3,348 research outputs found
Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification
No full textAlu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in ≥90% of diploid nuclei Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle
Methylation Status of Imprinted Genes and Repetitive Elements in Sperm DNA from Infertile Males
No full textStochastic, environmentally and/or genetically induced disturbances in the genome-wide epigenetic reprogramming processes during male germ-cell development may contribute to male infertility. To test this hypothesis, we have studied the methylation levels of 2 paternally (H19 and GTL2) and 5 maternally methylated (LIT1, MEST, NESPAS, PEG3, and SNRPN) imprinted genes, as well as of ALU and LINE1 repetitive elements in 141 sperm samples, which were used for assisted reproductive technologies (ART), including 106 couples with strictly male-factor or combined male and female infertility and 28 couples with strictly female-factor infertility. Aberrant methylation imprints showed a significant association with abnormal semen parameters, but did not seem to influence ART outcome. Repeat methylation also differed significantly between sperm samples from infertile and presumably fertile males. However, in contrast to imprinted genes, ALU methylation had a significant impact on pregnancy and live-birth rate in couples with male-factor or combined infertility. ALU methylation was significantly high-er in sperm samples leading to pregnancy and live-birth than in those that did not. Sperm samples leading to abortions showed significantly lower ALU methylation levels than those leading to the birth of a baby. Copyright (C) 2011 S. Karger AG, Base
Studies on polymorphic alu insertions and genomic diversity within the major histocompatibility complex
No full textAfter the initiation of the human genome sequencing project and the introduction of the field of 'bioinformatics', interest in human genetic diversity studies has been increased. Sequence diversity has helped define differences between genes and genomic regions that were previously unknown or difficult to determine. In this thesis I have undertaken to study sequence diversity in the human genome in three areas; 1) investigated diversity in the MHC as represented by the MICA alleles with respect to the known HLA alleles, 2) investigated the structure and diversity in the intergenic region from an MHC related (paralogous) genomic region and related the structural and diversity findings to the knowledge available on the MHC and the wider genome, and 3) described the identification of three and characterization of five new MHC class I polymorphic markers (Alu) and their polymorphic characteristics in worldwide populations and their associations with skin cancer.
1. Phylogenetic analysis of MICA alpha-domain (extracellular) sequences demonstrated relationships with HLA-B cross-reactive serogroups. The HLA-B and MICA loci are in linkage disequilibrium. The data indicated that MICA and HLA-B have evolved in concert from their common ancestors and that the transmembrane polymorphisms have arisen independently and more recently.
2. Sequence analysis of the CD1 genomic region confirmed the presence of five CD1 genes and revealed that there are four unrelated intergenic regions (IGRs). The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans.
3. Five polymorphic Alu insertions (POALINs) were identified (two from previous studies) and located within the 1.8 megabase of the MHC class I genomic region. All five POALINs are polymorphic, and are positively associated with the HLA-A and HLA-B alleles. The AluyHJ insertion was found most frequently associated with HLA-A1 or A24, AluyHG with HLA-A2, AluyHF with HLA-A2, A-10 or -A26 and AluyTF showed a marginal association with HLA-A29. The AluyMICB insertion was strongly associated with HLA-B17 (HLA-B57, HLA-B58) and HLA-B13. The presence of three Alu insertions (AluyHJ, AluyHG and AluyHF) was found in only one HLA class I haplotype (HLA-A1, -B57, -Cw6) in the 10th IHW cell lines. A novel positive association between the presence of AluyMICB and the 'MICAdel/MICBnull/HLA-B48' haplotype was determined. The AluyMICB insertion was also associated with at least three different MICB alleles (*0102, *0107N and *0105) and three different HLA-B alleles (B13, B48 and B57). Based on the analysis of associations between different polymorphic markers within the beta block, the MICB*0102 allele was inferred to be the ancestral form of the MICB*0105 and MICB*0107N alleles. The AluyMICB polymorphism can be used to further investigate haplotype relationship and consequently their lineage origins. Some of the MHC POALINs are haplospecific and associate strongly with certain groups of HLA class I alleles and MHC ancestral haplotypes. The AluyTF frequency was significantly associated with skin cancer (p<0.005).
