Yes / Background. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism. / This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/.

Made available in DSpace on 2016-08-10T10:54:12Z (GMT). No. of bitstreams: 1 RONALDO PORTELA.pdf: 1248321 bytes, checksum: 32236d6c47873061a20e4e8cd5b4e4b3 (MD5) Previous issue date: 2014-03-28 / Background: Syphilis is an infectious disease that in Brazilian prison systems has optimal conditions to increase the risk of transmission among inmates. Objectives: Determine prevalence and assess risk situations associated with transmission of syphilis among inmates of Prison Complex Aparecida de Goiânia/GO in the period from February to September 2011. Methods: cross-sectional study exploratory of quantitative approach. Study participants were 1.173 inmates that answered a questionnaire containing variables on behaviors risk for T. pallidum infection. The seroprevalence was found using serological tests screening VDRL and ELISA as confirmatory. Data were tabulated and the variables were analyzed using the EPI INFO and SPSS (Statistical Package for Social Sciences).Were calculated rates of seroprevalence by age and educational level, and relative risks (odds ratios) associated with risk behaviors. Results: The seroprevalence of syphilis found in this population was 2.22% (IC95%). Of the study participant s total, 83.4% were aged between 18 and 39 years. This age group contributes with 85% of cases found positive for serological screening. The seroprevalence among those with low education contributed 58.2% of the population seroprevalence. For the variables of risk situations the homosexual relationship had an odds ratio of 3.44 (IC 0,77 - 15,29 and p=0,084), use of tattoo 3.05 (IC 1,14 - 8,14 and p=0,019), condom use 1.84 (IC 0,40 - 8,43 and p=0,356)and use of injecting drugs 3.71 (IC 1,35 - 10,13 and p=0,006).Conclusions: The seroprevalence rate was lower than those found in studies performed in other prisons in the country. Of the variables evaluated in the study the use of tattoos and the use of injectable drugs were statistically significant risk factors associated with seropositivity to syphilis. The combination of risk situations evaluated in this study can contribute to the transmission of syphilis on the Brazilian prisons. / Introdução: A sífilis é uma doença infectocontagiosa que encontra no ambiente prisional brasileiro condições que podem aumentar o risco da sua transmissão entre a população carcerária. Objetivos: Determinar a soroprevalência e avaliar as situações de risco associadas à transmissão da sífilis entre os presidiários do Complexo Prisional de Aparecida de Goiânia/GO no período de fevereiro a setembro de 2011. Metodologia: Estudo de base exploratória transversal com abordagem quantitativa. Um total de 1.173 participantes do estudo responderam a um questionário contendo variáveis sobre comportamentos e situações de risco para infecção pelo T. pallidum. A soroprevalência foi encontrada após a triagem sorológica utilizando o teste VDRL e ELISA como confirmatório. Os dados obtidos foram tabulados e as variáveis foram analisadas através dos programas EPI INFO e SPSS (Statistical Package for Social Sciences). Foram calculadas as taxas de soroprevalência por faixa etária e nível de escolaridade, e os riscos relativos (odds ratio) associados a comportamentos de risco. Resultados: A soroprevalência encontrada para sífilis na população estudada foi de 2,22% (IC95%). Do total de participantes do estudo, 83,4% tinham idade entre 18 e 39 anos. Esta faixa etária contribui com 85% dos casos positivos encontrados na triagem sorológica. A soroprevalência entre àqueles que possuem escolaridade abaixo do nível médio contribuiu com 58,2% da soroprevalência encontrada na população. Para as variáveis de situações de risco a relação homossexual teve uma razão de chances de 3,44 (IC 0,77 - 15,29 e p= 0,084), uso de tatuagem 3,05 (IC 1,14 - 8,14 e p= 0,019), uso de preservativos 1,84 (IC 0,40 - 8,43 e p =0,356) e uso de drogas injetáveis 3,71 (IC 1,35 - 10,13 e p= 0,006). Conclusões: A taxa de soroprevalência encontrada foi menor do que as encontradas em estudos realizados em outros presídios do País. Das variáveis avaliadas no estudo o uso de Tatuagens e o uso de Drogas Injetáveis foram os fatores de risco estatisticamente significantes associados à soropositividade para a sífilis. A combinação das situações de risco avaliadas pode contribuir para a transmissão da sífilis nos presídios brasileiros.

