Дисертації з теми "SLF gene"
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1
Czyzyk, Rafal Sebastian. "Role of the SLF gene in self-compatability in Petunia hybrida." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.546518.
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2
Guo, Ling. "Identification of novel SLE susceptibility genes by microarray analysis and candidate gene association study." Oklahoma City : [s.n.], 2008.
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3
Guerra, Sandra. "Gene polymorphisms in SLE." Thesis, King's College London (University of London), 2013. https://kclpure.kcl.ac.uk/portal/en/theses/gene-polymorphisms-in-sle(96ff1bac-bcca-40f1-bfd7-24bc87dc7a25).html.
Повний текст джерелаАнотація:
Systemic lupus erythematosus (SLE) is an autoimmune disease, with a strong genetic component. It is characterised by hyperactive T and B cells, chronic inflammation and the production of antinuclear autoantibodies. SLE affects mostly women of child baring age, with a 9:1 ratio, women to men and has been reported to be more prevalent in people of non-European ancestry. In the era of genome-wide association studies (GWAS), elucidating the genetic factors present in SLE has been very successful, with over 28 confirmed disease susceptibility loci mapped and a number of candidate genes identified. During this thesis I fine mapped IL18 as it had previously been reported to be associated with SLE, SNP rs360719. After fine mapping and subphenotype analysis in UK and African American cohorts, I was unable to replicate the published association. Although genetic data did not confirm IL18 to be associated with SLE, I demonstrated increased IL-18 serum levels in SLE renal patients compared to SLE patients. I further analysed IL10, another previously associated SLE candidate loci in our current SLE GWAS cohort (4000 cases and 9000 controls) of European ancestry. I again was unable to replicate the previous association, however using other SLE GWAS data showed SNP rs3024505 to be associated in Northern European samples. Further analysing our SLE GWAS, I located IKZF3 as a candidate loci. I identified an associated block of 56 SNPS and located the association to a single SNP rs2941509, p=1.46xlQ-8. Furthermore, I demonstrated an allelic imbalance in this SNP, with the protective G allele being expressed 1.5 times greater than the risk A allele, in controls. These data here demonstrated in this thesis indicates the importance of fine mapping candidate loci and verification of previously associated loci. This thesis contributes to the current knowledge of SLE by demonstrating discrepancies in published association data and showing the importance of larger studies.
4
Wang, Dali. "Adaptive Double Self-Organizing Map for Clustering Gene Expression Data." Fogler Library, University of Maine, 2003. http://www.library.umaine.edu/theses/pdf/WangD2003.pdf.
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5
Anderson, George. "Existentially self deceptive storytelling : a new genre." Thesis, University of Westminster, 2009. https://westminsterresearch.westminster.ac.uk/item/90vq0/existentially-self-deceptive-storytelling-a-new-genre.
Повний текст джерелаАнотація:
This thesis is an exploration into the function and form of storytelling. Its initial assumption is that consciousness is a genetically transmitted mechanism which generates a concept of self by creating a story. In this formulation, the consciousness is called narrative-consciousness. Since the concept of self necessarily suggests its opposite and this in turn involves awareness of existential futility, the purpose of the story, generated by the narrative-consciousness, is seen to be, in the first instance, the hiding of this unavoidable and potentially damaging awareness. This thesis suggests that to achieve this goal the story must be based on the process of self-deception. The thesis shows that, in general, self-deception involves three significant components in its bid to separate any two paradoxical ideas: unease, process and hiding and that each of these maps onto a particular component in the final narrative of the self. The narrative created by a consciousness hiding, in particular, the awareness of existential angst is given the specific name existentially-selfdeceptive- story, with an acronym ESDeS. The thesis goes on to suggest that such a narrative-consciousness could produce written stories that follow the same pattern, in which case the stories are called existentially-self-deceptive-novels, with an acronym ESDeN. Such a story or genre is then shown to be part of a continuum consisting of up to three distinct ways of dealing with existential futility. The thesis labels these Story-1, Story-2 and Story-3 respectively but reserves the name ESDeN for a subset of Story-2. Analyses of three of these stories, Heart of Darkness, Chance and Thinks concludes that the genre necessarily includes genre-markers, bracketing deaths and repetition, can also include other optional components such as the self-deceptive process or the parent-child mechanism but that its defining characteristic is its division between an overt plot and a covert plot which contains a collusive death of a character identified with existential angst. A covert plot is necessarily available but it is, by definition, not easily discovered. Its successful hiding is made possible, primarily, by foregrounding the overt content of the novel at the expense of the covert. In this sense, the only necessary requirement of the overt content is it should distract and it does this best when the reader cooperates by investing time in interpretation: that is, in order to disguise the ultimate the reader concentrates on the proximal. Finally, the thesis mirrors the endings of each ESDeN by drawing attention to the fact that this collusion will not work for long: just as self-deception cannot withstand too much contrary evidence, the covert plot will not stand too many rereadings. Inevitably, for the true ESDeS, another point of recognition will occur and this will necessitate the renewal or replacement of the ESDeS.
6
Sun, Wei [Verfasser]. "RNA isoform analyses of Drosophila Dscam gene and Xenopus tropicalis clustered Protocadherin genes provide insights for neuronal self-avoidance / Wei Sun." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1098185358/34.
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7
Zhang, Yan, and 张彦. "Association studies of systemic lupus erythematosus (SLE) : from novel susceptibility loci to gene-gene interaction." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/211557.
Повний текст джерелаАнотація:
Systemic lupus erythematosus (SLE) is characterized as an autoimmune disorder with unclear etiology.
To identify the genetic effect of SLE, a genome wide association study (GWAS) and its further replication were conducted on SLE patients in Asian populations and ethnically matched controls. Before this study, most of the confirmed association loci were identified by GWAS studies in European populations. Apart from the established associations, we identified a SLE susceptible single nucleotide polymorphisms (SNP) located in ETS1 (rs1128334, P=2.3E-11, OR=1.29) in four different cohorts. This locus is probably an Asian-specific susceptibility locus since no Caucasian study has reported further validation in the last years. A new susceptibility variant in UHRF1BP1 (rs13205210, P=4.4E-09, OR=1.49) independent from the previously confirmed SNPs in Caucasian study was also confirmed to be associated with SLE in the Hong Kong Chinese population.
Meta-analysis was performed by introducing another Chinese Han GWAS data set from Anhui province, China. Three loci, TET3-DGUOK, CD80, DRAM1, were confirmed to be associated with SLE. Two loci with suggestive signals in Hong Kong GWAS and further replication were also confirmed by the meta-analysis: PTTG1-MiRNA146a, YDJC.
In order to identify the genetic effect for females who have predominant chance to suffer from SLE, X chromosome specific meta-analysis based on the Hong Kong and Anhui GWAS data and further replication study were performed by considering the difference between females and males. A signal in PRPS2, and three independent signals in the Xq28 were confirmed with the replication in three different cohorts by considering both females and males.
Gene-gene interactions were also investigated genome-widely in a hypothesis free manner based on the meta-analysis results. The further validation processes were preceeded based on each independent GWAS data set. Four pairswise interacting loci were found and cross validated by three methods including logistic regression and Multifactor Dimensionality Reduction (MDR) and information gain theory based on the Anhui GWAS data set. Further studies are still needed to better explain the real features of genetic epistasis and the potential biological roles.
By incorporating two GWAS from the same population, the population difference is efficiently avoided. Together with the putative gene-gene interactions, this study presents a comprehensive analysis based on the GWAS data conducted on SLE. It may shed new light on the disease mechanisms of SLE.published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy
8
Raciti, Daniela. "A large-scale gene discovery screen identifies over hundred solute carrier (SLC) genes with organ specific expression patterns in the Xenopus embryo /." Zürich : ETH, 2007. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=17204.
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9
Morimoto, Takuya. "Insights into the evolution and establishment of the Prunus-specific self-incompatibility recognition mechanism." 京都大学 (Kyoto University), 2017. http://hdl.handle.net/2433/225645.
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10
Jonsson, Per. "Improving Clustering of Gene Expression Patterns." Thesis, University of Skövde, Department of Computer Science, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-482.
