A Febre Purpúrica Brasileira - FPB- foi reconhecida como uma doença pediátrica fulminante, caracterizada por febre alta com rápida progressão para púrpura, choque e óbito. A grande maioria dos pacientes apresentaram conjuntivite prévia. O presente trabalho procura descrever os aspectos epidemiológicos desta doença, desde o surgimento dos primeiros casos, em 1984, em Promissão, Estado de São Paulo, Brasil. Foram registrados 277 casos distribuídos no Brasil em cinco Estados: São Paulo, Paraná, Mato Grosso, Mato Grosso do Sul e Minas Gerais. Fora do Brasil, dois casos foram relatados na Austrália. Cerca de 89% dos casos ocorreram em crianças de até cinco anos de idade. A letalidade foi de 38%. Estima-se o período de incubação médio de 15 dias. O quadro inicial se caracteriza por conjuntivite e febre. O agente etiológico é a bactéria Haemophilus influenzae biogrupo aegyptius, clone invasivo, tendo sido isolado de sangue, liquor, lesão hemorrágica de pele, secreção de conjuntiva e de orofaringe em 1986, de pacientes no Estado de São Paulo. Os estudos moleculares identificaram características diferentes das descritas até então para o Haemophilus aegyptius isolado de surtos de conjuntivite. Os principais surtos da doença ocorreram em Serrana, Valparaíso no Estado de São Paulo e Maracaju no Estado do Mato Grosso do Sul. Em algumas localidades com surtos de conjuntivite se observou a presença de moscas (Diptera). Acredita-se que estes insetos tenham participação na disseminação da doença. O diagnóstico precoce da FPB é um fator importante para redução da letalidade. / Brazilian Purpuric Fever- BPF- has been recognized as a fulminant pediatric disease characterized by fever with rapid progression to purpura, shock and death. The vast majority of BPF patients have previously presented conjunctivitis. The present work aims to describe epidemiological aspects of BPF since the appearance of the first cases, in 1984, in Promissão, State of São Paulo, Brazil. It has been reported 277 cases in Brazil, distributed by five states: São Paulo, Paraná, Mato Grosso, Mato Grosso do Sul and Minas Gerais. Outside Brazil, only two cases have been reported in Australia. About 89% from the total number of cases occurred in children aging five years old or less. The case fatality rate was 38%. The average incubation period is estimated as being 15 days. The initial clinical symptoms are conjunctivitis and fever. The etiologic agent of BPF is the bacteria Haemophilus influenzae biogroup aegyptius, invasive clone. It has been isolated from blood, cerebrospinal fluid, hemorrhagic skin lesion and conjunctival and orofarynx secretions in 1986, from São Paulo State patients. Molecular studies have identified different features from those described to H.aegyptius which have been isolated during conjunctivitis outbreaks since then. The most important BPF outbreaks occurred in Serrana and Valparaíso, State of São Paulo and in Maracaju, State of Mato Grosso do Sul. The presence of gnats (Diptera) has been observed in some of the places where conjunctivitis outbreaks have occurred. It is believed that these insects are associated with BPF transmission. Early diagnosis is an important factor in fatality reduction.

We propose subword spotting, a generalization of word spotting where the search is for groups of characters within words. We present a method for performing subword spotting based on state-of-the-art word spotting techniques and evaluate its performance at three granularitires (unigrams, bigrams and trigrams) on two datasets. We demonstrate three applications of subword spotting, though others may exist. The first is assisting human transcribers identify unrecognized characters by locating them in other words. The second is searching for suffixes directly in word images (suffix spotting). And the third is computer assisted transcription (semi-automated transcription). We investigate several variations of computer assisted transcription using subword spotting, but none achieve transcription speeds above manual transcription. We investigate the causes.

We propose subword spotting, a generalization of word spotting where the search is for groups of characters within words. We present a method for performing subword spotting based on state-of-the-art word spotting techniques and evaluate its performance at three granularitires (unigrams, bigrams and trigrams) on two datasets.We demonstrate three applications of subword spotting, though others may exist. The first is assisting human transcribers identify unrecognized characters by locating them in other words. The second is searching for suffixes directly in word images (suffix spotting). And the third is computer assisted transcription (semi-automated transcription). We investigate several variations of computer assisted transcription using subword spotting, but none achieve transcription speeds above manual transcription. We investigate the causes.

