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Lactobacillus plantarum WCSF1 has two putative prolinases (PepR1 and PepR2), and they share only 48.5% amino acid sequence identity. To investigate the differences in enzymatic characters between two enzymes, the genes are cloned and expressed in E. coli using non-tagged pKK223-3 and His-tagged pET32b(+) systems. Culture conditions of overexpressed recombinant prolinases (r-PepR1 and r-PepR2) are optimized as pH7.0-7.5 LB media at 16°C with 1 mM IPTG induction. Recombinant prolinases with His-tag give higher yields and are more cost-efficient over non-tagged recombinant prolinases. After purification, these recombinant enzymes show similar hydrolysis activities towards Pro-Gly substrate, proving their nature as prolinases. Structural analyses using CD spectrum and computer modelling show that r-PepR1 and r-PepR2 share structural similarity in their secondary structure having the highest β-sheets over other components; and dynamic light scattering and gel filtration chromatography analyses indicate their quaternary structure being homotetrameric. Structural similarity can be linked to enzyme function feature. The two enzymes have the same enzymatic functionality may be due to their structural similarity. Despite for their structural similarities and the same enzymatic functionality, they show differences in their substrate specificity, optimum temperature and pH, kinetic parameters (Km and kcat values), thermal stability, and proteolysis mode. r-PepR1 has its optimal activity at 25°C pH7.5 against substrate Pro-Met, whereas r-PepR2 is most active at 30°C pH8.0 against Pro-Gly. r-PepR1 has a low thermal stability with a TM (the midpoint temperature of the unfolding transition) at 29°C, whereas r-PepR2 has a higher TM at 48°C. However, r-PepR1 is aggregated and inactivated at near physiological temperature (42°C). The catalytic mode of r-PepR1 could be a metallo-protease since its activity reduces by 38% with a metal-chelating agent EDTA; while the activity of r-PepR2 is inhibited by 47% with a serine protease inhibitor PMSF, suggesting it is a serine protease. These isozymes cooperatively and complementarily work together to hydrolyze proline-containing peptides, showing broader specificity, broader range of working pH and temperature, and higher efficiency, suggesting that the proline recycling are mediated through these two enzymes to adapt a wide rage of environmental conditions.

Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.

Diplodia ear rot (DER) of maize (Zea mays L.) caused by the fungus, Stenocarpella maydis (Berk.) Sutton has increased in incidence in localized fields over the past decade. My research focused on screening for resistance by examining the development of DER following inoculations prior to flowering, analyzing a diallel cross for DER resistance, and examining the genetic variability of the fungus from isolates collected from the U.S. and the Republic of South Africa. DER developed in maize following inoculations with a spore suspension prior to flowering in both greenhouse and field evaluations. A spore suspension gave a better differentiation of resistance responses than dried preparations of colonized millet, colonized ground popcorn, or kernels from a diseased maize ear, all applied in the whorl 10 to 15 days prior to flowering (V12 for inbreds), and natural occurrence of disease. General combining ability was significant for both 1994 and 1995 growing seasons in an analysis of the F₁ of the diallel cross, indicating that additive gene action may be responsible for resistance and could be introduced into commercial cultivars. Specific combining ability was significant in 1995 and indicates that dominant gene action or epistasis may play role in DER resistance. There were minimal numbers of isozyme polymorphisms found in my S. maydis collection. Two isolates were polymorphic for esterase, two isolates were polymorphic for hexokinase and malate dehydrogenase and one isolate was polymorphic for hexose kinase. Fungi that have limited isozyme polymorphisms often are biotrophs or fungi with formae speciales which are usually limited to one host. These groups of fungi usually have races and this may indicate that a gene-for-gene interaction exists. These findings suggest that i) the whorl inoculation separates genotypes into resistant, intermediate, and susceptible groupings; ii) additive gene action is predominant form of inheritance, and iii) there are few isozyme polymorphisms in the population of S. maydis sampled. / Ph. D.

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pHi for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes.

