Quantitative fluorescence polymerase chain reaction (QF-PCR) is a molecular genetic method based on the amplification of microsatellites (Short tandem repeats, STR) and measurement of the peak heights of amplicons in the electropherogram. Currently, the QF-PCR deemed reliable, fast, and inexpensive method that is gradually replacing conventional cytogenetic analysis of aneuploidy (examination of long-term cultures of amniotic fluid). However, in certain cases it is impossible to determine the parental origin and meiotic aneuploidy by QF-PCR. The aim of this work was to verify the new dinucleotide STR markers on chromozomes 13, 16, 18, 21, and 22 and further increase the diagnostic efficiency of QF-PCR retaining other STR markers on chromozome 15, 16, 22 and to determine the population and the analytical characteristics of these markers. For all dinucleotide STR markers stutter occurred in high frequency and therefore there were found not to be suitable for routine diagnostics. STR markers for chromozomes 15, 16 and 22 were tested on 100 patients. We selected four informative markers for both chromozome 16 and 22, and three markers for chromozome 15. Thus, I expanded set of diagnostic STR markers in this thesis.

Despite the prevalent utilization of capillary electrophoresis (CE) in the analysis of short tandem repeats (STRs) to generate deoxyribonucleic acid (DNA) profiles for forensic comparisons, the method is not without its inherent drawbacks. Massively parallel sequencing (MPS) is still a relatively novel technology in the forensics field, but contains the capacity to address current challenges faced by the traditional CE approach - such as degraded samples, low template DNA, and artifacts - while also providing additional information such as isoalleles, same-length alleles with sequence variation, and ancestry, mixture, and phenotyping-informative single nucleotide polymorphisms (SNPs). One of the principal ongoing challenges faced by both technologies is the presence of artifacts such as stutter, a byproduct of slipped strand mispairing during amplification of STRs, which can further complicate interpretation of DNA profiles. Understanding and predicting the behavior of stutter is important in establishing appropriate thresholds to distinguish these artifacts from true alleles. With complex MPS data, new approaches in characterizing stutter have been established such as the BLMM and simplified sequence. In this study, twenty-one oral samples from individuals belonging to the same family were constructed into libraries containing 58 STR regions and 98 identity SNPs using Verogen’s Forenseq™ DNA Signature Prep Kit and sequenced on the MiSeq FGx™ Forensics Genomics System. Isoallele and stutter sequences were extracted from the data and simplified using the longest uninterrupted stretch (LUS), block length of missing motif (BLMM) and simplified sequence approaches. It was found that the stutter ratio for the 11 isoallele pairs at the D13S317 locus did not follow the linear correlation with increasing LUS. Instead, the isoallele with the higher LUS demonstrated equal or lower stutter ratios. Additionally, three different stutter patterns were identified for the same locus. Based on the provided pedigree, ten different relations were defined and the amount of allele sharing between the individuals in the pedigree was analyzed with and in the absence of isoallelic information to determine its impact on predicting relatedness. It was found that there was an average of 1.3% difference across the ten defined categories when isoalleles were taken into consideration. However, the difference in the percentage of shared alleles was not found to be significant for each of the relations category between the results before and after the consideration of isoallelelic data.