Повний текст джерелаАнотація:
The central question investigated in this project was whether clustering of gene expression patterns could be done more biologically accurate by providing the clustering technique with additional information about the genes as input besides the expression levels. With the term biologically accurate we mean that the genes should not only be clustered together according to their similarities in expression profiles, but also according to their functional similarity in terms of functional annotation and metabolic pathway. The data was collected at AstraZeneca R&D Mölndal Sweden and the applied computational technique was self-organising maps. In our experiments we used the combination of expression profiles together with enzyme classification annotation as input for the self-organising maps instead of just the expression profiles. The results were evaluated both statistically and biologically. The statistical evaluation showed that our method resulted in a small decrease in terms of compactness and isolation. The biological evaluation showed that our method resulted in clusters with greater functional homogeneity with respect to enzyme classification, functional hierarchy and metabolic pathway annotation.
11
Besemer, John David. "Heuristic and self-training methods for improving gene prediction in prokaryotes." Diss., Georgia Institute of Technology, 2002. http://hdl.handle.net/1853/25388.
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12
Pan, Fang. "Self-assembly of amphiphilic peptides and their potential in gene transfection." Thesis, University of Manchester, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.496700.
Повний текст джерелаАнотація:
The surfactant-like peptides are amphiphillic molecules containing polar or charged amino acids moieties as hydrophilic heads and non-polar ammo acids as hydrophobic tails, their surfactant-like amphiphilicity is attractive to a wide range of biotechnological applications including regenerative medicine and drug delivery. Short and designed peptides are easy to synthesise. They are becoming increasingly popular for self-assembly studies as model systems. It is of bith fundamental and practical significance to explore how these designed peptide molecules aggregate in solution and adsorb at the interface.
13
Wood, Kris Cameron. "Nanostructured gene and drug delivery systems based on molecular self-assembly." Thesis, Massachusetts Institute of Technology, 2007. http://hdl.handle.net/1721.1/39350.
Повний текст джерелаАнотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2007.Includes bibliographical references.
Molecular self-assembly describes the assembly of molecular components into complex, supramolecular structures governed by weak, non-covalent interactions. In recent years, molecular self-assembly has been used extensively as a means of creating materials and devices with well-controlled, nanometer-scale architectural features. In this thesis, molecular self-assembly is used as a tool for the fabrication of both gene and drug delivery systems which, by virtue of their well-controlled architectural features, possess advantageous properties relative to traditional materials used in these applications. The first part of this thesis describes the solution-phase self-assembly of a new family of linear-dendritic "hybrid" polymers with plasmid DNA for applications in gene therapy. It begins with an overview of the design of next-generation, non-viral gene delivery systems and continues through the synthesis and validation of hybrid polymer systems, which possess modular functionalities for DNA binding, endosomal escape, steric stabilization, and tissue targeting. This part of the thesis concludes with applications of these systems to two areas of clinical interest: DNA vaccination and tumor targeted gene therapy.
(cont.) The second part of this thesis describes the directed self-assembly of polymeric thin films which are capable of degrading in response to either passive or active stimuli to release their contents. It begins with a description of passive release thin films which degrade by basic hydrolysis to release precise quantities of model drug compounds. These systems can be engineered to release their contents on time scales ranging from hours to weeks and can also be designed to release multiple drugs either in series or in parallel. Later, field-activated thin films which release their contents in response to an external, electrical stimulus are described and characterized in detail. Together, these approaches combine rapid and inexpensive processing, the ability to conformally coat any surface regardless of composition, size, or shape, and the ability to release multi-drug or multi-dose schedules, and as such they may find applications in a range of areas.
by Kris Cameron Wood.
Ph.D.
14
Junior, Antonio Paulo Galdeano Damiance. "Desenvolvimento de modelos dinâmicos para a formação de clusters aplicados em dados biológicos." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/55/55134/tde-23012007-103117/.
Повний текст джерелаАнотація:
Com o advento da tecnologia de microarray, uma grande quantidade de dados de expressão gênica encontra-se disponível. Após a extração das taxas de expressão dos genes, técnicas de formação de clusters são utilizadas para a análise dos dados. Diante da diversidade do conhecimento que pode ser extraído dos dados de expressão gênica, existe a necessidade de diferentes técnicas de formação de clusters. O modelo dinâmico desenvolvido em (Zhao et. al. 2003a) apresenta diversas características interessantes para o problema de formação de clusters, entre as quais podemos citar: a não necessidade de fornecer o número de cluster, a propriedade de multi-escala, serem altamente paralelos e, principalmente, permitirem a inserção de regras e mecanismos mais complexos para a formação dos clusters. Todavia, este modelo apresenta dificuldades em determinar clusters de formato e tamanho arbitrários, além de não realizar a clusterização hierárquica, sendo estas duas características desejáveis para uma técnica de clusterização. Neste trabalho, foram desenvolvidas três técnicas para superar as limitações do modelo dinâmico proposto em (Zhao et. al. 2003a). O Modelo1, o qual é uma simplificação do modelo dinâmico original, porém mais eficiente. O Modelo2, que a partir da inserção de um novo conjunto de elementos no modelo dinâmico, permite a formação de clusters de formato e tamanho arbitrário. E um algoritmo para a clusterização hierárquica que utiliza o Modelo1 como bloco de construção. Os modelos desenvolvidos foram aplicados em dados biológicos, segmentando imagens de microarray e auxiliando na análise do conjunto expressão de genes de St. Jude Leukemia.With the advent of microarray technology, a large amount of gene expression data is now available. Clustering is the computational technique usually employed to analyze and explore the data produced by microarrays. Due to the variety of information that can be extracted from the expression data, many clustering techniques with different approaches are needed. In the work proposed by (Zhao et. al. 2003a), the dynamical model for data clustering has several interesting features to the clustering task: the number of clusters does not need to be known, the multi-scale property, high parallelism, and it is flexible to use more complex rules while clustering the data. However, two desirable features for clustering techniques are not present: the ability to detect different clusters sizes and shapes, and a hierarchical representation of the clusters. This project presents three techniques, overcoming the restrictions of the dynamical model proposed by (Zhao et. al. 2003a). The first technique, called Model1, is more effective than the original model and was obtained simplifying it. The second technique, called Model2, is capable of detecting different clusters sizes and shapes. The third technique consists in a hierarchical algorithm that uses Model1 as a building block. The techniques here developed were used with biological data. Microarray image segmentation was performed and the St. Jude Leukemia gene expression data was analyzed and explored.
15
Arnold, Jennifer Monique. "Changes in connexin 32 protein and gene expression following cocaine self-administration." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0005/NQ42783.pdf.
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16
Arnold, Jennifer Monique Carleton University Dissertation Psychology. "Changes in connexin 32 protein and gene expression following cocanine self-administration." Ottawa, 1999.
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17
Lubovac, Zelmina. "Evaluation of clusterings of gene expression data." Thesis, University of Skövde, Department of Computer Science, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-484.
Повний текст джерелаАнотація:
Recent literature has investigated the use of different clustering techniques for analysis of gene expression data. For example, self-organizing maps (SOMs) have been used to identify gene clusters of clear biological relevance in human hematopoietic differentiation and the yeast cell cycle (Tamayo et al., 1999). Hierarchical clustering has also been proposed for identifying clusters of genes that share common roles in cellular processes (Eisen et al., 1998; Michaels et al., 1998; Wen et al., 1998). Systematic evaluation of clustering results is as important as generating the clusters. However, this is a difficult task, which is often overlooked in gene expression studies. Several gene expression studies claim success of the clustering algorithm without showing a validation of complete clusterings, for example Ben-Dor and Yakhini (1999) and Törönen et al. (1999).
In this dissertation we propose an evaluation approach based on a relative entropy measure that uses additional knowledge about genes (gene annotations) besides the gene expression data. More specifically, we use gene annotations in the form of an enzyme classification hierarchy, to evaluate clusterings. This classification is based on the main chemical reactions that are catalysed by enzymes. Furthermore, we evaluate clusterings with pure statistical measures of cluster validity (compactness and isolation).
The experiments include applying two types of clustering methods (SOMs and hierarchical clustering) on a data set for which good annotation is available, so that the results can be partly validated from the viewpoint of biological relevance.
