Thesis--Københavns universitet. / Bibliography: p. [130]-141.

Thesis--Københavns universitet. / Bibliography: p. [130]-141.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "May 1996." Typescript. Vita. Includes bibliographical references (leaves 128-138). Also available on the Internet.

Thesis (Ph.D.(Physiology))--University of the Witwatersrand, Faculty of Health Sciences, 2012. / The acute phase response is implemented by infected hosts in response to exposure to pathogens, including bacteria and viruses. Acute phase responses comprise physiological and behavioural changes, such as fever and a range of “sickness behaviours”, including lethargy and anorexia as well as impairment in learning and memory. Similar to other sickness behaviours, the effect of infection on learning and memory processes has been attributed to the release of pro-inflammatory cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6). However, the exact role of IL-1β and IL-6 in mediating infection-induced cognitive impairment is not clear. Unlike fever, anorexia and lethargy, which may benefit an infected host, the physiological benefit of cognitive impairment during illness is doubtful. To initiate an acute phase response experimentally, moieties of typical bacteria (Gramnegative and Gram-positive) and viruses frequently are employed. Moieties from the atypical Mycoplasmas seldom have been used. Consequently, there is a dearth of information on the physiological mechanisms that underlie acute phase responses following Mycoplasma infection, despite the prevalence of the disease in the general population. Mycoplasma pneumoniae frequently causes community-acquired pneumonia, which may have serious extra-pulmonary complications, including cognitive deficits. Therefore, I investigated fever and sickness behaviours as well as cytokine responses in simulated, atypical Mycoplasma infection. I implemented an animal model of simulated Mycoplasma infection and characterised fever and sickness behaviours, including lethargy and anorexia as well as impairment in learning and memory during acute and recurrent acute simulated infection. I also characterized the response in the periphery and in the brain of individual pro-inflammatory cytokines, IL-1β and IL-6, to administration of fibroblast-stimulating lipopeptide-1 (FSL-1), which simulates Mycoplasma infection. Using rats, I recorded fever and lethargy with biotelemetry and assessed effects of simulated Mycoplasma infection on learning and memory using a Morris Water Maze. In addition, I examined the histology of tissue from the hippocampus, a key brain area involved in spatial learning and memory, to assess residual effects of simulated Mycoplasma infection on learning and memory. I showed that bolus administration of a pyrogenic moiety from Mycoplasma, fibroblaststimulating lipopeptide-1 (FSL-1), dose-dependently induced fever, lethargy, anorexia and body mass stunting in rats. However, FSL-1 administration did not induce concomitant impairment in spatial learning and memory. Importantly, at the time of testing in the Maze, I found the concentrations of IL-1β to be up-regulated in both the hypothalamus and the hippocampus, while the concentrations of IL-6 were unaffected. I also showed that recurrent acute injections of FSL-1, at 10 d intervals, induced recurrent fevers, lethargy and anorexia without the development of pyrogenic tolerance to any of the sickness responses measured. However, there was no residual body mass stunting in rats and also no growth retardation, despite the recurrent simulated infection. Equally importantly, there were neither lasting detrimental effects on spatial learning and memory nor any residual histological damage to the hippocampus of rats. My findings in simulated Mycoplasma infection are important, firstly because Mycoplasma infection is prevalent in both developing and developed countries and frequently causes outbreaks, and secondly because Mycoplasma infection affects children and adolescents of school-going age. My findings also are encouraging: although lasting detrimental effects, including impairment in learning and memory as well as body mass stunting may occur in other infections, these appear not to be inevitable outcomes in Mycoplasma infection.

Thesis (D Phil. (Microbiology))--2010. / Mycoplasma genitalium is the smallest existing self-replicating prokaryote, lacks a cell wall and has a genome consisting of only 580 kilo base pairs. It has characteristic pear/flask-like morphology with a terminal tip organelle used for attachment. Many researchers, mainly in developed countries, have investigated the role the organism plays in the aetiology of male urethritis and the majority of studies show an association between M. genitalium and male urethritis. In this study, the modified Koch’s postulates were applied to answer the question whether M. genitalium is a true pathogen, or merely a passenger, invading already inflamed or damaged cells. A total of 300 urine specimens were collected from adult males with symptoms and/or signs of urethritis and 75 from asymptomatic men. In the first study, three molecular assays; viz, a commercial conventional PCR test, a real-time PCR (q- PCR) test and a transcription mediated amplification (TMA) assay were evaluated for the detection of M. genitalium. The comparison between the assays was based on the extended gold standard concept, where a specimen was deemed positive when any two nucleic acid amplification tests were positive. In the second study, the specimens were tested for four common urethral pathogens (N. gonorrhoeae, C. trachomatis, T. vaginalis and M. genitalium) using TMA assays. Finally, the bacterial loads for M. genitalium were determined using the q-PCR assay. v All three assays tested were highly specific (98-99%) for the detection of M. genitalium. However, where q-PCR and TMA demonstrated high sensitivities (96% and 100%), the sensitivity of the conventional PCR assay was low (78%). One or more pathogens were detected in a total of 129 (43%) men with urethritis. M. genitalium was the most frequently detected pathogen in men with urethritis (129; 43%), and significantly more (p= 0.04) than in asymptomatic men (7; 9.0%). There is a strong association with M. genitalium bacterial load and clinical urethritis. Patients with urethral discharge had significantly higher M. genitalium concentrations than those with only burning on micturition (p<0.001), and the bacterial concentrations in men with symptoms and/or signs of urethritis were significantly higher than that in asymptomatic men (p=0.02). As the number of organisms increased, the severity of the symptoms increased; an indication of the role that the organism plays in disease progression. In conclusion, by applying the modified Koch postulates, it was shown that Mycoplasma genitalium is by no means a passenger, but rather an important cause of adult male urethritis that should be taken into account when making diagnosis and when designing treatment strategies.

