrix vesicles (MVs) from biomineralizing systems; and protein/denaturating-agents interactions. On the case of amyloid formation, we have investigated the aggregation of G-domain of septin-6 (SEPT6G) and the protein complex formed with G-domain of septin-2 (SEPT2G-SEPT6G). At temperatures lower than 15°C, both SEPT6G and SEPT2G-SEPT6G were found predominantly as dimers. At 25°C, SEPT2G-SEPT6G heterodimer is still stable while aggregates of SEPT6G grow. Both coexist in solutions of SEPT2G-SEPT6G dimers, with the percentage of dimers decreasing the higher the temperature. As for the study of MVs, we have shown that DPPC and DPPC:DPPS (9:1) liposomal mimetics have the same structural characteristics at the absence or presence of Calcium. The interaction with human annexin V protein (A5), related to biomineralization processes, affects the model membrane by the creation of nanopores. The addition of tissue-nonspecific alkaline phosphatase (TNAP) does not change the structural properties of the proteoliposome when A5 is present. The addition of SDS surfactant (30 mM) does not alters the conformation of bovine serum albumin (BSA), and we have observed the formation of SDS micelles coexisting with free protein in solution. The addition of 50 mM of SDS, on the other hand, induces the partial unraveling of the protein, as seen by the analysis of SAXS data via the pearl necklace\'\' model. The effect of adding 3M and 8M urea is, respectively, the partial and total unraveling of BSA, with ensuing aggregation of the protein dependent on the temperature (T > 30°C). The introduction of SDS 6mM promotes further unraveling in proteins that were previously partially unraveled by urea. The resulting effective potential for the interaction between BSA and lysozyme at total concentration of 100mg/ml and 7.0 pH has been obtained from the analysis of SAXS curves. In order to obtain this result we have used a simplified analysis (first order approximation) in which were considered the effective potentials for the interactions between BSA-BSA, lysozyme-lysozyme and lysozyme-BSA. We have varied the BSA:LISO molar ratio up to 1:42. At the studied pH, BSA has a surface residual charge of -11e, and lysozyme has +9e. As we changed the BSA:LISO molar ratio, we have found two regimens for the resulting effective potential: i) up to BSA:LISO 1:2, the effective charge of the system is virtually zero and the resulting potential is attractive; and ii) for BSA:LISO between 1:3 and 1:42 the effective charge increases, and the resulting potential is repulsive. Therefore, both lysozyme and BSA coexist without forming aggregates, by a delicate balance of attractive and repulsive forces.

Neste trabalho estudamos por espalhamento de raios-X a baixos ângulos (SAXS) quatro diferentes sistemas de interesse biológico. Visamos investigar a auto-agregação de proteínas e de complexos proteicos que darão origem a fibras amilóides, interação proteína-proteína, simulando ambientes altamente concentrados, interação proteína-membrana simulando vesículas de matriz extracelular (MVs) de sistemas de biomineralização e interações proteína-agentes desnaturantes. No caso de formação de amilóides, investigamos a agregação do domínio GTPase da septina 6 (SEPT6G) e do complexo formado com o domínio GTPase da septina 2 (SEPT2G-SEPT6G). A temperaturas de até 15°C, tanto SEPT6G quanto SEPT2G-SEPT6G apresentam-se predominantemente diméricas em solução. Já a 25°C, o heterodímero SEPT2G-SEPT6G permanece estável enquanto agregados maiores de SEPT6G evoluem e coexistem em solução com SEPT6G-SEPT6G dimérica, sendo que a proporção de dímeros diminui com a temperatura. No estudo das MVs, mostramos que miméticos lipossomais de DPPC e DPPC:DPPS (9:1) possuem as mesmas características estruturais na ausência e presença de cálcio na solução. A interação da proteína anexina V humana (A5), envolvida em processos de biomineralização, impacta na membrana modelo induzindo a formação de nanoporos. A adição da fosfatase alcalina tecido não-específico (TNAP) não altera as propriedades estruturais do proteolipossomo na presença de A5. A ação do surfactante dodecil sulfato de sódio (SDS) a 30 mM não altera a conformação da albumina soro bovina (BSA), de maneira que é observada a formação de micelas de SDS coexistindo com a proteína livre em solução. Já a adição de 50 mM de SDS induz um desenovelamento parcial da proteína, identificado pela análise das curvas de SAXS via modelo de \"colar de pérolas\". A ação de uréia a 3 M e 8 M promove um desenovelamento parcial e total da BSA, respectivamente, com subsequente agregação de proteína dependente da temperatura (T > 30°C). A adição de 6 mM de SDS em proteínas parcialmente desenoveladas pela ação da uréia promove um desenovelamento mais acentuado. O potencial efetivo resultante da interação entre duas proteínas distintas, BSA e lisozima a concentração total de 100 mg/mL em solução, pH 7.0, foi obtido da análise de curvas de SAXS. Para isto, utilizou-se uma análise simplificada (em primeira aproximação) considerando um potencial efetivo de interação entre BSA-BSA, lisozima-lisozima e lisozima-BSA. Variamos a razão molar BSA:LISO até 