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1
Goh, Siew-Lee. "Functional studies of MEIS1, a HOX co-factor". Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=103200.
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HOX proteins are evolutionarily conserved homeodomain-containing transcription factors involved in hematopoiesis and patterning during embryogenesis. Their tasks as master regulators of embryonic development are achieved in large part through their ability to interact with co-factors of the PBX and MEIS/PREP families, which constitute the broader three amino-acid loop extension (TALE) class of homeodomain proteins. HOX, MEIS, and PBX have been implicated in leukemic hematopoiesis due to their association with hematological malignancies. The oncogenic function of MEIS1 in accelerating the onset of acute myeloid leukemia induced by HOX was mapped to its C-terminal transactivation domain, which is responsive to PKA signaling. This thesis extends our understanding regarding the mechanism by which MEIS1A executes its C-terminal transactivation function in vivo. We describe the involvement of CREB and its co-activators CBP and TORC in conferring the PKA-responsiveness of the ME1S1A C terminus. CREB mutants that fail to bind CBP or TORC also fail to promote PKA induction mediated by the C terminus of ME1S1A. TORC was further shown to be capable of bypassing the need for PKA to activate transcription by MEIS1, an ability endowed by its physical interaction with MEIS1. Chromatin immunoprecipitation (ChIP) demonstrated a concerted recruitment of endogenous MEIS1, TORC2, and CREB proteins on ME1S1 target genes. In addition, this thesis also characterizes the promoter of the murine Meis1 gene. Meis1 possesses multiple transcription start sites upstream of its translation initiation site. We identified a ME1S·PBX consensus recognition site within the Meis1 promoter and showed that PBX1 binds to this sequence in vitro. Our ChIP assay results further suggest an autoregulatory mode for the Meis1 gene as revealed by a co-occupancy of endogenous CREB, TORC2, PBX1, and MEIS1 itself on the Meis1 promoter. Collectively, this thesis proposes a mechanistic action conferred by CREB, CBP and TORC in the PKA-inducible transactivation of ME1S1A, and provides new information on the Meis1 promoter.
2
Svingen, Terje, i n/a. "Hox Transcription Factors: Their Involvement in Human Cancer Cells and In Vitro Functional Specificity". Griffith University. School of Biomolecular and Biomedical Science, 2005. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20050830.135356.
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Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.
3
Svingen, Terje. "Hox Transcription Factors: Their Involvement in Human Cancer Cells and In Vitro Functional Specificity". Thesis, Griffith University, 2005. http://hdl.handle.net/10072/365774.
Pełny tekst źródłaStreszczenie:
Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Physical Sciences
Full Text
4
Phelan, Michael Leo. "A molecular analysis of functional differences between hox proteins". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0026/NQ37012.pdf.
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5
Choo, Siew Woh. "The in vivo specificity of HOX proteins in Drosophila". Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609425.
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6
Iaroshenko, V. (Vladislav). "Role of human HOX-proteins in different cancer types". Bachelor's thesis, University of Oulu, 2019. http://jultika.oulu.fi/Record/nbnfioulu-201905221901.
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Abstract. HOX-proteins are master regulators of embryogenesis. They determine the body plan along the anterior-posterior axis. Apart from their fundamental function in ontogenesis, they are also connected with different cancer types in adult organisms. Their aberrant expression may influence cell growth and support the development of cancer in many different ways. HOX-proteins appear to be a noticeable diagnostic and therapeutic target.
7
Litim-Mecheri, Isma. "Spécificité des protéines Hox : Rôle des motifs connus et découverte de nouveaux motifs". Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22096.
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Les gènes Hox sont responsables de l’identité des segments le long de l’axe antéro-postérieur. Ils sont évolutivement conservés et codent des facteurs de transcription. In vitro, toutes les protéines Hox se lient à des séquences nucléotidiques très similaires via un domaine de liaison à l’ADN très conservé, l’homéodomaine (HD). Cette faible spécificité de liaison à l’ADN in vitro contraste avec leur spécificité d’action in vivo. Une manière d’expliquer ce paradoxe est que les protéines Hox agissent en interaction avec des protéines cofacteurs, dont le mieux caractérisé est Extradenticle (Exd) chez la drosophile, Pbx chez les mammifères (collectivement appelés PBC). L’interaction Hox-PBC s’appui sur un motif conservé chez la presque totalité des protéines Hox, situé en amont de l’HD, le motif Hexapeptide (HX). Des travaux récents au sein de notre équipe ont montré l’existence d’un nouveau mode d’interaction Hox-PBC, non-générique, médié par un motif spécifique à certains groupes de paralogies seulement, et situé en en C-terminal de l’HD. Ceci souligne que les interactions Hox-PBC, qui spécifient la fonction des protéines Hox, reposent sur de modes multiples d’interaction.Mes travaux de thèse ont porté sur le mode d’action des protéines Hox, en étudiant la fonction de trois motifs ayant un rôle démontré ou potentiel dans le recrutement du cofacteur Exd par la protéine Hox de drosophile AbdominalA (AbdA). L’approche empruntée visait à analyser de manière globale la manière dont chacun de ces motifs pris isolément, ou en interaction, définit la fonction d’AbdA. Les conclusions de ce travail soulignent l’absence de pléiotropie fonctionnelle et un haut degré d’interactivité entre ces motifs. Le second volet de ma thèse a été d’initier la découverte de nouveaux motifs fonctionnels au sein des protéines Hox. J’ai abordé cette question en sélectionnant des modules phylogénétiquement conservés. Afin d’évaluer leur fonction, ces motifs ont été mutés et l’impact de leur mutation a été analysé in vitro et in vivo. Les résultats obtenus ont permis l’identification d’au moins un domaine protéique qui contribue de manière prédominante à la fonction de la protéine DfdHox genes are responsible for the identity of segments along the antero-posterior axis. They are evolutionarily conserved and encode transcription factors. In vitro, all Hox proteins bind to a similar nucleotide sequence via a highly conserved DNA binding domain, the homeodomain (HD). This low specificity of DNA binding in vitro contrasts with their specificity in vivo. One way to explain this paradaox is that Hox protein function with protein cofactors, best represented by Extradenticle (Exd) in Drosophila, Pbx in mammals (collectivaly refered as PBC). Hox-PBC interaction relies on a motif located upstream of the HD, conserved in most Hox proteins, the Hexapeptide (HX). Recent work in our group identified a novel mode of Hox-PBC interaction, non-generic, specific to a subset only of Hox paralog groups, and relying on a motif located C-terminal to the HD. This highlight plasticity in Hox-PBC interaction.My PhD work aimed at investigating the mode of action of Hox protein, by studying the function of three protein motifs, with known or putative role in Exd recruitment by the Drosophila Hox protein AbdominalA (AbdA). The approach taken aimed at analyzing, using a large functional window, how these motifs, taken in isolation or collectively, define AbdA protein activity. Conclusions highlight the absence of pleitropy and a high degree of functional interaction for these protein motifs. The second part of my PhD work has been to initiate the search for novel functionally important protein motifs within Hox proteins. This was approached by selecting phylogenetically conserved motifs, and addressing their function in vitro and in vivo following motif mutations. At least one functional domain was isolated, that contributes predominantly to Dfd protein function
8
Weicksel, Steven E. "hox Gene Regulation and Function During Zebrafish Embryogenesis: A Dissertation". eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/692.