Typescript. / Thesis (Ph. D.)--University of Hawaii, 1970. / Bibliography: leaves 142-149. / ix, 149 l illus., tables

Düsseldorf, Universiẗat, Diss., 2002.

Mycoplasma hyopneumoniae é uma das menores bactérias encontradas na natureza, apresentando genoma altamente reduzido e ausência de parede celular. Este organismo é o agente causador da pneumonia enzoótica suína, a qual apresenta distribuição mundial, causando importantes perdas econômicas. Na última década, várias espécies de Mycoplasma tiveram seus genomas completamente seqüenciados, incluindo quatro cepas de M. hyopneumoniae. Apesar da grande quantidade de dados gerados, pouco se sabe sobre as seqüências nucleotídicas que controlam a expressão gênica nestes microrganismos. A grande variabilidade encontrada nas regiões promotoras, o baixo conteúdo de GC presente no genoma e a carência de promotores experimentalmente caracterizados, são fatores que dificultam o reconhecimento in silico das seqüências reguladoras no gênero Mycoplasma. Assim sendo, este trabalho tem como objetivo identificar seqüências nucleotídicas envolvidas com o início da transcrição em M. hyopneumoniae, gerando dados que possibilitem a construção de uma matriz capaz de fazer a predição de promotores nesta espécie. Inicialmente, os sítios de início de transcrição (TSSs) de 23 genes de M. hyopneumoniae foram definidos. Os resultados mostraram que os TSSs identificados localizavam-se entre 2 e 144 pb de distância do início dos genes, sendo compostos em sua grande maioria por um resíduo de adenosina. Um padrão semelhante ao elemento −10 de promotores σ70 foi encontrado a montante dos TSSs. No entanto, não foi possível identificar conservação de uma provável região −35, porém, um sinal periódico AT-rico foi observado. Aproximadamente metade dos genes analisados continha o motivo 5'-TRTG-3', que é idêntico ao elemento −16, comumente encontrado em bactérias gram-positivas. A partir da determinação dos promotores, foi construída uma matriz de pontuação posição-específica que foi utilizada para localizar promotores putativos a montante de todas as seqüências codificantes (CDSs) de M. hyopneumoniae. Duzentos e um sinais foram encontrados associados a 169 CDSs. A maioria destas seqüências estava localizada até 100 nucleotídeos de distância dos códons de iniciação. Este estudo mostra que o número de seqüências promotoras preditas no genoma de M. hyopneumoniae é mais freqüente que o esperado ao acaso, indicando que a maioria das seqüências detectadas são provavelmente sítios de ligação funcionais. / Mycoplasma hyopneumoniae is one of the smallest bacteria found in nature, presenting a small genome and absence of cell wall. It is present in the majority of swine herds throughout the world, and is considered an important pathogen in the swine industry. In the last decade, many species of this genus had their genome completely sequenced, including four strains of M. hyopneumoniae. Nevertheless, very little is understood of the nucleotide sequences that control transcription initiation in these microorganisms. Like its relatives, M. hyopneumoniae lacks several major regulators of gene expression, including two component regulatory systems and multiple σ factors, thus it appears that the signals for promotion and regulation of transcription may differ significantly from other bacteria. In addition, the low GC content and the dearth of experimentally characterized promoters in this genus severely limit the recognition of the controlling sequences. Therefore, this study aims to identify nucleotide sequences involved in transcription initiation in M. hyopneumoniae, and thus generate data to enable the construction of a matrix capable of predicting promoters in this species. Initially, the transcription start sites (TSSs) of 23 genes of M. hyopneumoniae were experimentally defined. The results showed that the identified TSSs were located between 2 and 144 bp away from the gene starts, being composed mostly of an adenosine residue. A pattern that resembles the σ70 promoter −10 element was found upstream of the TSSs. However, no −35 element was distinguished. Instead, an AT-rich periodic signal was identified. About half of the experimentally defined promoters contained the motif 5'-TRTG-3', which was identical to the −16 element usually found in gram-positive bacteria. The defined promoters were utilized to build position-specific scoring matrices in order to scan putative promoters upstream of all coding sequences (CDSs) in the M. hyopneumoniae genome. Two hundred and one signals were found associated with 169 CDSs. Most of these sequences were located within 100 nucleotides of the start codons. This study has shown that the number of promoter-like sequences in the M. hyopneumoniae genome is more frequent than expected by chance, indicating that most of the sequences detected are probably biologically functional.