The evaluation of the clusters indicates that clusters obtained from hierarchical average linkage clustering have much higher relative entropy values and lower compactness and isolation compared to SOM clusters. Clusters with high relative entropy often contain enzymes that are involved in the same enzymatic activity. On the other hand, the compactness and isolation measures do not seem to be reliable for evaluation of clustering results.
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Agarwal, Ankit. "Novel amphiphilic block copolymers and their self-assembled injectable hydrogels for gene delivery." [Ames, Iowa : Iowa State University], 2007.
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19
Bailey, Susannah Ines. "Self-inactivating retroviral vectors for gene therapy of X-Linked severe combined immunodeficiency." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1444526/.
Повний текст джерелаАнотація:
X-Linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the gene encoding the common cytokine receptor gamma chain, yc, resulting in profound defects in both cellular and humoral immunity. Although allogeneic bone marrow transplantation has proved highly successful, HLA-mismatched procedures are associated with significant morbidity and mortality. This disease is therefore a good candidate for gene therapy and sustained correction of 19 SCID-X1 patients have been reported in two clinical trials. However, the occurrence of severe adverse advents in one trial has highlighted the potential side-effects of retroviral gene transfer and reinforced the need to develop safer gene therapy vectors. A series of self-inactivating (SIN) gammaretroviral and lentiviral vectors for the treatment of SCID-X1 have consequently been developed. To reduce the potential for insertional mutagenesis mediated by the duplicated viral LTR sequences, alternative internal regulatory elements have been incorporated into the vector backbone. These include both endogenous (human elongation factor la - EFS) and viral (spleen focus forming virus - SFFV) promoters. In vitro, the SIN retroviral vectors were able to regulate yc expression on the cell surface of SCID-X1 cell lines and restore the lymphoid differentiation potential of Il2rg" haematopoietic progenitor cells. Functional correction of the immunological defect in the SCID-X1 mouse model was also achieved at similar levels for the both the SIN retroviral vectors and the LTR-regulated clinical vector. To further improve upon safety, lentiviral vectors were developed incorporating the endogenous human IL2RG promoter to regulate physiological expression of yc. In vitro and in vivo analysis of the promoter indicated a degree of haematopoietic tissue specificity and restoration of functional yc-receptor complexes was achieved following transduction of a SCID-X1 T cell line with a lentiviral vector incorporating this promoter however phenotypic correction of the yc-deficient mouse was unsuccessful. These results demonstrate that SIN retroviral vectors for SCID-X1 are effective in restoration of the immune defect in the yc-deficient murine model. The SIN design together with an endogenous (EFS) promoter might provide a potentially less mutagenic but equally effective vector for gene therapy of SCID-X1.
20
Freude, Lisa [Verfasser]. "Ig gene repertoire analysis in an SLE patient in clinical remission / Lisa Freude." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2010. http://d-nb.info/102450235X/34.
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21
Weinhold, Florian. "Self/other representations in Aleksei Balabanov's 'Zeitgeist movies' : film genre, genre film and intertextuality." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/selfother-representations-in-aleksei-balabanovs-zeitgeist-movies-film-genre-genre-film-and-intertextuality(29460f94-0440-431c-8d59-53133c73489f).html.
Повний текст джерелаАнотація:
This thesis uses the prism of genre to explore the character of self/other representations in five 'genre films' made by the Russian filmmaker Aleksei Balabanov and released between 1997 and 2006. It provides the first book-length study of Balabanov and aims to shed new light on the complexity of genre films and their representation techniques in an influential area of post-Soviet Russian cinema. The thesis aims to deconstruct the widespread perception of Balabanov as a populist director of 'mere genre movies', which are replete with xenophobic self/other representations. The films under investigation are linked through their developments of genre, evolving themes, an overarching narrative and multiple dialogicity among themselves, with their audiences and with Hollywood. They are shown to reflect the changing post-Soviet Russian Zeitgeist and its historical context. They do so by self-consciously deploying Hollywood genres and blending them with transgeneric modes/styles under the influence of renowned cinematic and literary inter-/transtextual works. The study examines the relationship between Balabanov's articulation of post-Soviet Russian identity vis-à-vis representations of dominant others, such as America, the Caucasus, Western Europe, Ukraine and, importantly, what the films portray as society's ruling criminal elites (primarily the New Russian 'gangsters').Combining the concepts of film genre with inter-/transtextuality within close film-textual analyses, the thesis focuses on the filmic texts and their visual, sound and narrative elements, which together indicate particular genre blends and their parabolic/allegorical potential. The analytical chapters investigate how these impinge upon the ideological orientation of Balabanov's approach to self/other representations. Film genre thus provides a method for exploring the articulations of an evolving post-Soviet Russian identity in Balabanov's work. The thesis reveals the director's self-consciously ambiguous perspectives on Russia's self, its own otherness in a globalised/ing world and the corrupting influences of the country's state-Socialist militarist past, previous and current military conflicts and the country's capitulation to the capitalist market. The application of a conceptual framework drawn from film genre studies enables the thesis to explore how these popular genre films become a platform for presentations of an internally divided Russian national self in its interactions with its various constitutive others, themselves characterised by diversity and inner heterogeneity. As a result, the thesis provides a long-overdue methodological interpretation of the most controversial segment of Balabanov's oeuvre and challenges received bi-partite views of this hitherto largely misrepresented auteur.
22
Azzi, Joelle. "The Role of p130/DREAM in Silencing Self-renewal Genes in Post-mitotic Neurons." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/37719.
Повний текст джерелаАнотація:
The recently identified DREAM complex assembles when Rb-like protein (p130, p107) recruits E2F4, DP (dimerization partner) and MuvB (multivulval complex B (Lin9, Lin37, Lin52, Lin54, and RbBp4)) during G0 and quiescence to repress cell cycle-dependent genes. DREAM assembly requires phosphorylation of the MuvB subunit Lin52 mediated by Dyrk1a, a kinase that has been linked to Down syndrome and neurodegenerative diseases. Our lab previously demonstrated an essential role for the Rb-like pocket proteins in the regulation of neural precursor population and that E2F4 is also involved in the regulation of the expression of the pluripotent gene Sox2. Here, we performed in utero electroporation experiments to overexpress the DREAM complex components and assess their roles during neurogenesis. Our results showed that the overexpression of DREAM components (Lin52 and p130) and Dyrk1a promotes commitment to differentiation at the expense of self-renewal. We also showed that Dyrk1a requires p130 or p107 to regulate neurogenesis. Furthermore, using harmine treatment which is an inhibitor of Dyrk1a the kinase that induces DREAM assembly, our results revealed that DREAM regulates the expression of self-renewal markers affecting the cell fate decision. Performing ChIP experiments, we detected a binding enrichment of the DREAM components on the promoters of not only classical cell cycle genes but on the self-renewal genes like Sox2 and EZH2. Taken together, our study confirmed that DREAM complex plays an important role in the cell fate determination during the regulation of neurogenesis through the control of the self-renewal genes.
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Vendrell, Arasa Alexandre. "SCF cdc4 regulates msn2 and msn4 dependent gene expression to counteract hog1 induced lethality." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7153.
Повний текст джерелаАнотація:
L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1.També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut.
Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation.
We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.
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Sun, Qian. "Application of Polyelectrolyte Layer-By-Layer Self-Assembly on Polymer-Based Gene Delivery System." Thesis, University of Nottingham, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519429.
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Li, Chen. "Attenuated Cocaine Seeking After Adolescent-Onset of Cocaine Self-Administration in Male Rats: Behavior, Environment, and Genes." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/100.
