Microvariation at the human D1S80 locus

Microvariant allelic polymorphisms have been known since 1966 when Harris, Hubby and Lewontin described the huge store of genetic variation detectable at the polypeptide level. Later Jeffreys used MVR (minisatellite variant repeat) analysis to describe the variation hidden within minisatellite VNTRs and to propose a mutational mechanism.^ The questions I have asked follow these traditions: (1) How much microvariant polymorphism exists at the discrete allele minisatellite D1S80 locus? (2) Do alleles or groups of alleles associate randomly with the flanking markers to form haplotypes? (3) What mechanisms might explain mutations at this locus? What are the phylogenetic relationships among the alleles?^ The minisatellite locus D1S80 (1p35-36), GenBank sequence (Accession # D28507), is a highly polymorphic Variable Number of Tandem Repeat (VNTR) based on a 16 base core. D1S80 alleles are electrophoretically separable into discontinuous sets of equivalent length alleles. Sequence variation or minor length variation within these classes was expected: I have sought to determine the nature of this microvariant heterogeneity by sequencing nominal and variant alleles.^ Alleles were analyzed by Single-Strand Conformation Polymorphism (SSCP) analysis. Sequences were determined to ascertain whether sequence variation or size variation is the major cause of altered electrophoretic migration of microvariant D1S80 alleles. Twenty three alleles from 14 previously typed individuals were sequenced. The individuals were from African American, Caucasian, or Hispanic databases.^ A Tsp509 I restriction site, previously reported as a Hinf I flanking polymorphism, and a 3$\sp\prime$ flanking region BsoF I restriction site polymorphism were identified. There appears to be a strong association of the 5$\sp\prime$ flanking region Hinf I(+) and Tsp509 I(-) site and the 3$\sp\prime$ flanking region BsoF I(-) site with the 18 allele, while the 24 tends to be associated with the Hinf I(-), Tsp509 I(+) and BsoF I(+) sites.^ The general conclusion for this locus is clearly the closer you look, the more you find. D1S80 allelic polymorphisms are primarily due to variation in the number of repeat units and to sequence variation among repeats. The sequenced based gene tree depicts two major classes of alleles which conform to the two most common alleles, reflecting either equivalent age or population size bottlenecks. ^

Practical secure information flow in programming languages

If we classify variables in a program into various security levels, then a secure information flow analysis aims to verify statically that information in a program can flow only in ways consistent with the specified security levels. One well-studied approach is to formulate the rules of the secure information flow analysis as a type system. A major trend of recent research focuses on how to accommodate various sophisticated modern language features. However, this approach often leads to overly complicated and restrictive type systems, making them unfit for practical use. Also, problems essential to practical use, such as type inference and error reporting, have received little attention. This dissertation identified and solved major theoretical and practical hurdles to the application of secure information flow. ^ We adopted a minimalist approach to designing our language to ensure a simple lenient type system. We started out with a small simple imperative language and only added features that we deemed most important for practical use. One language feature we addressed is arrays. Due to the various leaking channels associated with array operations, arrays have received complicated and restrictive typing rules in other secure languages. We presented a novel approach for lenient array operations, which lead to simple and lenient typing of arrays. ^ Type inference is necessary because usually a user is only concerned with the security types for input/output variables of a program and would like to have all types for auxiliary variables inferred automatically. We presented a type inference algorithm B and proved its soundness and completeness. Moreover, algorithm B stays close to the program and the type system and therefore facilitates informative error reporting that is generated in a cascading fashion. Algorithm B and error reporting have been implemented and tested. ^ Lastly, we presented a novel framework for developing applications that ensure user information privacy. In this framework, core computations are defined as code modules that involve input/output data from multiple parties. Incrementally, secure flow policies are refined based on feedback from the type checking/inference. Core computations only interact with code modules from involved parties through well-defined interfaces. All code modules are digitally signed to ensure their authenticity and integrity. ^

The use of a computer graphic organizer for persuasive composition writing by Hispanic students with specific learning disabilities

Many culturally and linguistically diverse (CLD) students with specific learning disabilities (SLD) struggle with the writing process. Particularly, they have difficulties developing and expanding ideas, organizing and elaborating sentences, and revising and editing their compositions (Graham, Harris, & Larsen, 2001; Myles, 2002). Computer graphic organizers offer a possible solution to assist them in their writing. This study investigated the effects of a computer graphic organizer on the persuasive writing compositions of Hispanic middle school students with SLD. A multiple baseline design across subjects was used to examine its effects on six dependent variables: number of arguments and supporting details, number and percentage of transferred arguments and supporting details, planning time, writing fluency, syntactical maturity (measured by T-units, the shortest grammatical sentence without fragments), and overall organization. Data were collected and analyzed throughout baseline and intervention. Participants were taught persuasive writing and the writing process prior to baseline. During baseline, participants were given a prompt and asked to use paper and pencil to plan their compositions. A computer was used for typing and editing. Intervention required participants to use a computer graphic organizer for planning and then a computer for typing and editing. The planning sheets and written composition were printed and analyzed daily along with the time each participant spent on planning. The use of computer graphic organizers had a positive effect on the planning and persuasive writing compositions. Increases were noted in the number of supporting details planned, percentage of supporting details transferred, planning time, writing fluency, syntactical maturity in number of T-units, and overall organization of the composition. Minimal to negligible increases were noted in the mean number of arguments planned and written. Varying effects were noted in the percent of transferred arguments and there was a decrease in the T-unit mean length. This study extends the limited literature on the effects of computer graphic organizers as a prewriting strategy for Hispanic students with SLD. In order to fully gauge the potential of this intervention, future research should investigate the use of different features of computer graphic organizer programs, its effects with other writing genres, and different populations.

