Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.

Orientadores: Maria de Fatima Sonati, Maricilda Palandi de Mello / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:17:59Z (GMT). No. of bitstreams: 1 Wenning_MarciaReginadeSouzaCossa_D.pdf: 2460425 bytes, checksum: bd59f41846de3b0439b17450b12154a1 (MD5) Previous issue date: 2007 / Resumo: A Doença da Hb H resulta da remoção ou inativação de três dos quatro genes da cadeia a da hemoglobina normalmente presentes no genoma diplóide e é caracterizada por anemia hemolítica crônica de intensidade moderada a grave. Os pacientes apresentam microcitose, hipocromia e poiquilocitose, com cerca de 25 a 30% de Hb Bart¿s (?4) ao nascimento e 5-30% de Hb H (ß4) na vida adulta. Embora a base molecular da doença tenha influência nos níveis de Hb H produzidos, uma heterogeneidade em relação a esse aspecto tem sido observada mesmo em pacientes com genótipos a idênticos, sugerindo que outros fatores contribuem para esta diversidade além dos determinantes talassêmicos. No presente trabalho, procuramos identificar transcritos diferencialmente expressos nos reticulócitos de dois pacientes com Doença da Hb H, irmãos, de origem étnica mista (chinesa e africana), um do sexo masculino, 21 anos de idade, com 18% de Hb H (MKS), e outro do sexo feminino, 19 anos, com 5% desta Hb (FKS), ambos com genótipo -a3.7/--SEA. O método envolveu a técnica de Differential Display Reverse Transcription-Polymerase Chain Reaction (DDRT-PCR) e a realização de Hibridização Subtrativa Supressiva (SSH). A validação dos achados foi feita pela Reação em Cadeia da Polimerase quantitativa em Tempo Real (qRT-PCR). Quatro perfis diferenciais de expressão foram selecionados, todos mais representados no paciente com maior nível de Hb H. Dois foram detectados por ambas as abordagens metodológicas: um transcrito altamente homólogo à parte do gene da PIP5KIIA (fosfatidilinositol 4-fosfato-5 quinase tipo II a) e outro ao gene da cadeia ß da hemoglobina humana. Os outros dois transcritos, selecionados apenas pela SSH, apresentararam similaridade ao gene FAM46C (Family with sequence similarity 46, member C), que corresponde a uma proteína hipotética de função indeterminada, e ao gene EIF4E-BP1 (eukariotic translation initiation factor 4E ¿ binding protein 1), que codifica uma proteína regulatória da tradução com capacidade de inibição do fator eIF4E (eukariotic translation initiation factor 4E). Na tentativa de identificar os mecanismos responsáveis pelo aumento dos transcritos da PIP5KIIA e da globina ß nos reticulócitos de MKSalguns outros genes, relacionados às vias de atuação ou ao processo de transcrição dos primeiros, tiveram sua expressão também avaliada pela qRT-PCR. Os resultados obtidos, embora não conclusivos, sugeriram que a diferença nos níveis de Hb H apresentada pelos pacientes aqui estudados está correlacionada com a taxa de síntese de cadeias ß, e que a enzima PIP5KIIA, provavelmente via sinalização celular pelo fosfatidilinositol, de alguma maneira participa de sua regulação. O significado destes achados e o papel dos transcritos dos genes FAM46C e EIF4E-BP1 devem ser futuramente investigados para uma melhor compreensão dos mecanismos de regulação da expressão dos genes da globina ß em pacientes com talassemia a / Abstract: Hb H disease results from the inactivation of three of the four a-globin genes normally present on diploid genome and it is characterized by a moderate to severe chronic hemolytic anemia. Patients usually present microcytosis, hypochromia and poikilocytosis, with 25 to 30% of Hb Bart¿s (?4) at birth and 5 to 30% of Hb H (ß4) in adult life. Although the molecular base of this disease influences the Hb H levels produced, some heterogeneity has been observed in relation to this aspect, even in patients with identical a genotypes, thus suggesting that other factors contribute to this diversity besides a-thalassemic determinants. The aim of the present study was to identify differentially expressed transcripts in the reticulocytes from two patients with Hb H disease, siblings, from Chinese and African origins, a 21-year- old male (MKS) with 18% of Hb H and a 19-year-old female (FKS) with 5% of Hb H, both with genotype -a3.7/--SEA. The methodology involved two techniques: the Differential Display Reverse Transcription-Polymerase Chain Reaction (DDRT-PCR) and the Suppression Subtractive Hybridization (SSH). Quantitative real time PCR (qRT-PCR) experiments were used to confirm some results. Four differentially expressed profiles were obtained, all of them better represented in the subject with the highest Hb H level. Two transcripts were detected by both methodological approaches, one being highly similar to PIP5KIIA gene (Phosphatidylinositol ¿ 4 phosphate 5-kinase, type II a) and the other one similar to human ß-globin gene. Two others transcripts were selected only by SSH and they showed to be to FAM 46C (Family with sequence similarity 46, member C) and EIF4E-BP1 (eukariotic translation initiation factor 4E-binding protein 1) gene homologues. In order to identify the mechanisms that are responsible for the transcripts PIP5KIIA and ß- globin increase in the reticulocytes from MKS patient, some other genes related to the transcriptional process also had its expression evaluated by qRT-PCR. Although not conclusive, our results suggest that the difference between the Hb H levels, showed by the subjects here studied, is correlated with ß-globin synthesis rate and that PIP5KIIA may participate of its regulation, probably by cell signalizing through Phosphatidylinositol. Studies, particularly involving a higher number of patients, and experiments aimed at elucidating the PIP5KIIA function in erythroid cells, should help to understand this process. The initiation factor -4E binding protein (EIF4E-BP1) and its capacity to bind to eIF4E, acts as negative regulator of cell growth. Its overexpression was detected in the patient with the highest HbH level. The significance of these findings and the role of the FAM46C and EIF4E-BP1 gene transcripts should be further investigated so that the regulating of the ß-globin gene expression in a-thalassemic patients can be better understood / Doutorado / Medicina Experimental / Doutor em Fisiopatologia Medica