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Darbdavio prievolė mokėti darbo užmokestį kyla iš darbo teisinių santykių, o darbuotojo gaunamas darbo užmokestis yra svarbiausia socialinė garantija užtikrinanti jo pragyvenimo lygį. Nors šiandieną darbo užmokestis nėra vienintelis atlygis, kurį darbuotojas gauna mainais už atliktą darbą, tačiau jis vis dėlto išlieka vienu svarbiausių. Pagrindinis darbo užmokesčio mokėjimą Lietuvoje reguliuojantis teisės aktas yra Lietuvos Respublikos darbo kodeksas. Pagal Lietuvos Respublikos darbo kodekso 186 straipsnį, darbo užmokestis yra atlyginimas už darbą, atliekamą darbuotojo pagal darbo sutartį, kuris yra mokamas pinigais. Darbo užmokesčio išmokėjimo pareiga atsiranda darbo sutarties pagrindu. Jo dydis ir mokėjimo sąlygos yra nustatomos susitarimu, o tam tikri minimalūs standartai – įstatymais ir juos įgyvendinančiais teisės aktais. Darbo užmokestis yra periodinė išmoka, ir kai ši darbuotojui priklausanti išmoka nustatytu laiku neišmokama, nesvarbu visa ar iš dalies, pažeidžiamos darbuotojo teisės. Už pareigos laiku mokėti darbo užmokestį nesilaikymą, darbdaviui nustatytos sankcijos, kurios įtvirtintos Lietuvos Respublikos darbo kodekse, tai vidutinis darbo užmokestis už uždelsimo laiką (Lietuvos Respublikos darbo kodekso 141 straipsnis) bei delspinigiai (Lietuvos Respublikos darbo kodekso 207 straipsnis). Darbe bus pasirinktais aspektais aptariamos sankcijos darbdaviui už pavėluotai sumokėtą darbo užmokestį, numatytos Lietuvos Respublikos darbo kodekso 141 straipsnyje ir 207... [toliau žr. visą tekstą] / Employer's obligation to pay wage arises naturally from employment relationship. Receiving wage is the most important social guarantees for employee, which ensures a secure standard of living. Although today wage is not the only reward, which employee receives in exchange for the work completed, but it still remains one of the most important. The primary piece of legislation regulating the payment of wage in Lithuania is the Labour Code. According to Article 186 of the Labour Code, the wage is compensation for work performed by employee under employment contract, which is payable in cash. The obligation of wage payment is resulted by the nature of employment contract. The amount that shall be paid and payment terms are defined by mutual agreement, and certain minimum standards are laid by legislation. Wage is a periodical payment and when this payment which belongs to employee is not paid on time, whether in whole or in part, it shall be treated as a violation of employee’s rights. For failure to implement employer’s obligation to pay wage in timely manner, there are sanctions for the employer established in the Labour Code, namely, the average salary for the time delay (Article 141 of the Labour Code) and interest (Article 207 of the Labour Code). In this thesis based on selected aspects of the aforementioned sanctions for failure to pay wage in timely manner will be analysed. Furthermore, application of these sanctions in the Lithuanian Supreme Court case law, problems... [to full text]

Quantitative fluorescence polymerase chain reaction (QF-PCR) is a molecular genetic method based on the amplification of microsatellites (Short tandem repeats, STR) and measurement of the peak heights of amplicons in the electropherogram. Currently, the QF- PCR deemed reliable, fast, and inexpensive method that is gradually replacing conventional cytogenetic analysis of aneuploidy (examination of long-term cultures of amniotic fluid). However, in certain cases it is impossible to determine the parental origin and meiotic aneuploidy by QF-PCR. The aim of this work was to verify the new dinucleotide STR markers on chromosomes 13, 16, 18, 21, and 22 and further increase the diagnostic efficiency of QF-PCR retaining other STR markers on chromosome 15, 16, 22 and to determine the population and the analytical characteristics of these markers. For all dinucleotide STR markers stutter occurred in high frequency and therefore there were found not to be suitable for routine diagnostics. STR markers for chromosomes 15, 16 and 22 were tested on 100 patients. We selected four informative markers for both chromosome 16 and 22, and three markers for chromosome 15. Thus, I expanded set of diagnostic STR markers in this thesis. Key words: QF-PCR, STR markers, prenatal diagnosis, trisomy.