The Effect of Sample and Sample Matrix on DNA Processing: Mechanisms for the Detection and Management of Inhibition in Forensic Samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.

The Use of a Computer Graphic Organizer for Persuasive Composition Writing by Hispanic Students with Specific Learning Disabilities

Many culturally and linguistically diverse (CLD) students with specific learning disabilities (SLD) struggle with the writing process. Particularly, they have difficulties developing and expanding ideas, organizing and elaborating sentences, and revising and editing their compositions (Graham, Harris, & Larsen, 2001; Myles, 2002). Computer graphic organizers offer a possible solution to assist them in their writing. This study investigated the effects of a computer graphic organizer on the persuasive writing compositions of Hispanic middle school students with SLD. A multiple baseline design across subjects was used to examine its effects on six dependent variables: number of arguments and supporting details, number and percentage of transferred arguments and supporting details, planning time, writing fluency, syntactical maturity (measured by T-units, the shortest grammatical sentence without fragments), and overall organization. Data were collected and analyzed throughout baseline and intervention. Participants were taught persuasive writing and the writing process prior to baseline. During baseline, participants were given a prompt and asked to use paper and pencil to plan their compositions. A computer was used for typing and editing. Intervention required participants to use a computer graphic organizer for planning and then a computer for typing and editing. The planning sheets and written composition were printed and analyzed daily along with the time each participant spent on planning. The use of computer graphic organizers had a positive effect on the planning and persuasive writing compositions. Increases were noted in the number of supporting details planned, percentage of supporting details transferred, planning time, writing fluency, syntactical maturity in number of T-units, and overall organization of the composition. Minimal to negligible increases were noted in the mean number of arguments planned and written. Varying effects were noted in the percent of transferred arguments and there was a decrease in the T-unit mean length. This study extends the limited literature on the effects of computer graphic organizers as a prewriting strategy for Hispanic students with SLD. In order to fully gauge the potential of this intervention, future research should investigate the use of different features of computer graphic organizer programs, its effects with other writing genres, and different populations.

D1S80 DNA profiling in five African populations

The highly polymorphic DlS80 locus has no known genetic function. This variable number of tandem repeat (VNTR) has been valuable in forensic identification. We have obtained allelic and genotypic frequencies for five African populations (Benin, Cameroon, Egypt, Kenya and Rwanda), which could be employed as databases to identify individuals. The polymerase chain reaction, followed by vertical polyacrylamide gel electrophoresis and silver staining was our method of analysis. Allele frequencies were used to infer genetic associations using Phylip 3.5, Principal Component and G-test statistical programs. Tests for Hardy-Weinberg equilibrium were employed. Fst estimates and power of discrimination values were also determined for each of our populations. Our analyses of 28 additional populations demonstrated that the D1 S80 locus alone provided for the discrimination of major racial groups. Genetic homogeneity between the African groups was observed. We have generated a database useful for human differentiation and phylogenetic studies.

Development of microsatellite markers in Cannabis Sativa for fingerprinting and genetic relatedness analyses

Microsatellite markers were developed for Cannabis sativa L. (marijuana) to estimate the level of polymorphism, usefulness for DNA typing (genotype identification), and to measure the genetic relationships between the different plants. Twelve different oligonucleotide probes were used to screen an enriched microsatellite library of Cannabis sativa in which 49% of the clones contained microsatellite sequences. Characterization of microsatellite loci in Cannabis revealed that GA/CT was the most abundant class of isolated microsatellites representing 50% overall. Eleven polymorphic SSR markers were developed, derived from dinucleotide motifs and eight from trinucleotide motifs. A total of 52 alleles were detected averaging 4.7 alleles/locus. The expected heterozygosity of the eleven loci ranged between 0.368 and 0.710 and the common probability of identical genotypes was 1.8 x 107. The loci identified 27 unique profiles of the 41 Cannabis samples. The eleven microsatellite markers developed in this study were found to be useful for DNA fingerprinting and for assessing genetic relationships in Cannabis.

The effect of sample and sample matrix on DNA processing: Mechanisms for the detection and management of inhibition in forensic samples

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments were conducted to establish the inhibition tolerance of quantification and amplification kits using common inhibitory substances in order to determine if current laboratory practices are optimal for identifying potential problems associated with inhibition. DART mass spectrometry was used to determine the amount of inhibitor carryover after sample purification, its correlation to the initial inhibitor input in the sample and the overall effect in the results. Finally, a novel alternative at gathering investigative leads from samples that would otherwise be ineffective for DNA typing due to the large amounts of inhibitory substances and/or environmental degradation was tested. This included generating data associated with microbial peak signatures to identify locations of clandestine human graves. Results demonstrate that the current methods for assessing inhibition are not necessarily accurate, as samples that appear inhibited in the quantification process can yield full DNA profiles, while those that do not indicate inhibition may suffer from lowered amplification efficiency or PCR artifacts. The extraction methods tested were able to remove >90% of the inhibitors from all samples with the exception of phenol, which was present in variable amounts whenever the organic extraction approach was utilized. Although the results attained suggested that most inhibitors produce minimal effect on downstream applications, analysts should practice caution when selecting the best extraction method for particular samples, as casework DNA samples are often present in small quantities and can contain an overwhelming amount of inhibitory substances.^