DNA methylation patterns of candidate genes regulated by thymine DNA glycosylase in patients with TP53 germline mutations

Li-Fraumeni syndrome (LFS) is a rare, autosomal dominant, hereditary cancer predisposition disorder. In Brazil, the p.R337H TP53 founder mutation causes the variant form of LFS, Li-Fraumeni-like syndrome. The occurrence of cancer and age of disease onset are known to vary, even in patients carrying the same mutation, and several mechanisms such as genetic and epigenetic alterations may be involved in this variability. However, the extent of involvement of such events has not been clarified. It is well established that p53 regulates several pathways, including the thymine DNA glycosylase (TDG) pathway, which regulates the DNA methylation of several genes. This study aimed to identify the DNA methylation pattern of genes potentially related to the TDG pathway (CDKN2A, FOXA1, HOXD8, OCT4, SOX2, and SOX17) in 30 patients with germline TP53mutations, 10 patients with wild-type TP53, and 10 healthy individuals. We also evaluated TDG expression in patients with adrenocortical tumors (ADR) with and without the p.R337H TP53 mutation. Gene methylation patterns of peripheral blood DNA samples assessed by pyrosequencing revealed no significant differences between the three groups. However, increased TDG expression was observed by quantitative reverse transcription PCR in p.R337H carriers with ADR. Considering the rarity of this phenotype and the relevance of these findings, further studies using a larger sample set are necessary to confirm our results.

Nucleotide excision repair pathway assessment in DNA exposed to low-intensity red and infrared lasers

Low-intensity lasers are used for prevention and management of oral mucositis induced by anticancer therapy, but the effectiveness of treatment depends on the genetic characteristics of affected cells. This study evaluated the survival and induction of filamentation of Escherichia coli cells deficient in the nucleotide excision repair pathway, and the action of T4endonuclease V on plasmid DNA exposed to low-intensity red and near-infrared laser light. Cultures of wild-type (strain AB1157) E. coli and strain AB1886 (deficient in uvrA protein) were exposed to red (660 nm) and infrared (808 nm) lasers at various fluences, powers and emission modes to study bacterial survival and filamentation. Also, plasmid DNA was exposed to laser light to study DNA lesions produced in vitro by T4endonuclease V. Low-intensity lasers:i) had no effect on survival of wild-type E. coli but decreased the survival of uvrA protein-deficient cells,ii) induced bacterial filamentation, iii) did not alter the electrophoretic profile of plasmids in agarose gels, andiv) did not alter the electrophoretic profile of plasmids incubated with T4 endonuclease V. These results increase our understanding of the effects of laser light on cells with various genetic characteristics, such as xeroderma pigmentosum cells deficient in nucleotide excision pathway activity in patients with mucositis treated by low-intensity lasers.

B cells expressing IL-10 mRNA modulate memory T cells after DNA-Hsp65 immunization

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43−) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Optimization of random amplified polymorphic DNA protocol for molecular identification of Lophius gastrophysus

Lophius gastrophysus has important commercial value in Brazil particularly for foreign trade. In this study, we described the optimization of Random Amplified Polymorphic DNA (RAPD) protocol for identification of L. gastrophysus. Different conditions (annealing temperatures, MgCl concentrations, DNA quantity) were tested to find reproducible and adequate profiles. Amplifications performed with primers A01, ² A02 and A03 generate the best RAPD profiles when the conditions were annealing temperature of 36ºC, 25 ng of DNA quantity and 2.5 mM MgCl2. Exact identification of the species and origin of marine products is necessary and RAPD could be used as an accurate, rapid tool to expose commercial fraud.