The major goal of this study was to achieve sustained cultivation of virulent T. pallidum in vitro. The putatuive binding of host proteins to the outer membrane (OM) of intact, virulent T. pallidum subsp. pallidum has been investigated. A major breakthrough was the development of a filtration assay, usinglow protein-binding membrane filters, for the measurement of substances bound to or incorporated into th eOM of T. pallidum. This avoided the conventional manipulations which can damage the fragile OM of T. pallidum. Using this filtration assay, studies on the binding of host serum proteins demonstrated that intact treponemes did not bind host proteins as previously reported. It also indicated that previous studies were probably performed with damaged by this research. The studies on the binding of polyclonal and monoclonal antibodies to intact and detergent treated treponemes provided evidence of the low level binding of antibody to intact treponemes which was greatly enhanced but the removal of the outer membrane with 0.1% Triton X. This research research corroborated that of others which suggests that the outer membrane of T. pallidum contains very little protein or surface exposed antigen.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-01T15:54:13Z No. of bitstreams: 2 Dissertação - Rafael Alves Guimarães - 2016.pdf: 4476558 bytes, checksum: 46bc5281f5c2ee7c26f5e3c06af53750 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-01T15:54:40Z (GMT) No. of bitstreams: 2 Dissertação - Rafael Alves Guimarães - 2016.pdf: 4476558 bytes, checksum: 46bc5281f5c2ee7c26f5e3c06af53750 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-01T15:54:40Z (GMT). No. of bitstreams: 2 Dissertação - Rafael Alves Guimarães - 2016.pdf: 4476558 bytes, checksum: 46bc5281f5c2ee7c26f5e3c06af53750 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Crack users are at high risk for syphilis, due to multiple risk behaviors, such as inconsistent condom use, exchanging sex for money and/or drugs and multiple sexual partners. The aim of this study was to investigate the epidemiology of syphilis in institutionalized crack users in Goiânia, Goiás. Between 2012 and 2013, a cross-sectional study was conducted with 600 users of a reference unit for chemical dependency treatment of Goiás. All participants were interviewed regarding socio-demographic characteristics and risk factors for syphilis. Blood samples were then collected for the detection of anti-Treponema pallidum antibodies using enzyme-linked immunosorbent assay (ELISA). Positive samples were retested by the Venereal Disease Research Laboratory (VDRL). Active syphilis was considered when there was a positive ELISA result and VDRL titers ≥ 1:8. Of the 600 samples tested by ELISA, 13.8% (95% CI: 11.3 to 16.8%) were positive and 4.5% (95% CI: 3.1 to 6.5%) had titles VDRL ≥ 1:8. The prevalence of exposure to Treponema pallidum and active syphilis was three times greater in women than in men (p < .001). In multiple regression analysis, female sex (adjusted prevalence ratio [APR]: 3.73; p < .001), exchange sex for money and/or drugs (APR: 3.22; p = .040) and a history of genital ulcers (APR: 4.12; p = .010) were associated with active syphilis. Furthermore, it was observed that the prevalence of infection decreased by 12% for each year of formal study (APR: 0.88; p = .030). The results of this research show high prevalence of syphilis in the investigated crack users and indicate the need to implement strategies for prevention and control of this infection in these individuals, including health education activities, diagnosis of infection in clinical treatment for chemical dependency and treatment of positive cases. / Usuários de crack apresentam elevado risco para sífilis, devido aos múltiplos comportamentos de risco, como uso inconsistente do preservativo, troca de sexo por dinheiro e/ou drogas e múltiplas parcerias sexuais. O objetivo desse estudo foi investigar a epidemiologia da sífilis em usuários de crack institucionalizados em Goiânia, Goiás. Entre 2012 e 2013, um estudo de corte transversal foi conduzido em 600 usuários de uma unidade de referência em tratamento de dependência química de Goiás. Todos os participantes foram entrevistados sobre