The work deals with the fuzzy classification of DNA sequences. In the first part the theory summarized information about Fuzzy logic and methods of its use in the classification of biological sequence data. The second part is practically deal with the classification algorithm for assessing the similarity of sequences. Specifically, the dividing of coding and non-coding parts of the sequence and the use of fuzzy classification in DNA barcoding.

No description available.

EN Heme containing gas sensor proteins play important role in bacterial physiology in regulating many processes such as cell differentiation, virulence, biofilm formation or intercellular communication. For their structure, typical modular architecture is characteristic where various sensor domains (usually at the N-terminus) regulate the activity of the catalytic or functional domains (usually at the C-terminus). In this dissertation thesis, we focused on three representatives from the group of oxygen sensing proteins, namely histidine kinase AfGcHK, diguanylate cyclase YddV, phosphodiesterase EcDOS and also on protein RR, which is the interaction partner of AfGcHK. The main aim of the thesis was to study intra-protein/inter-domain signal transduction in two representatives of heme sensor proteins with a globin fold of the sensor domain (AfGcHK, YddV) and in one representative with PAS fold of the sensor domain (EcDOS). Another objective was to describe inter-protein signal transduction in the two component signaling system AfGcHK-RR and structurally characterize these two interacting partners. Emphasis was also placed on the study of the interaction between model sensor domains and different signaling molecules and also on function of individual amino acids involved in the binding of these...

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Οι αυτοαναπαραγόμενοι επισωματικοί φορείς γονιδιακής μεταφοράς αποτελούν πολλά υποσχόμενους φορείς γονιδιακής θεραπείας. Στην παρούσα εργασία δημιουργήθηκε ο φορέας Zif-VP64-EP2 στα πλαίσια των μελετών γονιδιακής θεραπείας για τις αιμοσφαιρινοπάθειες. Πρόκειται για ένα κυκλικό πλασμίδιο, που φέρει το γονίδιο ενός τεχνητού μεταγραφικού παράγοντα της γ-σφαιρίνης του Zif-VP64 υπό την επενέργεια του ισχυρού υποκινητή pSFFV καθώς και τo γονίδιο της eGFP με το S/MAR στοιχείο από την περιοχή 5’ του γονιδίου της ανθρώπινης ιντερφερόνης β υπό την επενέργεια του υποκινητή pCMV. Διαπιστώθηκε ότι το Zif-VP64-EP2 μεσολαβεί γονιδιακή μεταφορά σε διαμολυσμένα κύτταρα της ανθρώπινης κυτταρικής σειράς Κ562. Η μακράς διάρκειας διαμολυσμένη καλλιέργεια (3 μήνες) καταδεικνύει ότι το όχημα είναι λειτουργικό και διατηρείται ως επισωματικό σε Κ562 κύτταρα διαμολυσμένα κύτταρα με το Zif-VP64- ΕΡ2. / Self-replicating episomal vectors for gene transfer are a new and very promising experimental approach to gene therapy. In this study, it was created the vector Zif-VP64-EP2, within the context of developing self-replicating episomal vectors for the gene therapy of hemoglobinopathies. Zif-VP64-EP2 is a circular plasmid which includes the gene of an artificial transcription factor for gamma globin: Zif-VP64 under the control of pSFFV promoter and the gene of eGFP with the S/MAR element from the region 5’ of the human interferon β gene under the control of pCMV promoter. It was established that Zif-VP64-EP2 was retained within the transfected Κ562 hematopoietic progenitor cell. Its episomal situation for a long time (3 months) and its normal expression in K562 human cells constitutes a proof of the utility of Zif-VP64- ΕΡ2 system in gene therapy applications.

