Dissertations / Theses on the topic 'Mutant frequency'

I used the hypoxanthine guanine phosphoribosyl transferase (hprt) clonal assay to determine the in vivo frequency of mutant T cells (FMC) from the peripheral blood, synovial fluid, and synovial tissue of rheumatoid arthritis (RA) patients and controls. The results demonstrate that there is an increased FMC in the peripheral blood of RA patients compared to controls. There was also a significant elevation in the corrected FMC (cFMC), which takes in to account the cloning efficiency of the T cells, in the peripheral blood of RA patients compared to controls. There is an elevated FMC and cFMC in synovial fluid of RA patients compared to the peripheral blood of controls. However. the FMC and cFMC from the peripheral blood of unselected RA patients from the outpatient clinic is not significantly different than from the synovial fluid of RA patients suggesting that the synovial fluid does not contain the necessary mitogenic and mutagenic factors to induce T cell genetic damage. The FMC and cFMC from RA and osteoarthritis (OA) synovium is approximately ten fold greater than the FMC and cFMC from the peripheral blood of the same patients which suggests that the mitogenic and genotoxic environment of the inflamed synovium is capable of inducing T cell mutations. There was no significant difference in the cloning efficiency of T cells (CE), FMC, and cFMC from the peripheral blood of RA patients with 'active' or 'inactive' disease. No correlation between the cFMC from the peripheral blood of RA patients and clinical disease parameters and patient medication was found. (Abstract shortened by UMI.)

A thesis submitted in fulfilment of the requirements for the degree of Masters in Molecular Genetics and Biomedicine
Motile ciliary dysfunctions cause specific Ciliopathies that affect mainly the respiratory tract, fertilization and left-right body establishment. The embryonic organ where left-right decisions are first taken is called the organizer, a ciliated organ where a leftward cilia driven fluid-flow is generated. The organizer is named node in the mouse and Kupffer’s vesicle (KV) in zebrafish. The correct left-right axis formation is highly dependent on signaling pathways downstream of such directional fluid-flow. Motile cilia need to be coordinated and Ciliary Beat Frequency (CBF) is characteristic of different types of cilia depending on their function. Using zebrafish as a model, our group has been studying cilia length regulation and motility in wild-type and deltaD-/- mutant embryos. Recently, we showed that Notch signalling was directly involved in the control of cilia length in the KV cells given that the deltaD-/- mutant present shorter KV cilia. The goal of this project was to characterize the CBF of deltaD-/- KV cilia vs. wild-type cilia and reveal how potential differences in CBF impact on KV fluid flow, using spectral analysis associated with highspeed videomicroscopy. By decomposing and comparing the obtained CBF with Fast Fourier Transform, we identified two major populations of motile cilia in wild-type as well as in deltaD-/- mutant embryos. However, we found the CBF populations had differential relative contributions and different distributions between wild-type and mutant embryos. Furthermore, by measuring the velocity of native particles we studied the KV fluid-flow and concluded that the dispersion of the flow velocity was much wider in the deltaD-/- mutants. On the other hand, based on a gene expression study of motility genes downstream of DeltaD, we concluded that motility related genes (dnah7, rsph3 and foxj1a) were deregulated in the mutants. During this project we generated data that led to new hypotheses that will allow us to test the causality between the described correlations.