características sociodemograficas e comportamentos de risco para sífilis. A seguir, foram coletadas amostras sanguíneas para detecção de anticorpos anti-Treponema pallidum pelo ensaio imunoenzimático (ELISA). As amostras positivas foram retestadas pelo Venereal Disease Research Laboratory/Laboratório de Pesquisa de Doenças Venéreas (VDRL). Considerou-se sífilis ativa positividade no ELISA e títulos de VDRL ≥ 1:8. Das 600 amostras testadas pelo ELISA, 13,8% (IC 95%: 11,3-16,8%) foram positivas e 4,5% (IC 95%: 3,1- 6,5%) apresentaram títulos de VDRL ≥ 1:8. A prevalência de exposição ao Treponema pallidum e de sífilis ativa foi três vezes maior em mulheres do que em homens (p < 0,001). Em análise de regressão múltipla, sexo feminino (Razão de Prevalência Ajustada [RPaj]: 3,73; p < 0,001), troca de sexo por dinheiro e/ou drogas (RPaj: 3,22; p = 0,040) e antecedentes de úlceras genitais (RPaj: 4,12; p = 0,010) foram associados à sífilis ativa. Além disso, observou-se que a prevalência da infecção diminuiu 12% a cada ano de estudo formal (RPaj: 0,88; p = 0,030). Os resultados dessa investigação evidenciam elevada prevalência de sífilis nos usuários de crack investigados e indicam a necessidade de implementação de estratégias de prevenção e controle dessa infecção nesses indivíduos, incluindo ações de educação em saúde, diagnóstico da infecção em clínicas de tratamento para dependência química e tratamento dos casos positivos.

Objectifs: Le but de cette étude clinique était de comparer un groupe d’adultes ayant un parodonte sain avec un groupe d’adultes atteints de parodontite chronique en terme de risque carieux et mesures cliniques et microbiologiques de la carie. Méthodes: Quatre-vingt-seize individus ont été divisés en deux groupes en fonction de leur état de santé parodontal et ont été appariés pour l'âge, le sexe et l'origine ethnique. Trente-huit sujets étaient atteints de parodontite chronique définie comme ayant au moins quatre dents avec ≥ 1 site avec une profondeur de sondage ≥ 4 mm et une perte d'attache clinique ≥ 2 mm, et 58 sujets présentaient un parodonte sain. Par la suite, les groupes ont été subdivisés en deux groupes en fonction de leur statut carieux : les participants ayant au moins une lésion carieuse non traitée sur une surface dentaire et ceux n’ayant pas de lésion carieuse non traitée. Les données ont été recueillies par le biais d’un questionnaire, un examen clinique et des échantillons de plaque supra- et sous-gingivale. L’évaluation de la charge buccale de Streptococcus mutans et de six agents pathogènes parodontaux a été réalisée par la technique d'amplification de la réaction en chaine de la polymérase (PCR). Les données ont été analysées à l'aide d’analyses statistiques descriptives et bivariées. Résultats: Les individus atteints de parodontite chronique étaient 3,5 fois plus susceptibles d'avoir des caries que les individus en bonne santé (OR 3,5 ; IC: 1,5 - 8,3 ; P = 0,006). Les sujets à la fois atteints de parodontite chronique et de caries dentaires ont eu un niveau d’éducation significativement plus faible que les sujets ayant un parodonte sain et sans caries dentaires (OR 6,0 ; IC: 1,7 à 21,7 ; P = 0,04). La proportion de sujets ayant une charge buccale élevée de Porphyromonas gingivalis (P. g.) et Treponema denticola (T. d.) était significativement plus élevée chez les patients atteints de parodontite chronique et de carie que chez les patients sains présentant des caries (P. g.: OR 8,6 ; IC: 2,4 - 30,3 ; P = 0,004 et T. d.: OR 10,0 ; CI: 2,6 - 38.1 ; P = 0,003). Conclusions: Les résultats de cette étude suggèrent que, chez les sujets adultes atteints de la parodontite chronique, la fréquence des caries est plus élevée que chez les sujets ayant un parodonte sain. De plus, le faible niveau d'éducation influence négativement le statut parodontal des individus. / Aim: The aim of this clinical study was to compare adults with a healthy periodontium and those with chronic periodontitis, in terms of caries’ risk and caries’ clinical and microbiological measures. Methods: Ninety-six healthy adults were divided into chronic periodontitis (n= 38) and healthy periodontium (n=58) based on their periodontal status, and matched for age, gender, and ethnic background. Chronic