Entre as muitas aplicações das tecnologias de identificação biológica humana, estão as finalidades forenses. O objetivo desta pesquisa foi verificar frequências alélicas de Short Tandem Repeat (STR) e os parâmetros estatísticos de interesse em genética de populações e forense para desenvolver o primeiro banco de dados populacional de DNA na Faculdade de Odontologia de Bauru, Universidade de São Paulo, (FOB/USP) para futuros usos forenses. Frequências alélicas de 15 locos autossômicos e do marcador de gênero amelogenina foram determinadas utilizando amostras de 200 μL de saliva doados por 296 alunos de graduação da FOB/USP, com idade ≥ 18 anos, após aprovação ética. Os testes laboratoriais foram feitos com kits comerciais. Resultados e parâmetros estatísticos foram obtidos por meio de programas clássicos: GeneMapper-ID-X, MS Excel 2002 versão 10.6871.6870, GenAlEx 6.5 e Arlequin 3.5, comparando quatro populações (brasileira, portuguesa, norte-americana e a população deste estudo). Os locos mais polimórficos foram D18S51 (17 alelos) e FGA (15 alelos), seguidos pelo D21S11 (13 alelos) e os menos polimórficos foram D16S539 e TH01 (7 alelos cada). A análise comparativa com amostra da população brasileira proveniente de estudos anteriores (n > 100.000) pelo teste goodness of fit X2 index não mostrou diferenças significativas entre estes grupos (p = 0,9999). Outros parâmetros estatísticos foram calculados comparando as populações: local (deste estudo), portuguesa e norte-americana. A análise de variância molecular (AMOVA) entre as três populações, entre as pessoas da mesma população e para cada pessoa de cada população mostrou que existe uma elevada variância individual (99%), que esta variância é mantida uniformemente entre as pessoas da mesma amostra/região (1%) e entre as três populações estudadas (0%). O estudo confirmou o elevado grau de polimorfismo e a alta heterozigosidade (96,5%) da população. Houve diferença significativa quanto ao gênero (79,7% mulheres) quando comparado à população brasileira em geral (50,4%), explicada pelas características do corpo discente da FOB/USP composto por 80,6% de pessoas do gênero feminino. Interessante foi a observação de uma microvariante alélica no loco D18S51, fora da escada padrão e da escala de abrangência do kit, correspondente ao alelo 29, ainda não definida na base de dados internacional (STRBase, atualizada em 07/08/2015). Esta microvariante deverá ser confirmada por testes familiares e sequenciamento de DNA para verificar a possibilidade de outra ocorrência familiar ou duplicação de nucleotídeos. No futuro, os dados obtidos neste estudo devem ser incorporados ao banco de dados da população brasileira e podem ser considerados como referência genética da população regional, ajudando a elucidar casos forenses. Após a confirmação, a potencial nova microvariante alélica contribuirá para a base de dados internacional STRBase. / There are many ways of applying biological human identification technologies, among these are forensic applications. The objective of this study was to verify allele frequencies for 15 autosomal short tandem repeat (STR) loci and develop the first human DNA population database at the Bauru School of Dentistry, University of São Paulo (FOB/USP), for future forensic uses. Allele frequencies for these STR loci and an amelogenin gender marker were determined using 200 μL samples of saliva donated by 296 undergraduates from FOB/USP who were ≥ 18 years old at the time of the sample collect after signing a consent form with ethical approval. For laboratory tests, commercial kits were used. Results and statistical parameters were obtained using the following software: GeneMapper IDX (version 1.5), MS Excel 2002 (version 10.6871.6870), GenAlEx (version 6.5) and Arlequin (version 3.5) to compare four populations (Brazil, Portugal, U.S. and this study population). Results indicated that the most polymorphic loci were D18S51 (17 alleles) and FGA (15 alleles), followed by D21S11 (13 alleles); the least polymorphic loci were D16S539 and TH01 (7 alleles each). Various Brazilian populations (n > 100,000) from other studies were compared with this studys Brazilian population using a goodness-of-fit chi-squared test, and no significant differences in these frequencies were observed between these two population groups (p = 0.9999). Other forensic and population genetic statistical parameters were calculated comparing this studys population with Portugal and U.S. populations. For example, an analysis of molecular variance (AMOVA) among all populations, among people of the same population and for each person for each population, showed that people have high individual variance (99%) and that this variance is maintained evenly between people of the same sample/region (1%) and among the three populations studied (0%). This study reinforced the conclusion of other allele frequency population studies for the 15 autosomal STR loci tested, confirming high polymorphic grade and high heterozygosity (96.5%). There were significantly more women in the study (79.7%) when compared to the general Brazilian population (50.4%) since the student body of FOB/USP is 80.6% female. Interestingly, an off-ladder D18S51 allele micro-variance corresponding to the allele 29, not yet identified, was found which should be confirmed using paternity and sequencing tests to verify the possibility of either familial occurrence or nucleotide duplication. In the future, due to the small differences found, the parameters obtained in this study should be incorporated into the Brazilian population database and be considered for a regional population genetic prototype database potentially aiding forensic cases with comparisons and calculations. After confirmation, the potentially new micro variant allele found could be included in the international STRBase.