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Hox genes encode a conserved family of homeodomain containing transcription factors essential for metazoan development. The establishment of overlapping Hox expression domains specifies tissue identities along the anterior-posterior axis during early embryogenesis and is regulated by chromatin architecture and retinoic acid (RA). Here we present the role nucleosome positioning plays in hox activation during embryogenesis. Using four stages of early embryo development, we map nucleosome positions at 37 zebrafish hox promoters. We find nucleosome arrangement to be progressive, taking place over several stages independent of RA. This progressive change in nucleosome arrangement on invariant sequence suggests that trans-factors play an important role in organizing nucleosomes. To further test the role of trans-factors, we created hoxb1b and hoxb1a mutants to determine if the loss of either protein effected nucleosome positions at the promoter of a known target, hoxb1a. Characterization of these mutations identified hindbrain segmentation defects similar to targeted deletions of mouse orthologs Hoxa1 and Hoxb1 and zebrafish hoxb1b and hoxb1a morpholino (MO) loss-of-function experiments. However, we also identified differences in hindbrain segmentation as well as phenotypes in facial motor neuron migration and reticulospinal neuron formation not previously observed in the MO experiments. Finally, we find that nucleosomes at the hoxb1a promoter are positioned differently in hoxb1b-/- embryos compared to wild-type. Together, our data provides new insight into the roles of hoxb1b and hoxb1a in zebrafish hindbrain segmentation and reticulospinal neuron formation and indicates that nucleosome positioning at hox promoters is dynamic, depending on sequence specific factors such as Hox proteins.
9
Weicksel, Steven E. "hox Gene Regulation and Function During Zebrafish Embryogenesis: A Dissertation". eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/692.
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Hox genes encode a conserved family of homeodomain containing transcription factors essential for metazoan development. The establishment of overlapping Hox expression domains specifies tissue identities along the anterior-posterior axis during early embryogenesis and is regulated by chromatin architecture and retinoic acid (RA). Here we present the role nucleosome positioning plays in hox activation during embryogenesis. Using four stages of early embryo development, we map nucleosome positions at 37 zebrafish hox promoters. We find nucleosome arrangement to be progressive, taking place over several stages independent of RA. This progressive change in nucleosome arrangement on invariant sequence suggests that trans-factors play an important role in organizing nucleosomes. To further test the role of trans-factors, we created hoxb1b and hoxb1a mutants to determine if the loss of either protein effected nucleosome positions at the promoter of a known target, hoxb1a. Characterization of these mutations identified hindbrain segmentation defects similar to targeted deletions of mouse orthologs Hoxa1 and Hoxb1 and zebrafish hoxb1b and hoxb1a morpholino (MO) loss-of-function experiments. However, we also identified differences in hindbrain segmentation as well as phenotypes in facial motor neuron migration and reticulospinal neuron formation not previously observed in the MO experiments. Finally, we find that nucleosomes at the hoxb1a promoter are positioned differently in hoxb1b-/- embryos compared to wild-type. Together, our data provides new insight into the roles of hoxb1b and hoxb1a in zebrafish hindbrain segmentation and reticulospinal neuron formation and indicates that nucleosome positioning at hox promoters is dynamic, depending on sequence specific factors such as Hox proteins.
10
Rinaldi, Lucrezia. "Novel functions for Hox proteins in the development of the spinal cord". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0261/document.
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Les gènes Hox codent des facteurs de transcription à homéodomaines conservés qui coordonnent la spécification de l'identité régionale du corps pendant le développement des animaux bilatériens. Au nombre de 39 chez les vertébrés supérieurs, ils sont organisés en 4 complexes (A à D) sur les chromosomes. Treize groupes de gènes paralogues occupent des positions, des modes d'expression et des fonctions similaires. En plus de leurs fonctions spécifiques, des études récentes ont mis en évidence des fonctions "génériques", communes en dehors des groupes de paralogie. L'objectif de cette thèse était de rechercher de nouvelles fonctions génériques des protéines Hox vertébrées, en se concentrant sur le développement de la moelle épinière chez l’embryon de poulet et de souris. Nous avons identifié deux de ces fonctions, impliquant en particulier les gènes Hox du complexe B. La première concerne le contrôle de l’autophagie, qui a été précédemment établi dans le corps gras de Drosophile. Mon travail a établi la dynamique spatio-temporelle de l'autophagie et des protéines Hox au cours du développement de la moelle épinière. Ces dynamiques ont permis de suggérer un rôle générique pour les protéines Hox dans la répression de l'autophagie, qui a été confirmé par gain de fonction chez l’embryon de poulet. La deuxième fonction générique des protéines Hox concerne le contrôle de la neurogenèse. L'étude de l'expression des gènes Hox a mis en évidence une expression prédominante des gènes Hox du complexe B dans la zone intermédiaire du tube neural, où ils activent l'expression du gène Lzts1, dont le produit module la signalisation AKT pour finalement contrôler la différenciation neuronaleHox genes encode conserved homeodomain transcription factors that coordinate the specification of regional body identity during the development of bilaterian animals. There are 39 Hox genes in higher vertebrates, organized into four complexes (named A to D) on the chromosomes. Thirteen groups of paralogue genes occupy similar positions within the complexes and exhibit similar modes of expression and functions. In addition to their specific functions, for which these transcription factors are well known, some recent studies are suggestive of "generic" functions, i.e. functions common outside paralogy groups. The goal of this thesis was to look for generic functions of vertebrate Hox proteins, focusing on the development of the spinal cord in chick and mouse. We identified two such functions, implying B cluster Hox genes. The first regards the potential of Hox proteins to control autophagy, which was previously established for Drosophila Hox proteins in the fat body. My work established the spatio temporal dynamic of autophagy during spinal cord development in chick and mouse embryos. This dynamic identified complementary autophagy and Hox patterns, suggesting a generic role for Hox proteins in the repression of autophagy, which could be confirmed by gain of function in chick embryo. The second Hox generic function regards the control of spinal cord neurogenesis. The study of Hox expression highlighted a predominant expression of B cluster Hox genes in the neural tube Intermediate Zone (IZ), where they activate the expression of the Lzts1 gene, the product of which modulates AKT signaling to ultimately control neuronal differentiation
11
Comelli, Laura. "Localization and dynamics of homeotic oncogenic protein HOXC13 in pre-initiation complex of human DNA replication origins". Doctoral thesis, Scuola Normale Superiore, 2010. http://hdl.handle.net/11384/85938.
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In metazoan cells the DNA replication origins are not well defined. Differently
from what observed for bacteria cells and for budding yeast, in metazoan the origins
does not show a conserved sequence and they appear to be specified by many factors. In
order to better understand the mechanisms involved in the origin specification, many
studies have been done to identify the proteins involved in the recognition and
activation of the origins. From these kind of analysis is emerging that, beside the wellknown
proteins of the pre replicative complex, also other factors might be involved.
Between these, the HOX proteins seem to be able to play a role in the origin activity.
One of the first studies of this involvement was done by our group and leads to the
identification of three homeotic proteins able to specifically bind in vitro the human
lamin B2 origin. Thus, in the study conducted during this PhD program, was
investigated the involvement of one of these homeotic proteins, namely HOXC13, with
human DNA replication origins and with replicative complexes.
We found an interaction of HOXC13 with two crucial factors of the pre
Replication Complex (pre-RC), ORC1 and Cdc6 and that HOXC13 binds a good
fraction of the origins, in particular the early replicating ones, like the lamin B2 origin
and other known human origins. The HOXC13 protein is bound to origin chromatin, at
least for the lamin B2 origin, at a precise site within the pre-RC at specific moments of
the cell cycle. Interaction with the origin occurs within the area protected by the pre-RC
in G1, very close to the start sites of leading strand synthesis and to the binding sites of
ORC1, ORC2, Cdc6, topoisomerase (topo) I and topo II. The protein is absent from the
origin in M and appears on it at the beginning of G1, reach a peak at G1/S and as
synthesis starts, the interaction of HOXC13 with the origin fades, in parallel with the
transition from this large pre-RC to a smaller and differently organized post-RC.
Recently also other HOX proteins have been identify as proteins involved in
regulation processes of DNA replication, suggesting that the interaction of HOXC13
with the origins might occur in a multi-homeotic proteins complex. Depletion of one of
these proteins however is compatible with the continuation of the cell cycle and,
according with what observed for the other homeotic proteins, we found that also the
depletion of HOXC13 does not alter cell cycle progression or S phase entry. This is
probably due to the redundancy of homeotic proteins and indicates a relatively generic
function for the HOX proteins. Among the identified elements influencing the choice and the activity of a
sequence as DNA replication origin, much relevance is assumed by the chromatin
structure and topology of DNA. Therefore, we analysed the effects of chromatin
structure disruption using Tricostatin A, a histone deacetylase inhibitor. The alteration
of chromatin caused by this treatment not only sharply reduces origin function, but also
disturbs the binding of replication complex members like HOXC13 and the well known
Cdc6 to the DNA replication origins, while does not affect the binding of other
unrelated proteins like USF1. On the basis of this finding, we infer that an appropriate
chromatin organization and DNA topology strongly influence the binding between
factors of the pre Replication Complex and DNA replication origins. This influence
could be a key element in origin specification.
The described interactions are not restricted to a single origin nor to a single
homeotic protein, leading us to conclude that HOX proteins, probably in the context of a
multi-protein homeotic effectors, contribute to recruit and stabilize the replicative
complexes onto early replicating origins, in presence of specific chromatin and
topological configurations.
The relevance of HOXC13 in DNA replication is also underlined by its
involvement in oncogenesis, clearly demonstrated in acute myeloid leukaemia when
HOXC13 is fused with NUP98 protein.
12
Mace, Kimberly Ann. "Split ends cooperates with Hox proteins to maintain epithelial integrity in the epidermis during embryonic development /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2003. http://wwwlib.umi.com/cr/ucsd/fullcit?p3099927.
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Ferretti, Elisabetta. "Molecular studies towards understanding the role of Prep1 (Pbx regulation protein 1) in segmental expression of Hox proteins in the vertebrate hindbrain". Thesis, Open University, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.396926.
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Zouaz, Amel. "Etude de la régulation de l'activité transcriptionelle de la protéine Abdominal-A". Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4100.
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Les gènes Hox codent des facteurs de transcription à homéodomain (HD). Bien que ce dernier reconnaisse des séquences similaires in vitro, les protéines Hox achèvent des fonctions hautement spécifiques in vivo. Des séquences protéiques en dehors de l’HD influencent la spécificité d’action des protéines Hox par le recrutement de cofacteurs, dont le mieux caractérisé est Extradenticle (Exd) chez la drosophile. Des travaux récents au sein de notre équipe ont démontré la contribution fonctionnelle de trois motifs de AbdA, aussi bien dans des fonctions Exd-dépendantes qu’à des fonctions Exd-indépendantes. Mon travail de thèse a porté sur la caractérisation de la contribution des motifs protéiques de AbdA dans la sélection puis dans la régulation des gènes cibles en utilisant une approche combinée ChIPseq/RNAseq, dans un contexte Exd-indépendant. Le code ADN identifié nous a renseigné sur la présence d’inputs transcriptionnels additionnels. Ces derniers correspondant à des facteurs de transcription déjà connus, leur présence dans un complexe protéique avec AbdA a été démontrée par des analyses de spectrométrie de masse. Un second volet de mon travail de thèse a été l’identification de modifications post-traductionnelles pouvant rendre compte d’un mécanisme de régulation de l’activité des protéines Hox. Des analyses prédictives in silico, confirmées par des approches biochimiques et des analyses in vivo ont démontré la SUMOylation de AbdA. Ces résultats préliminaires posent les bases pour des travaux futures qui auront pour objectif d’identifier les résidus d’AbdA SUMOylés et d’élucider le rôle de cette modification dans la régulation de l’activité de la protéine AbdAHox genes encode homeodomain-containing transcription factors (HD). Although the HD binds to similar DNA sequences in vitro, Hox proteins display a high functional specificity in vivo. Protein motifs outside of the HD influence Hox specificity through recruiting additional cofactors, with the best characterized being Extradenticle (Exd in Drosophila). Recent evidence from our group has uncovered the functional contribution of AbdA intrinsic motifs to AbdA Exd-dependent functions as well as AbdA Exd-independent functions. My PhD work has aimed to characterize the contribution of AbdA motifs to target gene selection and regulation using a combined approach of ChIPseq/RNAseq in an Exd-independent context. The DNA code identified provides us with new insights about additional transcriptional inputs from additional DNA-binding proteins lying in the vicinity of AbdA recognition sites. Mass spectrometry analysis establishes the occurrence of these additional DNA binding proteins in a multi-protein complex with AbdA. Deciphering the involvement of post-translational modifications in the regulation of Hox protein activity was another aspect of my PhD work. In silico predictive analysis, followed by biochemical approaches and in vivo assays reveal the potential for SUMOylation of AbdA as a potentially important regulatory component of AbdA activity. These preliminary results set the bases for further work aimed at identifying SUMOylated residues on AbdA and the functional relevance of such post-translational modification on AbdA activity regulation
15
Li-Kroeger, David. "Integration of regional and neural transcription factors controls EGF signaling from sensory organ precursor cells during Drosophila development". University of Cincinnati / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1337351052.
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Vanderperre, Solène. "Analyse d'interactions Hox/Cofacteur à l'échelle super-résolutive et contrôle transcriptionnel de l'autophagie chez la drosophile". Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEN048.