periodontitis was defined as having at least four teeth with ≥1 site with a pocket depth ≥4 mm and clinical attachment loss ≥2 mm. Each group were subsequently subdivided in 2 groups according to their caries status: participants having at least one untreated decayed surface and those with no untreated caries. Data were collected by means of self-administrated questionnaire, clinical examination, and supra- and subgingival plaque sampling. Assessments of oral levels of Streptococcus mutans and six periodontal pathogens were conducted by PCR amplification techniques. Data were analyzed using descriptive and bivariate statistical tests. Results: Individuals with chronic periodontitis were 3.5 times more likely to have caries than healthy individuals (OR 3.5; CI: 1.5 – 8.3; P = 0.006). Subjects with both chronic periodontitis and dental caries had a significantly lower level of education than periodontally healthy subjects without dental caries (OR 6.0; CI: 1.7 – 21.7; P = 0.04). A significant higher proportion of subjects with high oral levels of Porphyromonas gingivalis (P. g.) and Treponema denticola (T. d.) was found among subjects with chronic periodontitis and untreated caries compared to periodontally healthy subjects with untreated caries (P. g.: OR 8.6; CI: 2.4 – 30.3; P = 0.004 and T. d.: OR 10.0; CI: 2.6 – 38.1; P = 0.003). Conclusion: The results from this study suggest that, adults with chronic periodontitis are more prone to caries disease than those adults with a healthy periodontium. Furthermore, low educational level could have a negative impact on the periodontal status of individuals.

Objectifs: Le but de cette étude clinique était de comparer un groupe d’adultes ayant un parodonte sain avec un groupe d’adultes atteints de parodontite chronique en terme de risque carieux et mesures cliniques et microbiologiques de la carie. Méthodes: Quatre-vingt-seize individus ont été divisés en deux groupes en fonction de leur état de santé parodontal et ont été appariés pour l'âge, le sexe et l'origine ethnique. Trente-huit sujets étaient atteints de parodontite chronique définie comme ayant au moins quatre dents avec ≥ 1 site avec une profondeur de sondage ≥ 4 mm et une perte d'attache clinique ≥ 2 mm, et 58 sujets présentaient un parodonte sain. Par la suite, les groupes ont été subdivisés en deux groupes en fonction de leur statut carieux : les participants ayant au moins une lésion carieuse non traitée sur une surface dentaire et ceux n’ayant pas de lésion carieuse non traitée. Les données ont été recueillies par le biais d’un questionnaire, un examen clinique et des échantillons de plaque supra- et sous-gingivale. L’évaluation de la charge buccale de Streptococcus mutans et de six agents pathogènes parodontaux a été réalisée par la technique d'amplification de la réaction en chaine de la polymérase (PCR). Les données ont été analysées à l'aide d’analyses statistiques descriptives et bivariées. Résultats: Les individus atteints de parodontite chronique étaient 3,5 fois plus susceptibles d'avoir des caries que les individus en bonne santé (OR 3,5 ; IC: 1,5 - 8,3 ; P = 0,006). Les sujets à la fois atteints de parodontite chronique et de caries dentaires ont eu un niveau d’éducation significativement plus faible que les sujets ayant un parodonte sain et sans caries dentaires (OR 6,0 ; IC: 1,7 à 21,7 ; P = 0,04). La proportion de sujets ayant une charge buccale élevée de Porphyromonas gingivalis (P. g.) et Treponema denticola (T. d.) était significativement plus élevée chez les patients atteints de parodontite chronique et de carie que chez les patients sains présentant des caries (P. g.: OR 8,6 ; IC: 2,4 - 30,3 ; P = 0,004 et T. d.: OR 10,0 ; CI: 2,6 - 38.1 ; P = 0,003). Conclusions: Les résultats de cette étude suggèrent que, chez les sujets adultes atteints de la parodontite chronique, la fréquence des caries est plus élevée que chez les sujets ayant un parodonte sain. De plus, le faible niveau d'éducation influence négativement le statut parodontal des individus. / Aim: The aim of this clinical study was to compare adults with a healthy periodontium and those with chronic periodontitis, in terms of caries’ risk and caries’ clinical and microbiological measures. Methods: Ninety-six healthy adults were divided into chronic periodontitis (n= 38) and healthy periodontium (n=58) based on their periodontal status, and matched for age, gender, and ethnic background. Chronic periodontitis was defined as having at least four teeth with ≥1 site with a pocket depth ≥4 mm and clinical attachment loss ≥2 mm. Each group were subsequently subdivided in 2 groups according to their caries status: participants having at least one untreated decayed surface and those with no untreated caries. Data were collected by means of self-administrated questionnaire, clinical examination, and supra- and subgingival plaque sampling. Assessments of oral levels of Streptococcus mutans and six periodontal pathogens were conducted by PCR amplification techniques. Data were analyzed using descriptive and bivariate statistical tests. Results: Individuals with chronic periodontitis were 3.5 times more likely to have caries than healthy individuals (OR 3.5; CI: 1.5 – 8.3; P = 0.006). Subjects with both chronic periodontitis and dental caries had a significantly lower level of education than periodontally healthy subjects without dental caries (OR 6.0; CI: 1.7 – 21.7; P = 0.04). A significant higher proportion of subjects with high oral levels of Porphyromonas gingivalis (P. g.) and Treponema denticola (T. d.) was found among subjects with chronic periodontitis and untreated caries compared to periodontally healthy subjects with untreated caries (P. g.: OR 8.6; CI: 2.4 – 30.3; P = 0.004 and T. d.: OR 10.0; CI: 2.6 – 38.1; P = 0.003). Conclusion: The results from this study suggest that, adults with chronic periodontitis are more prone to caries disease than those adults with a healthy periodontium. Furthermore, low educational level could have a negative impact on the periodontal status of individuals.

The objective of this study is to identify and describe the cases reported / investigated of Congenital Syphilis in the Santa Maria’s University Hospital (HUSM), Rio Grande do Sul, Brazil, in the period of 2010 to 2014. Is a retrospective, cross-sectional, study in which secondary data found in records of notification/investigation of Congenital Syphilis Information System notification where evaluated. 128 records notification/investigation Congenital Syphilis where evaluated. The survey instrument was developed based on research of Congenital Syphilis records and pregnant women, where with the data collected has built up a database, which was submitted to descriptive statistical analysis. The analyzed variables consisted on the genitor: sociodemographic; realization or not of the prenatal care; period of maternal diagnosis of the Syphilis, nontreponemal test achievement in prenatal and childbirth; Prenatal treponemic test and delivery; treatment performed in pregnancy, partner treatment. The variables relating to infant were: not treponemal test, cerebrospinal fluid change, not treponemic test cerebrospinal fluid; radiological diagnosis; clinical diagnosis; in child treatment regimen; developments. Results: 128 cases of congenital syphilis were reported during the study. Pregnant women analyzed were the age from 20 to 35 years old, all of them of white color, educational level of 5th to 8th grade incomplete, underwent the prenatal care, had the diagnosis carried out in the third-trimestrer or childbirth, with inadequate treatment and with partners who were not treated. Newborns were male, white, have been notified within seven days after the birth date, treponemal test in peripheral blood reagent and asymptomatic in most cases. Three cases with non treponemal test in the reagent liquor six cerebrospinal change and a case with abnormal X-ray of the long bones, and more than half of the cases received treatment with Penicillin Cristalina for 10 days. Conclusion: Persisting vertical transmission, there is failure in the health care network focused on prenatal quality. / O objetivo deste estudo é identificar e descrever os casos notificados/investigados de sífilis congênita no Hospital Universitário de Santa Maria (HUSM) no Estado do Rio Grande do Sul-Brasil, no período de 2010 a 2014. Constitui um estudo retrospectivo, transversal, no qual avaliou dados secundários encontrados nas