Fatores associados ao baixo peso ao nascer na Maternidade do Hospital da Universidade Federal de Santa Catarina, em Florianópolis

Trabalho de Conclusão de Curso - Universidade Federal de Santa Catarina. Curso de Medicina. Departamento de Saúde Pública

Microscopia eletrônica de varredura do endotélio da córnea de avestruz

The aim of this study was to examine the endothelial surface morphology and perform a morphometric analysis of the corneal endothelial cells of ostrich (Struthio camelus) using scanning electron microscopy. Polygonality, mean cell area, cell density and coefficient of variation of mean cell area were analyzed. The normal corneal endothelium consisted of polygonal cells of uniform size and shape with few interdigitations of the cell borders. Microvilli appeared as protusions on the cellular surface. The average cell area was 269±18µm² and the endothelial cell density was 3717±240cells mm-2. The coefficient of variation of the cell area was 0.06, and the percentage of hexagonal cells was 75%. The parameters evaluated did not differ significantly between the right and the left eye from the same ostrich. The results of this study showed that the ostrich corneal endothelial cells appear quite similar to those of the other vertebrates.Objetivou-se examinar a superfície posterior do endotélio corneano e realizar análise morfométrica das células endoteliais da córnea de avestruz (Struthio camelus) valendo-se da microscopia eletrônica de varredura. Avaliaram-se o número de lados, a área celular média, a densidade celular e o coeficiente de variação da área celular. O endotélio corneano de avestruz constitui-se de células poligonais uniformes em tamanho e forma, e com poucas interdigitações das bordas celulares. Visibilizaram-se microvilosidades na superfície celular. A área celular média foi de 269±18µm² e a densidade celular foi de 3717±240 células mm-2. O coeficiente de variação foi de 0,06 e o percentual de células hexagonais de 75%. Não foram observadas diferenças significativas entre os parâmetros avaliados entre os olhos esquerdo e direito. Este estudo demonstrou que o endotélio corneano de avestruz é semelhante ao descrito em outros vertebrados

The Barretos Cancer Hospital Animal Facility: implementation and results of a dedicated platform for preclinical oncology models

The Barretos Cancer Hospital Animal Facility (BCHAF) is a unique facility in Brazil exclusively dedicated to working with animal models for cancer research. In this article, we briefly present our modern facility and the main experiments performed, focusing on mutant strains of mice (PTCH-knockout and ApcMin mice), xenograft models, and patient-derived xenografts (PDXs). Our results show the progress and challenges in establishing these models and the need for having an appropriate representation of our cancer population to better understand tumor biology and to identify cancer biomarkers, which could be putatively targeted, allowing for personalized therapy.This study was funded by the Public Ministry of Labor Campinas (Research, Prevention and Education of Occupational Cancer) and by Pio XII Foundation, Barretos Cancer Hospital internal funds, Grant Number: 13/2021