Orientador: Reginaldo Bruno Gonçalves
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Esta tese, apresentada na forma de 3 artigos, teve por objetivos: (1) analisar a freqüência dos genes de produção de mutacinas I, II, III e IV, em genótipos de S. mutans isolados de indivíduos cárie-ativos e livres de cárie, (2) analisar a freqüência dos genes de produção das mutacinas I, II, III, IV, N, B-Ny 266, 1140 e genes homólogos às bacteriocinas, identificadas em outras espécies bacterianas, em cepas de S. mutans isolados de crianças, bem como detectar a expressão diferencial dos genes identificados, em células de S. mutans crescidas na condição planctônica e séssil, (3) analisar a expressão dos genes de produção das mutacinas I, II e proteínas kinases CiaH, Dgk e ComD, em diferentes fases do crescimento planctônico e séssil. O rastreamento e a freqüência dos genes estruturais de diferentes mutacinas em isolados de S. mutans, foram realizados pela técnica de PCR e a análise da expressão gênica, pela técnica de RT-PCR semi-quantitativa. Os estudos, apresentados nesta tese, demonstraram o papel das mutacinas como um fator de virulência, altamente diversificado entre a espécie S. mutans, e relacionado com o risco de cárie. Este fator de virulência, pode ser regulado por mecanismos quorum-sensing, sendo assim, dependente da condição de crescimento planctônica ou séssil. A regulação da produção de mutacinas, por mecanismos quorum-sensing, pode representar uma vantagem seletiva à espécie produtora, principalmente em ambiente complexo, como o biofilme dental e lesões de cárie. Futuramente, mais estudos serão necessários para identificar novos determinantes genéticos, necessários para a síntese de substâncias semelhantes às mutacinas, bem como, identificar os mecanismos e componentes, que modulam a expressão deste importante fatorde virulência em S. mutans
Abstract: This thesis, comprised of 3 manuscripts was designed (1) to analyse the frequency of biosynthesis genes of the mutacins types I, II, III and IV, in S. mutans isolated from caries-affected and caries-free individuals, (2) to analyse the frequency of biosynthesis genes of the mutacins types I, II, III, IV, N, B-Ny 266, 1140 and genes homologues to bacteriocins identified in other bacterial species, in S. mutans isolated from children, in addition, to detect the differential expression of these genes, in S. mutans cells grown in planktonic and sessil conditions, (3) and to analyse the expression of the mutacins I and II production genes and kinase proteins genes (ciaH, dgk e comD), in different phases of the planktonic and sessile growth. The screening and frequency of the mutacins structural genes in S. mutans isolates were realized by PCR technique and the analysis of genetic expression, by RT-PCR semiquantitative method. The studies, presented in this thesis, demonstrate the role of mutacins as a virulence factor, highly diverse among S. mutans, and related to risk of dental caries. The mutacins production may be regulated by quorum-sensing mechanisms and is dependent on planktonic and sessile conditions. The modulation by quorum-sensing mechanisms may represent a selective advantage for producer S. mutans strain, mainly in complex environments as the dental biofilm and caries. Hereafter, more studies will be necessary to identify new genetic determinants for synthesis of mutacin-like substances and elucidate the mechanisms and components that modulate the genetic expression of this important virulence factor in S. mutans
Doutorado
Microbiologia e Imunologia
Doutor em Biologia Buco-Dental

Methylmalonic aciduria results from defects in the enzyme methylmalonyl-CoA mutase and from defects in the synthesis of the enzyme's cofactor adenosylcobalamin. Two patients who excrete methylmalonic acid have been shown to have a homozygous nonsense mutation in the methylmalonyl-CoA epimerase gene (MCEE). To further understand the causes of methylmalonic acid excretion, the MCEE gene was sequenced in 229 patients who excreted methylmalonic acid for which no cause was known. Mutations were detected in five patients. Fusion of fibroblast lines from two patients with a homozygous nonsense mutation in MCEE did not result in correction of [14C]propionate incorporation toward control values while the defect in these fibroblasts was complemented by mut, cblA, and cblB fibroblasts. Transfection with wild-type MCEE cDNA resulted in correction of the biochemical phenotype in cells from both patients. These experiments support the hypothesis that a defective epimerase enzyme can be a cause of elevated methylmalonic acid excretion.

