APLF (Aprataxin and Polynucleotide kinase/phosphatase-Like Factor) is an FHA (forkhead-associated)-domain-containing nuclear protein that facilitates the repair of single-strand and double-strand breaks (SSBs and DSBs). Specifically, the APLF-FHA domain mediates interactions with XRCC1 and XRCC4, factors involved in SSB and DSB repair, respectively. A novel substitution was identified in pancreatic cancer patients, where a conserved histidine was substituted to leucine (H42L) in the APLF-FHA domain. The functional and biological characterization of this “variant of unknown significance” was investigated. This thesis shows that the H42L substitution affects APLF phosphorylation, impairs APLF retention at laser-induced DNA breaks, disrupts protein-binding to XRCC1 but not to XRCC4, and reduces cell survival following irradiation and etoposide exposure. Collectively, these data suggest that the H42L substitution impacts APLF participation in global DNA damage repair. The findings from this work could provide insight into the role of APLF in genomic integrity and, moreover, in cancer predisposition.

APLF (Aprataxin and Polynucleotide kinase/phosphatase-Like Factor) is an FHA (forkhead-associated)-domain-containing nuclear protein that facilitates the repair of single-strand and double-strand breaks (SSBs and DSBs). Specifically, the APLF-FHA domain mediates interactions with XRCC1 and XRCC4, factors involved in SSB and DSB repair, respectively. A novel substitution was identified in pancreatic cancer patients, where a conserved histidine was substituted to leucine (H42L) in the APLF-FHA domain. The functional and biological characterization of this “variant of unknown significance” was investigated. This thesis shows that the H42L substitution affects APLF phosphorylation, impairs APLF retention at laser-induced DNA breaks, disrupts protein-binding to XRCC1 but not to XRCC4, and reduces cell survival following irradiation and etoposide exposure. Collectively, these data suggest that the H42L substitution impacts APLF participation in global DNA damage repair. The findings from this work could provide insight into the role of APLF in genomic integrity and, moreover, in cancer predisposition.

No description available.

The Dawdle (DDL) gene in Arabidopsis encodes a protein with a Fork Head-Associated domain. A mutation in the DDL gene causes pleiotropic phenotypes and reduced the levels of several microRNAs. However, it is not completely known whether the FHA domain of the DDL is necessary for its function. Furthermore, the interactors involved in the same signaling pathway as DDL have not been identified yet. To determine the role of FHA domain in DDL function, four Tiller mutant lines were isolated and characterized. Results from phenotypic characterization suggest that the FHA domain may require for DDL function. For interactors, the interactions between DDL and At1G68010, At2G28200, At3G68010 and At4G18372 were identified by Yeast Two Hybrid, and then confirmed by the Split Luciferase assays. In addition, T-DNA mutants for each interactor were isolated and analyzed phenotypically suggesting that the genes carrying those T-DNAs should function in the same signaling pathway as DDL.

No description available.

No description available.

No description available.

