Orientadores : Thales Rocha de Mattos Filho, Francisco Carlos Groppo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-04T03:41:21Z (GMT). No. of bitstreams: 1 Motta_RogerioHeladioLopes_D.pdf: 659292 bytes, checksum: 254fb5f4f10c3501c081c5129d2db30f (MD5) Previous issue date: 2005 / Doutorado / Farmacologia, Anestesiologia e Terapeutica / Doutor em Odontologia

OBJECTIVE: The principal objective of this study was to measure various aspects of immunity in children with invasive infections due to Haemophilus influenzae type b and Staphylococcus aureus. These serious infections are a significant cause of childhood morbidity and mortality in all populations and affect healthy as well as compromised children. Evidence suggests that imbalances or deficiencies in certain aspects of immunity such as IgG subclasses, the capacity to make specific subclass antibodies, antibody affinities, complement isotypes, immunoglobulin allotypes or mannose binding protein may place certain children at risk for developing invasive disease. Investigation of these factors in a group of children with infection necessitated that normal ranges be established for children of comparable ages from the same population. A secondary objective of this study has therefore been to establish normal percentiles for the IgG subclasses in age, race and sex matched healthy controls. METHODS: Patients admitted to the Red Cross War Memorial Children's Hospital with septic meningitis due to Haemophilus influenzae type b and osteomyelitis/septic arthritis due to Haemophilus influenzae type b or Staphylococcus aureus formed the study population. Section A of this thesis describes the methods for establishing, validating and standardizing ELISAs for measuring the IgG subclasses (lgGl, IgG2, IgG3 and IgG4) and subclass antibodies specific to Haemophilus influenzae polyribosylribitol phosphate, Staphylococcus aureus teichoic acid and tetanus toxoid. The relative affinity of antibodies in these ELISAs was determined by the incorporation of diethylamine (DEA). In order to determine the immunoglobulin allotypes ELISAs were developed to measure the G1m(f), G2m(n) and Km(3) allotypes. The frequency of these allotypic markers in the different ethnic groups was established. The relationship between immunoglobulin allotypes and IgG subclass values were investigated in both patient and control groups. RESULTS: ELISA assays to measure IgG subclasses; IgG, IgG 1 and IgG4 tetanus toxoid antibodies; IgG, IgG 1 and IgG2 H. influenzae type b polyribosylribitol phosphate capsular polysaccharide antibodies; IgG, IgG1 and IgG2 S. aureus teichoic acid antibodies and G1m(f), G2m(n) and Km(3) allotypes were successfully established. Where possible the assays were standardized with reference sera and specimens were exchanged with international laboratories. Age, race and sex related percentile charts and tables of normal ranges for IgG and IgG subclasses of Black and Coloured children were established. The IgG and IgG 1 values were higher than those previously reported for children in developed countries. Black children with H. influenzae meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls and although similar trends were seen for IgG and IgG4 levels they were not statistically significant. Coloured children with H. influenzae meningitis and Coloured and Black children with H. influenzae osteomyelitis/septic arthritis also showed a similar tendency of lower IgG and IgG subclass levels than the controls but these trends were also not significantly different. All patients responded to tetanus toxoid antigen suggesting normal immunocompetence to protein antigens. H. influenzae type b capsular polysaccharide antibodies were low in children with H. influenzae type b meningitis and osteomyelitis/septic arthritis and did not increase during the illness. IgG and IgG 1 teichoic acid antibodies were raised in patients with S. aureus osteomyelitis/septic arthritis although no further rise in these antibodies was seen when measured several weeks after the illness. The antibody affinity ELISAs showed that IgG 1 tetanus toxoid antibody had a greater affinity than IgG4 tetanus toxoid antibody, the IgG 1 and IgG2 H. influenzae capsular polysaccharide antibodies were of similar affinity and the IgG 1 teichoic acid antibody was of higher affinity than the IgG2 antibody. The G1m(f) and G2m(n) positive allotypes were uncommon in Black but common in the Coloured populations whereas Km(3) was common in both groups. There was a significantly decreased frequency of the G2m(n) positive allotype in Coloured patients with H. influenzae type b meningitis and H. influenzae type b osteomyelitis/septic arthritis which was not found in patients with S. aureus osteomyelitis/septic arthritis. In both Coloured and Black children with H. influenzae meningitis there was a significantly decreased frequency of the Km(3) allotype. No differences in C4 isotypes and mannose binding protein levels were evident in the patient and control groups. CONCLUSION: This study has developed simple, specific and reproducible ELISAs to measure IgG subclasses and subclass antibodies specific to tetanus toxoid, H. influenzae polyribosylribitol phosphate and S. aureus teichoic acid. Age, sex and race related normal ranges for IgG subclasses in the local Black and Coloured populations have been established. Black children with H. influenzae type b meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls. There was a clear association between a decrease of the G2m(n) allotype and the Km(3) allotype and susceptibility to invasive infections caused by H. influenzae.