1:42. No pH estudado, BSA tem uma carga residual superficial de -11e, enquanto a lisozima possui +9e. Conforme variamos a razão molar BSA:LISO, observamos dois regimes para o potencial efetivo resultante: i) até BSA:LISO 1:2, a carga efetiva do sistema é praticamente nula com um potencial resultante de caráter atrativo e ii) para razões entre BSA:LISO 1:3 a 1:42, a carga efetiva aumenta e o potencial resultante tem caráter repulsivo. Assim, lisozima e BSA coexistem sem agregar, através de um delicado balanço de forças atrativas e repulsivas no sistema. / In this work we have used small-angle x-ray scattering (SAXS) to study four systems of biological interest. We aim to investigate the self aggregation of proteins and protein complexes that would form amyloid fibers; protein/protein interaction, simulating high concentrations; protein/cell-membrane interaction, simulating extracellular matrix vesicles (MVs) from biomineralizing systems; and protein/denaturating-agents interactions. On the case of amyloid formation, we have investigated the aggregation of G-domain of septin-6 (SEPT6G) and the protein complex formed with G-domain of septin-2 (SEPT2G-SEPT6G). At temperatures lower than 15°C, both SEPT6G and SEPT2G-SEPT6G were found predominantly as dimers. At 25°C, SEPT2G-SEPT6G heterodimer is still stable while aggregates of SEPT6G grow. Both coexist in solutions of SEPT2G-SEPT6G dimers, with the percentage of dimers decreasing the higher the temperature. As for the study of MVs, we have shown that DPPC and DPPC:DPPS (9:1) liposomal mimetics have the same structural characteristics at the absence or presence of Calcium. The interaction with human annexin V protein (A5), related to biomineralization processes, affects the model membrane by the creation of nanopores. The addition of tissue-nonspecific alkaline phosphatase (TNAP) does not change the structural properties of the proteoliposome when A5 is present. The addition of SDS surfactant (30 mM) does not alters the conformation of bovine serum albumin (BSA), and we have observed the formation of SDS micelles coexisting with free protein in solution. The addition of 50 mM of SDS, on the other hand, induces the partial unraveling of the protein, as seen by the analysis of SAXS data via the pearl necklace\'\' model. The effect of adding 3M and 8M urea is, respectively, the partial and total unraveling of BSA, with ensuing aggregation of the protein dependent on the temperature (T > 30°C). The introduction of SDS 6mM promotes further unraveling in proteins that were previously partially unraveled by urea. The resulting effective potential for the interaction between BSA and lysozyme at total concentration of 100mg/ml and 7.0 pH has been obtained from the analysis of SAXS curves. In order to obtain this result we have used a simplified analysis (first order approximation) in which were considered the effective potentials for the interactions between BSA-BSA, lysozyme-lysozyme and lysozyme-BSA. We have varied the BSA:LISO molar ratio up to 1:42. At the studied pH, BSA has a surface residual charge of -11e, and lysozyme has +9e. As we changed the BSA:LISO molar ratio, we have found two regimens for the resulting effective potential: i) up to BSA:LISO 1:2, the effective charge of the system is virtually zero and the resulting potential is attractive; and ii) for BSA:LISO between 1:3 and 1:42 the effective charge increases, and the resulting potential is repulsive. Therefore, both lysozyme and BSA coexist without forming aggregates, by a delicate balance of attractive and repulsive forces.

Orientador: Celso Luis Marino / Resumo: The forest-breeding program in Brazil has the general objective of providing most adapted plants to different environments for various Brazilian regions, for fulfilling timber demands meant for multiple uses in the country. One of the main problems found in different forest breeding programs are the difficulty to identify the different species and hybrids. The use of molecular biology techniques in plant breeding programs is found very effective in the optimization of the time and the direction of these programs, particularly among those plants of the same subgenus. The process of selection and hybrid plants selected for planting in most cases; significantly increase the gain in terms of production and adaptability. The use of molecular markers to characterize the molecular variability of forest species has revolutionized genetic analysis in recent years. The bulk segregant analysis (BSA) is a technique used to identify molecular markers linked to monogenic, dominant or recessive characters. BSA technique in combination with Amplified Fragment Length Polymorphisms (AFLP) technique is an efficient methodology for the detection of polymorphism from genomic restriction fragments through PCR amplification; which helps in analyzing large number of loci for testing without the need for previous information of their sequence in respect to their dominance and reproducibility. The most recent and promising applications of molecular biological methods for the detection of small DNA fra... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor