The present study investigated the expression profiles of nine genes involved in immune defense of the Pacific white shrimp Litopenaeus vannamei and their responses to oral administration of beta-1,3-glucan. The nine immune related genes were beta-glucan binding protein-high density lipoprotein (BGBP-HDL), lipopolysaccharide/beta-glucan binding protein (LGBP), hemocyanin, prophenoloxidase (proPO), transglutaminase (TGase), penaeidin-3 (PEN-3), crustin, cytosolic manganese superoxide dismutase (cMnSOD), and lysozyme. A series of experiments were carried out in the study including: (1) cDNA cloning and characterization of proPO and LGBP; (2) tissue mRNA expressions of the nine genes in adult shrimp; (3) expression and localization of the nine genes during larval and postlarval ontogenic development; (4) the effects of dietary beta-1,3-glucan on the expression of the nine genes. The cDNA cloning study showed that the proPO cDNA contains an open reading frame of 2061 bp and encodes a 686 amino-acid peptide. The protein sequence of the proPO has a similarity of 85% with those of Penaeus monodon and P. semisulcatus and has an identity of between 58 and 77% with other crustaceans. Northern blot analysis revealed that proPO was constitutively expressed mainly in hemocytes. Its transcripts were observed in hemocytes and many other tissues when detected with RT-PCR. The results of in situ hybridizations showed that the hemocytes that infiltrated in tissues were responsible for the positive signals. The LGBP cDNA contains an open reading frame of 1104 bp and encodes a 367 amino-acid protein with a 17 a. a. signal peptide. The protein sequence of the LGBP has a similarity of 97% with LGBP of L. stylirostris, >90% identity with BGBP of P. monodon and LGBP of Fenneropenaeus chinensis and has an identity of between 63 and 86% with other crustaceans. Northern blot analysis revealed that LGBP was constitutively expressed mainly in hepatopancreas. The results of in situ hybridizations showed that the hepatopancreatic F cells might be the major cell type for LGBP production. Using the complete cDNAs of proPO and LGBP and partial fragments of the other seven genes, their tissue expressions were analyzed by conventional RT-PCR, quantitative real time PCR (qPCR) and in situ hybridization. The results demonstrated that BGBP-HDL, LGBP and hemocyanin were mainly expressed in the hepatopancreas and their expressions levels were about 10 to 30% those of

Traditional hybrid recommender systems typically follow a manually created fixed prediction strategy in their decision making process. Experts usually design these static strategies as fixed combinations of different techniques. However, people&#039 / s tastes and desires are temporary and they gradually evolve. Moreover, each domain has unique characteristics, trends and unique user interests. Recent research has mostly focused on static hybridization schemes which do not change at runtime. In this thesis work, we describe an adaptive hybrid recommender system, called AdaRec that modifies its attached prediction strategy at runtime according to the performance of prediction techniques (user feedbacks). Our approach to this problem is to use adaptive prediction strategies. Experiment results with datasets show that our system outperforms naive hybrid recommender.

<p>In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping.</p><p>P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools.</p><p>The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples.</p><p>To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension.</p><p>The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants.</p>

Thesis (Ph.D.)--University of Alberta, 2010. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Virology, Medical Microbiology and Immunology. Title from pdf file main screen (viewed on January 10, 2010). Includes bibliographical references.

In this thesis, different genetic tools are used to investigate pre- and postzygotic barriers to gene exchange and their role in speciation in the pied flycatcher (Ficedula hypoleuca) and the collared flycatcher (F. albicollis). This species complex consists of four genetically distinct clades that apparently diverged in allopatry (I). Sequencing of introns from autosomal and Z-linked genes from the two species reveals signs of selection on the Z-chromosome. Sexual selection acting on Z-linked genes might explain this pattern (II). By using large-scale genotyping of single nucleotide polymorphisms (SNPs), introgression is observed at autosomal- but not Z-linked loci, mostly from the pied- to the collared flycatcher. Male plumage characters and genes involved in hybrid fitness are largely mapped to the Z-chromosome (III). By studying mate choice of female hybrids I show that there is a link between female preferences and the Z chromosome (IV). The rate of introgression in island versus clinal hybrid zones is consistent with regional differences in hybrid fertility. Asymmetric gene flow from allopatry on the islands may oppose reinforcement, leading to introgression and a partial breakdown of postzygotic isolation. Adaptive introgression may explain the high rate of introgression observed at one of the genetic markers (V). For late breeding female collared flycatchers it appears to be adaptive to use pied flycatchers as social fathers but conspecific males as genetic fathers. Additionally, females in mixed species pairs may reduce hybridization costs by producing an excess of male hybrid offspring that are more fertile than females (VI). In conclusion, the Z-chromosome appears to play a major role in flycatcher speciation. Sexual selection and reinforcement are important mechanisms in the divergence of these birds. However, gene flow from allopatry, introgression of adaptive genes and adaptive hetrospecific pairing by late breeding collared flycatcher females may work in the opposite direction.

Šiame moksliniame darbe nagrinėjama paprastojo (Quercus robur L.) ir bekočio (Q. petraea [Matt.] Liebl.) ąžuolų hibridizacija Trako miške (Alytaus raj.), bei su ja susiję ąžuolų pomiškio plitimo, lapijos nuokritų biocheminės sudėties bei įtakos dirvožemio mikrobiotai procesai. Šis darbas nuo kitų Europoje atliktų tyrimų išsiskiria savo specifika, kadangi tyrimų objektas apima bekočio ąžuolo arealo pakraštį. Nustatyta, kad tarprūšiniai hibridai Trako miške plito greičiau nei bekočiai. Trako miško motinmedžių ir jų palikuonių palyginamoji taksonominė analizė parodė, kad tarprūšinių paprastojo ir bekočio ąžuolų hibridų morfologinių lapų požymių variacija juvenaliniame amžiuje buvo didesnė nei vienos kurios tėvinių rūšių. Molekulinė-genetinė analizė patvirtino rūšinės priklausomybės nustatymo pagal morfologinius lapų požymius tinkamumą ir efektyvumą. Įrodyta, kad hibridizacijos procesams būdingas asimetrinio kryžminimosi pobūdis ir introgresijos procese dalyvauja ribotas individų skaičius. Vyrauja grįžtamasis kryžminimasis su viena iš tėvinių rūšių, šiuo atveju su bekočiu ąžuolu. Didesnė lignino koncentracija paprastojo ąžuolo lapuose sąlygojo mažesnį bakterijų ir mikroorganizmų, ypač mikromicetų, aktyvumą šio ąžuolo rizosferoje. Palyginus su paprastuoju ąžuolu, bekočio ir hibridinių ąžuolų lapų nuokritos ir rizosferos organinės medžiagos buvo intensyviau skaidomos. Todėl šių ąžuolų aplinkoje sąlygos plisti pomiškiui yra palankesnės negu po paprastaisiais ąžuolais. / This study analyzes hybridization of pedunculate (Quercus robur L.) and sessile (Q. petraea [Matt.] Liebl.) oak in forest Trakas (Alytus district), including the following processes related to hybridization: the prevalence of oak undergrowth, leaf litter fall and its biochemical composition, impact on soil microbiota. This study differs from other studies carried out in Europe by its specifics, since the research object is situated at the edge of the natural distribution of sessile oak. It was found the increase of the number of hybrid oaks in the undergrowth. A comparative taxonomic analysis of the parental trees of the Trakas forest showed that the variation of morphological traits of leaves of interspecific hybrids of pedunculate and sessile oaks was higher at juvenile age if compared with any paternal species. Molecular-genetic analysis confirmed suitability and effectiveness of the use of leaf morphological traits for species discrimination. Studies confirmed that hybridization process had a character of asymmetric crossings, a limited number of individuals participate in the introgression process. Backcross with one of the parental species – sessile oak prevails. The leaves litter fall of pedunculate oak was distinguished for a higher content of lignin, which determines decreased activeness of bacteria and, especially, micromicetes in the rhizosphere. The decomposition rate of leaves litter fall and organic compounds in rhizosphere under sessile and hybrid oaks were... [to full text]

Activity-induced immediate-early gene (IEG) transcription foci can be labelled with fluorescent probes, permitting high temporal and spatial resolution in mapping neuronal circuits. Previous quantification approaches have assumed a Boolean function of transcription foci, assuming that cells are either active or inactive. Due to multiple amplification steps in the in situ hybridization process, it was thought that information relating to magnitudes of firing rates was lost. However, the current data suggest that transcription foci actually exhibit non-Boolean intensity and size values which vary according to behavioural condition. Systematic characterization of transcription foci intensity and size revealed incremental variations such that: home-cage < one-environment exposure < five-environment exposure < maximal electroconvulsive shock. Visual differences in transcription foci may result from a quantifiable relationship between spiking patterns and transcription rates. The exact stoichiometry between neuronal spiking and transcription is not yet clear, but these results suggest that Boolean applications of IEG imaging may neglect accurate neuronal activation properties. / xvi, 125 leaves : ill. ; 29 cm

The use of microfluidic tools to develop two novel approaches to surface-based oligonucleotide hybridization assays has been explored. In one of these approaches, immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by the use of fluorescently labeled target. The fluorescence intensity profile evolved as a gradient that could be related to concentration, and was a function of many factors including hybridization reaction rate, convective delivery speed, target concentration and target diffusion coefficient. It was possible to acquire kinetic information from the static fluorescence intensity profile to distinguish target concentration, and the length and base-pair mismatches of target sequences. Numerical simulations were conducted for the system, and fit well with the experimental data. In a second approach, a solid-phase nucleic acid assay was developed using immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that had been coated with oligonucleotides having two different sequences. The hybridization of one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary sequence that was covalently attached to a glass substrate of a microfluidic channel was shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic potential for quantitative replacement of the sensing chemistry by control of stringency. A second oligonucleotide sequence conjugated to the immobilized QDs provided for the selective detection of target nucleic acids. The microfluidic environment offered the ability to manipulate flow conditions for control of stringency and increasing the speed of analytical signal by introduction of convective delivery of target sequences to the immobilized QDs. This work introduces a stable and adaptable immobilization strategy that facilitates solid-phase QD-bioprobe assays in microfluidic platforms.