Повний текст джерелаАнотація:
Recreational drug use peaks in the developmental stage of adolescence in humans. In this dissertation, we used a rodent model of adolescence and behavioral assessments of intravenous (i.v.) cocaine self-administration and reinstatement of cocaine-seeking to explore age differences in these cocaine-related behaviors, and then tested for the influence of environmental enrichment and for correlations between behavior and expression of plasticity genes. Although taking similar amount of cocaine, male rats trained to self-administer cocaine during adolescence (adolescent-onset) showed attenuated cue-induced reinstatement of cocaine seeking compared with adults. This attenuated cue-induced reinstatement did not generalize to a natural reward, sucrose pellets. Then we asked whether the attenuated reinstatement may be due to rapid developmental re-organization of reinforcement circuits (high plasticity) in adolescent-onset groups. To stimulate or inhibit neuroplasticity, subjects experienced environmental enrichment or impoverishment during abstinence. Environmental manipulations had no effect in adolescent-onset groups, whereas the enriched environment attenuated cue-induced reinstatement in adults compared with their impoverished counterparts. Thus, we turned to internal factors that may contribute to age-differences in reinstatement of cocaine seeking. Using in situ hybridization to quantify the mRNA for two neuroplasticity-related genes, activity-regulated cytoskeletal-associated gene (arc) and brain-derived neurotrophic factor (bdnf), we identified that overall, arc expression in the nucleus accumbens (NAc) and bdnf expression in the medial prefrontal cortex (mPFC) was higher in adolescent-onset than in adult groups. Together our data suggest that adolescence in rodents may be a period of relative biological resistance to some long-term drug effects.
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Bian, Xue-Yu. "Towards cloning the self-incompatibility genes from Phalaris coerulescens." Title page, contents and abstract only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phb577.pdf.
Повний текст джерелаАнотація:
Bibliography: leaves 97-114. "Self-incompatibility (SI) is an important genetic mechnism to prevent the inbreeding of flowering plants and also an excellent system for studying cell-cell recognition and signal transduction."
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Özdogan, Alper. "Clustering Genes by Using Different Types of Genomic Data and Self-Organizing Maps." Thesis, University of Skövde, School of Life Sciences, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-2265.
Повний текст джерелаАнотація:
The aim of the project was to identify biologically relevant novel gene clusters by using combined genomic data instead of using only gene expression data in isolation. The clustering algorithm based on self-organizing maps (Kasturi et al., 2005) was extended and implemented in order to use gene location data together with the gene expression and the motif occurrence data for gene clustering. A distance function was defined to be used with gene location data. The algorithm was also extended in order to use vector angle distance for gene expression data. Arabidopsis thaliana is chosen as a data source to evaluate the developed algorithm. A test data set was created by using 100 Arabidopsis genes that have gene expression data with seven different time points during cold stress condition, motif occurrence data which indicates the occurrence frequency of 614 different motifs and the chromosomal location data of each gene. Gene Ontology (http://www.geneontology.org) and TAIR (http://arabidopsis.org) databases were used to find the molecular function and biological process information of each gene in order to examine the biological accuracy of newly discovered clusters after using combined genomic data. The biological evaluation of the results showed that using combined genomic data to cluster genes resulted in new biologically relevant clusters.
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McLean, Megan Elizabeth. "Synthesis and characterization of covalently-linked dendrimer bioconjugates and the non-covalent self-assembly of streptavidin-based megamers." Texas A&M University, 2004. http://hdl.handle.net/1969.1/1319.
Повний текст джерелаАнотація:
This work details the attachment of dendrimers to proteins, peptides and single stranded DNA (ssDNA). Dendrimers based on melamine satisfy many of the synthetic demands in the field of bioconjugate chemistry including: monodispersity, synthetic flexibility and scalability. The solution-phase syntheses of both ssDNA-dendrimer and peptide-dendrimer bioconjugates is described, and thorough characterization by matrix-assisted laser desorption ionization/ time-of-flight (MALDI-TOF) mass spectrometry, UV-vis spectroscopy, fluorescence spectroscopy, and polyacrylamide gel electrophoresis is discussed.
Non-covalent DNA-dendrimer complexes have been shown to facilitate antisense gene delivery, but are vulnerable to dissociation and subsequent enzymatic degradation within the cell. In an effort to prepare biocompatible antisense agents capable of effectively shielding ssDNA from intracellular nuclease digestion, disulfide-linked ssDNA-dendrimers were prepared and rigorously characterized to rule out the possibility of an electrostatic-based interaction.
Hybridization assays were performed to determine if the covalently-attached dendrimer affected the ability of the attached ssDNA strand to anneal with a complementary sequence to form double-stranded DNA (dsDNA)-dendrimers. Results indicate that ssDNA-dendrimer conjugates readily anneal to complementary ssDNA strands either in solution or attached to gold surfaces. Nuclease digestions of conjugates in solution suggested that enzymatic manipulation of dsDNA-dendrimers is possible, offering promise for DNA-based computation and other fields of DNA-nanotechnology.
Much larger bioconjugates consisting of dendrimers, proteins and peptides were prepared with the goal of obtaining molecular weights sufficient for enhanced permeability and retention (EPR) in tumors. While the dendrimer provides the advantages of a purely synthetic route for drug delivery, the protein portion of the bioconjugate provides a monodisperse, macromolecular scaffold for the non-covalent self-assembly of the dendrimers. The strategy presented herein is based on the strong interaction between biotin and the 60 kD tetrameric protein streptavidin. Each monomer of streptavidin is capable of binding 1 biotin molecule, thus when biotin functionalized peptide-dendrimers are added to streptavidin they bind to form a cluster of dendrimers, or a megamer.
The biotinylated peptides that link the dendrimers to the streptavidin core provide a way to actively target specific cell types for drug delivery. Megamer formation through the addition of tetrameric streptavidin was successful as indicated by MALDI-TOF, UV-vis titration and gel electrophoresis assays.
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Unzueta, Elorza Ugutz. "De novo design of self-assembling protein nanoparticles towards the gene therapy of colorectal cancer." Doctoral thesis, Universitat Autònoma de Barcelona, 2013. http://hdl.handle.net/10803/125920.
Повний текст джерелаАнотація:
Hoy en día, el cáncer sigue siendo la segunda causa de muerte en el mundo. Por lo tanto, existe una gran necesidad de encontrar nuevas terapias que resulten más efectivas para su tratamiento. Las terapias actuales, lejos de ser efectivas, producen una gran toxicidad sistémica y ofrecen un bajo porcentaje de supervivencia a los pacientes, siendo la principal causa de muerte la aparición de focos metastáticos, especialmente en el cáncer de colon. Por lo tanto, mejorar la especificidad celular y evitar la aparición de focos metastaticos son los mayores retos a los que se enfrentan las futuras terapias contra el cáncer. En este contexto, la terapia génica aparece como una alternativa muy prometedora ya que ofrece la posibilidad de personalizar las terapias además de disminuir su toxicidad. Dado que la bioseguridad es uno de los parámetros que más preocupa en este tipo de terapias, las proteínas multifuncionales aparecen como uno de los vectores de terapia génica más prometedores no solo por su alta biocompatibilidad y su baja toxicidad, sino también por su gran plasticidad. Por otro lado, la necesidad de controlar el tamaño de las partículas generadas para conseguir una adecuada biodistribucion in vivo ha sido ampliamente descrita en la literatura.
En este trabajo, hemos explorado la posibilidad de modular el autoensamblaje de proteínas multifuncionales en nanoparticulas de un tamaño predefinido con tal de obtener el máximo potencial de su capacidad de entrega de ácidos nucleicos o drogas terapéuticas. En este contexto, hemos descrito parejas de tags arquitectónicos (de los cuales uno de ellos es una poli-histidina) que son capaces de inducir el autoensamblaje de las proteínas que las contienen en nanoparticulas con propiedades estructurales predefinidas. También hemos estudiado la interacción y el traffiking intracelular de estas nanoparticulas cuando las ponemos en contacto con células en cultivo, donde hemos visto que su internalización no resulta toxico para las células de mamíferos. Además, también hemos estudiado la estabilidad estructural que presentan estas nanoparticulas cuando se administra por vía intravenosa en modelos de ratón, mostrando que las interacciones intermoleculares que se generan durante el proceso de ensamblaje de las nanoparticulas in vitro, son suficientemente fuertes como para asegurar su estabilidad estructural in vivo.