Heterologisen geenin DNA-sekvenssin optimointi sekä Fab-fragmentin DNA-sekvenssin vaikutus Escherichia colin kasvuun ja tuotto-ominaisuuksiin

Synonyymisillä kodoneilla tarkoitetaan kodoneja, jotka ovat erilaisia, mutta koodaavat samaa aminohappoa. Synonyymisten mutaatioiden, eli kodonin vaihtaminen samaa aminohappoa koodaavaan toiseen kodoniin, on pitkään ajateltu olevan yhdentekeviä, mutta synonyymit muutokset DNA-sekvenssissä voivat kuitenkin vaikuttaa esimerkiksi proteiinin laskostumiseen ja proteiinin toimintaan solussa. Eri organismit käyttävät synonyymisiä kodoneja eri frekvensseillä. Tätä ilmiötä kutsutaan kodonikäytön poikkeamaksi. Kodonikäytön poikkeamien on osoitettu olevan tärkein yksittäinen prokaryoottien geeniekspressioon vaikuttava tekijä, ja usein proteiineja on hankala tuottaa vieraassa isännässä, jos kodonikäytön poikkeamat ovat liian suuria. Erilaisia kodonioptimointistrategioita on kehitetty näiden ongelmien ratkaisemiseksi. Kodonikäyttöä optimoitaessa on otettava huomioon lisäksi erilaiset paikalliset muuttujat, jotka lisäävät optimoinnin monimutkaisuutta. Työn lähtökohtana oli synteettinen ihmisen vasta-aineen Fab-fragmentin geeni. Geeni oli optimoitu kahdella eri strategialla, jotka tuottivat eri DNA-sekvenssit, mutta saman aminohapposekvenssin. Toinen varianteista tuotti aktiivista Fab-fragmenttia, toinen ei. DNA-sekvenssin vaikutuksen tutkimiseksi, toimivan geenin osia korvattiin toimimattoman variantin vastaavalla osalla. Kaikkiaan seitsemän geenivariantin kykyä ilmentää Fab-fragmenttia sekä liukoisena proteiinina että filamenttifaagin pinnalla vertailtiin. Lisäksi tutkittiin varianttien vaikutusta isäntäsolun kasvukinetiikkaan. Muunneltuja variantteja verrattiin alkuperäiseen toimivaan varianttiin. Faagituotossa havaittiin Fab-fragmentin kevyen ketjun DNA-sekvenssin synonyymisten muutosten vaikuttavan faagien immunoreaktiivisuuteen. Erityisen olennainen oli kevyen ketjun vakioisen alueen muuttaminen, joka myös aiheutti 45 % laskun faagien kokonaismäärässä ja 54 % laskun faagien immunoreaktiivisuudessa. Liukoista proteiinia tuotettaessa kevyen ketjun vakioisen alueen muutos laski Fab-määrän tasolle, jota ei voitu mitata. Tämän lisäksi, avoimen lukukehyksen alun kodonien synonyymiset mutaatiot aiheuttivat 48-kertaisen laskun aktiivisen liukoisen proteiinin määrässä verrattuna alkuperäiseen toimivaan varianttiin.

Use of real-time PCR to evaluate two DNA extraction methods from food

The DNA extraction is a critical step in Genetically Modified Organisms analysis based on real-time PCR. In this study, the CTAB and DNeasy methods provided good quality and quantity of DNA from the texturized soy protein, infant formula, and soy milk samples. Concerning the Certified Reference Material consisting of 5% Roundup Ready® soybean, neither method yielded DNA of good quality. However, the dilution test applied in the CTAB extracts showed no interference of inhibitory substances. The PCR efficiencies of lectin target amplification were not statistically different, and the coefficients of correlation (R²) demonstrated high degree of correlation between the copy numbers and the threshold cycle (Ct) values. ANOVA showed suitable adjustment of the regression and absence of significant linear deviations. The efficiencies of the p35S amplification were not statistically different, and all R² values using DNeasy extracts were above 0.98 with no significant linear deviations. Two out of three R² values using CTAB extracts were lower than 0.98, corresponding to lower degree of correlation, and the lack-of-fit test showed significant linear deviation in one run. The comparative analysis of the Ct values for the p35S and lectin targets demonstrated no statistical significant differences between the analytical curves of each target.

