OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.

Este estudo comparou o desempenho de quatro protocolos de extração de DNA a partir de homogeneizados de diferentes órgãos provenientes de vacas infectadas experimentalmente com a B. abortus 2308. Os protocolos de extração comparados foram o método de GT (lise com isotiocianato de guanidina), Boom (lise com GT e tratamento com suspensão carreadora Diatomaceous earth), PK (lise com proteinase K) e Santos (lise por fervura e congelamento com nitrogênio líquido). Foram constituídos os grupos padrão ouro positivo e negativo baseados na bacteriologia clássica e compostos por: 54 cotilédones (27 pos. e 27 neg.), 39 linfonodos supra mamários (12 pos. e 27 neg.), 44 pré-escapulares (17 pos. e 27 neg.), 33 fígados (6 pos. e 27 neg.), 37 baços (10 pos. e 27 neg.), e 34 úberes (7 pos. e 27 neg.). Todas as amostras foram submetidas aos quatro protocolos de extração e a um mesmo processo de amplificação com os primers B4 e B5. Nos resultados consolidados por órgãos, a proporção de positivos no cotilédone foi maior do que a encontrada no linfonodo supramamário (p=0,0001), linfonodo pré-escapular (p<0,0001), fígado (p=0,0006), baço (p<0,0001) ou úbere (p=0,0019). Os resultados acumulados para os métodos de extração mostraram que o protocolo de Santos teve maior sensibilidade relativa do que o método de Boom (p=0,003) e GT (p=0,0506), e foi igual ao PK (p=0,2969). As demais comparações de proporções não resultaram em diferenças estatisticamente significantes. No estudo verificou-se amostas positivas a PCR e negativas ao isolamento e viceversa. Assim, apesar das desvantagens do método bacteriológico clássico, a melhor estratégia para o diagnóstico direto da infecção de vacas por B. abortus em homogeneizado de órgãos é a utilização conjunta do isolamento e da PCR, examinando os cotilédones e utilizando os métodos de extração de DNA Santos ou PK. / This study compared the performance of four protocols of DNA extraction from suspensions of different tissues from experimentally infected cows with 2308 strain. The compared extraction protocols were GT protocol (lyse with guanidine isotiocianate), Boom (lyse with GT and treated with the carrying suspension Diatomaceous earth), PK (lise with proteinase K) and Santos (lyse by boiling and freezing with liquid nitrogen). Based on classical bacteriology, positive and negative gold standard groups were built and consisted of 54 cotyledons (27 pos. and 27 neg.), 39 supramammary lymph nodes (12 pos. and 27 neg.), 44 prescapulars (17 pos. and 27 neg.), 33 livers (6 pos. and 27 neg.), 37 spleens (10 pos. and 27 neg.), and 34 udders (7 pos. and 27 neg.). All the samples were submitted to the four DNA extraction protocols and the same amplification process using the primers B4 and B5. According to consolidated results by tissue, the proportion of positives in cotyledon was bigger than supramammary lymph node (p=0,0001), prescapular lymph node (p<0,0001), liver (p=0,0006), spleen (p<0,0001) and udder (p=0,0019). The consolidated results for the extraction methods show that Santos protocol had bigger relative sensitivity than Boom method (p=0,003) and GT (p=0,0506), and was equal than PK (p=0,2969). There were not significant statistical differences in the others comparisons of proportions. In the study, PCR-positive and isolation-negative samples and vice-versa were verified. However, the disadvantages of the classic bacteriological method, the best strategy for direct diagnosis of the infection of cows for B. abortus in homogenized of tissues is combined use of isolation and PCR, examining the cotyledons and using the methods of DNA extraction from Santos or PK.