Orientador: Jaime A. Cury
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: As relações quantitativas entre frequência do consumo de sacarose, cárie dental e contagem de Estreptococos do "grupo mutans" não estão bem estabelecidos. Assim, foi realizado um estudo in situ utilizando-se um delineamento experimental do tipo cnlzado (4x4) em 04 etapas de 28 dias. doze voluntários usando dispositivos intra-orais palatinos, contendo 04 blocos de esmalte dental humano (3x3 mm), participaram desta pesquisa. Os voluntários gotejaram sobre os blocos dentais solução de sacarose a 20% na freqüência de 0 (zero) a 8x/dia. Os blocos dentais estavam protegidos por uma tela plástica e os voluntários utilizaram para sua higiene bucal dentifrício não fluoretado, mas a água consumida pelos mesmos era fluoretada (0.70 ppm). Após cada etapa a placa dental formada sobre os blocos foi coletada, pesada, homogeneizada e analisada em termos de contagem de Estreptococos do "grupo mutans" (UFC/mg) usando meio seletivo SB20. Os blocos dentais limpos, embutidos, seccionados e polidos para a determinação da dureza Knoop (KHN) do esmalte. Foram feitas indentações a 10 'mu'm de superfície utilizando microdurômetro SHIMADZU HM 2000 e carga de 25 g por 30 segundos. Os resultados microbiológicos observados em termos de média + desvio padrão da média de UFC/mg foram respectivamente em relação a exposição a sacarose de 0 (zero), 2, 4, 8x/dia: 26,72 '+ ou ¿' 13,36A; 46,72 '+ ou ¿' 30,81A; 102,44 '+ ou ¿' 53,34A e 52,18 '+ ou ¿' 21;48A, sendo que médias seguidas de mesma letra não diferem estatisticamente a nível de 5%. Quanto a dureza do esmalte diferenças significativas (p<0,05) com relação a área total só foram observadas quando a exposição a sacarose 8x/dia, resultado este semelhante quando se analisa a cada distância da superfície dental. Conclui-se que perdas de mineral só foram significativas quando da exposição a sacarose 8x/dia, não havendo entretanto relação com a contagem de Estreptococos do "grupo mutans"
Abstract: The quantitative relationship among frequency of sucrose intake, dental caries and S. mutans counts are not well established. Therefore, it was performed an in situ study utilizing an experimental design of the crossover type (4x4) in four phases of 28 days. Twelve volunteers using intra-oral palatal appliances, containing 4 blocks of human dental enamel (3x3 mm) participated in this research. The volunteers dropped on the dental blocks, 20% sucrose solution in a frequency from 0 (zero) to 8x/day. The dental blocks were protected by a plastic cover and the volunteers used for their bucal hygiene, non fluoridated dentifrice, but the water consumed by them was fluoridated (0.70 ppm). After each phase, the dental plaque formed on the blocks was collected, weighed, homogenized and assessed for S. mutans count (CFU/mg) using selective media. The dental blocks were clean, embedded, cut and polished to the Knoop hardness determination (KHN) of the enamel. It was done indentations at 10 'mu¿m of the surface using SHIMADZU H 2000 microhardness tester and 25 g load for 30 sec. The microbiological results in average '+ or ¿' standard deviation of the media of CFU/mg were, respectively in relation to the sucrose exposure of 0 (zero), 2, 4, 8x/day: 26.72 '+ or ¿'13.36A; 46.72 '+ or ¿' 30.81A; 102.44 '+ or ¿'53.34A and 52.18 '+ or - ' 21.48A . The media followed by the same letter are not statistically different at the 5% level. In the enamel hardness test, significative differences (p<0.05) in relation to the total area only were observed when the sucrose exposure was 8x/day, similar results were obtained when we assessed at each distance the dental surface. We can conclude that mineral loss only was significative when the sucrose exposure was 8x/day, although there was not a relationship to the S. mutans counts
Mestrado
Biologia e Patologia Buco-Dental
Mestre em Odontologia

Orientador: Marcelo de Carvalho Ramos
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Objetivo: Deternllnar a freqüência do alelo recessivo ccr-5 em população de indivíduos infectados pelo HIV -1. Material e Métodos: Foram estudados 187 pacientes, que freqüentam os ambulatórios da Disciplina de Moléstias Infecciosas do Hospital das Clínicas da UNICAMP. Foram selecionados pacientes de ambos os sexos com testes positivos para o anti-HIVl por ELISA e Westem-Blot, em qualquer fase clínica da doença. Para amplificação foram utilizados os seguintes primers: 5'-CCTGGCTGTCGTCCATGCTG-3' e 5'CTGATCTAGAGCCATGTGCACAACTCT-3'. Após a desnaturação a 94°C por 5min, seguiram-se 34 ciclos de: 94°C por lmin; 57°C por lmin; 72°C por lmin e extensão final a 72°C por 7min. A seguir, foi realizada a digestão enzimática com 1 J.lg de cada amostra, incubadas por 60min à 37°C com 10U de EcoRI. Foram também deternllnadas a carga viral pela técnica do NASBA e deternllnação de subpopulações linfocitárias por citometria de fluxo (Ortho Diagn.). Resultados e Conclusões: Do total de 187 indivíduos analisados, 15 apresentaram padrões de restrição do produto de PCR compatíveis com heterozigose para a deleção delta 32 (freqüência=8,02%). Essa freqüência é semelhante à encontrada em populações caucasianas européias. Com respeito à carga viral e quantidade de células CD4 e CD8, os dados foram prejudicados pela administração de medicação antiretroviral na maioria desses indivíduos. Todos os pacientes que apresentavam a mutação e que pud~ram ser classificados clinicamente possuíam estadiamento B2 ou maior
Abstract: Previous studies have shown a relationship between a 32-base-pair deletion within the J3-chemokine receptor 5 (ccr5) gene and the acquisition and progression of the Acquired Human Immunodeficiency Syndrome (AIDS). Several. populations have been tested concerning this deletion, and it was found that Caucasians have a higher probability of bearing this defect, whereas in black and japanese populations it is virtually absent. In Brazil, no studies have been done to estimate this frequency in HIV infected populations, so faro In an urban brazilian population 93% of individuaIs tested showed normal CCR-5 anele and 7% were heterozygous CCRS/ ~ccr5. In this study 187 HIV-infected persons were tested to determine the prevalence of this mutation. Genomic DNA samples were amplified using primers producing a 735bp fragment, which was submmited to c1eavage with EcoRl. Normal homozygous aneles showed two fragments of 332 and 403 bp, and in heterozygous aneles an additional371 bp fragment was found. No homozygous individuaIs were found in this population, and the frequency ofheterozygous CCR5/~ccr5 was found to be 8,02%
Mestrado
Ciencias Basicas
Mestre em Clinica Medica