Extraction of DNA from honey and its amplification by PCR for botanical identification

The physiochemical and biological properties of honey are directly associated to its floral origin. Some current commonly used methods for identification of botanical origin of honey involve palynological analysis, chromatographic methods, or direct observation of the bee behavior. However, these methods can be less sensitive and time consuming. DNA-based methods have become popular due to their simplicity, quickness, and reliability. The main objective of this research is to introduce a protocol for the extraction of DNA from honey and demonstrate that the molecular analysis of the extracted DNA can be used for its botanical identification. The original CTAB-based protocol for the extraction of DNA from plants was modified and used in the DNA extraction from honey. DNA extraction was carried out from different honey samples with similar results in each replication. The extracted DNA was amplified by PCR using plant specific primers, confirming that the DNA extracted using the modified protocol is of plant origin and has good quality for analysis of PCR products and that it can be used for botanical identification of honey.

Análise da pureza genética e discriminação de cultivares de vinca (Catharanthus roseus (L.) G.Don) usando "random amplified polymorphic DNA" em DNA extraído de sementes e folhas

A técnica "Random Amplified Polymorphic DNA" (RAPD) surgiu como uma ferramenta útil para testar a pureza genética e a discriminação de cultivares em muitas espécies. É uma técnica simples, rápida, relativamente de baixo custo e permite o uso de DNA extraído de sementes secas, o que é muito importante em um programa de análise de sementes. O uso desta tecnologia no teste da pureza genética pode ser muito interessante para algumas espécies, como a vinca (Catharanthus roseus (L.) G.Don), pois pouco se conhece a respeito da seqüência de seu DNA. É interessante, também, pelo fato de existir grande número de "primers" comercialmente disponíveis, que podem ser prontamente utilizados para gerar dados. Essa técnica pode ser mais facilmente utilizada para gerar padrões de bandas polimórficas suficientes para discriminar genótipos diferentes. Todavia, no presente estudo, os padrões RAPD de bandas obtidas de amostras de DNA extraído de sementes de vinca em "bulk" foram não-consistentes, o mesmo ocorrendo com o uso de DNA extraído de sementes individuais de um mesmo cultivar, o que evidencia que a técnica não é aplicável para testar a pureza genética e a discriminação de cultivares de vinca. Entretanto, os padrões de bandas RAPD gerados a partir de DNA extraído de tecido foliar de plântulas foram mais reproduzíveis e poderiam ser considerados na caracterização de cultivares.

Rekombinanttitekniikalla tuotettujen vasta-aineiden käyttö immunodiagnostiikassa ; rekombinantti-DNA-tekniikalla tuotetun Fab-fragmentin käyttö sydänperäisen troponiini I:n immunomäärityksessä