Mycoplasma hyopneumoniae é uma das menores bactérias presentes na natureza, apresentando um genoma reduzido com alto conteúdo de A+T e ausência de parede celular. Este organismo é o agente etiológico da pneumonia enzoótica suína, uma doença crônica que afeta rebanhos em todo mundo sendo responsável por grandes perdas econômicas. Para investigar a patogênese de M. hyopneumoniae é importante entender seus mecanismos genéticos, porém, apesar do sequenciamento do genoma de várias linhagens (7448, J, 232 e 168), pouco se sabe sobre os mecanismos que regulam e controlam a expressão gênica neste microrganismo, principalmente no que se relaciona às proteínas regulatórias. Os fatores de transcrição são proteínas que se ligam ao DNA e propiciam a capacidade de controlar a expressão gênica sob diferentes estímulos metabólicos ou condições de crescimento. O conhecimento que se tem sobre essas proteínas em M. hyopneumoniae é escasso, então, desta forma, este trabalho tem como objetivo a análise de potencias fatores de transcrição de M. hyopneumoniae. Para isso foram selecionadas proteínas tradicionalmente descritas em bancos de dados como fatores de transcrição (HrcA – MHP7448_0010; transcriptional regulator – MHP7448_0279; e a proteína hipotética MHP7448_0551), além de uma proteína hipotética que apresenta similaridades estruturais com fatores sigma (MHP7448_0639). Estas proteínas foram clonadas por recombinação homóloga in vivo no vetor de expressão pGEX 4T1 e transformadas em Escherichia coli para expressão das proteínas recombinantes. Porém, para empregar essa técnica foi necessário realizar a mutagênese sítio-dirigida para a substituição do códon UGA (triptofano em micoplasmas) para UGG (triptofano no código genético universal) nas proteínas que apresentavam este códon. As mutações foram inseridas através da técnica de overlap extension PCR, utilizando primers mutagênicos. Foram obtidos clones para três proteínas (MHP7448_0551, MHP7448_0639 e MHP7448_0010), que foram eficientemente expressas em E. coli. Depois de solubilizadas com 0,5% de sarcosil, essas proteínas foram submetidas a um processo de purificação por cromatografia de afinidade. A purificação foi bem sucedida para duas das três proteínas testadas, e novos testes serão realizados com a proteína ainda não purificada (MHP7448_0010). Uma vez purificados, os potenciais fatores de transcrição serão utilizados na identificação das regiões do DNA que essas proteínas regulam e que estão envolvidas no controle da expressão gênica em M. hyopneumoniae. / Mycoplasma hyopneumoniae is one of the smallest bacteria found in nature, presenting a reduced genome with a high A+T content and no cell wall. This bacterium is the etiological agent of swine enzootic pneumonia, a chronic disease that affects herds throughout the world and is responsible for great economic losses. To investigate the pathogenesis of M. hyopneumoniae it is important to understand their genetic mechanisms, however, despite the genome sequencing of several strains (7448, J, 232 and 168), little is known about the mechanisms that regulate and control the gene expression in this bacterium, especially about regulatory proteins. Transcription factors are proteins that bind to DNA providing the ability to control gene expression under different metabolic stimuli or growth conditions. The knowledge about these proteins in M. hyopneumoniae is scarce; therefore, the aim of this study is the analysis of potential transcription factors of M. hyopneumoniae. We selected proteins traditionally reported in databases as transcriptional factors (HrcA – MHP7448_0010; transcriptional regulator – MHP7448_0279; and the hypothetical protein MHP7448_0551), and a hypothetical protein which has structural similarities with sigma factors (MHP7448_0639). These proteins were cloned by in vivo homologous recombination in the expression vector pGEX 4T1 and were transformed into Escherichia coli to express the recombinant proteins. Nevertheless, for the application of this system it was necessary to perform site-directed mutagenesis to replace the codon UGA (tryptophan in mycoplasma) to UGG (tryptophan in the universal genetic code) in the proteins that presented this codon. The mutations were introduced by overlap extension PCR technique, using mutagenic primers. Clones were obtained for three proteins (MHP7448_0551, MHP7448_0639 and MHP7448_0010), which were efficiently expressed in E. coli. After solubilization with 0.5% sarkosyl, these proteins were subjected to purification by affinity chromatography. Purification was successful for two of the three tested proteins, and further tests will be carried out with the protein not purifyed (MHP7448_0010). After purification, the potential transcription factors will be used to identify the regions of DNA that these proteins regulate and which are involved in the control of gene expression in M. hyopneumoniae.