This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

碩士
國立陽明大學
醫學生物技術研究所
87
The main purpose of the research project is to study the effect of ionizing radiation to HPRT gene. There are two separate, but related, subjects studied in this project. The first part was to investigate the mutant frequency of hprt gene induced by ionizing radiation in TK6 human lymphoblast cells. The second part was to study the difference of radiosensitivity between three human nasopharyngeal carcinoma (NPC) cell lines obtained from the NPC prevalent regions, i.e. Taiwan (CG1), China (CNE1) and Hong Kong (HK1). The purpose of this study is to find the correlation between the radiosensitivity and the hprt gene mutant frequency. The results of the first study showed that radiation-induced hprt gene mutant frequency of TK6 cells was (5.01  1.82) ×10-6 and was significantly higher than that of spontaneous mutant frequency which was (1.43  0.53) ×10-6 (p<0.01). This finding indicates that mutant frequency at HPRT gene locus can be induced by ionizing radiation. The results of the second study showed that D10 were 5.5 Gy for CNE1 cell, 6.75 Gy for CG1 cell and 7.0 Gy for HK1 cell, respectively. This result indicates that the CNE1 cells were the most radiosensitive among the three cell lines. The results also proved that mutant frequency at HPRT gene locus induced by ionizing radiation was increased both in CNE1 and HK1 cells. However, CNE1 cells was less mutable compared with HK1 cells. This finding indicates that the negative correlation exists between the radiosensitivity and the induced mutant frequency, i.e. the more radiosenstive the cells were the lower mutant frequency they were induced. This hypomutability for more radiosensitive cells found in NPC cells was consistent with the study found in bladder cancer cell lines reported by other investigators.

Oncogenesis is tightly related to the occurrence of somatic mutations. Somatic mutations are constantly produced by DNA replication processes, as the activity of DNA polymerases and DNA repair systems is highly efficient, but not perfect. The great part of spontaneous somatic mutations is irrelevant to cancer, but a very small fraction of them is oncogenic. Therefore, the individual predisposition to develop somatic mutations may be considered as a risk factor for tumor development. In order to investigate whether there is any association between the rate of occurrence of somatic mutations and the individual risk of developing cancer, we used the flow cytometry PIGA mutant assay to measure the mutant frequency in peripheral blood granulocytes of 177 healthy subjects and of a cohort of 195 patients affected by Philadelphia-negative classical MPN.

碩士
國立陽明大學
放射醫學科學研究所
89
Abstract The head and neck cancers, include tongue, larynx, pharynx, tonsils and nasopharyngeal carcinoma, are common diseases in Asia. In Taiwan, about 8% of cancer mortality, i.e. 2400 people each year, belongs to the head and neck cancer. In this study, colony and immunoflurescent staining assays were used to detect mutant frequency and molecular characterization of the HPRT gene in lymphocytes of patients with head and neck cancers before and after radiotherapy. No significant difference was found for HPRT variant frequency (Vf) measurement between the colony assay and the immunofluorescence staining assay (P=0.754). A mean variant frequency of (3.37±2.14) ×10-5 of the HPRT gene in patients before radiotherapy. The variant frequency of HPRT gene is significantly increased in five patients (45%) during treatment, and in seven patients (78%) after treatment. The HPRT Vfs of patients, were elevated 3 folds to 13 folds, respectively, (p＜0.01) at the end of radiotherapy. These data suggest that the HPRT variant frequency of head and neck cancer patients was increased about 6 folds after radiotherapy.

