Orientadores: Oswaldo Di Hipolito Junior, Plinio Trabasso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-07T03:11:34Z (GMT). No. of bitstreams: 1 Alcure_MonicaLeal_D.pdf: 2751232 bytes, checksum: 9ec1e6f0e9b7aa9c25ff089f07e417eb (MD5) Previous issue date: 2006 / Resumo: As infecções superficiais e sistêmicas provocadas por Candida são freqüentes causas de morbidade e mortalidade em pacientes submetidos a transplante de células progenitoras hematopoiéticas (TCPH). Entre os fatores de risco destaca-se a colonização prévia a qual parece ser um pré-requisito para o desenvolvimento de infecção. Desta forma, a cavidade oral pode atuar como um reservatório para esses microorganismos. O presente estudo teve como objetivo avaliar, prospectivamente, o fluxo salivar e os efeitos da profilaxia antifúngica com fluconazol (200mg/dia) no número de pacientes colonizados por Candida spp. e no número de unidades formadoras de colônias (UFC) das espécies Candida encontradas na saliva dos pacientes submetidos a TCPH. Objetivou, ainda, realizar testes susceptibilidade antifúngica dos isolados, determinar o cariótipo eletroforético por meio de eletroforese em campo pulsátil (PFGE) nos isolados de Candida albicans e verificar a similaridade entre as cepas colonizadoras e as responsáveis pelos quadros de infecção. Foram incluídos no estudo 35 pacientes, 21 do TCPH Alogênico e 14 do TCPH Autólogo, com mediana de idade de 44 anos (21-71) sendo 22 (63%) do gênero masculino e 13 (37%) do gênero feminino. O fluxo salivar variou de modo estatisticamente significativo nos grupo Alogênico (p=0,0003) e no grupo Autólogo (p=0,009) no decorrer do estudo. Colonização por Candida ocorreu em 57% dos pacientes sendo que durante o transplante, estando o paciente sob profilaxia com fluconazol, houve redução de 60% no número de pacientes colonizados. Nesse período também ocorreu uma redução da média do número de UFC/ml seguida de expressivo aumento nas coletas subseqüentes, alcançando valores maiores que os obtidos na coleta inicial. O número de UFC/ml apresentou uma relação inversamente proporcional com o fluxo salivar (p=0,005). Não foi possível estabelecer relação entre o número de UFC/ml de saliva e a presença de candidose oral. Candida albicans foi a espécie mais freqüentemente encontrada representando 63% de todos os isolados seguida da Candida parapsilosis. Resistência aos antifúngicos foi encontrada em 6 isolados de Candida não albicans sendo 2 Candida parapsilosis, 2 Candida guilliermondii, 1 Candida krusei e 1 Candidq sp. Foram encontrados 9 diferentes perfis de cariótipo eletroforético de Candida albicans. Em 6 pacientes a mesma cepa encontrada na coleta inicial foi identificada ao longo do estudo. Quatro (11,4%) pacientes desenvolveram candidose oral no período pós-enxertia. Em todos os casos somente isolados de Candida albicans foram identificados. Todos esses pacientes foram colonizados inicialmente pela , mesma espécie sendo que em 3 pacientes, a mesma cepa colonizadora foi a responsável pela infecção. A maior ocorrência de candidose oral no período pós-enxertia pode estar relacionada com fatores inerentes ao hospedeiro. A persistência da mesma cepa sugere que a profilaxia com fluconazol durante o transplante não erradica a cepa colonizadora e que a colonização prévia parece ser um pré-requisito para o desenvolvimento de infecção / Abstract: Superficial and systemic Candida infections are usually responsible for morbidity and mortality in hematopoietic stem cell transplant (HSCT) recipients. Previous colonization is an important risk factor and seems to be a pre-requisite for infection and the oral cavity may be a reservoir for these microorganisms. The aim of this study was to ana1yze, prospectively, the unstimulated salivary flow rate and the fluconazole prophylactic regimen effects (200mg/day) in the number of Candida colonized patients and in the colonyforming unit (CFU) count of the Candida species identified in the saliva of HSCT recipients. The objective was also performed antifungal susceptibility tests in clinical isolates, determine the electrophoretic karyotype by pulsed-field gel electrophoresis (pFGE) in the Candida albicans isolates and verify the similarity between colonization and infection strains. Thirty-five patients were enrolled, 21 Allogeneic and 14 Autologous HSCT recipients. The median age was 44 years old (range 21-71). Twenty-two (63%) were male and 13 (37%) were female. The salivary flow rate varied in the Allogeneic (p=0,0003) and in the Autologous groups (p=0,009). Candida colonization was found in 57% of the patients. There was a 60% reduction of the number of colonized in patients under prophylactic fluconazole