Mycoplasma hyopneumoniae é uma das menores bactérias encontradas na natureza, apresentando genoma altamente reduzido e ausência de parede celular. Este organismo é o agente causador da pneumonia enzoótica suína, a qual apresenta distribuição mundial, causando importantes perdas econômicas. Na última década, várias espécies de Mycoplasma tiveram seus genomas completamente seqüenciados, incluindo quatro cepas de M. hyopneumoniae. Apesar da grande quantidade de dados gerados, pouco se sabe sobre as seqüências nucleotídicas que controlam a expressão gênica nestes microrganismos. A grande variabilidade encontrada nas regiões promotoras, o baixo conteúdo de GC presente no genoma e a carência de promotores experimentalmente caracterizados, são fatores que dificultam o reconhecimento in silico das seqüências reguladoras no gênero Mycoplasma. Assim sendo, este trabalho tem como objetivo identificar seqüências nucleotídicas envolvidas com o início da transcrição em M. hyopneumoniae, gerando dados que possibilitem a construção de uma matriz capaz de fazer a predição de promotores nesta espécie. Inicialmente, os sítios de início de transcrição (TSSs) de 23 genes de M. hyopneumoniae foram definidos. Os resultados mostraram que os TSSs identificados localizavam-se entre 2 e 144 pb de distância do início dos genes, sendo compostos em sua grande maioria por um resíduo de adenosina. Um padrão semelhante ao elemento −10 de promotores σ70 foi encontrado a montante dos TSSs. No entanto, não foi possível identificar conservação de uma provável região −35, porém, um sinal periódico AT-rico foi observado. Aproximadamente metade dos genes analisados continha o motivo 5'-TRTG-3', que é idêntico ao elemento −16, comumente encontrado em bactérias gram-positivas. A partir da determinação dos promotores, foi construída uma matriz de pontuação posição-específica que foi utilizada para localizar promotores putativos a montante de todas as seqüências codificantes (CDSs) de M. hyopneumoniae. Duzentos e um sinais foram encontrados associados a 169 CDSs. A maioria destas seqüências estava localizada até 100 nucleotídeos de distância dos códons de iniciação. Este estudo mostra que o número de seqüências promotoras preditas no genoma de M. hyopneumoniae é mais freqüente que o esperado ao acaso, indicando que a maioria das seqüências detectadas são provavelmente sítios de ligação funcionais. / Mycoplasma hyopneumoniae is one of the smallest bacteria found in nature, presenting a small genome and absence of cell wall. It is present in the majority of swine herds throughout the world, and is considered an important pathogen in the swine industry. In the last decade, many species of this genus had their genome completely sequenced, including four strains of M. hyopneumoniae. Nevertheless, very little is understood of the nucleotide sequences that control transcription initiation in these microorganisms. Like its relatives, M. hyopneumoniae lacks several major regulators of gene expression, including two component regulatory systems and multiple σ factors, thus it appears that the signals for promotion and regulation of transcription may differ significantly from other bacteria. In addition, the low GC content and the dearth of experimentally characterized promoters in this genus severely limit the recognition of the controlling sequences. Therefore, this study aims to identify nucleotide sequences involved in transcription initiation in M. hyopneumoniae, and thus generate data to enable the construction of a matrix capable of predicting promoters in this species. Initially, the transcription start sites (TSSs) of 23 genes of M. hyopneumoniae were experimentally defined. The results showed that the identified TSSs were located between 2 and 144 bp away from the gene starts, being composed mostly of an adenosine residue. A pattern that resembles the σ70 promoter −10 element was found upstream of the TSSs. However, no −35 element was distinguished. Instead, an AT-rich periodic signal was identified. About half of the experimentally defined promoters contained the motif 5'-TRTG-3', which was identical to the −16 element usually found in gram-positive bacteria. The defined promoters were utilized to build position-specific scoring matrices in order to scan putative promoters upstream of all coding sequences (CDSs) in the M. hyopneumoniae genome. Two hundred and one signals were found associated with 169 CDSs. Most of these sequences were located within 100 nucleotides of the start codons. This study has shown that the number of promoter-like sequences in the M. hyopneumoniae genome is more frequent than expected by chance, indicating that most of the sequences detected are probably biologically functional.