碩士
國立陽明大學
放射醫學科學研究所
92
Radiobiological effect under low doses of most studies have been obtained by back extrapolating the cell survival from high doses. It does not demonstrate an accurate response of the effects of most environmental exposures, which tend to be of low dose and protracted over time. For this reason, the effect of high and low dose-rate (HDR, LDR), low doses ionizing radiation (IR) on the mutant frequency (MF) and the mutation spectrum at the hypoxanthine phosphoribosyl transferase (hprt) locus was studied in Chinese hamster ovary (CHO-10B2) cell line. Cells were cultured in the medium containing 6-thioguanine (6-TG) for 14 days to select for hprt mutants following 0, 0.2, 0.5, 0.75 and 1.0 Gy Co-60 γ- ray exposure. The mutation spectrum was determined for exon deletion analysis by multiplex polymerase chain reaction (M-PCR). Intact mutants were further analyzed by sequencing. In addition, cell cycle distribution analysis and mitotic cells were confirmed by flow cytometry and mitotic index (MI), respectively. The results showed that hprt MF was increased with radiation doses under both HDR and LDR irradiation. However, the higher MF induced by 0.2 Gy suggests that hyper-radiosensitivity (HRS) and inverse dose-rate effect likely exist around this dose range. The dose response for the induction of hprt large deletions were significantly increased with radiation dose (r2=0.99, p=0.0064) compared to the point mutation (r2=0.29, p=0.35) induced by LDR IR which is the major of spontaneous mutations. In addition, exons deletions were found mostly at both ends of hprt gene. Terminal deletions were the predominant mutation type (r2=0.95, p=0.02). The higher deletion mutation at 0.2 Gy under LDR irradiation was also found. Furthermore, base sequencing of intact mutations seemed to indicate that hprt exons 3-9, and bases A or T were mostly substituted and about 22% were silent mutations.

碩士
國立陽明大學
放射醫學科學研究所
88
The main purpose of this project is to study the difference of the radiation-induced hprt gene mutant frequency and mutation spectra among three human nasopharyngeal carcinoma (NPC) cell lines obtained from the NPC prevalent regions, i.e. Taiwan (CG1 cells), Hong Kong (HK1 cells) and China (CNE1 cells). The results showed that mutant frequency at hprt gene locus induced by ionizing radiation was increased in all of the three NPC cell lines. A total of 57 CNE1 mutants (5 spontaneous, 52 radiation-induced) were isolated and genetic alterations at the locus were studies by multiplex-PCR. The analysis showed that majority of both spontaneous (4/5) and radiation-induced (37/52) mutants were terminal deletions. The dose response for the induction of deletion mutations best fit a quadratic dose response（r2＝0.99）. Terminal deletions also increased quadratically with radiation dose （r2＝0.99）, while the dose response for intragenic deletion best fit linear dose response （r2＝0.94）. Sequence analysis of hprt cDNA of CNE1 cells showed that the radiation-induced mutation types were transversion. 2GCN9 was found with base substitution at position 513 ( T＞A) so the amino acid was changed from serine to aspartic acid. The position 353 ( base 1 is the A of the initiation codon) of 6GCN4 was G to T so the amino acid changed from serine to aspartic acid. In conclusion, the present results concerning the hprt mutants induced by ionizing radiation of three human NPC cell lines showed the following: First, the radiosensitivity of three human NPC cells versus the induced hprt mutant frequency is negatively correlated. Second, it may valuable in understanding the mechanism(s) of high-dose radiation induced DNA damage, through the analysis of hprt mutant spectrum and cDNA sequencing. Third, the radiosensitivity difference among NPC patients may be elucidated by analyzing the molecular spectra of NPC cells.