The objectives of this investigation have been to characterize black gram (Phaseolus Mongo L.) and rice (Oryza sativa L.) proteins and to study changes in their nutritional value due to fermentation. black gram, the legume chosen for this work, is one of the most important legume crops throughout a large part of the tropics. The protein content of 60 mesh, dehydrated, defatted black gram meal was 28.5 percent. Sodium carbonate (0.5- 1.0 percent), tetra-sodium pyrophosphate (0.5 percent), and sodium dodecyl sulfate (SDS) (0.5-5 percent) proved to be the potential protein solubilizers as they extracted more than 76 grams of Lowry's protein per 100 grams Kjeldahl protein. On the considerations of contaminating residue in the final product and disruption of native structure of the proteins, these chemical agents were unsuitable. Sodium sulfate at the 10 percent level was judged to be the best protein solubilizer. Proteins separated on polyacrylamide gel using a phenol-acetic acid-mercaptoethanol-urea (PAMU) system were run on the flat bed gel containing SDS. The proteins were separated in 13 constituents and the molecular weights of the major ones were 140,000 and 55,000. Solubilized proteins contained 81 percent globulins, 13 percent albumins, 4 percent prolamins, and 2 percent glutelins . Sulfur containing amino acids and threonine were deficient in total proteins xv of the seeds with 27. 6 and 78.8 as their respective chemical scores . Chemical scores of the albumin, globulin, prolamin, and glutelin fractions were 64, 0, 56, and 70.7, respectively. The predicted biological values in human nutrition varied from O for globulins to 110 for glutelins, and was 14.9 for total proteins in the seeds. The constituents of the protein fractions were isoelectrically focused in the acidic pH range with the exception of two globulins for which the isoelectric points were 8.42 and 8.65. The trypsin inhibitor from black gram was isolated using affinity chromatography gel with 19. 5 fold purification. The inhibitor had 75 amino acid residues and contained one disulfide bridge. Chemical studies assigned an important role for the hydrogen bonds and demonstrated vital importance of the disulfide bridge in retaining the inhibiting activity. The inhibitor was stable and retained 35 percent of the activity when heated at 100°C for 60 minutes at pH 11. Chemical modification of amino acid residues suggested the involvement of lysine and arginine residues at the active site of the inhibitor. Lysine and arginine moieties at the active site have been proposed to be present as alanyllysine and histidylarginine. Inhib i tion of bovine pancreatic trypsin by the inhibitor was kinetically studied . The kinetic constants Km and Vm ax were 2.7 x l0- 5M and 6 x l0 - 3M/min, respectively. The dissociation constant for the enzyme-inhibitor complex (Ki) was 4 x l0- 7M, whereas that for the enzyme-inhibitor-substrate complex (K. 1 , ) was 1.89 x l0- 6M. The inhibition was a mixture of partial competitive and pure-noncompetitive systems. Rice and black gram form the integral parts of a fermented snack food of the Indian subcontinent~idli. Amino acid composition of black gram and rice were complementary. Leucine, lysine, and sulfur containing amino acids were the most limiting amino acids in rice with 65. 1, 66.3, and 67.9 as their respective scores. The estimates of biological values of rice proteins in human nutrition qualified albumins as superior and prolamins as inferior proteins. The PAMUsy stem in polyacrylamide gel electrophoresis was more efficient in resolving protein subunits than the SOS gel system. The PAMUsy stem was not sensitive to the ionic strength of the sample. Mobilities of rice and black gram proteins in SOS and PAMUsy stems were based on the related parameters. In the PAMUsy stem, the mobilities of most proteins seemed to depend on their molecular size. The PAMUsy stem on gel electrophoresis was judged superior to the SOS system. Fermentation of the black gram-rice blend was kinetically studied for changes in physicochemical characteristics and nutritional functionality. Trypsin inhibiting activity was unaffected, but chymotrypsin inhibiting activity was reduced to 3 percent after 20 hours fermentation , Significant increases were noted in the contents of sulfur containing amino acids during fermentation . These amino acids seemed to be bioavailable. In vitro digestion with pepsin and pancreatin indicated improvement in digestibility of proteins after fermentation. The digestibility was further enhanced by steaming.

The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB - GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.