Diatoms associated with foraminifers of the genus Amphistegina were assessed using a combination of morphological and molecular techniques. These included: 1) microscopic identification of diatoms cultured from the host, 2) sequencing of portions of the small subunit of the ribosomal RNA gene (18S) and the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase [i.e., RubisCO] gene (rbcL) from DNA extracted directly from the Amphistegina hosts and also from diatoms cultured from these hosts, and 3) denaturing gradient gel electrophoresis (DGGE) profiles of rbcL and internal transcribed spacer 1 (ITS1) PCR amplicons from DNA extracted directly from hosts and from cultures. Consistent with previous culture studies, multiple species of pennate diatoms of the genera Nitzschia, Fragilaria (including Nanofrustulum), Amphora, and Navicula, were cultured from >900 host specimens collected from >20 sites in the western Atlantic and four sites in the Pacific. Diatoms of the genus Nitzschia grew in about half of all successful cultures. The genetic identities of selected cultures were consistent with those based on morphological taxonomy. Diatom sequences from DNA extracted directly from the cytoplasm of the Amphistegina hosts were species specific and distinct from sequences obtained from cultured diatoms and from sequences in GenBank of diatom taxa previously reported as endosymbionts. Multiple phylogenetic analyses revealed that the 18S and rbcL diatom sequences from specimens of A. gibbosa collected from the Atlantic sites and of Amphistegina spp. from Hawai’i were most similar to the 18S and rbcL sequences of an unnamed Fragilariaceae diatom in GenBank (Accession # JX413542.1 for 18S and JX413559.1 for rbcL) and other closely related diatoms in that family. Of diatom taxa previously reported as endosymbionts of larger foraminifers, Nanofrustulum shiloi was the most similar, but not identical, to the sequences from hosts collected from the Atlantic and Hawai’i. The 18S and rbcL diatom sequences from the Atlantic host species, A. gibbosa, were all nearly identical, but small intra-species differences (subclades) were observed from specimens collected from the deepest (75 m) site in the Florida Keys and also from the eastern-most site, Young Island near St. Vincent. The 18S and rbcL diatom sequences from the two host species from Hawai’i, A. lobifera and A. lessonii, were more variable but still within the family Fragilariaceae. The diatom sequences from A. radiata collected from two sites in Papua New Guinea (PNG) were most similar to diatoms of the family Plagiogrammaceae and therefore distinct from sequences obtained from other Amphistegina species in this study, as well as from all diatoms previously reported as endosymbionts. A small difference was observed between the diatom sequences from host specimens collected from a Pacific site as compared to a Bismarck Sea site. The ITS1 DGGE profiles of DNA extracted directly from A. gibbosa specimens at different depths, locations, and seasons in the western Atlantic were nearly identical. Differences were seen between rbcL DGGE profiles of DNA extracted directly from the different Amphistegina host species. The rbcL DGGE profiles directly from all hosts were clearly different from those extracted from diatoms cultured from the same host specimens, as well as from Nitzschia laevis, a commonly reported diatom endosymbiont in past culture-based studies. My findings are consistent with ultrastructural studies of endosymbionts of Amphistegina published in the early 1980s and congruent with recent molecular studies of endosymbionts in other diatom-bearing foraminifers, all of which indicate specificity. Nevertheless, the consistency with which several diatom taxa have been reported in culture studies from all oceans indicates the possibility of some relationship with Amphistegina spp., either as important food items, epiphytes, or minor opportunistic symbionts that can thrive in culture media.

There is an increasing demand for DNA analysis because of the sensitivity of the method and the ability to uniquely identify and distinguish individuals with a high degree of certainty. But this demand has led to huge backlogs in evidence lockers since the current DNA extraction protocols require long processing time. The DNA analysis procedure becomes more complicated when analyzing sexual assault casework samples where the evidence contains more than one contributor. Additional processing to separate different cell types in order to simplify the final data interpretation further contributes to the existing cumbersome protocols. The goal of the present project is to develop a rapid and efficient extraction method that permits selective digestion of mixtures. Selective recovery of male DNA was achieved with as little as 15 minutes lysis time upon exposure to high pressure under alkaline conditions. Pressure cycling technology (PCT) is carried out in a barocycler that has a small footprint and is semi-automated. Typically less than 10% male DNA is recovered using the standard extraction protocol for rape kits, almost seven times more male DNA was recovered from swabs using this novel method. Various parameters including instrument setting and buffer composition were optimized to achieve selective recovery of sperm DNA. Some developmental validation studies were also done to determine the efficiency of this method in processing samples exposed to various conditions that can affect the quality of the extraction and the final DNA profile. Easy to use interface, minimal manual interference and the ability to achieve high yields with simple reagents in a relatively short time make this an ideal method for potential application in analyzing sexual assault samples.