Gene flow and hybridization are pervasive in nature, and can lead to different evolutionary outcomes. They can either accelerate divergence and promote speciation or reverse differentiation. The process of divergence and speciation are strongly influenced by both neutral and selective forces. Disentangling the interplay between these processes in natural systems is important for understanding the general importance of interspecific gene flow in generating novel biodiversity in plants. This thesis first examines the importance of introgressive hybridization in the evolution of the genus Pinus as a whole, and then focusing on specific pine species, investigates the role of geographical, environmental and demographical factors in driving divergence and adaptation. By examining the distribution of cytoplasmic DNA variation across the wide biogeographic range of the genus Pinus, I revealed historical introgression and mtDNA capture events in several groups of different pine species. This finding suggests that introgressive hybridization was common during past species’ range contractions and expansions and thus has played an important role in the evolution of the genus. To understand the cause and process of hybrid speciation, I focused on the significant case of hybrid speciation in Pinus densata. I established the hybridization, colonization and differentiation processes that defined the origin of this species. I found P. densata originated via multiple hybridization events in the late Miocene. The direction and intensity of introgression with two parental species varied among geographic regions of this species. During the colonization on Tibetan Plateau from the ancestral hybrid zone, consecutive bottlenecks and surfing of rare alleles caused a significant reduction in genetic diversity and strong population differentiation. Divergence within P. densata started from the late Pliocene onwards, induced by regional topographic changes and Pleistocene glaciations. To address the role of neutral and selective forces on genetic divergence, I examined the association of ecological and geographical distance with genetic distance in Pinus yunnanensis populations. I found both neutral and selective forces have contributed to population structure and differentiation in P. yunnanensis, but their relative contributions varied across the complex landscape. Finally, I evaluated genetic diversity in the Vietnamese endemic Pinus krempfii. I found extremely low genetic diversity in this species, which is explained by a small ancestral population, short-term population expansion and recent population decline and habitat fragmentation. These findings highlight the role of hybridization in generating novel genetic diversity and the different mechanisms driving divergence and adaptation in the genus Pinus.

In this thesis techniques are presented that aim to determine individual DNA signatures by controlled synthesis of nucleic acid multimers. Allele-specific extension reactions with an improved specificity were applied for several genomic purposes. Since DNA polymerases extend some mismatched 3’-end primers, an improved specificity is a concern. This has been possible by exploiting the faster extension of matched primers and applying the enzymes apyrase or Proteinase K. The findings were applied to methods for resequencing and viral and single nucleotide polymorphism (SNP) genotyping. P53 mutation is the most frequent event in human cancers. Here, a model system for resequencing of 15 bps in p53 based on apyrase-mediated allele-specific extension (AMASE) is described, investigated and evaluated (Paper I). A microarray format with fluorescence detection was used. On each array, four oligonucleotides were printed for each base to resequence. Target PCR products were hybridized and an AMASE-reaction performed in situ to distinguish which of the printed oligonucleotides matched the target. The results showed that without the inclusion of apyrase, the resulting sequence was unreadable. The results open the possibilities for developing large-scale resequencing tools. The presence of certain types of human papillomaviruses (HPV) transforms normal cells into cervical cancer cells. Thus, HPV type determination is clinically important. Also, multiple HPV infections are common but difficult to distinguish. Therefore, a genotyping platform based on competitive hybridization and AMASE is described, used on clinical sample material and evaluated by comparison to Sanger DNA sequencing (Papers II and III). A flexible tag-microarray was used for detection and the two levels of discrimination gave a high level of specificity. Easy identification of multiple infections was possible which provides new opportunities to investigate the importance of multiply infected samples. To achieve highly multiplexed allele-specific extension reactions, large numbers of primers will be employed and lead to spurious hybridizations. Papers IV to VI focus on an alternative approach to control oligomerization by using protease mediated allele-specific extension (PrASE). In order to maintain stringency at higher temperatures, Proteinase K, was used instead of apyrase, leading to DNA polymerase degradation and preventing unspecific extensions. An automated assay with tag-array detection for SNP genotyping was established. First PrASE was introduced and characterized (Paper IV), then used for genotyping of 10 SNPs in 442 samples (Paper V). A 99.8 % concordance to pyrosequencing was found. PrASE is a flexible tool for association studies and the results indicate an improved assay conversion rate as compared to plain allele-specific extension. The highly polymorphic melanocortin-1 receptor gene (MC1R) is involved in melanogenesis. Twenty-one MC1R variants were genotyped with PrASE since variants in the gene have been associated to an increased risk of developing melanoma. A pilot study was performed to establish the assay (Paper VI) and subsequently a larger study was executed to investigate allele frequencies in the Swedish population (Paper VII). The case and control groups consisted of 1001 and 721 samples respectively. A two to sevenfold increased risk of developing melanoma was observed for carriers of variants. / QC 20101028