Made available in DSpace on 2016-08-10T10:38:31Z (GMT). No. of bitstreams: 1 PATRICIA BONILHA DE TOLEDO PIZA.pdf: 970259 bytes, checksum: e296c4994d48ecfafd048f3cc57617e4 (MD5) Previous issue date: 2012-08-31 / In Brazil sexual violence is a crime. Rape and vulnerable rape are heinous crimes. It is estimated that only 10% of rapes are recorded and the SENASP point 42.946 police incidents in 2010. Most victims are women aged below 14 years. The absence of expert evidence difficult the sentencing of the offender. In victim, exams corpus delicti of carnal knowledge and research of semen when positive do not identify the perpetrator and negativity is not a factor of no sexual assault. Genetic analysis by PCR-STR and Y identifies the presence of male DNA and the genetic profile of the perpetrator. This paper aims to analyze samples for traces of sex crimes to obtain molecular profiles of Y-STR. We selected 19 cases of sex crimes with no suspect, totaling 20 female victims aged 11 months to 81 years, which resulted in 48 samples questioned, of which 44 were subjected to differential extraction and 4 to organic extraction, totaling 92 products extraction (44 SF, 44 NSF and 4 Organic). The DNA quantitation by real-time PCR detected the presence of male DNA in 62% of the samples. Of these, 21 samples were selected and standardized to a concentration of Y-DNA 0.1 ng to 1.25 ng / PCR reaction for Y-STR. Amplification was performed for 17 joint multiplex Y-STR markers and electrophoresis capillary was preceded in genetic analyzer and the results were analyzed by programs. Of the 21 samples amplified, 12 had results for Y-STR and their haplotype been classified as full (Y-STR 17), minimum (11 Y-STR) and incomplete (absence of one or more of the Y-STR minimum haplotype) resulting 10 minimal haplotypes and complete and 02 incomplete. After comparing the minimum haplotypes and complete intra case and between their criminal cases, it was evident that the sexual assaults were committed in each case, by a single assailant, not featuring serial crimes. It is easy to recognize the importance that the Y-STR haplotype analysis assumes the sexual crimes as expert evidence, especially when it becomes the only genetic information of the offender being questioned samples obtained. The results presented in this study demonstrate the importance of being analyzed the largest number of polymorphic markers Y-STR haplotype to compose an informative, giving preference to multiplex sets that amplify multiple loci simultaneously, including polymorphic markers with products up to 200 base pairs. / No Brasil a violência sexual é crime. O estupro e o estupro de vulnerável são crimes hediondos. Estima-se que apenas 10% dos estupros são registrados e dados da SENASP apontam 42.946 ocorrências policiais em 2010. A maioria das vítimas é do sexo feminino com idade abaixo de 14 anos. A ausência de prova pericial dificulta a condenação do agressor. Na vítima, os exames de corpo de delito de conjunção carnal e pesquisa de sêmen quando positivos não identificam o agressor e a negatividade não é fator de inexistência de agressão sexual. A análise genética, através da PCR e Y-STR permite identificar a presença de DNA masculino e o perfil genético do agressor. Este trabalho objetiva analisar amostras de vestígios de crimes sexuais para a obtenção de perfis moleculares de Y-STR. Foram selecionados 19 casos de crimes sexuais com ausência de suspeito, totalizando 20 vítimas do sexo feminino com idade entre 11 meses a 81 anos, que resultaram em 48 amostras questionadas, das quais 44 foram submetidas à extração diferencial e 4 à extração orgânica, totalizando 92 produtos de extração (44 FE, 44 FNE e 4 Orgânica). A quantificação de DNA pela PCR em tempo real detectou a presença de DNA masculino em 62% das amostras extraídas. Destas, 21 amostras foram selecionadas e normalizadas a uma concentração de Y-DNA entre 0,1 ng a 1,25 ng/reação de PCR para Y-STR. A amplificação foi realizada com conjunto multiplex para 17 marcadores Y-STR e a eletroforese capilar foi procedida em analisador genético; os resultados foram analisados por programas específicos. Das 21 amostras amplificadas, 12 apresentaram resultados para Y-STR e seus haplótipos foram classificados como completo (17 Y-STR), mínimo (11 Y-STR) e incompleto (ausência de 1 ou mais Y-STR do haplótipo mínimo), resultando em 10 haplótipos mínimos e completos e 02 incompletos. Após confrontar os haplótipos mínimos e completos intra caso e entre os respectivos casos criminais, ficou evidenciado que as agressões sexuais foram cometidas, em cada caso, por um único agressor, não caracterizando crimes em série. É de facil reconhecimento a importância que o haplótipo de Y-STR assume nas análises de crimes sexuais como prova pericial, principalmente quando este se torna a única informação genética do agressor a ser obtida de amostras questionadas. Os resultados apresentados neste estudo demonstraram a importância de serem analisados o maior número de marcadores polimórficos Y-STR para compor um haplótipo informativo, dando-se preferência a conjuntos multiplex que amplificam simultaneamente vários loci, incluindo marcadores polimórficos com produtos de até 200 pares de bases.