Se ha descrito que el receptor CXCR4 es un elemento clave en la formación de focos metastaticos durante el desarrollo tumoral de diferentes tipos de cáncer incluyendo el cáncer de colon, para el cual actualmente no existe todavía ningún vehículo que reconozca de forma específica las células metastaticas. En este contexto, en este estudio hemos explorado la posibilidad de funcionalizar las nanoparticulas proteicas con ligandos que reconocen el receptor CXCR4 con tal de dirigirlas de forma específica a las células que expresan este receptor. Entre los ligandos testados, hemos visto que el péptido T22 es un ligando inusualmente eficiente para el reconocimiento selectivo e internalización en células que expresan el receptor CXCR4 y que las nanoparticulas funcionalizadas con este ligando se biodistribuyen de forma selectiva a las células CXCR4+ in vivo en modelos murinos de cáncer colorectal. Finalmente, también hemos explorado la posibilidad de usar nanoparticulas proteicas funcionalizadas como virus artificiales para la entrega especifica de ácidos nucleicos en las células diana. Este trabajo muestra como las nanoparticulas que contienen dominios de unión a ácidos nucleicos, cuando son incubados junto a un DNA externo, son capaces de generar estructuras que imitan las estructuras virales, encapsulando el DNA en la parte interna de las estructura y protegiéndolo a su vez del ataque de nucleasas externas. Sin embargo, es necesario añadir un paso de hidrolisis con DNasa y RNasas en la purificación de nanoparticulas que contienen dominios de unión a ácidos nucleicos ya que se ha visto que unen ácidos nucleicos bacterianos provenientes del sistema de expresión bacteriano utilizado para su producción recombinante, afectando muy negativamente sobre su funcionalidad como virus artificiales.
Por lo tanto, dado la gran biocompatibilidad que a priori se espera de las proteínas, sus propiedades arquitectónicas regulables y la posibilidad de funcionalizarlos con ligandos específicos, hace que las nanoparticulas proteicas autoensamblables sean una herramienta muy prometedora para la entrega dirigida de ácidos nucleicos y drogas terapéuticas en las células de cáncer metastatico de colon y en general en las células de mamífero.Cancer is ranked as the second leading cause of death worldwide. Consequently there is a huge necessity of finding more effective cancer therapies. Currently available cancer therapies, far from being effective, present high systemic toxicity and low patient survival rates being the main mortality cause the appearance of metastatic foci, especially in colon cancer. Thus, improving cell specificity and avoiding metastases generation are the mayor challenges for future cancer therapies. In this context, gene therapy appears as very promising alternative therapy since cell targeted personalized therapies can be performed with low systemic toxicity. Since biosafety is the current mayor concern in this type of therapies, multifunctional proteins appear as the most promising gene therapy vectors because of their high biocompatibility and biosafety, low toxicity and really complete tuneability. Moreover, the necessity of effectively controlling nanoparticles size for their efficient biodistribution and delivery has been widely described in the literature. In the present work has been explored the possibility of effectively modulating the self-assembling of multifunctional protein building blocks into predefined size distribution nanoparticles in order to get the full potential of those protein-only nanoparticles for their application in therapeutic drugs and nucleic acids delivery approaches. In this regard, we have described cationic architectonic tag pairs ( one of them being a poly-histidine) that when incorporating to proteins, they induce the self-assembling of protein monomers into nanoparticles with predefined structural properties. It has also been studied the interaction and intracellular trafficking of these kind of protein nanoparticles in cultured cells proving not to be toxic for mammalian cells. Moreover, the structural stability of generated nanoparticles upon intravenous administration in mice has been also studied proving in vitro generated intermolecular interactions during protein assembling process strong enough to ensure nanoparticles structural stability in vivo. It has been shown that CXCR4 chemoquine receptor is a key element in metastasis formation during cancer evolution in different types of tumors, including colorectal cancer, for which metastatic intracellular targeting vehicles are currently missing. In this context, the possibility of effectively functionalizing protein nanoparticles with CXCR4 specific ligands has been deeply explored in this study. Among tested peptide ligands, T22 peptide has shown to be an unusually powerful tag for selective intracellular targeting in CXCR4+ cells having T22-empowered protein nanoparticles of optimal size selectively biodistribute in CXCR4+ cells in vivo. Finally, the suitability of functionalized self-assembling protein nanoparticles for their use as artificial viruses has been also extensively explored. Protein nanoparticles that contain nucleic acid binding domains have shown an appealing capacity to generate virus-like structures when combined with external DNA, completely shielding cargo DNA in the inner part of the structure and protecting it from external nucleases hydrolysis. However, the necessity of an additional DNase/RNase hydrolysis treatment during the nanoparticles purification process has been described since nanoparticles with nucleic acid binding domains have been shown to bind nucleic acids from the bacterial host used for their recombinant expression, resulting strongly detrimental for their functionality as artificial viruses. All together, the high biocompatibility expected for proteins, their regulatable architectonic properties and their efficient targeting possibility, make self-assembling protein-only nanoparticles a very promising material for the therapeutic delivery of drugs and nucleic acids in metastatic colorectal cancer cells and in general in mammalian cells.
30
Fernando, Marie Michelle Agatha. "Association of the HIN200 gene cluster on 1q23 and the MHC on 6p21 with SLE." Thesis, Imperial College London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.516503.
Повний текст джерела
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Rose, Margaret Anne. "Plotting the networked self : cyberpunk and the future of genre." Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=83839.
Повний текст джерелаАнотація:
Cyberpunk's attempt to imagine the futures that the expanding communications networks will shape, as explored in Sterling's Islands in the Net and Stephenson's The Diamond Age, discovers that the boundaries between the machine and human, the natural and artificial, and the past and present have never been as clear as the modern realist schematic has drawn them. Gothic literature represents transgressions of these boundaries as threatening to the self, and Mary Shelley's Frankenstein is the node where the gothic is dismembered and sutured into science fiction, and the modern self faces its monstrous double. Yet if boundaries are represented as sites of interface, gothic threats become opportunities for growth and generation. Individual texts, even realist ones, have always sutured together intertextual ingredients. Jane Eyre offers an alternative model for constructing the subject through sorting texts, a technique which emerges through cyberpunk as the essential survival skill of the future self.
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Brown, David Spaulding. "CD4+ T Cell Responses: A Complex Network of Activating and Tolerizing Signals as Revealed by Gene Expression Analysis: A Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/230.
Повний текст джерелаАнотація:
Immunologic self-tolerance is maintained by both central and peripheral mechanisms. Furthermore, regulation of mature lymphocyte responses is governed by inhibitory as well as stimulatory signals. TCR recognition of cognate peptide bound to MHC molecules provides the initial stimulus leading to T lymphocyte activation and determines the antigen specificity of any subsequent response. However, lymphocytes must discriminate between foreign and self antigens presented by self-MHC molecules to maintain self tolerance and avoid pathological autoimmunity. Consequently, TCR ligation alone is reported to result in abortive activation, T cell anergy, apoptosis, and tolerance. Under normal physiological conditions, costimulatory signals modify lymphocyte responsiveness to TCR ligation to prevent autoimmunity while enabling robust responses to foreign antigen. Members of the CD28/B7 superfamily provide the critical secondary signals essential for normal immune cell function.
CD28 is an essential positive costimulatory molecule with critical functions in thymic development, lineage commitment, and regulation of peripheral lymphocyte responses to antigenic stimuli. CD28 ligation by APC-expressed B7 molecules alters proximal signaling events subsequent to MHC/TCR interactions, and initiates unique signaling pathways that alter mRNA stability and gene transcription. Furthermore, CD28 signaling is required for regulatory T cell development and function. Thus, CD28 has a central role in both potentiating lymphocyte activation mediated by TCR engagement and regulating peripheral tolerance. In contrast, Ctla-4 mediates an inhibitory signal upon binding B7 molecules on an antigen-presenting cell. Its importance in governing lymphocyte responses is manifested in the fatal lymphoproliferative disorder seen in Ctla-4-/- mice. The lymphocyte proliferation is polyclonal, antigen and CD28 dependent, and arises from defects in peripheral CD4+T cell regulation. The high percentage of peripheral T lymphocytes expressing activation markers is accompanied by lymphocyte infiltration into numerous non-lymphoid tissues and results in death by 3-4 weeks. While still controversial, Ctla-4 signaling has been reported to be essential for induction of peripheral T lymphocyte tolerance in vivo and in some model systems is proposed to regulate both T lymphocyte anergy induction and the immune suppressive effects of some regulatory T cells in the prevention of autoimmunity.
