HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients.

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway

Biochem. J. (2011) 438,485–494 doi:10.1042/BJ20110836

Crystal structure of the zinc-, cobalt-, and iron-containing adenylate kinase from Desulfovibrio gigas: a novel metal-containing adenylate kinase from Gram-negative bacteria

J Biol Inorg Chem (2011) 16:51–61 DOI 10.1007/s00775-010-0700-8

Molybdenum Induces the expression of a protein containing a new heterometallic Mo-Fe cluster in desulfoVibrio alaskensis

Biochemistry. 2009 Feb 10;48(5):873-82. doi: 10.1021/bi801773t.

NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo–Cu cluster

Biomol NMR Assign (2007) 1:81–83 DOI 10.1007/s12104-007-9022-3

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

J Biol Inorg Chem (2007) 12:777–787 DOI 10.1007/s00775-007-0229-7

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

J Biol Inorg Chem (2006) 11: 433–444 DOI 10.1007/s00775-006-0090-0

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

J Biol Inorg Chem (2004) 9: 839–849 DOI 10.1007/s00775-004-0584-6

Formation of a stable cyano-bridged dinuclear iron cluster following oxidation of the superoxide reductases from Treponema pallidum and Desulfovibrio vulgaris with K(3)Fe(CN)(6)

Inorg. Chem., 2003, 42 (4), pp 938–940 DOI: 10.1021/ic0262886

Production of bacterial biopolymers from industrial fat-containing wastes

Dissertation presented in partial fulfilment of the Requirements for the Degree of Master in Biotechnology

Synthesis and characterization of sugar containing hydrophilic and hydrophobic polymers with potential application in medicine

Dissertation toobtaina Master of Science degree in Bioorganics

Amyotrophic Lateral Sclerosis:Mammalian Cell Models,Copper-Zinc Superoxide Dismutaseand Biological Characteristics

"Amyotrophic Lateral Sclerosis (ALS) is the most severe and common adult onset disorder that affects motor neurons in the spinal cord, brainstem and cortex, resulting in progressive weakness and death from respiratory failure within two to five years of symptoms onset(...)

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

Bioremediation and CO2 scavenging using molybdenum-containing enzymes

Carbon dioxide valorization, will not only help to relieve the greenhouse effect but might also allow us to transform it in value-added chemicals that will help overcoming the energy crisis. To accomplish this goal, more research that focus on sequestering CO2 and endeavors through a carbon-neutral or carbon-negative strategy is needed in order to handle with the dwindling fossil fuel supplies and their environmental impact. Formate dehydrogenases are a promising means of turning CO2 into a biofuel that will allow for a reduction of greenhouse gas emissions and for a significant change to the economic paramount. The main objective of this work was to assess whether a NAD+-independent molybdenum-containing formate dehydrogenase is able to catalyze the reduction of CO2 to formate. To achieve this, a molybdenum-containing formate dehydrogenase was isolated from the sulfate reducing bacteria Desulfovibrio desulfuricans ATCC 27774. Growth conditions were found that allowed for a greater cellular mass recovery and formate dehydrogenase expression. After growth trials, kinetic assays for formate oxidation and CO2 reduction were performed and kinetic parameters determined. For the formate oxidation reaction, a KM of 49 μM and a turnover constant of 146 s-1 were determined. These kinetic parameters are in agreement with those determined by Mota, et al. (2011). Finally, we found that this molybdenum-containing enzyme was able to catalyze the reduction of CO2 to formate with a turnover constant of 4.6 s-1 and a KM of 13 μM. For the first time a NAD+-independent molybdenum-containing formate dehydrogenase was found to catalyze CO2 reduction, allowing its use as a biocatalyst in energetically efficient CO2 fixation processes that can be directed towards bioremediation or as an alternative and renewable energy source. Characterizing these enzymes may lead to the development of more efficient synthetic catalysts, make them readily available and more suited for practical applications.

Different culture media containing methyldopa for melanin production by Cryptococcus species

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.