碩士
國立臺灣大學
職業醫學與工業衛生研究所
90
Part I The association between metabolic polymorphisms and cigarette smoking-induced cancers has been documented. However, the role of DNA repair polymorphism in carcinogenesis is less clear. To investigate if the polymorphisms of metabolic traits and DNA repair modulate smoking-related DNA damage, we used sister chromatid exchange (SCE) as a marker of genetic damage to explore the relationship of microsomal epoxide hydrolase (mEH), glutathione S-transferase M1 (GSTM1), and x-ray cross-complementing group 1 (XRCC1) and cigarette smoking-induced SCE. Sixty-one workers without significant exposure to mutagens were recruited. Questionnaires were completed to obtain detailed occupational, smoking, and medical histories. SCE frequency in peripheral lymphocytes was determined using a standard cytogenetic assay and GSTM1, mEH (exon 3, eoxn 4), XRCC1 (codon 399) genotypes were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP). Smokers had higher SCE frequency than nonsmokers (8.4 vs. 7.1, p < 0.05). Among workers who had smoked equal to or greater than 10 cigarettes each day, those with XRCC1 Arg/Gln + Gln/Gln had higher SCE frequency than those with XRCC1 Arg/Arg after adjusting for potential confounders (9.0 vs. 7.9, p < 0.05). The interaction of XRCC1 and cigarettes smoked per day on SCE frequency was also observed (p = 0.02). There was no significant interaction between cigarettes smoked per day with GSTM1 and mEH on SCE frequency. Our results support previous epidemiological studies that XRCC1 may play a role in cigarette smoking-induced lung cancer. Part II The presence of mutant Asp13-K-ras protein, p53 overexpression and anti-p53 antibody have been reported to be associated with vinyl chloride monomer (VCM)-related cancers. The aim of this study was to compare the relationship between VCM-induced p53 and K-ras oncoproteins, and to further investigate the role of polymorphisms of metabolic and DNA repair genes on VCM-induced oncoprotein expression. We examined the plasma samples of 218 male workers occupationally exposed to VCM. Plasma mutant p53 protein and anti-p53 antibody were detected with enzyme-linked immunosorbent assay (ELISA), and Asp13-K-ras proteins were detected using enhanced chemiluminescence Western blotting. Genotypes of cytochrome P450 2E1 (CYP2E1), aldehyde dehydrogenase 2 (ALDH2), glutathione S-transferase T1 (GSTT1) and X-ray repair cross-complementing group 1 (XRCC1, exon 10) were identified using the polymerase chain reaction (PCR). The results revealed that the plasma mutant p53 protein was positive in 10.1% of workers, anti-p53 antibody was positive in 5% of workers and Asp13-K-ras protein was positive in 10.1% of workers. High VCM exposure group (>40 ppm-years) had significantly higher mutant oncoprotein (mutant p53 protein, anti-p53 antibody or Asp13-K-ras protein) expression as compared to low VCM exposure group (<40 ppm-years) (OR=2.0, 95%CI=1.0-3.8). Among high exposure workers, subjects with XRCC1 Gln/Gln genotypes demonstrated significantly higher risk of mutant oncoproteins expression as compared to those with XRCC1 Arg/Arg or Arg/Gln variants (OR=8.5, 95%CI=1.9-38.9) after adjusting for potential confounders. Moreover, there was an interaction between VCM exposure and XRCC1 polymorphisms on oncoprotein expression (p=0.06). In our further analysis, amongst low exposure workers, subjects with CYP2E1 c2c2 genotypes demonstrated greater risk than subjects with CYP2E1 c1c1 or c1c2 genotypes (OR=3.1, 95%CI=0.3-37.8). However, there was no interaction between VCM exposure and genotypes of CYP2E1, GSTM1, and ALDH2 on oncoproteins. We found that p53 overexprssion was significantly associated with VCM cumulative dose (OR=2.5, 95%CI=1.0-6.2). After adjusted for smoking, age, drinking, and hepatitis infection, amongst high exposure workers, subjects with XRCC1 Gln/Gln demonstrated significantly greater risk of p53 overexpression than subjects with Arg/Arg or Arg/Gln (OR=17.0, 95%CI=4.3-198.5). Amongst low exposure workers, subjects with CYP2E1 c2c2 genotypes have significantly higher risk than CYP2E1 c1c1 and c1c2 genotypes (OR=19.9, 95%CI=1.6-253.2). Mutant Asp13-K-ras oncoprotein was not associated with VCM cumulative exposure dose; however, it was significantly associated with current high exposure experience within 5 years of sample collection (OR=2.9, 95%CI=1.1-7.9). After further analysis, we found that subjects experiencing high exposure job within 5 years with XRCC1 Gln/Gln had greater risk of K-ras oncoprotein than those with XRCC1 Arg/Arg or Arg/Gln (OR=1.7, 95%CI=0.1-23.3) although didn’t reach statistical significance. Similarly, subjects with ALDH2 1-2 or 2-2 genotypes demonstrated greater risk than those with ALDH2 1-1 genotypes (OR=2.5, 95%CI=0.6-11.2). Further, subjects experiencing high exposure job within 5 years with GSTT1 non-null genotype revealed significantly higher risk than those GSTT1 null type. Our results suggest that mutant p53 protein and mutant K-ras oncoprotein revealed differential expression in VCM-exposed workers. Mutant K-ras oncoprotein was associated with current exposure, and it became undetected when exposure ends. However, mutant p53 protein was associated with cumulative dose. One possible explanation is mutant p53 gene provide selective growth advantage which making mutant p53 protein sustained in the cell persistently. In contratst, K-ras mutants may be demolished by p53 through the apoptosis. Furthermore, the relationship between p53 overexpression and DNA repair gene and metabolic genes was consistent with our previous study, but the association of mutant K-ras oncoprotein and DNA repair gene and metabolic traits are not clear in this study because of small sample size. Therefore, further studies are needed to elucidate the exact mechanism.