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La régulation transcriptionnelle est le sujet principal de nombreuses recherches et est un mécanisme indispensable pour assurer les fonctions cellulaires de tout organisme. Les avancées technologiques dans le monde de la microscopie ouvrent de nouvelles opportunités pour visualiser différentes étapes de ce mécanisme. Notamment les dynamiques et la localisation d’un FT a l’échelle super-résolutive. Cependant, un unique FT n’est pas suffisant pour réguler finement l’activation ou la répression de la transcription d’un gène. En effet, différents complexes de FT coopèrent pour atteindre une telle précision de régulation. La visualisation de la fixation d’un complexe (binaire) sur sa séquence régulatrice cible serait donc un atout pour mieux déchiffrer la régulation transcription elle.La première partie de mon travail de Thèse a consisté à mettre en place des outils permettant de visualiser en microscopie confocale et super-résolution, la fixation de complexes Hox-cofacteur sur des séquences ADN cibles spécifiques. Ces outils ont été appliques pour quantifier l’enrichissement de différents complexes Hox/Exd au niveau d’un enhancer connu (appelé fkh250) du gène cible forkhead (fkh) dans les glandes salivaires de la larve de drosophile. J’ai combine la méthode de BiFC (confocale) ou BiFC-PALM (super-résolution) et le système ParB/INT pour visualiser simultanément les complexes Hox/Exd et l’enhancer fkh250, respectivement.Mes analyses confirment un enrichissement spécifique des complexes Hox/Exd sur les différents types d’enhancer fkh250. Surtout, des résultats préliminaires indiquent la possibilité de quantifier le nombre exact de complexes Hox/Exd fixes sur l’enhancer fkh250 a l’échelle super-résolutive.La deuxième partie de mon travail de thèse concerne l’analyse d’une nouvelle interaction entre les protéines Hox avec la Lamine C (LamC) pour une répression transcriptionnelle active des gènes lies a l’autophagie (atg) dans le corps gras de la larve de drosophile. Ce travail a permis de révéler un profil de co-expression typique des protéines Hox et de la LamC, au sein du noyau par imagerie confocale ≪ Lightning ≫ et l’importance de contrôler le positionnement des loci génomiques pour une régulation fine de la transcriptionTranscriptional regulation is essential for all cellular functions and is the subject of a number of studies. Technological advances in the field of microscopy open new opportunities to visualize different steps of this mechanism. In particular, it allows visualizing individual TFs at the super resolution scale in vivo.However, an isolated TF is not sufficient to tightly regulate the activation or repression of a target gene. Indeed, different complexes need to cooperate to achieve this level of accurate control. The observation of binary protein-protein interactions bound on a specific DNA sequence would be an asset to decipher the complex mechanism of transcription.The first part of my thesis project consisted to establish the tools allowing the visualization of different Hox-cofactor complexes on a specific target sequence, at confocal resolution and super-resolution. These tools were applied to quantify a specific enrichment of Hox/Exd complexes on a well characterized enhancer called fkh250. This enhancer is regulating the expression of a Drosophila salivary gland gene named forkhead (fkh). I combined Bimolecular Fluorescent Complementation (BiFC) (confocal resolution) or BiFC-PALM (super-resolution) with the ParB/INT system to simultaneously detect Hox/Exd complexes bound to the fkh250 enhancer,respectively.My results confirm a specific enrichment of Hox/Exd complexes on several fkh250 enhancers. Moreover, my preliminary results show the possibility to perform bi-colour PALM for revealing in the same nucleus the Hox/Exd complexes and its target DNA sequences.The second part of my project revealed a new interaction between Hox proteins and a nuclear matrix component, the Lamin C (LamC), in the context of transcriptional repression of autophagy related genes (atg) in Drosophila larval fat body. This work revealed a typical profile of co-expression of Hox and LamC in Drosophila fat body nuclei. This profile was imaged through confocal Lightning microscopy. These results also revealed the importance of genomic loci positionning for the fine control of transcription
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Weber, Christine. "Cdx-Hox protein interactions". Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27656.
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Cdx and Hox gene families both code for homeodomain-containing transcription factors and both are required for proper patterning of the anterior-posterior axis. Although Cdx is known to be upstream of certain Hox genes, preliminary data indicated Cdx1 interacts in vitro with Hoxd4 but not Hoxa9, and that this interaction is localized to the C-terminal region of both proteins. GST-pulldown assays with in vitro radiolabelled proteins or transfected cell lysates were used to investigate the extent of the Cdx-Hox interactions. The interaction was narrowed down to specific residues in the first half of the homeodomain, but an inhibitory N-terminus can abrogate interaction in vitro. A larger group of Hox proteins interact with GST-Cdx when they are expressed in a cellular environment. This could mean that the interaction requires other cofactors, such as Hox cofactors Pbx and Meis that also interact with GST-Cdx1 in vitro. This study is the first example of Cdx-Hox protein interactions and suggests that the Cdx-Hox complex could be involved in the regulation of downstream Hox target genes.
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Ronshaugen, Matthew Rand. "Evolution of Hox protein function /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3071026.
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Clitheroe, Crystal-Leigh. "In-silico analysis of Plasmodium falciparum Hop protein and its interactions with Hsp70 and Hsp90". Thesis, Rhodes University, 2013. http://hdl.handle.net/10962/d1003819.
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A lessor understood co-chaperone, the Hsp70/Hsp90 organising protein (Hop), has been found to play an important role in modulating the activity and co-interaction of two essential chaperones; Hsp90 and Hsp70. The best understood aspects of Hop so far indicate that residues in the concave surfaces of the three tetratricopeptide repeat (TPR) domains in the protein bind selectively to the C-terminal motifs of Hsp70 and Hsp90. Recent research suggests that P. falciparum Hop (PfHop), PfHsp90 and PfHsp70 do interact and form complex in the P. falciparum trophozooite and are overexpressed in this infective stage. However, there has been almost no computational research on malarial Hop protein in complex with other malarial Hsps.The current work has focussed on several aspects of the in-silico characterisation of PfHop, including an in-depth multiple sequence alignment and phylogenetic analysis of the protein; which showed that Hop is very well conserved across a wide range of available phyla (four Kingdoms, 60 species). Homology modelling was employed to predict several protein structures for these interactions in P. falciparum, as well as predict structures of the relevant TPR domains of Human Hop (HsHop) in complex with its own Hsp90 and Hsp70 C-terminal peptide partners for comparison. Protein complex interaction analyses indicate that concave TPR sites bound to the C-terminal motifs of partner proteins are very similar in both species, due to the excellent conservation of the TPR domain’s “double carboxylate binding clamp”. Motif analysis was combined with phylogenetic trees and structure mapping in novel ways to attain more information on the evolutionary conservation of important structural and functional sites on Hop. Alternative sites of interaction between Hop TPR2 and Hsp90’s M and C domains are distinctly less well conserved between the two species, but still important to complex formation, making this a likely interaction site for selective drug targeting. Binding and interaction energies for all modelled complexes have been calculated; indicating that all HsHop TPR domains have higher affinities for their respective C-terminal partners than do their P. falciparum counterparts. An alternate motif corresponding to the C-terminal motif of PfHsp70-x (exported to the infected erythrocyte cytosol) in complex with both human and malarial TPR1 and TPR2B domains was analysed, and these studies suggest that the human TPR domains have a higher affinity for this motif than do the respective PfHop TPR domains. This may indicate potential for a cross species protein interaction to take place, as PfHop is not transported to the human erythrocyte cytosol.