Proteases are important enzymes in the biotechnology due to their specific cleavage of substrates. HRV 3C, sortase A and TEV are some examples of cysteine proteases which become more of use lately in applications as removal of affinity tags (3C/TEV) and labelling of proteins (sortase). Here an investigation was made on the proteases by displaying them on two different prokaryotic hosts; E. coli and S. carnosus and to use these to cleave away affinity proteins (Affibody molecule) from other cells with an incorporated cleavage site. Constructs were cloned and incorporated into expressing strains which were then cultivated and induced. Analysis of surface expression was done by flow cytometer. Cleavage was made by cultivating combinations with cleavable bacteria and bacteria displaying proteases. A functional protease would lead to the presence of Affibody molecules in the supernatant. Flow cytomtery analysis was first made to inevstigate signal difference in Affibody binding by the addition of flurophores. Secondly SDS-PAGE was made on the centrifuged supernatant to investigate the presence of a product. Finally analysis of the bacteria was made by examining the reaction with soluble substrate and comparing activity with soluble enzyme. All of the enzymes were able to be displayed on the surface of bacteria with a clear separation from control. The cleavage analysis showed however varying results yet no clear evidence of product. Best flow cytometer results were seen for 3C but SDS-PAGE/MS did not show any cleaved product. For Sortase SDS-PAGE showed positive result but analysis with MS showed no product. TEV was concluded not to be funcional at all hence the failing to cleave soluble substrate  when condition seemed near optimal and faulty flow cytometer data. Even though the lack of success there is still many further studies that can be done on the proteases in order to prove its absence/presence  of activity.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.

ABSTRACT Few antibiotics are left that are effective against methicillin-resistant Staphylococcus aureus (MRSA) and even strains resistant to these agents have been isolated. Previous studies have identified five distinct MRSA clonotypes, which are present globally. No comprehensive national study has previously been undertaken to investigate the MRSA types in South Africa, and this study was aimed at elucidating the genotypic population structure of South African MRSA isolates. SmaI digested genomic DNA, separated by pulsed-field gel electrophoresis, was used to characterise 349 S. aureus isolates, obtained from various state and private diagnostic laboratories. PFGE results were complemented with those of spa typing and staphylococcal cassette chromosome mec (SCCmec) typing results. Two-hundred-and-five different PFGE patterns were identified, which were grouped into twenty-four clusters. Three were major lineages, containing more than 20% of the isolates with a similarity cut-off of 70%. Only thirty-seven spa types were identified (fourteen novel spa types), which clustered into six spa-Clonal Complexes after BURP analysis. SCCmec types I-IV were identified, including variants of each type. Data suggest that the Archaic clone (RSA05), oldest of the epidemic clones, represents one of the major clones in South Africa. Strains that were part of this complex (n=98 (28.2%); t064; SCCmec type I-pls) clustered together with strain E2125/ATCC BAA-38 (t051; SCCmec type I). Another major complex, RSA16 (n=90 (25.7%); t012; SCCmec type II/IIB) possessed a single-locus variant (SLV) spa type and the same or a SLV SCCmec types as EMRSA-16 (t018; SCCmec type II). The third major complex, RSA03 (n=74 (21.2%); t037; SCCmec type III/IIIE), had similar spa and SCCmec types to control strainANS46 (t037; SCCmec type III). One MRSA and twelve MSSA isolates were also identified as carrying genes for the toxin Panton-Valentine leukocidin, which was confirmed by DNA nucleotide sequencing.