MICA gene diversity is derived from two different evolving paths, therefore one or the other alone cannot reliably mark an ancestral haplotype. The CD1 duplicons originated well before the HLA class I duplicons. The MHC POALINs provide new lineage and linkage markers for the fine mapping study of different haplotypes and variations in linkage groups across 1.8 Mb of the MHC class I region. The POALINs may also prove useful in investigating the origins and history of human populations and in determining the role of human genetic diversity in disease risk
Attempts to detect retrotransposition and de novo deletion of Alus and other dispersed repeats at specific loci in the human genome
No full textDispersed repeat elements contribute to genome instability by de novo insertion and unequal recombination between repeats. To study the dynamics of these processes, we have developed single DNA molecule approaches to detect de novo insertions at a single locus and Alu-mediated deletions at two different loci in human genomic DNA. Validation experiments showed these approaches could detect insertions and deletions at frequencies below 10(-6) per cell. However, bulk analysis of germline (sperm) and somatic DNA showed no evidence for genuine mutant molecules, placing an upper limit of insertion and deletion rates of 2 x 10(-7) and 3 x 10(-7), respectively, in the individuals tested. Such re-arrangements at these loci therefore occur at a rate lower than that detectable by the most sensitive methods currently available
Pharmaceutical Management and Prescribing Pattern of Antimalarial Drugs in the Public Health Facilities in Dar-es-salaam, Tanzania
No full textDespite the efforts of minimizing utilization of limited financial resources in Tanzania, there are still some factors that affect the whole system of supply chain of antimalarial medicines. The goals of Integrated Logistic System (ILS) have not been realized since still there is drug shortage and poor stock recording system in the health facilities. Inadequate knowledge of the pharmaceutical management and inappropriate use of antimalarial drugs always contribute to the poor quality and availability of the drugs in the health facilities and irrational use of the antimalarial drugs. The main objective of the study was to assess the knowledge in practice of pharmaceutical management and prescribing pattern of antimalarial drugs in the public health facilities in Dar-es-salaam. Descriptive retrospective cross-sectional study design was used to survey nine (9) public health facilities in Dar-es-salaam region. The health facilities included 4 hospitals and 5 Health Centers. A total of 32 pharmaceutical health workers (drug store managers and drug dispensers) were interviewed using structured questionnaires. Two different structured questionnaires were used to interview drug store managers and drug dispensers separately. Average stock-out time of antimalarial drugs from January to December 2010 was assessed using a designed form. A total of 4320 prescriptions were examined and recorded from all the selected public health facilities. Adequate storage conditions and handling of medicines procedures were also assessed. Since Medical Stores Department (MSD) is the main supplying source of the antimalarial drugs to these facilities, it was also included in this study as the special site for assessing availability of antimalarial medicines from the source. Among all nine (9) drug store managers, six (6) had poor knowledge on quantification concept, and seven (7) did not apply Integrated Logistic System indicating 2 that it is still confusing to be applied. All of drug store managers had inadequate knowledge on the concept of procurement, and there were no effective and efficient procurement systems in all the health facilities. In most of the facilities, BIN Cards and Stock ledger books were the most commonly used stock recording systems. . There are no Electronic Drug Recording systems in these facilities. A tedious and bureaurocratic process of expired drug disposal was cited as a reason for pilling up of expired drugs in the health facilities. Percentage time out of stock for the antimalarial drugs were 25% for Artemether-Lumefantrine (ALU) and 25.7% for Quinine tablets and 6.4% for injections All drug stores in the health facilities scored satisfactory marks in adequacy storage conditions and handling of antimalarial medicines. However, all facilities had no cold storage facilities with temperature charts; and medicines were kept directly on the floor in seven (7) out of 9 main drug stores and six (6) out of 15 dispensing rooms. Regarding rational dispensing of medicines, only 22 and 12 of all (32) interviewed drug dispensers had good knowledge on recommended doses and dosage regimen of ALU respectively. Regarding dispensing of ALU in pregnancy as special group, 19 drug dispensers were not able to provide correct information for use of ALU during pregnancy. The average number of drugs prescribed per prescriptions was 2.4±0.014, and 98 % of prescriptions contained only one antimalarial drug, among them 88.9 % was ALU. About 96.9 % of the prescriptions showed wrong prescribing pattern for ALU in terms of its dosage. Despite the government efforts in increasing public awareness of ALU and ensuring constant availability of such drugs at MSD, there is a significant stock-out period due to poor pharmaceutical management of antimalarial medicines in the public health facilities. Although adherence to the national malaria treatment guidelines is satisfactory, there is significant irrational prescribing of antimalarials among the prescribers. Based on these findings, it is proposed that regular on-job training and continuing education should be provided to drug dispensers and prescribers in the public health facilities
Bioinformatics approaches for functional predictions in diverse informatics environments
No full textBioinformatics is the scientific discipline that collates, integrates and analyses data and information sets for the life sciences. Critically important in agricultural and biomedical fields, there is a pressing need to integrate large and diverse data sets into biologically significant information. This places major challenges on research strategies and resources (data repositories, computer infrastructure and software) required to integrate relevant data and analysis workflows. These challenges include:
The construction of processes to integrate data from disparate and diverse resources and legacy systems that have variable data formats, qualities, availability and accessibility constraints.
Substantially contributing to hypothesis driven research for biologically significant information.