Bordetella pertussis, o agente etiológico da coqueluche ou tosse comprida, que estabelece a infecção através da fixação bacteriana no epitélio do trato respiratório superior. Os principais mediadores de adesão da bactéria são a toxina pertússica (PT) e a hemaglutinina filamentosa (FHA). A FHA é a adesina majoritária e contém pelo menos 4 domínios: porção Nterminal, domínio de reconhecimento de carboidrato (CRD) (FHA1141-1279), trinca de aminoácidos Arginina-Glicina-Ácido aspártico (RGD) (FHA1097-1099) e o sítio de ligação a heparina (domínio Hep) (FHA442-863). Neste trabalho, foi realizado a amplificação de duas regiões do domínio de ligação à heparina, as regiões MAL80 (FHA299-873) e HEP (FHA430-873). As regiões foram amplificadas, clonadas no vetor de expressão pAE, expressas utilizando a cepa BL21 SI de E. coli e purificadas. A proteína HEP purificada de E. coli possui baixa afinidade por heparina e não é capaz de aglutinar hemácias. A proteína recombinante HEP purificada foi utilizada para a produção de anticorpos. Através do experimento de ELISA foi demonstrado que o anti-soro anti-HEP é capaz de reconhecer além da região HEP, a região MAL80 e a proteína FHA. Estes resultados foram confirmados por experimentos de Western. Neste período também foi realizada a amplificação do domínio HEP de FHA e da subunidade S1 da toxina pertússica (PT) de B. pertussis através do método de TAP Express. Este método envolve duas reações de PCR e no final do processo é obtido um fragmento que contém uma região promotora (CMV), uma seqüência codificadora e uma região terminadora (SV40), que está pronto para ser introduzido e expresso em animais. De posse deste material e da proteína recombinante HEP, foi realizado o desafio intracerebral com Bordetella pertussis e através do monitoramento dos camundongos foi observado que nenhum dos candidatos testados foi capaz de proteger os animais contra B. pertussis. Foi realizado também a expressão do domínio Hep em lactobacilos, visando um possível sistema de imunizações de mucosas. Os anticorpos produzidos nos camundongos imunizados com a proteína HEP expressa em E. coli, lactobacilos e por vacina de DNA foram capazes de inibir a hemaglutinação promovida pela proteína FHA. / Bordetelfa pertussis, the agent of whopping cough, establishes infection by attaching to the ciliated epithelial cells of the respiratory tract. The bacterial adherence is mediated by pertussis toxin and filamentous hemagglutinin (FHA). FHA is the major adhesin of B. pertussis and displays multipie adherence activities. FHA contains four distin\'ct domains that exhibit specific affinities for different ligands or receptor, the amino-terminal end, the RGD triplet (FHA1097-1099), the lectin domain (FHA1141-1279) and the heparin-binding domain (FHA442-863). In this study, two overlapping regions of the heparin-binding domain, Mal80 (FHA299-873) and Hep (FHA442-873), were amplified by peR and subcloned in pAE expression vectors for E. coli. The fusion proteins in pAE were transformed in E. coli BL21 SI, induced with NaCI 0,3 M and purified using a nickel-charged metal chelating resin. The purified protein has low heparin affinity and does not have hemagglutination activity. The purified protein HEP was used to produce polyclonal antibodies in mouse. The anti-HEP antibodies are able to recognize the HEP, MAL80 and FHA proteins in ELISA and western assays, but anti-FHA only recognized the FHA protein. The genetically detoxified S1 subunit of pertussis toxin and Hep domain were amplified by the TAP Express method. There are two PCR reactions involved in the TAP processo At the end of the process the fragment of interest will carry a CMV promoter and a SV40 terminator and is ready to be introduced into animals or cell by transfection. Groups were immunized with proteins and/or DNA, challenged i.c. with a lethal dose of live Bordetelfa pertussis and the survival was monitored. No groups were protected against the challenge. The recombinant protein HEP were also expressed in Lactobacilfus aiming the development of potential mucosal vaccines. The polyclonal antibodies produce in mouse immunized with DNA and protein Hep expressed in E. coli and Lactobacillus were able to inhibition the FHA hemagglutination activity.