Protein engineering and in vitro selection systems are powerful methods to generate binding proteins. In nature, antibodies are the primary affinity proteins and their usefulness has led to a widespread use both in basic and applied research. By means of combinatorial protein engineering and protein library technology, smaller antibody fragments or alternative non-immunoglobulin protein scaffolds can be engineered for various functions based on molecular recognition. In this thesis, a 46 amino acid small albumin-binding domain derived from streptococcal protein G was evaluated as a scaffold for the generation of affinity proteins. Using protein engineering, the albumin binding has been complemented with a new binding interface localized to the opposite surface of this three-helical bundle domain. By using in vitro selection from a combinatorial library, bispecific protein domains with ability to recognize several different target proteins were generated. In paper I, a bispecific albumin-binding domain was selected by phage display and utilized as a purification tag for highly efficient affinity purification of fusion proteins. The results in paper II show how protein engineering, in vitro display and multi-parameter fluorescence-activated cell sorting can be used to accomplish the challenging task of incorporating two high affinity binding-sites, for albumin and tumor necrosis factor-alpha, into this new bispecific protein scaffold. Moreover, the native ability of this domain to bind serum albumin provides a useful characteristic that can be used to extend the plasma half-lives of proteins fused to it or potentially of the domain itself. When combined with a second targeting ability, a new molecular format with potential use in therapeutic applications is provided. The engineered binding proteins generated against the epidermal growth factor receptors 2 and 3 in papers III and IV are aimed in this direction. Over-expression of these receptors is associated with the development and progression of various cancers, and both are well-validated targets for therapy. Small bispecific binding proteins based on the albumin-binding domain could potentially contribute to this field. The new alternative protein scaffold described in this thesis is one of the smallest structured affinity proteins reported. The bispecific nature, with an inherent ability of the same domain to bind to serum albumin, is unique for this scaffold. These non-immunoglobulin binding proteins may provide several advantages as compared to antibodies in several applications, particularly when a small size and an extended half-life are of key importance. / <p>QC 20121122</p>

No description available.

No description available.