The hypothesis proposed in this thesis is that in organisms from divergent origins, with differing data availability and analysis resources, in silico approaches can identify genomic targets in a range of disease systems. The particular aims were to:
1. Overcome data constraints that impact analysis of different organisms.
2. Make functional genomic predictions in diverse biological systems.
3. Identify specific genomic targets for diagnostics and therapeutics in diverse disease mechanisms.
In order to test the hypothesis three case studies in human cancer, pathogenic bacteria, and parasitic arthropod were selected, the results are as follows.
In case study 1 sequence information was integrated to make novel predictions, and generate novel findings for the role of the Alu repeat element in cancer. An under representation of Alu was found in cancerous transcript and most noncancerous Alu transcript found were of an unknown function. These findings led to an Alu-mediated siRNA model for the down regulation of Alu containing mRNA in cancer.
Case study 2, comparative genomic analyses identified venereal diagnostic targets that discriminated Campylobacter fetus subspecies venerealis from other Campylobacter species and subspecies. Plasmid borne virulence Type IV secretory pathway genes specificity however varied for biovars, compromising their use for diagnostics. These findings resulted in the targeted sequencing of Campylobacter fetus subspecies venerealis biovar genomes.
Case study 3, in cattle tick ectoparasite (Rhipicephalus microplus), a large highly complex and under researched genome, transcript sequence was analysed and tick vaccination targets identified. These vaccine candidates successfully imparted immunity in the bovine host. The developed high throughput vaccine target identification system is now being applied to other disease systems.
Through the shared bioinformatics approaches, novel functional targets and models in disease were determined.
This thesis has developed and demonstrated in silico approaches for:
1. The collation, annotation and integration of data from divergent organisms with variable data constraints.
2. Novel functional predictions in diverse biological systems.
3. Novel vaccine and diagnostic candidate identification, in diverse disease mechanisms, substantially contributing to hypothesis driven research
Identificació de nous marcadors moleculars en metàstasi hepàtica de càncer colorectal
No full text[cat] L’objectiu d’aquest treball és la identificació i caracterització de les principals alteracions en metàstasi hepàtica (MH) de càncer colorectal (CCR) tant a nivell d’expressió gènica com de metilació de l’ADN. Els resultats obtinguts han de servir per millorar la comprensió del procés metastàtic i aportar nous coneixements que puguin ser aplicats en el diagnòstic molecular i en la recerca de noves opcions terapèutiques.
Els resultats de la tesi estan estructurats en 2 articles:
“LIVER METASTASIS SIGNATURE AS PROGNOSTIC GENES OF DISTANT DISSEMINATION IN STAGE III COLORECTAL CANCER”
L’objectiu és la identificació de marcadors moleculars d’expressió gènica importants en el procés metastàtic. 19 mostres aparellades de tumor primari de còlon i MH han estat hibridades en un microarray de cDNA (Affymetrix). S’obté un llistat de 1374 sondes diferencialment expressades de forma significativa entre tumors i metàstasis.
S’ha creat una signatura metastàtica (algoritme de Golub) que consta de 7 gens específics de MH: MMP3, MMP1, CYP1B1, CLCA4, MS4A12, SPP1 i CEACAM7.
La capacitat de pronòstic de MH d’aquesta signatura en tumors i en mucosa normal de còlon també ha estat avaluada, mitjançant RT‐PCR quantitativa. Segons un anàlisi de regressió logística univariant, els gens MS4A12, CYP1B1 i CLCA4 estan significativament sobreexpressats en tumors metastàtics (n=61) en comparació amb els no metastàtics (n=40).
L’anàlisi estadístic Random Forest, a partir dels nivells d’expressió de MS4A12 i CYP1B1 (en tumors i en còlon normal), permet pronosticar disseminació al fetge amb una probabilitat d’encerts del 80%.
Ambdós gens mostren major expressió gènica en el component estromal que en el tumoral quan s’analitzen mostres de microdissecció per captura làser.
D’altra banda, s’ha desenvolupat un model murí per injecció intraesplènica de línies cel∙lulars humanes de CCR (KM12C i HCT116) capaces de metastatitzar específicament a fetge reproduint així el procés de disseminació en pacients.
“ALU ELEMENTS’ METHYLATION ANALYSIS IN METASTATIC COLORECTAL CANCER”
L’objectiu és la identificació d’alteracions en la metilació de l’ADN específiques de MH. S’han utilitzat dues metodologies: l’AUMA (Amplification of Unmethylated Alu) i l’array de metilació. En cap cas s’han trobat diferències de metilació entre tumors primaris i MH aparellades.
Tot i això, s’ha validat mitjançant seqüenciació per bisulfit i corbes de melting dues bandes d’AUMA amb canvis de metilació recurrents (28 i 33% dels casos) entre mucosa normal de còlon i tumors, anomenades banda B (19q13.32) i banda C (16p13.3).