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Veraksa, Alexey Nikolaevich. "Modulators of Hox protein function in Drosophila /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p9975051.
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Gava, Lisandra Marques 1982. "Caracterização e interação do domínio C-terminal da chaperona Hsp90 humana e das co-chaperonas Tom 70 e Hop". [s.n.], 2011. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314027.
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Orientador: Carlos Henrique Inácio RamosTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A função biológica das proteínas está relacionada à sua estrutura tridimensional adquirida pelo processo de enovelamento protéico. Neste contexto, proteínas denominadas, genericamente, de chaperonas moleculares exercem papel fundamental atuando no auxílio do enovelamento correto, no reenovelamento e na dissociação de agregados protéicos. A Hsp90 é uma das chaperonas moleculares mais importantes, é essencial para a viabilidade celular em eucariotos e está normalmente associada a proteínas atuantes no ciclo e sinalização celular, o que torna essa chaperona um alvo bastante interessante para abordagens terapêuticas de diversas doenças. A Hsp90 pode ser modulada por co-chaperonas diversas. Nesse trabalho foram caracterizadas as proteínas CHsp90 (domínio C-terminal da Hsp90 humana), e as co-chaperonas Hop e Tom70, além da interação entre C-Hsp90 e Tom70. Foram aplicadas técnicas de dicroísmo circular e emissão de fluorescência do triptofano; seguidas pela caracterização por ultracentrifugação analítica, gel filtração analítica, espalhamento dinâmico de luz, cromatografia de gel filtração acoplada a espalhamento de luz em multi-ângulos (SEC-MALS) e gel nativo. Para os ensaios de interação foram aplicadas técnicas de pull-down, SEC-MALS e calorimetria de titulação isotérmica. As proteínas foram produzidas puras e enoveladas, com estado oligomérico determinado como dímero para C-Hsp90 e monômero para Hop e Tom70, sendo que essas também foram encontradas como espécies diméricas. A estequiometria de interação entre a C-Hsp90 e Tom70 foi determinada em 1 monômero da Tom70 para 1 dímero da C-Hsp90, com KD de 360 ± 30 nM, ?Happ = -2,6 ± 0,1 kcal/mol e ?S = 21 ± 1 cal/mol.K, sugerindo que a interação é dirigida por entalpia e entropia. Os resultados obtidos nesse trabalho contribuem para uma melhor compreensão do sistema Hsp90, que está envolvido em diversos processos celulares essenciais e patológicos, como doenças neurodegenerativas, processos inflamatórios, infecções e câncer
Abstract: The biological function of proteins is related to its three dimensional structure acquired via protein folding process. In this context, the molecular chaperones play a key role acting as auxiliary protein on protein folding, refolding and dissociation of protein aggregates. Hsp90 is one of the most important molecular chaperones, is essential for cell viability in eukaryotes and is usually associated with proteins involved in cell cycling and cell signaling, which makes these chaperone a very interesting targeting for therapeutic approaches for several diseases. The chaperone activity of Hsp90 can be modulated by other proteins, called co-chaperones. In this work, we characterized the protein C-Hsp90 (Cterminal domain of human Hsp90) and the co-chaperones Hop and Tom70, and also the interaction between C-Hsp90 and Tom70. Circular dichroism and fluorescence emission of tryptophan was first applied for initial characterization of the proteins, followed by analytical ultracentrifugation, analytical gel filtration, dynamic light scattering, size exclusion chromatography - multi angle light scattering (SEC-MALS) and native gel. The interaction between C-Hsp90 and Tom70 were measured by techniques like pull-down, SEC-MALS and isothermal titration calorimetry. The proteins were produced pure and soluble and their oligomeric state were determined as dimer for C-Hsp90, and monomer for Hop and Tom70, these two co-chaperones were also found as dimeric species. The stoichiometry of interaction between C-Hsp90 and Tom70 was determined by SEC-MALS and ITC as been 1 dimer of C-Hsp90 to 1 monomer of Tom70, with a KD of 360 ± 30 nM, ?Happ = -2.6 ± 0.1 kcal/mol and ?S = 21 ± 1 cal/mol.K, suggesting that these interaction is driven by both, enthalpy and entropy. The results contribute to a better understanding of the important Hsp90 machinery, which is involved in many essential cellular and pathological processes, such as neurodegenerative diseases, inflammation, infection and cancer
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Rui, Edmilson. "Mutagenese da proteina HBx do virus da hepatite B e estudo da interação com RNA e proteinas humanas". [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314356.
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Orientador: Jorg KobargTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A hepatite B constitui um grave problema de saúde pública. Dados epidemiológicos estimam que aproximadamente 350 milhões de pessoas são portadores do vírus da hepatite B (HBV). Admite-se que a infecção evolui para a cura em média 95% dos casos, entretanto nos portadores crônicos a infecção pode evoluir para cirrose e carcinoma hepatocelular (HCC). Há um crescente acúmulo de evidências que relaciona a proteína HBx do HBV ao desenvolvimento do HCC. A maioria dos estudos sobre a função da proteína HBx sugere que ela é uma proteína reguladora de funções pleiotrópicas com a capacidade de induzir o crescimento tumoral. Isso é possivelmente devido à sua capacidade de interagir com uma vasta gama de proteínas celulares. No presente estudo investigamos os aspectos moleculares da estrutura e função da proteína HBx e os resultados foram contextualizados no processo de transformação celular. Observamos pela primeira vez a capacidade da proteína HBx em se ligar ao RNA contendo seqüências ricas em adenina e uracila (AU), que são presentes em alguns proto-oncogenes como c-myc e c-fos. A geração de proteínas truncadas permitiu mapear a região de interação entre HBx e RNA. Realizamos ensaios de mutação sítio-dirigida em HBx e substituímos todas as cisteínas por serinas. Nossos resultados sugerem que as cisteínas na proteína HBx são de menor importância para a sua interação com o RNA e com as proteínas humanas p53 e RXR. Descobrimos ainda uma nova proteína humana que interage com HBx: E4F. Este fator de transcrição humano está relacionado com o controle do ciclo celular, com a segregação cromossômica e com a embriogênese. Ensaios em leveduras e in vitro mostraram que HBx selvagem, bem como as suas formas mutadas, foram capazes de interagir e regular a função de E4F, indicando um possível novo mecanismo para a transformação celular e a regulação da transcrição viral, uma vez que E4F exibiu uma capacidade de interagir com a região ¿enhancer II¿ do genoma do HBV
Abstract: Viral hepatitis is an important global public health problem. Epidemiological data show that worldwide 350 million people are chronically infected with the hepatitis B virus. About 95% of the infections cure spontaneously, but the chronically infected patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC). A large body of evidence points to the viral onco-protein HBx as the principal cause for the cellular transformation. The majority of studies on HBx function suggest that it is a regulatory protein with pleiotropic functions and its capacity to induce tumor growth may be due to its ability to interact with a diverse array of cellular proteins. In the present study we investigated molecular and structural aspects of the function of the HBx protein in order to be able to shed light on the process of cellular transformation. We were able to demonstrate that HBx protein has the ability to bind to an AU-rich RNA sequences present in the mRNAs of certain proto-oncogenes such as c-myc and c-fos. The generation of truncated proteins allowed to map the region of interaction of HBx with the RNA. Furthermore, we performed site directed mutagenesis studies of HBx protein and substituted all of its cysteine residues with serines. Our data suggest that the cysteine residues in the HBx protein are of minor importance for its interaction with RNA, p53 and RXR proteins. Finally, we discovered in E4F a new human interacting protein partner for the HBx protein. The transcription factor E4F has been functionally implicated in the control of the cell cycle, the chromosomal segregation and embriogenesis. In studies using the yeast we further showed that wild-type HBx, as well as its mutated forms, can physically interact with the E4F protein and regulate its function. This indicates a possible new mechanism of cellular transformation and the regulation of the viral transcription, since the human protein E4F demonstrated the capacity to bind to the enhancer II region of the HBV genome
Doutorado
Bioquimica
Doutor em Biologia Funcional e Molecular
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Angell, Yu Li. "Triazole based peptidomimetics for mimicking protein-protein hot spots". [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1432.