Rekombinanttivasta-aineet ovat synteettisesti valmistettuja vasta-aineita, jolloin niiden tuottamiseen ei tarvita eläintä. Rekombinantti-DNA-tekniikalla pystytään valmistamaan eri vasta-aineluokkia tai pelkästään niiden fragmentteja lähes mitä tahansa antigeeniä vastaan. Vasta-aineita voidaan etsiä eri antigeenejä vastaan ilmentämällä niitä esimerkiksi bakteriofagien tai solujen pinnalla, ja niiden sitomiskykyä kohteeseensa voidaan parantaa erilaisten mutaatioiden avulla. Rekombinanttivasta-aineita voidaan hyödyntää laajasti erilaisissa immunodiagnostisissa menetelmissä lääketieteessä, ympäristö- sekä elintarviketutkimuksissa. Tänä päivänä rekombinanttivasta-aineita käytetään myös terapiahoidossa. Immunomääritysten ongelmana voivat olla erilaiset häiriötekijät, jotka saattavat aiheuttaa väärän positiivisen tai negatiivisen tuloksen. Rekombinanttivasta-aineilla, erityisesti vasta-ainefragmenteilla voidaan vähentää määritysten häiriötä ja parantaa tulosten luotettavuutta. Rekombinanttivasta-aineiden hyötyjä ovat myös niiden nopea tuottaminen, helppo muokkaaminen sekä monipuolisuus erilaisia antigeenejä vastaan. Tutkimuksen kokeellisen työn tarkoitus oli kehittää uudelle rekombinantti-DNA-teknii-kalla tuotetulle osittain humanisoidulle Fab-fragmentille troponiini I -immunomääritys. Sydänperäinen troponiini I on sydäninfarktille spesifinen merkkiaine, jota voidaan mitata verestä. Työssä käytettävässä immunomäärityksessä sitojavasta-aineina käytettiin kahta biotinyloitua vasta-ainetta, joista toinen oli hiiren monoklonaalinen vasta-aine ja toinen oli kimeerinen Fab-fragmentti. Määrityksen leimana käytettiin uutta Fab-fragmenttia, joka kiinnitettiin kovalenttisesti europiumkelaatteja sisältävään nanopartikkeliin. Työssä pystyttiin kehittämään uudelle Fab-fragmentille partikkelipäällystysmenetelmä ja sitä hyödyntävä cTnI-immunomääritys. Määrityksen korkeaa taustaa saatiin merkittävästi vähennettyä käyttämällä määrityksessä polyetyleeniglykolilinkkeriä, joka esti leimavasta-aineen epäspesifistä sitoutumista. Immunomäärityksen herkkyyden parantamiseksi optimointeja tarvitaan lisää, mutta määritykselle on mahdollista kuitenkin saavuttaa tulevaisuudessa herkkä immunomääritys, jonka alttius häiriötekijöille on pieni.

Data analysis tools and methods for DNA microarray and high-throughput sequencing data

The recent rapid development of biotechnological approaches has enabled the production of large whole genome level biological data sets. In order to handle thesedata sets, reliable and efficient automated tools and methods for data processingand result interpretation are required. Bioinformatics, as the field of studying andprocessing biological data, tries to answer this need by combining methods and approaches across computer science, statistics, mathematics and engineering to studyand process biological data. The need is also increasing for tools that can be used by the biological researchers themselves who may not have a strong statistical or computational background, which requires creating tools and pipelines with intuitive user interfaces, robust analysis workflows and strong emphasis on result reportingand visualization. Within this thesis, several data analysis tools and methods have been developed for analyzing high-throughput biological data sets. These approaches, coveringseveral aspects of high-throughput data analysis, are specifically aimed for gene expression and genotyping data although in principle they are suitable for analyzing other data types as well. Coherent handling of the data across the various data analysis steps is highly important in order to ensure robust and reliable results. Thus,robust data analysis workflows are also described, putting the developed tools andmethods into a wider context. The choice of the correct analysis method may also depend on the properties of the specific data setandthereforeguidelinesforchoosing an optimal method are given. The data analysis tools, methods and workflows developed within this thesis have been applied to several research studies, of which two representative examplesare included in the thesis. The first study focuses on spermatogenesis in murinetestis and the second one examines cell lineage specification in mouse embryonicstem cells.

Desiccation tolerance and DNA integrity in Eugenia pleurantha O. Berg. (myrtaceae) seeds

The aim of this study was to assess the desiccation tolerance and DNA integrity in Eugenia pleurantha seeds dehydrated to different moisture contents (MCs). Seeds extracted from mature fruits were submmited to drying in silica gel and evaluated at every five percentual points of decrease from the initial MC (35.5%, fresh weight basis). The effects of dehydration on seeds were verified through germination tests and DNA integrity assessment. Undried seeds achieved 87% germination, value reduced to 36% after being dried to 9.8% MC. When dried slightly more, to 7.4% MC, seeds were no longer able to germinate, suggesting an intermediate behavior in relation to desiccation tolerance. It was observed DNA degradation in seeds with 7.4% MC, which might have contributed to the loss of seed germination.