Per limitar l’àrea afectada per aquests canvis de metilació, s’ha fet seqüenciació per bisulfit de les illes CpG veïnes a les bandes B i C concloent que es tracta d’alteracions poc extenses dins el genoma.
L’anàlisi transcripcional dels gens propers amb l’array d’expressió (Affymetrix) ens indica que no hi ha regulació de l’expressió gènica per metilació dins una regió de fins a 2Mb lluny de les bandes d’AUMA.
Tot i la manca de significació, la línia cel∙lular HCT116 desmetilada (tractament amb AZA i TSA) ha servit per analitzar una possible correlació entre la metilació de les bandes B/C i l’expressió de gens propers. La majoria de gens analitzats estaven sobreexpressats en la línia tractada.
Les conclusions principals d’aquesta tesi són dues:
‐ S’ha obtingut una signatura metastàtica de 7 gens específics de MH de CCR. Dos d’aquests gens, MS4A12 i CYP1B1, tenen capacitat per pronosticar MH, fet de gran importància clínica.
‐ No s’han trobat canvis de metilació específics de MH indicant que els canvis epigenètics succeeixen en estadis inicials de la tumorigènesis.[eng] Colorectal cancer (CRC) is the third most common cancer worldwide being liver metastasis (LM) a significant prognostic factor and the main cause of cancer related deaths. Despite of its clinical relevance, metastatic spread process is poorly understood.
The aim of this study is to clarify the mechanisms of colorectal cancer LM discovering new molecular markers (transcriptional and epigenetic ones) with significant role in the metastatic process to improve its diagnostics, treatment and prevention.
The results can be classified in 2 manuscripts:
1) LIVER METASTASIS SIGNATURE AS PROGNOSTIC GENES OF DISTANT DISSEMINATION IN STAGE III COLORECTAL CANCER
19 paired subjects (colorectal tumor/LM) have been used in cDNA microarrays (Affymetrix) in order to identify specific LM genes.
A metastatic signature of 7 specific liver metastasis related genes has been built: MMP3, MMP1, CYP1B1, CLCA4, MS4A12, SPP1 i CEACAM7. Two of them, MS4A12 and CYP1B1, have an important liver metastasis prognosis capability which could be very useful in clinics for CRC prognosis.
2) ALU ELEMENTS’ METHYLATION ANALYSIS IN METASTATIC COLORECTAL CANCER
Specific liver metastasis (LM) epigenetic alterations would be interesting for better dissemination process understanding.
Two different methodologies have been used: AUMA (Amplification of Unmethylated Alu) and methylation microarray. However, specific LM methylation changes have not been identified being, therefore, early events in adenoma‐carcinoma sequence
Unusual clustering of Alu repeats within the 5'-flanking region of the human lysozyme gene
No full textWe report the nucleotide sequence of the 2.2-kb 5'-flanking region of the human lysozyme gene. Four Alu repeats are located within this upstream region. Classification and dating of these four Alu elements, as well as of the four Alu elements present within the human lysozyme structural gene, were performed. Transposition of the eight Alu repeats found in the human lysozyme locus has apparently occurred at four different times during the primate genome evolution. Considering that Alu repeats are interspersed throughout human DNA with an average spacing of 4 kb, the presence of eight such repeats within the 8-kb lysozyme gene region and, in particular, of four of them in the 2.2-kb region upstream of the structural gene, appears quite unusual. © 1993 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted
A computational reconstruction of Papio phylogeny using Alu insertion polymorphisms
No full text© 2018 The Author(s). Background: Since the completion of the human genome project, the diversity of genome sequencing data produced for non-human primates has increased exponentially. Papio baboons are well-established biological models for studying human biology and evolution. Despite substantial interest in the evolution of Papio, the systematics of these species has been widely debated, and the evolutionary history of Papio diversity is not fully understood. Alu elements are primate-specific transposable elements with a well-documented mutation/insertion mechanism and the capacity for resolving controversial phylogenetic relationships. In this study, we conducted a whole genome analysis of Alu insertion polymorphisms unique to the Papio lineage. To complete these analyses, we created a computational algorithm to identify novel Alu insertions in next-generation sequencing data. Results: We identified 187,379 Alu insertions present in the Papio lineage, yet absent from M. mulatta [Mmul8.0.1]. These elements were characterized using genomic data sequenced from a panel of twelve Papio baboons: two from each of the six extant Papio species. These data were used to construct a whole genome Alu-based phylogeny of Papio baboons. The resulting cladogram fully-resolved relationships within Papio. Conclusions: These data represent the most comprehensive Alu-based phylogenetic reconstruction reported to date. In addition, this study produces the first fully resolved Alu-based phylogeny of Papio baboons
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