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Foos, Nicolas. "Etudes structurales d'un complexe HOX-PBC de drosophile. : Un exemple de régulateur transcriptionnel". Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4075.
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Les protéines Hox sont des protéines à homéodomaine. Elles sont très conservées et impliquées dans l'identité cellulaire selon les axes antéropostérieur, dorsoventral et proximodistal. Elles reconnaissent l'ADN pour réguler l’expression des gènes. La coopérativité avec des partenaires de la famille PBC est un modèle pour l'amélioration de la spécificité. Cette étude utilise Ubx (Hox) et Exd (PBC) de D. melanogaster comme modèles. Deux modes d'interaction entre Ubx et Exd : un par le motif « hexapeptide » d'Ubx et un autre par le motif UbdA objet de ce travail. UbdA se situe en C-terminal de l'hélice de reconnaissance de l'ADN d'Ubx. Nous avons résolu différentes structures de complexes Ubx-Exd-ADN avec deux types de séquences d'ADN. Ces structures montrent que le motif UbdA peut adopter différentes conformations permettant différents rôles : surface d'interaction avec Exd ou charnière entre l'HD et les domaines C-terminaux. En plus des motifs d'Ubx important pour la fonction de régulation, Ubx et Exd comportent des motifs intrinsèquement désordonnés appelés linker et bras N-terminal de l'homéodomaine, respectivement. Ces motifs interagissent avec l'ADN au niveau du sillon mineur et ont la capacité d'explorer l’environnement. Nous avons étudié l'extrémité en C-terminale d'Exd. Ce motif forme une quatrième hélice dont le repliement peut influer sur les contacts établis avec Ubx et avec l'ADN. L'ensemble de ces observations nous a permis d'apporter des éléments supplémentaires pour la compréhension de la spécificité de fonctions et de contribuer à alimenter les modèles de scannage de l'ADN de type monkey bar et d'« interface glissante »Hox proteins are homeodomain proteins belonging to the Transcription Factors superfamilly. These proteins are necessary for the determination of the cellular identity along the anteroposterior, dorsoventral and proximodistal axes. It's essential to recognize DNA targets with high specificity. One possible mechanism to acquire specificity implies the cooperativity between Hox and their PBC partners. Ubx (Hox) and Exd (PBC) proteins from D. melanogaster are the object of this work. One mechanisme of coopertivity involves the “hexapeptide” motif in Ubx and another one that involves its UbdA motif. The UbdA motif is located C-terminal to the recognition helix. We have solved seven different structures of Ubx-Exd-DNA complexes. Thanks to these structures, we show that UbdA can have a multipurpose role : it can provide an interaction surface to contact Exd and it can also act like a hinge between the C-terminal regions of Ubx and its homeodomain. UbdA and HX motifs from Ubx are not the only regions involved in the control of these proteins functions. Ubx and Exd also contain intrinsically disordered regions which are the linker region and the homeodomain N-terminal arm, for Ubx and Exd respectively. They interact with the DNA in the minor groove and can explore the space around. We studied the Exd 's C-terminal motif and determined that it has a flexible, helical fold. The folding of this fourth helix could modify the contacts established with Ubx and with the DNA. All these observations allow us to add supplementary information for the understanding of functional specificity and provide new arguments for the monkey-bar and for the « gliding interface » DNA- scanning models
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Scheufler, Clemens. "Struktur-Funktions-Analyse von Hop Tpr-vermittelte Protein-Protein-Wechselwirkungen /". [S.l. : s.n.], 2000. http://deposit.ddb.de/cgi-bin/dokserv?idn=962034444.
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Reyes, Samuel Onofre J. "Expanding beta-turn analogs for mimicking protein-protein hot spots". Thesis, [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1748.
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Reis, Dayane Eliara Bertolino. "Caracterização estrutural da Hsp70/Hsp90 organizing protein (Hop) de Plasmodium falciparum". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-28022018-095723/.