Engineered affinity proteins have together with naturally derived antibodies becomeindispensable tools in many areas of life-science and with the increasing number ofapplications, the need for high-throughput methods for generation of such different affinityproteins is evident. Today, combinatorial protein engineering is the most successful strategy toisolate novel non-immunoglobulin affinity proteins. In this approach, generally termed directedevolution, high-complexity combinatorial libraries are created from which affinity proteins areisolated using an appropriate selection method, thus circumventing the need for detailedknowledge of the protein structure or the binding mechanism, often necessary in more rationalapproaches. Since the introduction of the phage display technology that pioneered the field ofcombinatorial engineering, several alternative selection systems have been developed for thispurpose.This thesis describes the development of a novel selection system based onstaphylococcal surface display and its implementation in directed evolution approaches. In thefirst study, the transformation efficiency to the gram-positive bacteria Staphylococcus carnosus wassuccessfully improved around 10,000-fold to a level that would allow cell surface display ofcomplex combinatorial protein libraries. In two separate studies, the staphylococcal displaysystem was investigated for the applicability in both de novo selection and affinity maturation ofaffibody molecules. First, using a pre-selection strategy with one round of phage display, ahigh-complexity affibody library was displayed on staphylococcal cells. Using fluorescenceactivatedcell sorting, binders with sub-nanomolar affinity to tumor necrosis factor-alpha(TNF-α) were isolated. Second, a combined approach using phage display for de novo selectionof first-generation affibody binders and staphylococcal display in a subsequent affinitymaturation selection was applied to generate binders with low nanomolar affinity to the humanepidermal growth factor receptor-3 (ErbB3). Moreover, in an additional study, thestaphylococcal surface display system was improved by the introduction of a protease 3Ccleavage sequence in the displayed fusion products in order to facilitate straightforwardproduction of soluble proteins for further downstream characterization.Altogether, the presented studies demonstrate that the staphylococcal selection systemindeed is a powerful tool for selection and characterization of novel affinity proteins and couldbecome an attractive alternative to existing selection techniques. / <p>QC 20100726</p>

Even though our understanding of mechanisms such as protein folding and molecular recognition is relatively poor, antibodies and alternative affinity proteins with entirely novel functions are today generated in a routine manner. The reason for this success is an engineering approach generally known as directed evolution. Directed evolution has provided researchers with a tool for circumventing our limited knowledge and hence the possibility to create novel molecules that by no means could have been designed today. The approach is based on construction of high-complexity combinatorial libraries from which protein variants with desired properties can be selected. Engineered proteins are already indispensable tools in nearly all areas of life science and the recent advent of mainly monoclonal antibodies as therapeutic agents has directed even more attention to the field of combinatorial protein engineering. In this thesis, I present the underlying research efforts of six original papers. The overall objective of the studies has been to develop and investigate a new staphylococcal surface display method for protein engineering and protein characterization. The technology is based on display of recombinant proteins on surface of the Gram-positive bacteria Staphylococcus carnosus. In two initial studies, two key issues were addressed in order to improve the protein engineering method in regard to affinity discrimination ability and transformation efficiency. The successful results enabled investigation of the staphylococcal display system for de novo generation of affibody molecules from large combinatorial libraries. In this study, a high-complexity protein library was for the first time displayed on surface of Gram-positive bacteria and by means of fluorescence-activated cell sorting, specific affinity proteins for tumor necrosis factor-alpha were isolated. Moreover, in following papers, the staphylococcal display method was further improved and investigated for affinity determination, soluble protein production and epitope mapping purposes in order to facilitate downstream characterizations of generated affinity proteins. Taken together, in these studies we have demonstrated that the staphylococcal display system is a powerful alternative to existing technologies for protein engineering and protein characterization. / QC 20100809