regimen. In this same time, there also was a reduction of the median CFU/ml count followed by an important increase of the sequential samples, reaching values higher than the count found in the initial collection. There was a significant inverse relationship between salivary flow and Candida CFU/rnl count (p=0,005). It was not possible to correlate the CFU/rnl count and oral candidosis. Candida albicans was the most frequent specie identified (63%) followed by Candida parapsilosis. Antifungal resistance was found in 6 clinical isolates of non-albicans species of Candida, 2 Candida parapsilosis,2 Candida guilliermondii, 1 Candida krusei and 1 Candida sp. We found 9 pattems of Candida albicans electrophoretic karyotyping. In 6 patients the initial colonization strain was found during the following study. Four patients (11,4%) developed oral candidosis by Candida albicans in the post engraftment period. AlI patients were initially colonized by the same specie. In 3 of these patients the same colonization strain was identified in the infection period. The major occurrence of oral candidosis in the post engraftment period may be related with inherent host factors. The persistence of the same strain suggests that the fluconazole prophylaxis does not eradicate the colonization strain. The previous colonization appears be a pre-requisite for de infection development / Doutorado / Patologia / Doutor em Estomatopatologia

Fusarium virguliforme is a soil-borne pathogen that causes sudden death syndrome (SDS) disease in soybean. SDS is one of the most significant diseases of soybean in the United States. Fungal infection results in root and crown rot as well as SDS typical foliar symptoms including chlorosis, necrosis and complete defoliation. The use of soybean cultivars tolerant to SDS is still the most effective way to overcome the disease. On the other hand, the fungal isolates are known to have varied levels of aggressiveness on soybean indicated by the field and greenhouse experiments. Understanding the pathogen and its defense mechanism is the first step in exploring the pathogen-plant interaction. Therefore, the primary aim of this research was to elucidate the mechanism behind F. virguliforme response to soybean defense mechanisms. We further attempted to identify chromosome length polymorphism among F. virguliforme isolates and characterize the possible relationship to their level of aggressiveness. In order to fulfill the first objective, a series of differentially expressed genes were identified in F. virguliforme in the presence of soybean phytoalexin, glyceollin. The Fvgrx2 gene, a Saccharomyces cerevisiae grx2homologue, was selected for further analysis. This study demonstrates for the first time the identification and characterization of dithiol glutaredoxin gene in F. virguliforme . The role of FvGRX2 in the fungal defense to phytoalexin, glyceollin and induced oxidative burst was also investigated by generating anFvΔgrx2 knockout. In order to establish a link between the fungal karyotype and the level of fungal aggressiveness, the chromosome length polymorphism (CLP) was assessed for twenty-two F. virguliforme isolates exhibiting different levels of aggressiveness on soybean. The findings are instrumental in identifying novel pathogenicity such as the ones involved in phytotoxin production, fungicide resistance and aggressiveness.

Mosaicism is represented by two or more chromosomally different cell lines in an individual. Mosaics are most often caused by chromosome malsegregation during mitosis, resulting in the gain or loss of chromosomes, known as aneuploidy, but structural aberrations can also occur in mosaic form. The problem is the limitation of detection with standart cytogenetic methods. The present study was carried out to compare the efficiency of FISH, array CGH and cytogenetic techniques in detection of mosaicism. In the practical part the results of 45 patients with mosaicisms of aneuplody of gonosomes (26 patients) and mosaicisms of autosomes (19 patients) were compared. The data show that we have different peripheral blood karyotype and FISH results in 23 of 37 patients (62%). There was a case of failure of detection of the mosaicism on the karyotype and the FISH method revealed a abnormal cell lines with a percentage of less than 5%. The array CGH method confirmed the karyotype and FISH results in 10 out of 12 patients (83%) in peripheral blood tests. The work also dealt with artificially made mosaics. From the results, it is obvious that the FISH method has a more accurate percentage of mosaic capture compared to the karyotype. The results indicate that using the techniques in parallel allow in clinical...