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A malária é uma doença tropical negligenciada causada por protozoários do gênero Plasmodium spp, afeta populações em mais de 100 países ao redor do globo, apresentando 219 milhões de novos casos por ano sendo, portanto, um grave problema de saúde pública. Apresenta um ciclo complexo e digenético, necessitando do mosquito vetor e do hospedeiro vertebrado para se completar - ciclo este que envolve etapas de transformação e adaptação, já que o patógeno passa por 28 formas diferentes ao longo do ciclo, além de enfrentar situações de stress térmico, no momento do contágio e durante os picos febris. Sendo assim, é necessário que o protozoário garanta sua sobrevivência e possibilite a infecção do hospedeiro. Isso é realizado com a assistência de chaperonas moleculares, proteínas estas que são superexpressas no estágio intra-eritrocitário. Uma dessas proteínas é a Hsp90, uma Heat shock protein com diferentes funções, entre elas, maturação de proteínas clientes, encaminhamento de proteínas para translocação por membranas e marcação de proteínas para degradação. Para cumprir adequadamente as diversas funções, as Hsp90 contam com o auxílio de co-chaperonas, como a Hsp70/Hsp90 Organizing Protein (Hop) que modulam sua função. A Hop é uma co-chaperona do sistema foldossoma formado pelas Hsp70 e Hsp90 citoplasmáticas e que atua como proteína adaptadora transferindo proteínas clientes da primeira para a segunda chaperona molecular. A interação da Hop com Hsp70 e Hsp90 ocorre via domínios TPR, que se ligam ao motivo EEVD presente na extremidade C-terminal de ambas as chaperonas citoplasmáticas. É encontrada em diversos organismos, incluindo Plasmodium falciparum, o agente etiológico da malária. Sendo assim, conhecer a Hop de P. falciparum (PfHop), estrutural e funcionalmente, é importante para o entendimento do funcionamento das Hsp90 e Hsp70, proteínas essenciais para a sobrevivência do patógeno e, portanto, possíveis alvos terapêuticos. A PfHop recombinante foi obtida com pureza superior a 95%. A caracterização biofísica da mesma foi feita através de diferentes técnicas. Como outras Hops, a PfHop é majoritariamente constituída por hélices alfa. Os parâmetros hidrodinâmicos determinados sugerem que a PfHop se comporta como um equilíbrio monômero-dímero quando em solução. Dados de espalhamento de raios X a baixo ângulo mostram a PfHop como uma proteína dimérica e alongada. Este trabalho de dissertação de mestrado permitiu alcançar a caracterização estrutural da PfHop e com este conhecimento, espera-se avançar na caracterização funcional da mesma sobre a Hsp70 e Hsp90.Malaria is a neglected tropical disease caused by protozoa of the genus Plasmodium spp, affects populations in more than 100 countries around the globe, presenting 219 million new cases per year and is therefore a serious public health problem. It presents a complex and digenetic cycle, necessitating the vector mosquito and the vertebrate host to complete - this cycle involves transformation and adaptation stages, since the pathogen goes through 28 different forms along the cycle, besides facing situations of thermal stress , At the time of the contagion and during the feverish peaks. Thus, it is necessary that the protozoan guarantees its survival and makes possible a host infection. This is accomplished with the assistance of molecular chaperones, proteins that are overexpressed in the intra-erythrocyte stage. A life of proteins and Hsp90, a protection of thermal shock with different functions, among them, maturation of client proteins, routing of proteins for membrane translocation and labeling of proteins for degradation. To comply properly, for example, as Hsp90 rely on the help of co-chaperones, such as Hsp70 / Hsp90 Organizing Protein (Hop) that modulate their function. The Hop is a co-chaperone system folded by Hsp70 and Hsp90 cytoplasmic and which acts as an adapter protein transferring client proteins from the first to the second molecular chaperone. The interaction of Hop with Hsp70 and Hsp90 occurs via TPR domains, which bind to the EEVD motif present at the C-terminus of both as cytoplasmic chaperones. It is found in several organisms, including Plasmodium falciparum, the etiologic agent of malaria. Therefore, knowing a Hop of P. falciparum (PfHop), structurally and functionally, is important for the understanding of the functioning of Hsp90 and Hsp70, essential proteins for a pathogen survival and, therefore, in all the therapeutic aspects. A recombinant PfHop was obtained in greater than 95% purity. The biophysical characterization by the same brand made through different techniques. As there is Hops, a PfHop is mostly constituted by alpha helices. The indicated parameters are a PfHop behaves as a monomer-dimer balance when in solution. Higher low-angle X-ray scattering data on PfHop as a dimeric and elongated protein. This work of master\'s dissertation allowed to reach a structural characterization of the PfHop and with this knowledge, it is expected to advance in the functional characterization of the same in Hsp70 and Hsp90.
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Yuan, Ming. "Antiphagocytosis by Yersinia pseudotuberculosis : role of the YopH target proteins". Doctoral thesis, Umeå : Umeå University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-957.
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Zerbe, Brandon S. "Computational characterization of protein hot spots". Thesis, Boston University, 2012. https://hdl.handle.net/2144/31628.
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Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Protein hot spots provide a large portion of the binding free energy during interact ions, and detecting and characterizing these hot spot regions provides insight that can be used in the development of novel drugs for t he purpose of regulating pathological pathways. In t his dissertation, I first compare t he FTMap algorithm, which detects hot spots by identifying locations where different simulated solvent-sized molecules are consistently found to have favorable interactions, to experimental methods that detect hot spots by alternative means. Specifically, I show that FTMap detects the hot spots detected by alanine scanning, and I discover two roles for residues near hot spots in protein-protein interaction (PPI) complexes. Furthermore, additional insights into the binding energetics of PPis are uniquely provided by FTMap, and these insights are important for drug-design. FTMap is then shown to detect the hot spots identified by successful fragment-screening experiments, and the additional sites detected by FTMap are shown to provide insight into the optimal regions for ligand extension for the molecules identified by t he fragment-screening experiment. Since binding sites are composed of multiple hot spots, we have recently used FTMap for binding site detection. I examine the highly accurate binding site detection algorithm, show that the success of this algorithm is a consequence of only a portion of the scoring protocol, and develop a faster protocol for binding site detection based on this insight. I also quantitate the improvement in precision obtained by using multiple probes and argue t hat the principle biophysical considerations in hot spot detection are hydrophobicity and complexity. Finally, I develop a functional-group clustering algorithm, which is informative for evaluation of the binding locations of pre-determined chemical moieties. I then provide evidence that other approaches employing FTMap results may lead to insight into selectivity. I conclude with a discussion on the nature of hot spots, and I suggest that evolutionary studies of protein divergence should provide insight into the emergence of chemical-selectivity thus providing biophysical insight into the factors that drive selectivity within hot spots.
2031-01-01
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Giraud, Guillaume. "Dose et cofacteurs des protéines Hox lors du développement des organes de vol chez les insectes". Thesis, Lyon, 2020. http://www.theses.fr/2020LYSEN089.