Molecular Cytogenetic Characterization of Supernumerary Marker Chromosomes

Rearranjos cromossômicos envolvendo a presença de cromossomos marcadores extranumerário são achados citogenéticos freqüentes em pacientes que apresentam deficiência mental, alterações de crescimento, dismorfias e/ou malformações. A presença desse material é responsável por trissomia ou tetrassomia parcial de determinadas regiões cromossômicas, causando quadros clínicos distintos e inespecíficos. A variabilidade fenotípica está relacionada principalmente com os diferentes graus de mosaicismo, os genes presentes na região adicional, o cromossomo de origem, entre outros fatores. Sendo assim, a caracterização desse material cromossômico é de importância fundamental para a determinação do prognóstico e do aconselhamento genético dos pacientes e suas famílias. O presente estudo teve como objetivo a análise de cromossomos marcadores extranumerários por meio de técnicas de citogenética convencional e molecular. Foram selecionados onze pacientes que são acompanhados pelo o Serviço de Genética Médica do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto USP, todos com diagnóstico citogenético convencional por bandeamentos GTG de cromossomo marcador extranumerário. Para determinação da origem e caracterização dos cromossomos marcadores foram aplicadas as técnicas de Cariótipo Espectral (SKY) e de Hibridação in situ Fluorescente (FISH). Em dez pacientes foi possível determinar a origem e composição dos marcadores. Dois pacientes apresentam cromossomos marcadores identificados como duplicações invertidas do cromossomo 15, com cariótipos definidos, respectivamente, como 47,XY,+idic(15)(pterq15::q15pter) e 47,XX,+idic(15)(pterq21::q21p11.2), um paciente possui cromossomo marcador derivativo do cromossomo 15, com cariótipo 47,XX,+der(15)(pterq21) e dois pacientes, sendo uma menina e seu pai, possuem cromossomos marcadores derivativos do cromossomo 15 com cariótipos, respectivamente, 48,XX,+2der(15)(pterq12) e 48,XY,+2der(15)(pterq12). Dois pacientes possuem cromossomos marcadores derivativos do cromossomo 9, com cariótipos definidos, respectivamente, como 47,XX,+der(9)(pterq21) e 47,XX,+der(9)(pterq32) e um paciente apresenta cromossomo derivativo do cromossomo 4 [47,XX,+der(4)(p16q21)[9]/48,XX,+der(4)(p16q21),+mar[91]]. Um paciente possui um cromossomo marcador translocado derivativo do cromossomo 22 [47,XY,+der(22)t(11;22)(q25:q11.2)] e outro paciente, um cromossomo translocado derivativo do cromossomo 15 [47,XY,+der(15)t(15;16)(q13;q13)], ambos herdados de mães portadoras de translocações aparente balanceadas. Em um caso, não foi possível a caracterização dos cromossomos marcadores por meio das técnicas aplicadas. Há uma grande variação fenotípica associada à presença de cromossomos marcadores e muitas vezes o prognóstico e o aconselhamento genético são difíceis de determinar. As técnicas de citogenética molecular são ferramentas importantes para a caracterização dos cromossomos marcadores, tanto durante o pré-natal, como para uma família que já possui um membro afetado, auxiliando no mapeamento gênico de cada região envolvida para futura correlação cariótipo-genótipo-fenótipo.Chromosomal rearrangements involving supernumerary marker chromosomes are frequently found in patients with mental retardation, growth defects and malformations. The genetic materials presented in trisomy/tetrasomy are responsible by distinct and unspecific clinical symptoms. The phenotypic variation is related mainly to different mosaicismo degrees, genetic content and chromosomal origin. Thus, the characterization of marker chromosomes is important to determine the prognosis and genetic counseling to the patients and their families. The aim of this study was to analyze supernumerary marker chromosomes using conventional and molecular cytogenetic techniques. Eleven patients were included in this study, all assisted in Medical Genetic Division of Clinical Hospital of School of Medicine of Ribeirao Preto USP. They all presented supernumerary marker chromosomes detected by GTG band. The origin and composition were determined using Spectral Karyotype (SKY) and Fluorescence in situ Hybridization (FISH) techniques. To ten patients, the origin and composition were determined. Two patients presented inverted duplications of chromosome 15, and their karyotype were defined as 47,XY,+idic(15)(pterq15::q15pter) and 47,XX,+idic(15)(pterq21::q21p11.2), one patient had a derivative chromosome 15, with karyotype 47,XX,+der(15)(pterq21), and two patients, a girl and her father, had two derivatives chromosomes 15, with karyotypes 48,XX,+2der(15)(pterq12) e 48,XY,+2der(15)(pterq12), respectively. Two patients presented derivative chromosomes 9 and their karyotype were defined as 47,XX,+der(9)(pterq21) and 47,XX,+der(9)(pterq32), and one patient had a derivative chromosome 4, with karyotype 47,XX,+der(4)(p16q21)[9]/48,XX,+der(4)(p16q21),+mar[91]. One patient had a translocated marker chromosome, derivative 22, [47,XY,+der(22)t(11;22)(q25:q11.2)] and another patient had a translocated marker chromosome, derivative 15 [47,XY,+der(15)t(15;16)(q13;q13)]. In one case, was not possible to define the origin and composition of the marker chromosome using SKY and FISH techniques. A large phenotypic variation is associated with supernumerary marker chromosomes and many times, the prognosis and genetic counseling is difficult to determine. The molecular cytogenetic techniques are important tools to its characterization, during prenatal diagnosis or to a family with an affected person, helping the genetic mapping of each region to a future correlation karyotype-genotype-phenotype

Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions

Melanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffinembedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin

Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions

Melanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffin-embedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin.The authors would like to express their sincere thanks to the Sao Paulo Research Foundation (FAPESP) for its funding of this research (Vazquez's Grant #2012/04194-1, Vicente's Doctoral Fellowship Grant #2016/15941-3 and Vicente's International Research Fellowship Grant #2017/09612-0). The authors would like to thank Dr. Jeremy Squire for checking the English and for his critical review of this manuscript

Morfologia e morfometria das células do endotélio corneano de gatos (Felis catus) em diferentes faixas etárias obtidas pela microscopia celular

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Morfologia e morfometria das células do endotélio corneano de gatos (Felis catus) em diferentes faixas etárias obtidas pela microscopia celular

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