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Sequence alignment is one of the most intensely studied problems in bioinformatics, and is an important step in a wide range of analyses. An issue that has gained much attention in recent years is the fact that downstream analyses are often highly sensitive to the specific choice of alignment. One way to address this is to jointly sample alignments along with other parameters of interest. In order to extend the range of applicability of this approach, the first chapter of this thesis introduces a probabilistic evolutionary model for protein structures on a phylogenetic tree; since protein structures typically diverge much more slowly than sequences, this allows for more reliable detection of remote homologies, improving the accuracy of the resulting alignments and trees, and reducing sensitivity of the results to the choice of dataset. In order to carry out inference under such a model, a number of new Markov chain Monte Carlo approaches are developed, allowing for more efficient convergence and mixing on the high-dimensional parameter space. The second part of the thesis presents a directed acyclic graph (DAG)-based approach for representing a collection of sampled alignments. This DAG representation allows the initial collection of samples to be used to generate a larger set of alignments under the same approximate distribution, enabling posterior alignment probabilities to be estimated reliably from a reasonable number of samples. If desired, summary alignments can then be generated as maximum-weight paths through the DAG, under various types of loss or scoring functions. The acyclic nature of the graph also permits various other types of algorithms to be easily adapted to operate on the entire set of alignments in the DAG. In the final part of this work, methodology is introduced for alignment-DAG-based sequence annotation using hidden Markov models, and RNA secondary structure prediction using stochastic context-free grammars. Results on test datasets indicate that the additional information contained within the DAG allows for improved predictions, resulting in substantial gains over simply analysing a set of alignments one by one.

DNA methylation is an epigenetic modification that plays an important role in development and differentiation. The patterns of DNA methylation are largely established in early embryogenesis and maintained during development. Abnormal DNA methylation patterns have been associated with many human diseases, including cancer. Despite its importance, little is currently known about the mechanisms that determine DNA methylation patterns throughout the genome. To shed light on the molecular mechanisms that regulate DNA methylation, this study investigates whether DNA methylation patterns are established and maintained normally when human DNA is placed into a heterologous murine environment as opposed to its natural, endogenous chromosomal environment. Here, a previously generated transgenic mouse model, containing 117 kb of human DNA bearing the human α-globin cluster and all of its known regulatory elements, was analysed. The pattern of DNA methylation of the endogenous human α-globin cluster was compared with that of the transgenic cluster in the background of mouse embryonic stem cells (ESCs) and tissues. It was found that, although the normal human DNA methylation pattern was largely established and maintained in a mouse background, the region immediately around the human α-globin genes themselves is generally less methylated in mouse compared to human ESCs. It was found that regions adjacent and up to 2kb from the CpG islands (CGIs), so-called CGI shores, were unusually hypomethylated: this seems to be the result of an extension of CGIs in humanised mouse (hm) ESCs compared to human (h) ESCs. Furthermore, this hypomethylation appeared to increase during development in both erythroid and non-erythoid cells. To identify any cis-regulatory sequences responsible for the hypomethylated state of human CGI shores in the mouse, 2-4 kb human test sequences containing the CGI associated with the human α-globin 2 (α2) gene and its adjacent hypomethylated shore were re-integrated into the mouse α-globin locus via recombination-mediated cassette exchange (RMCE). Human CGI shores became hypomethylated in the context of the re-integrated test sequences, indicating that the appearance of hypomethylation is determined by the underlying human DNA sequence in the test fragments. In summary, the data presented here reveal that human CGIs become extended when placed in a mouse background leading to hypomethylation of human CGI shores in the mouse compared to the pattern of methylation at the normal endogenous human locus. These findings suggest that species-specific factors determine DNA methylation near CGIs. The transgenic mouse model provides an excellent system to dissect out species-specific regulation of CGI shore methylation. Furthermore, this study lays the foundation for future experiments addressing the role of DNA methylation in regulating human gene expression in the murine context, and examining the validity of transgenic mouse models for the study of human gene regulation.