In practice, most approaches for text error detection and correction are based on a conventional domain-dependent background dictionary that represents a fixed and static collection of correct words of a given language and, as a result, satisfactory correction can only be achieved if the dictionary covers most tokens of the underlying correct text. Again, most approaches for text correction are for only one or at best a very few types of errors. The purpose of this thesis is to propose an unsupervised approach to detecting and correcting text errors, that can compete with supervised approaches and answer the following questions: Can an unsupervised approach efficiently detect and correct a text containing multiple errors of both syntactic and semantic nature? What is the magnitude of error coverage, in terms of the number of errors that can be corrected? We conclude that (1) it is possible that an unsupervised approach can efficiently detect and correct a text containing multiple errors of both syntactic and semantic nature. Error types include: real-word spelling errors, typographical errors, lexical choice errors, unwanted words, missing words, prepositional errors, article errors, punctuation errors, and many of the grammatical errors (e.g., errors in agreement and verb formation). (2) The magnitude of error coverage, in terms of the number of errors that can be corrected, is almost double of the number of correct words of the text. Although this is not the upper limit, this is what is practically feasible. We use engineering approaches to answer the first question and theoretical approaches to answer and support the second question. We show that finding inherent properties of a correct text using a corpus in the form of an n-gram data set is more appropriate and practical than using other approaches to detecting and correcting errors. Instead of using rule-based approaches and dictionaries, we argue that a corpus can effectively be used to infer the properties of these types of errors, and to detect and correct these errors. We test the robustness of the proposed approach separately for some individual error types, and then for all types of errors. The approach is language-independent, it can be applied to other languages, as long as n-grams are available. The results of this thesis thus suggest that unsupervised approaches, which are often dismissed in favor of supervised ones in the context of many Natural Language Processing (NLP) related tasks, may present an interesting array of NLP-related problem solving strengths.

Antibacterial activities in the salivary glands of female mosquitoes Aedes aegypti were investigated in this study. The mean salivary bacteriolytic activity, during a period of 14-day of female mosquitoes exposed to five different concentrations of Gram-positive bacteria Micrococcus lysodeikticus in the sucrose meal, was detected by a lysoplate method. A logarithmic regression (R²=0.73) fits the different levels of bacteriolytic activity during the entire period. As the concentration of bacteria in the sugar meal increases, the level of mean salivary bacteriolytic activity increases. A maximum level of bacteriolytic factor may exist in the salivary gland when the concentration of M. lysodeikticus in the sucrose meal exceeds 0.6 g/100 ml. One way analysis of variance and multiple range analysis for the different levels of bacteriolytic activity further validate this finding. The mortality of mosquitoes in the different treatments was not affected by the quanti ties of this nonpathogenic bacteria in the sugar meal. Salivary bacteriostatic activity of female mosquitoes against Gram-negative bacteria Escherichia coli was not detectable in our study, despite positive preliminary results. / Graduation date: 1993

SecA is a central component of the general secretion system that is essential for growth and virulence of bacteria. A series of fluorescein analogs were tested against ATPase activities of Escherichia coli SecA. Rose Bengal (RB) and Erythrosin B are potent inhibitors abolishing the activities of three forms of SecA ATPase with IC50 in µM range. Both inhibit SecA intrinsic ATPase with two mechanisms depending on ATP concentrations, indicating they influence the two non-identical nucleotide binding sites differently. RB shows different inhibitory effects against three forms of SecA ATPase activities, suggesting that the inhibition is related to the conformation of SecA. RB with IC50 at sub-µM level is the most potent inhibitor of SecA ATPases and SecA-dependent protein translocation to date. The fluorescein analogs inhibit intrinsic ATPase of Bacillus subtilis SecA similarly, and also exhibit antibacterial effects in E. coli and B. subtilis. Our findings indicate the value of fluorescein analogs as probes for mechanistic studies of SecA and the potential development of new SecA-targeted antimicrobial agents. A series of SecA derivatives with truncated C-terminus within the first long α-helix of the helix-bundle extending the ATPase catalytic domain of N68 was analyzed. These SecA variants interact with lipids, and those containing the C-terminal portion of the long α-helix starting at residues #639 form the ring-like structure in liposomes, indicating the critical domains for forming the protein-conducting channel. The presence and length of the C-domain influence the response to RB of NBDII mutants and C-terminal truncates of SecA. Thus this region may interact with the inhibitors and is involved in the structure and regulation of SecA ATPase activity. B. subtilis SecA was analyzed for interspecies comparison. Despite sharing high homology, this SecA homolog cannot complement E. coli mutants with SecA defect. Phospholipids do not stimulate ATPase activities of B. subtilis SecA, but induce its conformational changes, leading to the lipid-specific domains and ring-like structures similar to E. coli SecA. These pore-ring structures may represent part of the protein-conducting channels. Therefore, the potential structural roles of SecA in the protein translocation machinery may be universal in both Gram-negative and Gram-positive bacteria.