Bordetella pertussis, o agente etiológico da coqueluche ou tosse comprida, que estabelece a infecção através da fixação bacteriana no epitélio do trato respiratório superior. Os principais mediadores de adesão da bactéria são a toxina pertússica (PT) e a hemaglutinina filamentosa (FHA). A FHA é a adesina majoritária e contém pelo menos 4 domínios: porção Nterminal, domínio de reconhecimento de carboidrato (CRD) (FHA1141-1279), trinca de aminoácidos Arginina-Glicina-Ácido aspártico (RGD) (FHA1097-1099) e o sítio de ligação a heparina (domínio Hep) (FHA442-863). Neste trabalho, foi realizado a amplificação de duas regiões do domínio de ligação à heparina, as regiões MAL80 (FHA299-873) e HEP (FHA430-873). As regiões foram amplificadas, clonadas no vetor de expressão pAE, expressas utilizando a cepa BL21 SI de E. coli e purificadas. A proteína HEP purificada de E. coli possui baixa afinidade por heparina e não é capaz de aglutinar hemácias. A proteína recombinante HEP purificada foi utilizada para a produção de anticorpos. Através do experimento de ELISA foi demonstrado que o anti-soro anti-HEP é capaz de reconhecer além da região HEP, a região MAL80 e a proteína FHA. Estes resultados foram confirmados por experimentos de Western. Neste período também foi realizada a amplificação do domínio HEP de FHA e da subunidade S1 da toxina pertússica (PT) de B. pertussis através do método de TAP Express. Este método envolve duas reações de PCR e no final do processo é obtido um fragmento que contém uma região promotora (CMV), uma seqüência codificadora e uma região terminadora (SV40), que está pronto para ser introduzido e expresso em animais. De posse deste material e da proteína recombinante HEP, foi realizado o desafio intracerebral com Bordetella pertussis e através do monitoramento dos camundongos foi observado que nenhum dos candidatos testados foi capaz de proteger os animais contra B. pertussis. Foi realizado também a expressão do domínio Hep em lactobacilos, visando um possível sistema de imunizações de mucosas. Os anticorpos produzidos nos camundongos imunizados com a proteína HEP expressa em E. coli, lactobacilos e por vacina de DNA foram capazes de inibir a hemaglutinação promovida pela proteína FHA. / Bordetelfa pertussis, the agent of whopping cough, establishes infection by attaching to the ciliated epithelial cells of the respiratory tract. The bacterial adherence is mediated by pertussis toxin and filamentous hemagglutinin (FHA). FHA is the major adhesin of B. pertussis and displays multipie adherence activities. FHA contains four distin\'ct domains that exhibit specific affinities for different ligands or receptor, the amino-terminal end, the RGD triplet (FHA1097-1099), the lectin domain (FHA1141-1279) and the heparin-binding domain (FHA442-863). In this study, two overlapping regions of the heparin-binding domain, Mal80 (FHA299-873) and Hep (FHA442-873), were amplified by peR and subcloned in pAE expression vectors for E. coli. The fusion proteins in pAE were transformed in E. coli BL21 SI, induced with NaCI 0,3 M and purified using a nickel-charged metal chelating resin. The purified protein has low heparin affinity and does not have hemagglutination activity. The purified protein HEP was used to produce polyclonal antibodies in mouse. The anti-HEP antibodies are able to recognize the HEP, MAL80 and FHA proteins in ELISA and western assays, but anti-FHA only recognized the FHA protein. The genetically detoxified S1 subunit of pertussis toxin and Hep domain were amplified by the TAP Express method. There are two PCR reactions involved in the TAP processo At the end of the process the fragment of interest will carry a CMV promoter and a SV40 terminator and is ready to be introduced into animals or cell by transfection. Groups were immunized with proteins and/or DNA, challenged i.c. with a lethal dose of live Bordetelfa pertussis and the survival was monitored. No groups were protected against the challenge. The recombinant protein HEP were also expressed in Lactobacilfus aiming the development of potential mucosal vaccines. The polyclonal antibodies produce in mouse immunized with DNA and protein Hep expressed in E. coli and Lactobacillus were able to inhibition the FHA hemagglutination activity.

This thesis explores the intersections of a Black Power leisure identity, real property ownership, the progression of economic agency and land development through the example of Black resorts, focusing on Azurest North, a summer community in Sag Harbor, New York developed in the 1950s by Azurest Syndicate Incorporated. The project traces the history of real estate syndicates during the mid-twentieth century as a way to circumvent the practices of Jim Crow housing discrimination. Independent mortgage financing and land development especially in the field of resort housing, also points to the emergence of what I call a Black Power leisure identity. This study also seeks to determine how the American pursuit of leisure during the twentieth century forged identity and how real estate property ownership has been used to maintain and secure community and individual identity.