Signaling pathways activated by TCR ligation and CD28 costimulation have been extensively characterized. In contrast, the mechanisms mediating Ctla-4 maintenance of tolerance remain largely unknown. Ctla-4 gene expression is tightly controlled during T cell development and activation, and its intracellular localization and expression on the cell surface is regulated by numerous pathways and intermediates. While a tailless Ctla-4 mutant is capable of inhibiting T cell activation, recent studies have shown that a ligand independent form of Ctla-4 is also capable of providing an inhibitory signal to T lymphocytes. In conjunction with the strictly controlled expression kinetics and the perfect amino acid homology between the intracellular domains of mouse and human Ctla-4, this data suggests that Ctla-4 may participate in the modulation or initiation of intracellular signaling pathways.
Positive and negative costimulatory receptors on the T cell modify lymphocyte responses by altering both quantitative and qualitative aspects of the lymphocyte response including threshold of activation, cytokine secretion, and memory responses. Positive costimulation augments T cell responses, in part, by downregulating the expression of genes that actively maintain the quiescent phenotype. This study was initiated to determine the role of Ctla-4 ligation in modifying the global gene expression profile of stimulated T cells and to determine if the Ctla-4 mediated maintenance of T cell tolerance was achieved, in part, by altering the transcription of quiescence genes necessary for the prevention of T cell activation subsequent to TCR and CD28 stimulation.
Previous studies investigating the influence of Ctla-4 ligation on transcriptional profiles of activated lymphocytes detected only quantitative alterations in the transcriptional regulation initiated by CD28 signaling. In contrast, our data suggests that quantitative effects of Ctla-4 ligation that differentially influence pathways acting downstream of stimulatory receptors results in a stable and qualitatively unique phenotype detectable at the level of the transcriptome. Thus, the cumulative effect of Ctla-4 signaling is unique and not constrained to reversing alterations in expression initiated by CD28. In addition, Ctla-4 ligation can be shown to influence T lymphocyte responsiveness and the resulting global expression profile within 4 hours after stimulation and prior to detectable Ctla-4 surface expression. In a subpopulation of T cells, TCR stimulation activates pathways that result in commitment to activation with 2-6 hours. In contrast, CD28 signaling must be maintained for 12-16 hours to ensure maximal responses at the population level. The period of sensitivity to Ctla-4 inhibition of activation is more constrained and does not extend beyond 12 hours. Together, these data support a potential role for Ctla-4 in modification of the early transcriptional response and may explain various alterations in phenotype resulting from Ctla-4 ligation that have been reported in secondary responses.
Identification of genes involved in lymphocyte activation, maintenance of selftolerance, and attenuation of immune responses opens the door to therapeutic manipulation of the pathways implicated. CD28 costimulation results in general amplification of TCR-initiated transcriptional responses, and specifically alters the expression profile of a subset of genes. In contrast, Ctla-4 ligation directly and specifically alters the expression of a select group of genes when ligated, and results in minimal suppression of the global CD28-mediated costimulatory transcriptional response. Ctla-4 regulated genes comprise a heterogeneous family, but include known quiescence factors, transcriptional regulators, and various determinants of cell cycle progression and senescence. The role of Ctla-4 in maintaining self-tolerance indicates that targeted manipulation of these gene products presents a novel therapeutic opportunity, and suggests that the mechanisms involved in Ctla-4-mediated maintenance of peripheral T cell tolerance and regulation of immune responsiveness is more nuanced than previously thought. In addition, this study provides the most comprehensive description of global gene expression during primary lymphocyte activation yet available. The integration of statistical and bioinfomatics analyses with large scale data mining tools identifies genes not previously characterized in lymphocytes and can direct future work by predicting potentially interacting gene products and pathways.
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Ryzhkov, Pavel. "Bioengineering of S-layers: molecular characterization of the novel S-layer gene sslA of Sporosarcina ureae ATCC 13881 and nanotechnology application of SslA protein derivatives." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2008. http://nbn-resolving.de/urn:nbn:de:bsz:14-ds-1204126129404-82781.
Повний текст джерелаАнотація:
S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.
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Sekulovic, Sanja. "Characterization of mouse hematopoietic stem cells primed to actively self-renew by NUP98-HOXA10hd fusion gene." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/35927.
Повний текст джерелаАнотація:
High-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Recently, it has been demonstrated that engineered NUP98-HOXA10hd expression stimulates >1,000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk lin⁻Sca-1⁺c-kit⁺ cells. In this thesis I coupled such ability of engineered NUP98-HOXA10hd expression, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. I discovered that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at near unit efficiency in cultures initiated with single cells. The clonally expanded HSCs showed preservation of normal proliferation kinetics in vitro and consistent balanced contributions long-term to the lymphoid and myeloid lineages in vivo without evidence of leukemogenic transformation. Preservation of a normal proliferating HSC phenotype allowed their re-isolation in large numbers at 25% purity. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.
Although there is growing excitement about the prospect of in vitro expansion of HSCs and their use to enhance the safety and application of transplant-based therapies, deleterious consequences of such manipulations remain unknown. Thus, I further examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. I observed ~10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert⁻/⁻ HSCs 6 months before. The second generation leukocytes also showed elevated expression of γH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type or Tert⁻/⁻ HSCs, suggesting that HSC replication post-transplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity upon prolonged self-renewal stimulation.
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Reynard, L. N. "Analysis of the role of the X/Y homologous gene family Xmr/Sly in murine spermiogenesis." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/15845/.
Повний текст джерелаАнотація:
Spermatogenesis is a complex, continuous developmental process that involves the formation of highly differentiated haploid sperm from diploid spermatogonial stem cells. Deletion analysis has established that genes on the Y chromosome are essential for normal sperm production in humans, mice and Drosophila. In mice, deletions of the Y chromosome long arm (Yq) are associated with abnormal sperm head formation, reduced fertilising ability, up-regulation of spermatid-expressed sex-linked genes, and an offspring sex ratio distortion in favour of females. The severity of the testicular phenotypes correlates with the extent of the deletion, with mice lacking the Yq being sterile. It has been suggested that deficiency of a Yq-encoded multicopy genetic element (the ‘spermiogenesis factor’) is responsible for these phenotypes, and one potential candidate gene is Sly, a member of the Xlr superfamily. This family also includes Xmr, an X-linked multicopy gene found to be up-regulated (along with other X- and Y-encoded genes) in the Yq deletion models. To access the candidacy of Sly as the ‘spermiogenesis factor’, the expression of this gene was examined in the testis at the transcript and protein levels. Sly encodes a protein that is expressed in round and early elongating spermatids. SLY interacts with the acrosomal protein DKKL1, suggesting that SLY may be involved in the development or function of the acrosome, a structure that contains digestive enzymes required for fertilisation. Secondly, the role of Xmr in spermatogenesis was investigated. Xmr has been reported to encode a meiotic protein that localises to the transcriptionally silent sex body in pachytene spermatocytes. However, evidence is provided that this meiotic protein is not XMR, with the antibody used in previous studies unable to recognise XMR. Instead, Xmr is predominantly transcribed in spermatids were it encodes a cytoplasmic protein of unknown function. Next, the up-regulation of X- and Y-linked genes in spermatids from the Yq deletion mice was studied in detail. This up-regulation is due, at least in part, to increased gene transcription, and this is accompanied by changes to the epigenetic profile of the sex chromosome and centromeric heterochromatin in round spermatids. In addition, the protein levels of the up-regulated genes are also increased in the testis from Yq deletion mice, and thus the testicular phenotypes exhibited by these mice may be the result of over-expression of one or more X- and Y-encoded proteins, rather than a direct effect of Yq-linked gene deficiency. Finally, mice transgenic for Sly were generated and characterised to determine if loss of this gene alone is responsible for the spermiogenic defects observed in mice with Yq deletions. Xmr transgenic mice were also produced to examine any potential regulatory interaction between Sly and Xmr and investigate if over-expression of Xmr contributes to the phenotypes exhibited by Yq deletion mice.
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Sanber, K. S. R. "Production of self-inactivating lentiviral vectors by constitutive packaging cell lines for gene therapy clinical applications." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1472300/.