fichas notificação/investigação do Sistema Informação de Agravos de Notificação (SINAN) de sífilis congênita. Foram avaliadas 128 fichas de notificação/investigação de Sífilis Congênita. O instrumento de pesquisa foi elaborado com base nas fichas de investigação de sífilis congênita e em gestantes, os dados coletados construiu-se um banco de dados o qual foi submetido à análise estatística descritiva. As variáveis analisadas consistiram relativas a genitora: sociodemografica; realização do pré-natal; período do diagnóstico materno, realização de teste não treponêmico no pré-natal e no parto; teste treponêmico pré-natal e parto; tratamento realizado na gestante, tratamento do parceiro. As variáveis referentes à criança são idade, cor, sexo teste não treponêmico, alteração liquórica, teste não treponêmico no liquor; diagnóstico radiológico da criança; diagnóstico clínico; esquema de tratamento na criança; evolução do caso. Como resultados: foram notificados 128 casos de sífilis congênita no período de estudo. As gestantes analisadas tinham faixa etária de 20 a 35 anos, da cor branca, nível de escolaridade de 5º a 8º serie incompleta, realizaram o pré-natal, diagnóstico realizado no 3º trimestre ou no parto, com tratamento inadequado e com parceiros que não foram tratados. Os recém-nascidos eram do sexo masculino, brancos, foram notificados em até sete dias da data de nascimento, teste treponêmico no sangue periférico reagente e assintomáticos na maior parte dos casos. Três casos com exame não treponêmico no liquor reagente, seis com alteração liquórica e um caso com alteração no Raio X dos ossos longos, sendo que mais da metade dos casos recebeu tratamento com penicilina cristalina por 10 dias. Com isso, conclui-se que persistindo a transmissão vertical, verificou-se falhas na rede de atenção à saúde voltada para um pré-natal de qualidade.

T. pallidum can survive a primary immune response and continue growing in the host for an extended period of time. T. pallidum is thought to bind serum fibronectin (FN) through Tp0483 on the surface to obscure antigens. A Tp0483 fragment (rTp0483) was adsorbed onto functionalized self-assembled monolayers (SAMs) with FN. FN capture by adsorbed rTp0483 depended greatly on surface chemistry with COO- groups being best for FN binding. Hemocompatibility was determined by analysis of plasma protein adsorption, intrinsic pathway activation, and platelet activation. rTp0483+FN bound an equal or lesser amount of fibrinogen (Fg), human serum albumin (HSA), and factor XII (FXII) compared to rTp0483 or FN alone and adsorption of rTp0483 prior to FN greatly decreased platelet activation. Inhibition of protein binding and platelet activation suggested an attenuated hematological response. Biocompatibility of rTp0483 and FN coated surfaces was characterized by macrophage uptake of protein coated polystyrene microspheres (PSMs), macrophage adsorption onto protein coated surfaces, cytotoxic effects of adsorbed rTp0483 and FN, and TNF-α and NO2- release in macrophages stimulated with rTp0483 and FN adsorbed and in solution. Addition of FN to rTp0483 on plain and COO- PSMs reduced phagocytosis compared to rTp0483 alone and on plain PSMs compared to FN alone. On plain PSMs addition of FN to adsorbed rTp0483 decreased TNF-α generation. Adsorption of rTp0483 before FN on large, flat COO- surfaces decreased macrophage adsorption and TNF-α and NO2- generation. High concentrations of rTp0483 were mildly cytotoxic to macrophages. FN binding by Tp0483 on T. pallidum likely plays a role in antigenic disguise and rTp0483+FN coatings may potentially inhibit FN and rTp0483 specific interactions with macrophages. Molecularly imprinted polymer coatings were also examined for biomaterial development. Fouling resistant 2-methacryloyloxyethyl phosphorylcholine (MPC) was imprinted with bovine serum albumin (BSA) protein templates to facilitate BSA specific binding. The BSA template was constructed and verified and BSA specific binding quantified using quartz crystal microbalance (QCM) and enzyme linked immunosorbent assay (ELISA). BSA imprinted coatings were determined to bind significantly more BSA than nonfouling MPC controls demonstrating the feasibility of targeted protein capture.