A grande biodiversidade nas florestas tropicais, a elevada exploração de florestas e as poucas espécies estudadas do ponto de vista genético levaram a este estudo que é essencial para o manejo sustentável e a conservação genética de espécies, sendo importante no controle da redução da diversidade genética natural permitindo que as espécies se mantenham vivas e reprodutivas ao longo dos tempos. Entre as diversas espécies arbóreas brasileiras submetidas a práticas de manejo, tem-se a candeia (Eremanthus erythropappus). Os objetivos foram quantificar e comparar a variabilidade genética intrapopulacional e sistema de reprodução de candeia e antes e após o manejo, através da técnica de eletroforese de isoenzimas em uma população natural de candeia. Para as progênies as heterozigosidades observadas foram altas 0,357 e 0,423 e heterozigosidades esperadas 0,403 e 0,425. Para os adultos foram 0,299 e 0,399. A porcentagem dos locos polimórficos variou de 76 a 100% entre progênies e adultos. O número de alelos por loco variou de 2,3 nos adultos e 2,57 em progênies antes e após o manejo. Nas progênies não houve diferença significativa entre estes valores indicando que a população encontra-se em EHW, tal fato pode ser comprovado pelo índice de fixação significativamente igual a zero (0,112 e 0,005) antes e após manejo, respectivamente. Os resultados do sistema de reprodução foram: taxa de cruzamento alta na análise antes (0,963) e pós manejo (0,967) indicando que a espécie é de reprodução mista; ocorreram cruzamentos entre aparentados tanto na população antes do manejo como na pós manejo, com estimativas entre 3 e 5%; as estimativas da correlação de autofecundação ( s rˆ ) foi alta nas duas populações (0,188 e 0,179), sendo ambas significativas, sugerindo que os indivíduos de autofecundação encontram-se aleatoriamente distribuídos nas progênies não existindo a tendência de algumas progênies apresentarem mais indivíduos de autofecundação do que outros. A correlação de paternidade foi significativamente diferente de zero (0,414 e 0,368), sugerindo que uma parte das progênies de cruzamentos foi gerada pelo mesmo parental materno e paterno, indicando haver a presença de cruzamentos biparentais dentro das populações. As estimativas do número médio de indivíduos polinizadores efetivos por árvore, foram baixas, em torno de 2 a 3 polinizadores por árvore. O sistema misto tem implicações na estimativa do tamanho efetivo de variância (Ne(v)), os valores estimados para este parâmetro foram de 1,99 e 2,08 para população antes do manejo e posterior ao manejo, respectivamente. O valor estimado para o coeficiente de coancestria dentro das progênies antes do manejo (0,229) e posteriores ao manejo (0,222) foram superiores em 83,2% e 77,6% ao esperado em progênies de meios-irmãos (0,125). O tamanho efetivo de endogamia foi de 178,4, 51 e 49 indivíduos em adultos, progênies antes e após o manejo, respectivamente. Os resultados indicam forte estrutura genética espacial na população, com árvores próximas até 200 m apresentando algum grau de parentesco, com 95% de probabilidade. Estes resultados indicam que a espécie é passível de manejo e que este não afetou a diversidade nesta geração. / The great biodiversity in the tropical forests, high level of forest exploitation and the few arboreal species which have been studied from the genetic point of view led to this study that is essential for the sustained handling and the genetic conservation of the species. They are important in controlling the natural genetic diversity reduction thus allowing the species to be alive and reproduce, in time. Among the various Brazilian arboreal species submitted to the handling practice there is the candeia (Eremanthus erythropappus). The objectives have been of quantifying and comparing the intrapopulational genetic variability and the mating system of candeia and before and after the allozyme electrophoresis technique has been applied in a natural population of candeia two different times (before and after the handling). In the progenies, the observed heterozigosity was high (0,357 and 0,423) and expected heterozogosity (0,403 and 0,425).For the adults the results were (0,299 and 0,399). The percentage of the polymorphic loci varied from 76 % to 100% among progenies and adults. The number of alleles per loci varied from 2,3 in adults and 2,57 in progenies before and after the handling. In the progenies there was no significant difference between those values indicating that the population is in.HWE. That fact can be proven by the fixation index significantly equal to zero (0,112 and 0,005) before and after handling, respectively. The results of the reproduction system were: the crossing rate was high in the analysis that was done before (0,963) and in the one that was done after the handling ( 0,967). The estimates of the self-fertilization correlation were high in the two populations (0,188 and 0,179), and both were significant suggesting that the selffertilization individuals are distributed at random in the progenies and there is no tendency of some progenies to present more individuals of self fertilization than others. The paternity correlation was significantly different from zero (0,414 and 0,368), suggesting that a part of the progenies of crossings were bred by the same maternal and paternal begetter thus indicating that there was the presence of biparental crossings within the populations. The mixed system is involved in the average effective number variance (Ne(v), the estimated values for that parameter were of 1,99 and 2,08 for the population before the handling and after the handling, respectively. The estimated value of the coefficient of coancestry within the progenies before the handling ( 0,229) and after the handling (0,222) were superior in 83,2% and 77,6% than the expected in progenies of half-sib (0,125).The effective size of inbreeding was of 178,4, 51 and 49 individuals in adults, progenies before and after the handling, respectively. The results indicate a strong spatial autocorrelation analysis in the population, with trees next to each other as far as approximately 200 m presenting some degree of relationship, with 95% of probability. Those results indicate that the species is unresistant to handling and that did not affect the diversity in this generation and future works must be considered to assure the sustainability of the handling that is done from the genetic point of view.