CRISPR-Cas9 is a recent discovered genetic editing mechanism, that shows a lot of versatility. This allows scientists to do genetic manipulation with relative ease when compared with others current genetic tools available. One possible application of the CRISPR-Cas9 system is to mimic human disease mutations by targeting orthologous genes in animal models, which allows a better characterization of the mechanisms behind a particular disease. Cilia are hair-like structures that protrude from the cell surface in organisms and can be classified as motile or non-motile. They are responsible for several important functions throughout the human body. Such functions include, generating fluid flow and sensing mechanical or chemical cues from the surrounding environment. If these are compromised it can lead to ciliopathies. Ciliopathies are a group of diseases and syndromic diseases characterized by malfunctioning of cilia. Motile cilia can lead to a disease known as primary ciliary dyskinesia (PCD). More than 35 genes have been linked with cilia motility in PCD patients. Some of these genes are associated with the inner dynein arms present in the axoneme. A better understanding of mutations in these genes would help the characterization of PCD. Using CRISPR-Cas9 we tried to cause a mutation in dnah7, a gene that encodes a protein present in inner dynein arms. Two SgRNAs were selected to disrupt dnah7 and injected into zebrafish embryos. These F0 embryos were screened for mutations outcrossed and left to sexually mature. When matured, the progeny was screened again to find any heritable mutations. Meanwhile, analyses of cilia beat frequency and pattern, the readouts of cilia function, were made in a set of wild type and ccdc40 MO injected zebrafish. Additionally, two SgRNAs were designed for targeting another PCD commonly mutated gene named rsph4a, a gene coding for a protein present in the radial spokes of the axonemes.

Summary: Background: An increased risk for development of hereditary breast cancer is associated with germline mutations in BRCA1/2 and the influence of NBN mutations is also supposed. The aim of this study is to specify the frequency of recurrent mutations in BRCA1/2 in unselected breast cancer patients and the frequency of most common pathogenic mutations in NBN in Czech republic, to assess current criteria for genetic testing and to consider the addition of NBN to the tested genes. Methods: Screening for recurrent mutations 5382insC and 300T>G in BRCA1 was performed by RFLP, screening for mutations in exon 11 of BRCA1 was performed by PTT, screening for mutations in a selected region of exon 11 of BRCA2 by DHPLC, and screening for mutations in exon 6 of NBN by HRMA. All the mutations were confirmed by direct sequencing. Results: In 679 unselected breast cancer patients 7 carriers of 5382insC, 3 of 300T>G, and 4 of other mutations in BRCA1 were identified. 2 locally prevalent mutations were found in BRCA2. In 730 controls only one 5382insC BRCA1 mutation was identified. Out of 5 NBN mutations found in 600 high-risk patients two were 657del5 and one R215W. A total of 8 NBN mutation carriers were identified among 703 breast cancer patients, 2 of them 657del5 carriers and three R215W carriers. In 915...