Treponema pallidum ssp. pallidum is the causative agent of syphilis, a sexually transmitted infection characterized by multi-stage disease and diverse clinical manifestations. Treponema pallidum undergoes rapid vascular dissemination to penetrate tissue, placental, and blood-brain barriers and gain access to distant tissue and organ sites. The rapidity and extent of T. pallidum dissemination is well documented, but the molecular mechanisms that underlie this process have yet to be fully elucidated. Tp0751 is a T. pallidum adhesin that interacts with vascular factors and mediates adherence to endothelial cells under shear flow. This dissertation explores the molecular interactions and functional outcomes of Tp0751-mediated vascular endothelium adhesion. The findings presented herein demonstrate that recombinant Tp0751 adheres to human macrovascular and microvascular endothelial cells, including cerebral brain endothelial cells. This interaction is confirmed using live T. pallidum, where spirochete- endothelial cells interactions are disrupted with Tp0751-specific antiserum. Further, the 67 kDa laminin receptor (LamR) is identified as an endothelial receptor using affinity chromatography coupled with mass spectrometry to isolate and identify Tp0751-interacting proteins from endothelial cells membrane extracts. Notably, LamR is a brain endothelial cell receptor for other neurotropic invasive pathogens. Evaluation of endothelial intercellular junctions reveals that recombinant Tp0751 and live T. pallidum disrupt junctional architecture. However, transwell solute flux assays reveal that Tp0751 and T. pallidum do not alter endothelial barrier integrity. The transendothelial migration of T. pallidum can be partially abrogated with an endocytosis inhibitor, implying a transcellular route for barrier traversal. However, a subpopulation of T. pallidum localizes to intercellular junctions, indicating paracellular traversal may also be employed. These findings enhance our understanding of the mechanics of T. pallidum attachment to endothelial cells and suggest that T. pallidum may use both paracellular and transcellular mechanisms to traverse the vascular endothelium without altering barrier permeability. A more complete understanding of this process will facilitate vaccine development for syphilis. / Graduate / 2020-06-18

<div>The eradication of yaws a neglected tropical disease caused by Treponema pallidum subsp. pertenue, which affects children living in very deprived hard to reach rural communities is constrained by the lack of rapid, accurate diagnosis. I sought to develop a molecular point-of-care test for the diagnosis of yaws. A Loop-mediated isothermal amplification (LAMP) assay with primers targeting the conserved gene, tp0967, with visual detection by lateral flow test strip was developed and optimized. The limit of detection was evaluated while 63 samples from clinical cases of yaws and 5 samples with PCR-confirmed syphilis were used to determine the sensitivity and specificity of the assay compared to the current molecular testing protocol. Reagents were dried in tubes and tested up to 14 days. The developed LAMP assay was found to be optimal when run at 65oC in a water bath for 30 minutes. The limit of detection was 2.7*104 DNA copies/ml. The sensitivity of the LAMP assay using unextracted and DNA extracted samples were 0.67 and 1.00 respectively. None of the syphilis samples tested positive in any of the assays. We show the development of a fast and sensitive LAMP assay for yaws detected by lateral flow test strip. Using extracted DNA, the assay sensitivity is at par with gold standard detection. The assay can be adapted to minimal sample processing required for in-field detection without DNA extraction.</div><div><br></div>