UDP-glucose pyrophosphorylase (UGPase) is an important enzyme of carbohydrate metabolism in all living organisms. The main aim of this thesis was to investigate the function and regulation of plant UGP genes as well as the UGPase proteins. Both in vivo and in vitro approaches were used, including the use of transgenic plants deficient in UGPase activity, and using purified proteins and their mutants to elucidate the structure/ function properties of UGPase. In both Arabidopsis and aspen, there are two highly similar UGP genes being actively transcribed, but not to the same extent. For both species, the UGP genes could be classified into two categories: a “house-keeping” gene and a subsidiary gene, with the former functioning universally in all the tissues to support the normal growth, whereas the latter usually expressed at a lower level in most of the organs/tissues tested. Besides, the two UGP genes were also found being differentially regulated under abiotic stress conditions, e.g. low temperature. By investigating the Arabidopsis T-DNA insertion mutants, which respectively have one or both of the UGP genes knocked out, we noticed that as little as 10% of the remaining UGPase activity could still support normal growth and development under controlled conditions, with little or no changes in carbohydrate contents, including soluble sugars (e.g. sucrose), starch and cell wall polysaccharides. Those plants, however, had a significantly decreased fitness under field conditions, i.e. the plants most deficient in UGPase activity produced up to 50% less seeds than in wt. Therefore, we concluded that UGPase is not a rate-limiting enzyme in carbohydrate synthesis pathways, but still is essential in viability of Arabidopsis plants. In order to characterize two Arabidopsis UGPase isozymes, both proteins were heterologously overexpressed in prokaryotic cells and purified by affinity chromatography. The two isozymes showed little differences in physical and biochemical properties, including substrate specificity, Km values with substrates in both directions of the reaction, molecular masses, isoelectric point (pI), and equilibrium constant. On the other hand, possibilities of distinct post-translational regulatory mechanisms were indicated, based on amino acid (aa) motif analyses, and on 3D analyses of derived crystal structures of the two proteins. We used the heterologous bacterial system also to overexpress barley UGPase and several of its mutants, both single mutants and those with whole domains/ exons deleted. As a result, we have identified several aa residues/ protein domains that may be essential for structural integrity and catalytic/ substrate-binding properties of the protein. For instance, we found that the last exon of UGPase (8 aa at the end of C-terminus) was important for the protein ability to oligomerize and that Lys-260 and the second-to-last exon were essential for pyrophosphate (but not UDP-glucose) binding. The data emphasized the critical role of central part of the active site (so called NB-loop) in catalysis, but also pointed out to the role of N-terminus in catalysis and oligomerization, but not substrate binding, and that of C-terminus in both catalysis/substrate binding and oligomerization.