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Ovarian cancer (OCa) remains the leading cause of death due to a gynecologic malignancy in North American women, and the pathogenesis of this disease is a consequence of the interplay between DNA, RNA and proteins. The genomes of these cancers are characterized by numerical and structural aberrations, resulting in copy number changes of the affected regions. The serine protease, Kallikrein 6 (KLK6), is a promising biomarker and is over-expressed in OCa. However, the mechanisms leading to the observed KLK6 overexpression are poorly understood; and to date, no study examining the chromosomal contributions to the overexpression have been conducted. Utilization of multi-colour Fluorescence in situ Hybridization (FISH)-based technologies to untreated primary serous OCa samples and cancer cell lines, showed that the KLK locus, on 19q13.3/4, is involved in both numerical and structural aberrations; was subject to high-level copy-number heterogeneity (p<0.001); and structural rearrangements of 19q were significantly co-related to grade (p<0.001). Patients with a loss of the KLK locus, or no structural rearrangement on 19q, experienced a trend towards longer disease free survival (DFS and better overall survival (OS), over those with a gain or amplification, or with breakage events on 19q. KLK6-specific immunohistochemistry (IHC) showed weak correlation with KLK6 copy-number, suggesting other mechanisms together with copy-number, drives its over-expression. Among these mechanisms are microRNA (miRNAs), also shown to be affected by the copynumber changes in OCas. Therefore, we investigated the role of miRNAs in OCa and their role in KLK6 regulation. Specifically, we examined the copy-number status and miRNA expression in a representative OCa cell line, OVCAR-3. miRNA expression profiling of OCa cell lines and primary tumours showed their differential expression, including the decrease in expression of the let-7 family members, which are predicted to target KLK6. Indeed, when hsa-let-7a was transiently transfected into OVCAR-3, a reduction of secreted KLK6 protein was detected. Thus, the contribution of numerical and structural aberrations of the OCa genome can directly affect the expression KLK6 through copy-number, but is also aided post-transcriptionally by miRNAs.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser och 1 appendix.

Spectral karyotyping (SKY) is a widely used methodology to identify genetic aberrations. Multicolor fluorescence in situ hybridization using chromosome painting probes in individual colors for all metaphase chromosomes at once is combined with a unique spectral measurement and analysis system to automatically classify normal and aberrant chromosomes. Based on countless studies and investigations in many laboratories worldwide, numerous new chromosome translocations and other aberrations have been identified in clinical and tumor cytogenetics. Thus, gene identification studies have been facilitated resulting in the dissection of tumor development and progression. For example, different translocation partners of the TEL/ETV6 transcription factor that is specially required for hematopoiesis within the bone marrow were identified. Also, the correct classification of complex karyotypes of solid tumors supports the prognostication of cancer patients. Important accomplishments for patients with genetic diseases, leukemias and lymphomas, mesenchymal tumors and solid cancers are summarized and exemplified. Furthermore, studies of disease mechanisms such as centromeric DNA breakage, DNA double strand break repair, telomere shortening and radiation-induced neoplastic transformation have been accompanied by SKY analyses. Besides the hybridization of human chromosomes, mouse karyotyping has also contributed to the comprehensive characterization of mouse models of human disease and for gene therapy studies. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Chromosomal aberrations are common causes of abnormal development of fetuses leading to the birth of malformed indvidual or to the intrauterine death. Half of miscarriages in the first trimester and a third in the second trimester are caused by fetal chromosomal abnormalities, mainly aneuploidies. If fetus is abnormally developed, invasive prenatal cytogenetic diagnosis should be recommended. Positive cytogenetic finding can be reason for induced abortion till the end of 24th week of gestation. We investigated 81 miscarriages, 46 fetuses from induced abortions and 80 fetuses with abnormal development from ongoing pregnancies. G-banding analysis was used as the main method for investigating miscarriages. Genomic DNA isolated from abnormally developed fetuses was screened by array CGH technique. We found 43,75 % chromosomal abnormal miscarried fetuses, majority of them with numerical aberrations (91,4 %). In group of induced abortions, 25,71 % fetuses carried chromosomal abnormality. The lowest rate 11,67 % of chromosoal aberrations was detected in group of prenatally diagnosed fetuses from ongoing pregnancies. Array CGH detected submicroscopic aberrations in 13,41 % fetuses with ultrasound findings. All together 25,74 % microscopic and causal submicroscopic chromosomal abnormalities were found to be...