Differential extraction has proven to be a challenging and time-consuming process, often requiring up to six hours of a forensic analyst’s concentration. With the ever-increasing backlog of sexual assault evidence kits, the forensic community is using new ways to diminish this backlog, including more streamlined evidence processing and sample analysis. The goals for processing sexual assault samples include efficient recovery of sperm deoxyribose nucleic acid (DNA), simplified sample processing, and the development of a profile eligible for forensic analysis. Cost and time can also be limiting factors. The Cotton Research Lab at Boston University has developed a novel method of differential extraction that combines separation of epithelial and sperm cell fractions, nuclease treatment to reduce female DNA carryover and a direct-cell lysis protocol. With the exception of a single centrifugation step, the entire protocol is conducted using a thermalcycler in the DNA extraction laboratory. Thus, the process is a Temperature Controlled Differential Extraction (TCDE), and has been effectively adapted for use with liquid, dried, and aged samples. The purpose of this research is to explore methods which further adapt the protocol for best use with forensic casework samples, namely vaginal swabs. Sexual assault evidence collection kits may contain a variety of items, and commonly include cotton swabs for the collection of fluids from intimate sources. To simulate casework-type samples, swabs were prepared with liquid epithelial cell preparations and various semen dilutions (ranging from 1:1 to 1:1000). Amendments were made to the TCDE protocol for best DNA recovery from a swab, and buffer changes were made to enhance compatibility with polymerase chain reaction (PCR)-amplification kits widely utilized in forensic labs. Finally, post-coital swabs from female donors were analyzed using the TCDE protocol with modifications for forensic casework samples. Preliminary testing of casework-type swabs with protocol modifications showed high yields of DNA and successful separation of epithelial and spermatozoa fractions. The epithelial fraction, when yielding a mixed profile, demonstrated a clear major female contributor, and the spermatozoa fractions showed little to no female carryover, often exhibiting single source male profiles. The TCDE protocol with modifications for casework-type samples requires approximately 2 hours and 30 minutes of an analyst’s time, from the moment the swab is removed from its evidence packaging to an extraction ready for DNA quant and short tandem repeat (STR) amplification. The method provides increased DNA recovery, can be used with various amplification kits, and generate probative profiles and is time efficient. This robust and promising new method that has the potential to be automated and to contribute to the effort to reduce the backlog in the analysis of sexual assault evidence kits.

No description available.

When there is a possibility of a low-template sample being processed in a forensic laboratory, it becomes important to retrieve all cells possible from the substrate they are collected on. The most common form of evidence received by forensic laboratories is epithelial cells collected on cotton material, swab or fabric, which may contain inhibitors. Data shows a likely mechanism for cellular adherence is the denaturation of surface proteins to expose residues hidden within. Proteins on cotton’s cell surface hydrogen bond to these residues, forming a strong attachment. An epithelial cell preparation was pipetted onto ISO adjacent cotton swatches. These swatches were incubated in 10mM Tris, 0.1 mM EDTA (TE) buffer with a constant temperature and agitation from a Thermal Mixer. The swatch was removed from the liquid and placed in a separate tube and digest separately. Each was quantified and used to calculate the percentage of cellular release. Variations of this baseline procedure were used to help determine the most efficient cellular release process. These variables included different temperatures and agitation speeds, sonication, resuspension and the addition of disaccharides. Results showed that the addition of a disaccharide is the most efficient method to achieve cellular release from cotton fabric. Specifically, drying 0.75 M D-(+)- Trehalose Dihydrate onto a cotton fabric swatch before the addition of the epithelial cell preparation. This procedure produced an average of 65.5% cellular release compared to a 26.0% release from our baseline procedure.

There is a transition nationwide on the analysis of forensic evidentiary stains containing biological material from traditional serology to Polymerase Chain Reaction (PCR) methodologies. The increased sensitivity of PCR, the limited number of alleles at each locus, and the necessity of producing unambiguous data for entry into the FBI's Combined DNA Index System make this study of extraction procedures of utmost importance. A "single tube" extraction procedure for blood stains collected onto FTA™ paper and a modified differential nonorganic extraction method from spermatozoa containing mixed stains were analyzed and compared. The extraction success was evaluated by amplification and typing of the amplified fragment length polymorphism, D1S80. These modifications of the nonorganic method utilized gave an improved separation of the spermatozoa-containing mixed stains.