Dissertation (Ph.D.)--University of South Florida, 2006. / Title from PDF of title page. Document formatted into pages; contains 117 pages. Includes vita. Includes bibliographical references.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2016-05-12T20:45:00Z No. of bitstreams: 2 Dissertação - Mariana de Andrade Ferreira - 2015.pdf: 2863875 bytes, checksum: 62c67ecc0abf8022b7bf54c6de62a20c (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-05-13T12:05:05Z (GMT) No. of bitstreams: 2 Dissertação - Mariana de Andrade Ferreira - 2015.pdf: 2863875 bytes, checksum: 62c67ecc0abf8022b7bf54c6de62a20c (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) / Made available in DSpace on 2016-05-13T12:05:05Z (GMT). No. of bitstreams: 2 Dissertação - Mariana de Andrade Ferreira - 2015.pdf: 2863875 bytes, checksum: 62c67ecc0abf8022b7bf54c6de62a20c (MD5) license_rdf: 19874 bytes, checksum: 38cb62ef53e6f513db2fb7e337df6485 (MD5) Previous issue date: 2015-08-28 / Staphylococcus aureus is an important foodborne pathogen, able to produce extracellular toxins and to express antimicrobial resistance. Among the foods involved in staphylococcal food poisoning, stands out the cheese, especially when manufactured under improper hygienic and sanitary conditions. The objectives of this study were to characterize Staphylococcus aureus isolated from artisanal and industrialized Minas frescal cheeses, to determine their antimicrobial susceptibility profile as well as the genetic similarity among the isolates. The isolates were also tested for staphylococcal enterotoxins (SE) genes and other virulence factors. Fifty-six artisanal raw milk cheeses sold at street fairs and 10 industrialized cheeses commercialized in supermarkets of Goiânia, Goiás were analyzed between June and August 2014. S. aureus was confirmed in 19 samples (33.9%) of artisanal cheese by detection of femA gene, in which 29 isolates were obtained. These isolates were submitted to the antimicrobial susceptibility test and classified into nine different profiles (A - I). Thirteen isolates (44.8%) were resistant to penicillin and three (10.3%) to tetracycline, with two (7.4%) resistant to both. The Multiplex PCR technique was performed to detect virulence genes that code for the production of hemolysins (Hla and Hlb), toxic shock syndrome toxin (TSST-1), exfoliative toxins (ETa and ETb) and enterotoxins (SEA - SEE, SEG - SEJ, SEM - SEO). Genes encoding TSST-1 and exfoliative toxins were not detected. All the isolates amplified for the hla gene and 14 (48.3%) for the hbl gene. The seh gene was the most frequently detected (n=11, 37.9%) followed by seo gene (n = 3; 10.3%), seg, sem and sen genes (n = 2, 6.9%) and sec and sei genes (n = 1, 3.4%). In one isolate (3.4%), four enterotoxins genes were detected, and in another, six (3.4%). The comparison performed by Pulsed Field Gel Electrophoresis technique revealed 18 different DNA banding patterns which were grouped into five clusters. The genotyping found high genetic similarity among the isolates. Identical isolates were obtained from different samples and one sample showed more than one genetically different isolate. It was identified up to four different isolates from the same sample. The high prevalence of S. aureus in a widely consumed product like Minas fresh cheese, as well as the detection of toxin encoding genes identified in this study, warns of the necessity to reduce the contamination levels in this type of cheese through monitoring and controling the production and trade of the product. / S. aureus é um importante patógeno de origem alimentar com capacidade de produção de toxinas extracelulares e resistência antimicrobiana. Entre os alimentos envolvidos em intoxicação alimentar estafilocócica, destaca-se o queijo, principalmente quando fabricado em condições higienicossanitárias impróprias. Os objetivos deste estudo foram caracterizar S. aureus isolados de queijo Minas frescal artesanal e industrializado, determinar o perfil de suscetibilidade a antimicrobianos, bem como determinar a similaridade genética entre os isolados. Os isolados foram também testados para genes de enterotoxinas estafilocócicas (SE) e outros fatores de virulência. Foram analisadas 56 amostras de queijo Minas frescal de fabricação artesanal e dez de fabricação industrial comercializados em feiras livres e supermercados de Goiânia-GO, coletadas entre junho e agosto de 2014. S. aureus foi confirmado em 19 amostras (33,9) de queijo artesanal através da detecção do gene femA, onde 29 isolados foram obtidos. Estes isolados foram submetidos ao teste de susceptibilidade aos antimicrobianos e classificados em nove diferentes perfis (A - I). Treze isolados (44,8%) foram resistentes à penicilina e três (10,3%) à tetracilina, sendo dois (7,4%) resistentes a ambos. A técnica de multiplex PCR foi realizada para a detecção de genes de virulência que codificam a produção de hemolisinas (Hla e Hlb), TSST-1, toxinas esfoliativas (ETa e ETb) e enterotoxinas (EEA - EEE, EEG - EEJ, EEM - EEO). Genes que codificam as toxinas TSST-1 e esfoliativa não foram detectados. Todos os isolados amplificaram para o gene hla e 14 (48,3%) para o gene hlb. O gene seh foi o mais frequentemente detectado (n=11; 37,9%), seguido do gene seo (n=3; 10,3%), genes seg, sem, sen (n=2; 6,9%) e os genes sec e sei (n=1; 3,4%). Um isolado (3,4%) amplificou genes para quatro enterotoxinas e outro (3,4%) para seis. A comparação dos 29 isolados de S. aureus feita por PFGE revelou 18 padrões de bandas diferentes de DNA agrupados em cinco clusters. A genotipagem demonstrou alta similaridade genética entre os isolados. Isolados idênticos foram obtidos de amostras diferentes e uma mesma amostra apresentou mais de um isolado geneticamente diferente. Identificou-se até quatro diferentes isolados da mesma amostra. A alta prevalência de S. aureus nas amostras de queijo Minas Frescal, bem como a detecção de genes para produção de toxinas, alertam para a necessidade de reduzir os níveis de contaminação neste tipo de queijo através de monitoramento e controle da produção e comércio do produto.