Повний текст джерелаАнотація:
Lentiviral vectors (LVs) are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the LV components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Generation of stable packaging cell lines (PCLs) that continuously produce LVs can potentially overcome these limitations. The WinPac-RDpro cell line was developed between Collins and Takeuchi laboratories in Division of Infection and Immunity, UCL by inserting a codon-optimized HIV-1 Gag-Pol expression cassette in a continuously expressed locus in 293FT cells using Cre recombinase-mediated cassette exchange (RMCE). Subsequently HIV-1 Rev and RDpro envelope expression cassettes were serially transfected. In this thesis, WinPac-RDpro cells were used to generate model producer cells by stably transfecting a plasmid expressing a SIN GFP-encoding LV. Vector titers in excess of 106 293T transducing units (TU)/ml could be repeatedly harvested from the final producer clones in a volume of >0.5 L even under reduced serum conditions. Titers could be increased to around 1 x 10^8 293T TU/ml by concentration using scalable tangential flow filtration (TFF). Additionally, these LVs efficiently transduced human T cells and CD34+ cells at low multiplicities of infection (MOI). Titers in excess of 10^6 TU/ml were achieved using an RMCE-based strategy that was aimed at introducing a SIN LV expression cassette at a pre-selected locus. Similar titers were also achieved by using a promoterless selectable marker cloned in cis to the vector genome expression cassette. Furthermore, the Cocal Virus G protein (COCV-G) was stably expressed in WinPac cells to generate WinPac-CVG cells. These packaging cells were able to support the production of COCV-G pseudotyped SIN LVs at high titers (up to 106 TU/ml) following transient supplementation of a SIN LV expression plasmid. The efficient and stable expression of SIN LV genomes in these cells is expected to facilitate high-titer production of vectors with favorable characteristics. In conclusion, the work presented here provides significant improvements to available LV production methods. This will be of use to all basic and clinical investigators who wish to produce large batches of LVs, and addresses an important issue that has hindered large-scale LV clinical testing and application.
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Pooley, Edward Charles. "Genetic association studies of serotonergic gene polymorphisms with obsessive-compulsive disorder, deliberate self-harm and obesity." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670094.
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Miyake, Jon Hatsuo. "Analysis of SBF, an snRNA enhancer binding protein, and cloning of the chicken rreb-1 gene /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1997. http://wwwlib.umi.com/cr/ucsd/fullcit?p9805798.
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Ter-Hovhannisyan, Vardges. "Unsupervised and semi-supervised training methods for eukaryotic gene prediction." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/26645.
Повний текст джерелаАнотація:
Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009.Committee Chair: Mark Borodovky; Committee Member: Jung H. Choi; Committee Member: King Jordan; Committee Member: Leonid Bunimovich; Committee Member: Yury Chernoff. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Howard, Gitanjali. "The Impact of Protagonist Race, Gender, and Genre on Latina Adolescent Personal Aspiration, Self-Esteem, and Self-Efficacy." Scholarship @ Claremont, 2017. http://scholarship.claremont.edu/scripps_theses/959.
Повний текст джерелаАнотація:
Inspired by the lack of minority female representation in the media, this study questions how 11-14 year old Latina adolescents from low SES backgrounds are influenced by protagonist race, gender, and genre in stories with respect to participant personal aspiration, gender atypical personal aspiration, self-esteem, and self-efficacy. Due to the particular lack of representation of non-whites and non-males in action/adventure stories, it is predicted in this intervention study completed each week over the course of 8 weeks, that Latina adolescents will experience the most positive increase in self-esteem, self-efficacy, and gender atypical personal aspiration when exposed to Latina female protagonists in action/adventure stories. They will also experience significant increases in the dependent variables from highest to lowest in the following conditions: Latina female biographies, Latino males in action/adventure, Latino male biographies, White females in Action/Adventure, and White female biographies. It is predicted that there will be a a decrease in self-esteem, self-efficacy, general aspiration, and gender-atypical personal aspiration for participants exposed to White male action/adventure stories, and to a lesser significant extent from the preceding condition, a decrease in self-esteem, self-efficacy, general aspiration, and gender-atypical personal aspiration for participants exposed to White male Biographies. This research is significant in understanding the influence of minority female representation in books, film, and the general media.
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Gonçalves, Paula Vieira Cristina Alexandra. "Population genetic studies of the S-locus gene family and other loci in self-compatible and self-incompatible populations of the plant Antirrhinum." Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/10925.
Повний текст джерелаАнотація:
In this work the mating system of several populations and species of Antirrhinum were established in the glasshouse. Levels of DNA diversity were estimated based on cyc and fil1 nuclear genes. Both genes are shown to belong to gene families. In these gene families, some members are very similar, which makes difficult to determine orthology. In the cases where orthology is not a problem, low levels of nucleotide diversity were found. Therefore the effect of the mating system on genetic diversity could not be tested. An unexpected finding of very little divergence between several Antirrhinum species, Digitalis, and the more distantly related genus Verbascum was also found for genes of the cyc and fill gene families. The generality of this pattern was addressed by extending these studies to fil2, far, globosa and Adh genes. Evidence is shown that these genes are also members of gene families in Antirrhinum. For fil2, far, and globosa, very similar sequences were found in Antirrhinum and Verbascum. For Adh I could not determine orthology because repeated gene duplication and loss of elements in this gene family has occurred in the Antirrhinum and Verbascum lineages. Several hypotheses that could account for the low diversity and divergence are discussed. In Antirrhinum, self-incompatibility is controlled by a gametophytic system. The gene responsible for pistil self-incompatibility is the S-locus that encodes basic glycoproteins with ribonuclease activity. High levels of variability are observed, consistent with frequency-dependent selection. The putative targets of selection are those regions, such as the hypervariable regions of this gene, that may be involved in specificity determination. In order to gather evidence on whether these regions are hypervariable because they are the target of selection, or merely regions of relaxed selective constraint, we have partially sequenced Antirrhinum S-alleles and analysed their level and pattern of nucleotide diversity. Within each allelic type, low levels of diversity were observed. Similar alleles were found in self-compatible and self-incompatible species, suggesting that the Antirrhinum group evolved recently.
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Collingsworth, Jean. "Transformational texts : genre, discourse and subjectivity in the self-help book." Thesis, London Metropolitan University, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549555.
Повний текст джерелаАнотація:
This research used selected structuralist and post-structuralist theory to investigate the notions of genre, discourse and subjectivity in the contemporary self-help book. It argues that these texts have links with classical and modern ethics of optimum living and are predicated on an ontology of transformative possibility which is expressed through typical rhetorical strategies. Furthermore, that the publications are a significant element in the therapeutic discourse prevalent in contemporary society. It suggests that as well as being a highly successful commodity, the self-help book is theoretically remarkable for two reasons. Firstly it operates as a redemptive paradigm for the reader; secondly it is an 'actantial' genre because each text participates as a 'protagonist' in the 'heroic' narrative of transformation which it articulates. Furthermore a self/subject dyad inhabits the genre because while advice literature is predicated on a humanistic discourse of essential, telic selfhood, critical analysis detects the underlying dynamics of socially-constructed subjectivity. Three levels of subject activity in the self-help book are distinguished: sub-stratum (ontological level of humanistic assumptions), inter-stratum (archetypal level: the reader as 'hero'; the book as 'helper' etc.) and super-stratum (the level of every-day matters). The research concludes that the selfhelp book is a paradoxical phenomenon for the cultural theorist because it asserts the survival of personal agency in the postmodern episteme which has seen the discrediting of grand narratives and the decentring of the human subject. Additionally, the lexicon of genre studies is extended by the coining of new terms to better describe the emergence of 'symbiotic' commercial materials and a generic twelve-step sequence of discourse emergence is also offered. This traces the discourse of post-traumatic stress from its diffuse beginnings to its present linguistic entrenchment in commercial publications. The research is thus original at two levels: it provides a detailed exploration of the self-help book qua text and extends the reach of critical theory.
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Ryzhkov, Pavel. "Bioengineering of S-layers: molecular characterization of the novel S-layer gene sslA of Sporosarcina ureae ATCC 13881 and nanotechnology application of SslA protein derivatives." Doctoral thesis, Technische Universität Dresden, 2007. https://tud.qucosa.de/id/qucosa%3A24121.