Blood stream infections are challenging infections to rapidly diagnose. The current clinical diagnostic methods for blood stream infections require culturing the blood sample prior to identifying the bacteria and any resistance the bacteria may contain. Removing the culturing step from the bacterial identification process of a blood stream infection provides a significant reduction in the processing time. However, eliminating the culturing step shifts the difficulty from processing time to concentration, since clinical concentration levels can be as low as 10 CFU/mL in blood. This dissertation developed and evaluated many aspects of the process required to identify bacteria from a blood stream infection without culturing the bacteria. Two new methods of separating the bacteria from the blood cells were developed: inducing clotting using a centrifugal-sedimentation on a hollow disk, and filtering whole blood. Inducing clotting achieved 69\% bacterial recovery from 7 mLs of whole blood in 117 s. Filtering whole blood achieved 100\% bacterial removal from 5 mLs of whole blood in $\approx 90$ s, but the bacteria were difficult to remove from the filter. Bacterial removal from the filter after blood filtration was also investigated. At a very low bacterial concentration of 200 CFU/mL, a blood lysis solution of 3\% Tween 80 followed by a 3\% Pluronic F108 backflush solution achieved 60\% removal of the bacteria from the filter. In addition to developing two new methods, a previously developed technique using centrifugal-sedimentation on a hollow disk underwent a stability analysis in order to decrease the occurrence of mixing. This analysis yielded the development of the analytical solution to the Navier-Stokes equations for a two-fluid flow with a moving wall boundary and a free surface. The analysis also experimentally identified a stability boundary that was found to be in good agreement with the Kelvin-Helmholtz instability model. After exploring the methods to recover bacteria from blood, experiments were performed to identify a bacterial lysing solution that could lyse \textit{E. coli}, \textit{E. cloacae} and \textit{K. pneumoniae} bacteria. The best bacterial lysing solution consisted of incubating the bacteria with 1 mg/mL lysozyme for 10 min followed by the addition of 6 M GHCl and 1\% SDS. This solution obtained a 46\% DNA recovery. The DNA were then fragmented by ultrasound to reduce the segment length for DNA labelling. In addition to lysing and fragmenting the DNA, a microfluidic device was prototyped and tested for incorporating the lysing, capturing, releasing, and fragmenting of the DNA all on a single device. Whole experiments were performed which extracted the bacteria from the blood, removed and collected the DNA from the bacteria, and fragmented the DNA. The best overall recovery from an experiment performing the whole process was 26.8\%. The 26.8\% recovery was achieved with a 68\% recovery of the bacteria from spinning and a 54.1\% removal of bacteria from off of the filter and a 72.9\% recovery of the DNA from the bacteria.

Soil microorganisms have a variety of beneficial and deleterious effects on plants, impacting such processes as plant growth, soil nutrient cycling, crop yield, disease resistance and tolerance to an array of biotic and abiotic stressors. The disruption of soil microbial community structures, particularly when beneficial soil biota are altered, has been shown to reduce crop yield and leave plants susceptible to disease. Long-term disruption of microbial communities may occur with repeated fumigation, being the application of gaseous pesticides, in agricultural soils. For this reason, we characterized bacterial, fungal, oomycete and nematode populations in paired fumigated and nonfumigated potato fields located in Idaho, Oregon, Washington and Minnesota. Samples were taken at three distinct timepoints: one before a fall fumigation event and two others at important stages in potato production, row closure and vine death. Soil biota populations were assessed by targeting the 16S, 18S and ITS1 gene regions. FunGuild, a database capable of guild and trophic assignment of fungal lineages, was used to sort fungal OTUs in different trophic modes. Fungal analyses indicated an increase in relative abundances of saprotrophic fungal populations and a decrease in pathotrophic fungal populations, both during row closure. Principally, the fungal genera of Humicola and Mortierella were responsible for the increase of saprotrophs while Alternaria decreased the most for pathotrophs. Other fungi occupying multiple trophic modes, such as Fusarium, also decreased during row closure. We found that fumigation treatments, in combination with various pesticide and fertilizer applications, alter both alpha- and beta- bacterial soil diversity although certain treatments, i.e. chloropicrin, may alter bacterial populations more than other treatment types such as metam-sodium. Nematode populations were likewise distinct at each location with soils from Boardman, OR, Minidoka, ID and Pine Point, MN with these having higher levels of nematodes associated with better soil health, i.e. Dorylaimidae. Conversely, nematodes associated with plant pathogenesis were found in higher relative abundances at Minidoka, ID and Quincy, WA. In this study, we characterize the populations of bacteria, fungi, oomycetes and nematodes with an emphasis on fungal taxa. We found that relative abundances of fungal trophic modes vary temporally. Additionally, we catalogue several other high abundance taxa with seasonal differential abundances whose functional capacity in potatoes remain uncharacterized.