Made available in DSpace on 2015-03-26T13:37:41Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1569948 bytes, checksum: 47e985324f875fabdd3773a51785089b (MD5) Previous issue date: 2010-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The root-knot nematodes, belonging to Meloidogyne spp., represent a restraint factor to the Brazilian coffee crop because they cause too many losses. There is only some information on the occurrence of these nematodes, especially in coffee plantations in Espírito Santo State (ES), thus it was aimed to study the distribution of Meloidogyne spp. in Coffea spp. in this State and to complement the investigation in Zona da Mata in Minas Gerais (MG). Samples of soil and roots were collected from 18 municipalities on ES and 5 municipalities on the region of Zona da Mata of MG. The identification of species of Meloidogyne spp. was performed through esterase phenotype and differential host test. On ES, different species of Meloidogyne spp. were detected in the analyzed samples. Meloidogyne incognita, I1 and I2 phenotypes, was found in 18% of the sampled farm estates, and in 81.25% of these infested farms C. canephora was cultivated. This species of root-knot nematodes is present in 55.5% of the sampled municipalities, and it is found mainly in the mountain region and in the north of the State. This species was found in a mixture with M. exigua in a coffee crop in Brejetuba, and it was found in a mixture with M. paranaensis in Sooretama. Meloidogyne exigua, E1 phenotype, was detected in 43.6% of the farms and only in C. arabica crops. This species was found in all sampled municipalities in the southern region and in 66% of the sampled municipalities in the mountain region where arabica coffee is grown in large scale. In the north of the state, M. paranaensis, P1 phenotype, was detected in the sampled municipalities and it is reported by the first time in the state of Espírito Santo. The species M. arenaria, A2 phenotype, and M. javanica (J3) were detected in Laranja da Terra parasitizing weeds in the area, but not in coffee trees. However, in Zona da Mata in Minas Gerais, it was found only M. exigua, E1 phenotype, parasitizing coffee trees in all the municipalities where collection was performed, except in Paula Cândido municipality, where no species of Meloidogyne was found. The populations were tested by differential host plants in order to determine physiological races. It was found M. incognita and M. exigua races 1 and 2 in Espírito Santo, and M. exigua race 2 in Zona da Mata in Minas Gerais. Nematodes from genus Tylenchus, Helicotylenchus, Rotylenchulus, Pratylenchus, Aphelenchoides, Aphelenchus, Xiphinema, Mesocrinonema, Psilenchus, Hemicriconemoides, Discocriconemella, Ditylenchus, Rotylenchus, were associated to coffee tree rizosphere. / Os nematoides das galhas pertencentes ao gênero Meloidogyne spp. representam um fator limitante à cafeicultura brasileira por causarem grandes perdas. Devido às escassas informações sobre a ocorrência desses nematoides, principalmente na cafeicultura capixaba, objetivou-se estudar a distribuição de Meloidogyne spp. em Coffea spp. no Estado do Espírito Santo e complementar os levantamentos na Zona da Mata de Minas Gerais. Amostras de solo e raízes foram coletadas em 18 municípios no Estado do Espírito Santo e em 5 municípios na Zona da Mata de Minas Gerais. A identificação das espécies de Meloidogyne spp. foi realizada pelo fenótipo da isoenzima esterase e pela gama de hospedeiros. No Estado do Espírito Santo foram detectadas diversas espécies de Meloidogyne nas amostras analisadas. Meloidogyne incognita, fenótipo I1 e I2, foi encontrada em 18% do total das propriedades amostradas, sendo que em 81,25% das propriedades infestadas se plantava C. canephora. Esta espécie está presente em 55,5% dos municípios amostrados sendo encontrada principalmente na região Serrana e Norte do Estado. Em uma propriedade do município de Brejetuba, ela foi encontrada em mistura com M. exigua, e no município de Sooretama, em mistura com M. paranaensis. Meloidogyne exigua, fenótipo E1, foi detectada em 43,6% das propriedades e apenas em lavouras de C. arabica. Esta espécie foi encontrada em todos os municípios amostrados da Região Sul e em 66,6% dos municípios amostrados da Região Serrana, regiões onde o café arábica é cultivado em larga escala. Meloidogyne paranaensis, fenótipo P1, foi detectado em todos os municípios amostrados da região Norte do Estado, e é relatada pela primeira vez no Estado do Espírito Santo. As espécies M. arenaria, fenótipo A2, e M. javanica (J3) foram detectadas em Laranja da Terra parasitando plantas daninhas presentes na área, mas não em cafeeiros. Já na Zona da Mata de Minas Gerais foi encontrada apenas M. exigua, fenótipo E1, parasitando as plantas de cafeeiro arábica em todos os municípios coletados exceto no município de Paula Cândido, onde nenhuma espécie de Meloidogyne foi encontrada. As populações foram submetidas ao teste com plantas hospedeiras diferenciadoras para determinação das raças fisiológicas. Foram encontradas as raças 1 e 2 de M. incognita e M. exigua no Estado do Espírito Santo, e a raça 2 de M. exigua na Zona da Mata de Minas Gerais. Nematoides dos gêneros Tylenchus, Helicotylenchus, Rotylenchulus, Pratylenchus, Aphelenchoides, Aphelenchus, Xiphinema, Mesocrinonema, Psilenchus, Hemicriconemoides, Discocriconemella, Ditylenchus, Rotylenchus, estavam associados com a rizosfera do cafeeiro.