INTRODUÇÃO: A Síndrome de Rubinstein-Taybi (RTS) é uma doença genética rara, caracterizada por dismorfismos craniofaciais típicos, polegares e háluces alargados, deficiência mental e baixa estatura. A incidência estimada é de 1: 125 000 a 1: 330000 nativivos. A SRT geralmente ocorre esporadicamente, mas pode ser herdada com um padrão de herança autossômico dominante. O diagnóstico da SRT é essencialmente clínico. OBJETIVOS: Realizar o estudo genético-clínico e citogenético em 30 pacientes brasileiros com SRT, e descrever de forma sistematizada a freqüência de dismorfismos faciais e malformações múltiplas encontradas. MÉTODOS: Neste estudo observacional retrospectivo e prospectivo, os pacientes foram seguidos no período de agosto de 2005 a junho de 2009. O cariótipo com bandeamento G foi realizado em todos os pacientes. RESULTADOS: A maioria dos pacientes avaliados foi do sexo feminino (60%). As seguintes características foram observadas em todos os pacientes da nossa casuística: atraso de desenvolvimento neuropsicomotor, ponta nasal voltada para baixo, columela proeminente, sorriso característico, dificuldades alimentares na infância, persistência dos coxins fetais, falanges distais dos polegares alargadas e pés planos. A baixa estatura e a microcefalia foi observada em 80% e 76% dos casos, respectivamente. As principais características craniofaciais observadas foram: fronte proeminente (86%), ponte nasal larga (60%), hipertelorismo (70%), sobrancelhas espessas e arqueadas (96%) cílios longos em 93%, prega epicântica (76%), fissura palpebral infra vertidas (76%), abertura bucal estreita (93%), retrognatismo (76 %), sorriso característico em 100%, palato alto e estreito (93%), anomalias dentárias (83%). Outras anomalias identificadas foram: estrabismo, erros de refração, obstrução do canal lacrimal, háluces e polegares alargados, angulação de polegares, anomalias do pavilhão auricular (rotação/posição/tamanho/forma), angulação do hálux, clinodactilia, sobreposição dos pododáctilos, falanges distais alargadas de outros dedos, marcha rígida, hipotonia, sopro cardíaco, cardiopatia congênita, criptoquidia, hemangioma plano e hipertricose. Uma paciente apresentou translocação recíproca de novo 46, XX, t (2; 16)(q36.3; p13.3). CONCLUSÕES: A raridade da SRT e o amplo espectro das manifestações clínicas pode atrasar o diagnóstico clínico. A média da idade ao diagnóstico dos nossos pacientes com SRT foi de três anos e oito meses. Todas as crianças devem receber avaliação por geneticista pediátrico, cardiologista, oftalmologista, neuropediatra, e odontopediatra / INTRODUCTION: Rubinstein-Taybi syndrome (RTS) is a rare genetic disorder characterized by distinctive craniofacial dysmorphisms, broad thumbs and toes and mental and statural deficiency. The prevalence of RTS has been estimated to be 1 in 125000 to 1: 330000 live births. RTS usually occurs sporadically although it can be inherited as an autosomal dominant disorder. The diagnosis of RTS is primarily based on clinical features. OBJECTIVES: We performed a clinical and cytogenetic assay in a group of 30 Brazilian RTS patients. We also decribed the frequencies of facial dysmorphisms and multiple malformations. METHODS: In this observational retrospective and prospective study, the patients were followed from August 2005 to June 2009. Chromosomal analysis was performed by G-banding karyotype. RESULTS: Most of the patients were female (60%).The following abnormalities were present in all of the patients: delayed psychomotor development, beaked nose, proeminent collumel, typical facies, broad thumbs and toes, flat feet, joint laxity, feeding problems during the childhood, and finger pads. Short stature was present in 80%, and microcephalia in 76% of the cases, respectively. Main craniofacial characteristics are frontal bossing (86%), wide nasal bridge (60%), ocular hyperthelorism (70%), high arched eyebrows (96%), long eyelashes (93%), epicathal folds (76%), downslanting palpebral fissures (76%), small opening of the mouth (93%), retrognathism (76%), grimacing smile (100%), high arched palate (93%), and dental anomalies (83%). Other findings were: strabism, refractive error, lacrimal obstruction, wide thumb and halux, angulated thumbs, external ears anomalies (rotation, implantation and morfology), angulated halux, clinodactyly, crowded toes, broad distal falanges of other fingers, stiff gait, hipotonia, cardiac murmur, congenital heart defect, undescendent testis, hypertrichosis, and hemangioma. One female patient has found to have a reciprocal de novo translocation t(2;16)(q36.3;p13.3) on G-banding karyotype CONCLUSIONS: The rarity of RTS and the wide spectrum of clinical manifestations, may delay the clinical diagnosis of RTS. The average age at the diagnosis of our patients was 3 years and 8 months. All children of RTS should receive an evaluation by a pediatric geneticist, cardiologist, ophthalmologist, pediatric neurologist, and pediatric dentist