Повний текст джерелаАнотація:
S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.
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Richter, Karin. "Molecular and cellular analysis of Lhx2 function in hematopoietic stem cells." Doctoral thesis, Umeå : Department of Molecular Biology and Umeå Centre for Molecular Medicine, Umeå University, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1389.
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Harvey, Alison. "Risky genes, healthy choices : public health as government of the somatic self." Thesis, University of East Anglia, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443179.
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Schmidt, Nicole. "A Self-Regulated Approach to Acquire Lexical Genre Conventions in Academic Writing." The University of Arizona, 2016. http://hdl.handle.net/10150/621898.
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Samsonova, Olga [Verfasser], and Thomas [Akademischer Betreuer] Kissel. "Self-assembling polycations for gene delivery : Effects of polymer structure and environmental pH / Olga Samsonova. Betreuer: Thomas Kissel." Marburg : Philipps-Universität Marburg, 2012. http://d-nb.info/1021498904/34.
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Burke, Arista. "Expression of the chimeric SAF gene from Human Papillomavirus in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha." Thesis, Stellenbosch : Stellenbosch University, 2011. http://hdl.handle.net/10019.1/6908.
Повний текст джерелаАнотація:
Thesis (MSc (Microbiology))--Stellenbosch University, 2011.ENGLISH ABSTRACT: The link between infection with Human Papillomavirus (HPV) and the development of cervical cancer has been established by several epidemiology studies. Cervical cancer is the second most common cancer among women and it occurs at a rate of 22.8 cases per 100 000 women in South Africa. Approximately 86% of newly reported cases of cervical cancer occur in developing countries where limited access to medical facilities hampers efforts to prevent and screen for HPV infection. Two commercial virus-like particle (VLP) vaccines consisting of HPV major structural protein L1, which protect against the most common high-risk HPV-types, are currently available. The high cost and type specificity of these commercially available vaccines have necessitated the development of a low cost, broad-spectrum HPV vaccine. Inclusion of the minor structural protein L2 has been shown to induce broadly cross-neutralizing antibodies and therefore a chimera was constructed that contains an epitope of L2 inserted within the L1 sequence. This construct, renamed SAF, was shown to be highly immunogenic and thus has the potential to be used as a prophylactic cervical cancer vaccine. Methylotrophic yeasts are known to be excellent producers of recombinant proteins due to their strongly inducible promoters that allow culturing of these yeasts to very high cell densities. Pichia pastoris and Hansenula polymorpha have been employed in several studies for heterologous protein production and levels of protein higher than 1 g/L have been reported. These yeasts also have GRAS status and can therefore be used to manufacture products for use in humans. In this study, the potential of H. polymorpha and P. pastoris to produce SAF intracellularly was evaluated. The effect of increased gene dosage and peroxisomal targeting on SAF production was examined as possible strategies to increase the yield of SAF. Peroxisomal targeting was achieved by fusing the SAF gene at the C-terminal end with the Peroxisomal Targeting Sequence 1 (PTS1) which consists of a short tri-peptide: –SKL. The functionality of PTS1 was confirmed using green fluorescent protein (GFP), fluorescence microscopy and peroxisome isolation. Peroxisomal targeting was shown to have a negative effect on SAF production levels in both H. polymorpha and P. pastoris. An increase in gene dosage had no discernable effect on SAF yield in H. polymorpha which is in contrast to previous research. The highest production levels were achieved by P. pastoris KM71 (24.86 mg/L) which compares well to levels of L1 achieved by other research groups. The most significant insight emerging from this work was that all the strains that produced SAF at detectable levels were equally efficient at the production of SAF. Increased biomass was therefore the biggest contributor to high SAF levels (mg/L) in the P. pastoris strains as significantly higher cell densities were achieved during culturing of these strains. With the necessary optimisation, the methylotrophic yeasts have the potential to be used as hosts for the production of a broad-spectrum HPV vaccine.
AFRIKAANSE OPSOMMING: Die skakel tussen infeksie met Mens Papilloomvirus (HPV) en die ontwikkeling van servikale kanker is deur verskeie epidemiologiese studies bevestig. Servikale kanker is die tweede mees algemene kanker onder vroue en dit kom voor teen ‘n tempo van 22.8 gevalle per 100 000 vroue in Suid Afrika. Ongeveer 86% van alle nuwe gevalle kom voor in ontwikkelende lande waar beperkte toegang tot mediese fasiliteite pogings om HPV infeksie te voorkom en te behandel, belemmer. Twee pseudovirale-partikel (VLP) entstowwe teen HPV is tans op die mark beskikbaar en hierdie entstowwe verleen immuniteit teen die mees algemene hoë-risiko HPV tipes. Die hoë koste en nou spektrum van hierdie entstowwe het dit nodig gemaak om ‘n goedkoop, wye-spektrum HPV entstof te ontwikkel. Navorsing het bewys dat die insluiting van die strukturele L2 proteïen in die VLP entstof, lei tot die indusering van neutraliserende teenliggame, wat wye spektrum antigenisiteit tot gevolg het. ‘n Chimeriese proteïen wat ‘n epitoop van L2 binne die L1 volgorde bevat is gekonstrueer, en hierdie proteïen is benoem SAF. SAF het hoë immunogenisiteit en kan dus potensieel as ‘n voorkomende servikale kanker entstof gebruik word. Metielotrofiese giste is bekend vir hulle vermoë om hoë vlakke rekombinante proteïene te produseer as gevolg van hulle induseerbare promotors wat groei tot baie hoë sel digthede toelaat. Pichia pastoris en Hansenula polymorpha is in menigte studies gebruik om heteroloë proteïene te produseer tot vlakke bo 1 g/L. Hierdie giste en die proteïen produkte wat hulle vorm word algemeen aanvaar as veilig vir menslike gebruik. In hierdie studie het ons die potensiaal van H. polymorpha en P. pastoris om SAF intrasellulêr te produseer, geevalueer. Die effek op SAF produksie van verhoogde geen dosering asook die teiken van SAF na die peroksisoom was ondersoek as moontlike strategieë om die opbrengs van SAF te verhoog. Die teiken van SAF na die peroksisoom is behaal deur die Peroksisomale Teiken Volgorde 1 (PTS1) aan die C-terminaal van SAF te heg. Die funksionaliteit van PTS1 was bevestig deur gebruik te maak van groen fluoroserende proteïen (GFP), fluoressensie mikroskopie en isolering van peroksisome. Teiken van SAF na die peroksisoom het ‘n negatiewe uitwerking gehad op SAF uitdrukking in beide H. polymorpha en P. pastoris. ‘n Verhoging in geen dosering het geen onderskeibare effek gehad op SAF opbrengs in H. polymorpha nie wat in teenstelling is met vorige navorsing. Die hoogste produksie vlakke is opgelewer deur P. pastoris KM71 (24.86 mg/L) wat goed vergelyk met vlakke van L1 wat deur ander navorsings groepe behaal is. Die belangrikste gevolgtrekking wat gemaak kan word uit hierdie studie is dat al die rasse wat SAF geproduseer het in meetbare hoeveelhede ewe effektief was. Verhoogde biomassa was dus die grootste bydraende faktor tot hoë SAF vlakke (mg/L) in die P. pastoris rasse as gevolg van die hoë sel digthede wat hierdie rasse kan bereik. Dit is duidelik dat metielotrofiese giste, met die nodige optimisering, oor die potensiaal beskik om as gasheer sisteme te dien vir die produksie van ‘n wye spektrum HPV entstof.
The NRF and the Department of Microbiology for financial support
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Ibraheim, Raed R. "Genome Engineering Goes Viral: Repurposing of Adeno-associated Viral Vectors for CRISPR-mediated in Vivo Genome Engineering." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1114.
Повний текст джерелаАнотація:
One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.
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Tang, Shiyuyun. "Improving algorithms of gene prediction in prokaryotic genomes, metagenomes, and eukaryotic transcriptomes." Diss., Georgia Institute of Technology, 2016. http://hdl.handle.net/1853/54998.
Повний текст джерелаАнотація:
Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack.
In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools.
Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods.
Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.