<p>The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex.</p>

<p>The objective of the study was to conduct an internal validation on miniSTR systems and apply it to cases received from the South African Missing Persons Task Team (SAMPTT). This was prompted by the fact that miniSTR systems have been shown to out perform some of the commercial kits available in the time of the study and provide an alternative to mtDNA when analysing degraded DNA from skeletal remains and that the DNA extracted from skeletal remains received from the SAMPTT would be degraded due to the remains generally being fragmented or charred and buried for many years. The miniSTR loci chosen for validation comprised the Combined DNA Index System (CODIS) thirteen core loci and were arranged into four triplexes and one uniplex.</p>

Made available in DSpace on 2016-08-29T15:34:36Z (GMT). No. of bitstreams: 1 tese_8966_Dissertação_Fernando Colnago20150826-123156.pdf: 3408650 bytes, checksum: 8e6f48a8678d513330c4dbecc0a5c9bb (MD5) Previous issue date: 2015-06-30 / Popularmente conhecida como maconha, a Cannabis sativa é a mais usada de todas as drogas ilícitas e seu comércio, em conjunto com outras drogas, é uma das atividades criminosas que mais consomem recursos públicos e está intimamente associado aos homicídios. No Estado do Espírito Santo (ES), 70% dos homicídios tem relação com o tráfico de entorpecentes, apresentando atualmente o segundo maior índice dos país. A despeito da atuação policial, não existem estudos detalhados sobre plantio, distribuição e consumo de Cannabis no Brasil, de uma forma geral, sabe-se apenas que a maior parte da droga é produzida no Paraguai ou cultivada dentro do próprio país e acredita-se que o Paraguai seja responsável pelo abastecimento das regiões sul, sudeste e centro-oeste do Brasil. A identificação genética de Cannabis apreendidas pela Polícia Civil pode auxiliar na determinação geográfica de plantios e possíveis rotas do tráfico, informação essencial para a ação da polícia no combate ao tráfico de drogas. Com objetivo de caracterizar as amostras de Cannabis apreendidas pela Polícia Civil do ES e verificar correlações que pudessem indicar origens comuns, foram analisados 18 loci STR de 165 amostras oriundas de 71 dos 78 municípios do Estado. Foram encontrados 89 alelos, variando de 2 (loci A501 e 9269) a 12 alelos (locus A301). Os loci H06 e A305 apresentaram 3 alelos cada e o locus B05 apresentou 4 alelos, similar ao encontrado em amostras do Paraguai. Os dados alélicos dos loci A305, B05 e H06 sugerem que a região sudeste do Brasil pode estar sendo abastecida por Cannabis de origem Paraguaia. Também foi verificado que 4 alelos apareceram apenas nas amostras 176 e 230. A amostra 230 era proveniente de uma apreensão realizada na cidade de Aracruz, onde a Polícia verificou a ligação do proprietário com o tráfico internacional de entorpecentes, e demonstrou ser geneticamente mais próxima de amostras da Alemanha, sugerindo que sua matriz tem origem no exterior podendo, dentre outras possibilidades, ter chegado ao ES através do tráfico pela internet. Os resultados obtidos fornecem mais informações a respeito da maconha comercializada no Brasil, podendo ser utilizados em estudos futuros para elaboração de um banco de dados baseado em STR para aplicação forense / Popularly known as marijuana, Cannabis sativa is the most used of all illicit drugs and, with other drugs, its trade is one of the criminal activities that most consume public resources and is closely related to homicides. In the Espírito Santo State (ES), 70% of homicides is related to drug trafficking, currently presenting the second highest rate in the country. Despite the police action, there is no detailed studies of planting, distribution and Cannabis consumption in Brazil, in general, we only know that most drug is produced in Paraguay or grown within the country and it is believed that Paraguay is responsible for supplying the Brazil’s south, southeast and centerwest region. Genetic analysis of Cannabis seized by the police can assist in determining, geographically, plantations and possible routes of trafficking, essential information for police’s action in drug trafficking combat. In order to characterize the Cannabis samples seized by the police at ES and verify correlations that could indicate common origins, we analyzed 18 STR loci in 165 samples from 71 of 78 municipalities in the State. We have found 89 alleles ranging from 2 (A501 loci and 9269) to 12 alleles (A301 locus). The loci H06 and A305 presented each, 3 alleles and the locus B05, 4 alleles, similar to that found in Paraguayan samples. The allelic data of loci A305, B05 and H06 suggest that Brazil 's southeast region can be being supplied by Paraguayan Cannabis. We also found four alleles that appeared only in samples 176 and 230. Sample 230 was seized in Aracruz city, where the police found owner's connection with international trafficking, and demonstrated to be genetically nearest to German samples, suggesting a foreign origin what could be explained by internet trafficking. The obtained results provide more information about marijuana’s market in Brazil and could be used in future studies to develop a STR database for forensic application.