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Au cours de l'évolution, les êtres vivants ont développé une étonnante diversité morphologique leur permettant de s'adapter à des environnements très variés. L'un des exemples les plus frappants est la radiation des appendices de vol chez les insectes à partir d'ancêtres possédant deux paires d'ailes très similaires. Plusieurs travaux montrent l'importance d'un gène Hox particulier, Ultrabithorax ( Ubx), pour la formation des organes de vol postérieurs. Ceux-ci correspondent à des organes balanciers appelés « haltères » chez les diptères. A l'inverse, la formation des organes de vol antérieurs est considérée comme indépendante des gènes Hox. Au cours de mes travaux de thèse, nous avons remis en question ce modèle, et montré que la protéine Hox Antennapedia (Antp) est non seulement produite da ns le primordium de l'a ile mais éga lement nécessaire à la formation des ailes antér ieures chez la mouche des fruits Drosophila melanogaster. De manière surprenante, la dose de Antp est bien en deçà de celle observée pour Ubx dans le primordium d'haltère, et l'augmentat ion artificielle de cette dose suffit à transformer l'aile en haltère. À l'inverse, la diminution contrôlée de la dose de Ubx induit une transformation de l'haltère en aile. Ces résultats montrent ainsi que ce n'est pas le type de protéine Hox, ici Antp ou Ubx, mais leur dose, qui contrôle le programme développemental de type a ile ou haltère chez la drosophile. L'analyse du rôle du facteur de transcription Homothorax (Hth) montre également que celui-ci est nécessaire respectivement à l'activation de Antp et à la répression de Ubx dans les primordia d'aile et d'haltère, permettant de mettre en place un profil d'expression spécifique. L'observation d'une corrélation entre variation de dose Hox et formation d'ailes identiques ou différentes dans d'autres lignages d'insectes permet de proposer un nouveau modèle dans lequel les modifications du niveau d'expression des gènes Antp et Ubx pourraient être plus largement responsables de la diversification morphologique des organes de vol au cours de l'évolution des insectes. Comment la protéine Ubx parvient à spécifier le programme développemental des haltères chez la drosophile reste encore mal compris au niveau moléculaire. En particulier, il est attendu que son activité dose-dépendante puisse reposer sur l'interaction avec d'autres partenaires transcriptionnels. J'ai donc initié deux approches complémentaires originales pour identifier de nouveaux cofacteurs de Ubx. Si l'une des approches n'a pu aboutir à son terme, l'autre a permis d'identifier un certain nombre de nouveaux partenaires et de révéler une stabilité surprenante du programme développemental de l'haltèreDuring evolution, animais have developed an astonishing morphological diversity, allowing them to adapt to a wide variety of environments. One of the most striking examples is the radiation of flight appendages in insects, starting from ancestors with two similar pairs of wings. Severa! studies showed the implication of a particular Hox gene, Ultrabithorax ( Ubx), in the formation of posterior flight organs. These organs correspond to the "halteres" in Diptera. Conversely, the formation of anterior flight organs is considered to be Hox independent.During my Ph.D work, we have questioned this mode!. We showed that the Hox protein Antennapedia (Antp) is produced in the wing primordium of the fruit fly Drosophi/a melanogaster and is required for the formation of forewings. Surprisingly, the express ion level of Antp is much lower than the level of Ubx in the haltere primordium, and the artificial increase of Antp level can transform the wing into a haltere. Conversely, the artificial decrease of the Ubx level can induce a haltere to wing transformation. These results show that this is not the type of the Hox protein, here Antp or Ubx, but the specific dose, which is important for the formation of a wing or a haltere in Drosophi/a. We also identified the transcription factor Homothorax (Hth) as an upstream regulator of Antp and Ubx in the wing and haltere primordia respectively. The observation of a correlation between Hox dose variation and wing morphology in other insect lineages supports a new mode! in which morphological diversification of flight organs during insect evolution could arise from changes in the Antp and Ubx expression level. How the Ubx protein could specify the haltere developmenta l program at the molecular level remains elusive. lt is expected that the dose-specific activity of Ubx in the haltere could rely on the interaction with other transcr iptiona l partners. 1 present two complementary approaches that 1 developed ta capture such cofactors. While one approach is still ongoing, the other led ta the identification of interesting putative Ubx-partners. This work also revea led a surpr ising robustness in the Ubx transcr iptional program that may expia in why halteres diverged very little during insect evolution
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Hudry, Bruno. "Application de la complémentation de fluorescence bi-moléculaire à l'étude du mode d'action des protéines Hox in vivo". Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22087/document.
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Comment le plan d’organisation d’un organisme est-il mis en place est une question centrale de la biologie du développement. Les séquençages complets de plusieurs génomes de métazoaires ont montré qu’un nombre restreint de molécules régulatrices soutiennent la diversité des plans d’organisation des animaux, suggérant que ces molécules sont utilisées de manière répétée dans des contextes différents. Cela soulève la question de la diversité d’action : comment ces molécules acquièrent-elle une diversité fonctionnelle ? De plus, la plupart des molécules régulatrices partagent des motifs (fonctionnels/structuraux) communs, soulevant la question de la spécificité : comment des molécules partageant des propriétés biochimiques similaires contrôlent-elles des programmes développementaux spécifiques ?Mon équipe d’accueil s’intéresse à ces questions en utilisant les facteurs de transcription Hox de la Drosophile comme paradigme d’étude.Durant ma thèse, j’ai développé trois lignes de recherches : (1) J’ai adapté la technique de complémentation bi-moléculaire de fluorescence (BiFC) de visualisation des interactions protéines-protéines à l’embryon de drosophile en développement.(2) J’ai employé la BiFC pour disséquer la formation des complexes Hox-protéines PBC. Mes résultats remettent en question le paradigme établit : (a) en soulignant la multiplicité des modes d’interaction Hox-PBC existants, (b) en démontrant que cette diversité peut être source de spécificité d’action.(3) La BiFC a ensuite été exploitée dans un crible par approche gènes candidats pour identifier de nouveaux partenaires des protéines HoxMy current laboratory aims to tackle the issue of specificity and diversity of regulatory molecules, taking the Drosophila Hox transcription factors as a paradigm for the analysis. During my PhD, I developed three connected research lines.Project 1: Visualization of protein interactions in living Drosophila embryos by the BiFC assayOur results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.Project 2: Investigation of Hox/PBC complex formation in vivo using BiFC Our findings challenge the current paradigm of Hox/Pbx complex assembly: (a) highlighting the existence of alternative modes of Pbx recruitment, (b) demonstrating that unique Hox-PBC interaction modes can provide specific regulatory function in absence of DNA-binding selectivity.To achieve this project BiFC was also performed with vertebrate Hox proteins in chicken embryos.Project 3: Realization of a candidate interaction screen based on BiFC to identify novel Hox protein partners in vivo(a) We have revealed that Hox proteins establish specific interactions with different subunits of the general mediator complex. These results constituted one of the rare studies making a direct link between the Hox regulators and components of the basal transcriptional machinery, in a physiological context.(b) We have discovered that Hox proteins can interact with importin proteins. This result allows us to assess the importance of controlling the nuclear localization of Hox proteins for controlling their regulatory activities during embryogenesis
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Melén, Karin. "Topology Prediction of Membrane Proteins: Why, How and When?" Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-6875.
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Membrane proteins are of broad interest since they constitute a large fraction of the proteome in all organisms, up to 20-30%. They play a crucial role in many cellular processes mediating information flow and molecular transport across otherwise nearly impermeable membranes. Traditional three-dimensional structural analyses of membrane proteins are difficult to perform, which makes studies of other structural aspects important. The topology of an α-helical membrane protein is a two-dimensional description of how the protein is embedded in the membrane and gives valuable information on both structure and function.
This thesis is focused on predicting the topology of α-helical membrane proteins and on assessing and improving the prediction accuracy. Reliability scores have been derived for a number of prediction methods, and have been integrated into the widely used TMHMM predictor. The reliability score makes it possible to estimate the trustworthiness of a prediction.
Mapping the full topology of a membrane protein experimentally is time-consuming and cannot be done on a genome-wide scale. However, determination of the location of one part of a membrane protein relative to the membrane is feasible. We have analyzed the impact of incorporating such experimental information a priori into TMHMM predictions and show that the accuracy increases significantly. We further show that the C-terminal location of a membrane protein (inside or outside) is the optimal information to use as a constraint in the predictions.
By combining experimental techniques for determining the C-terminal location of membrane proteins with topology predictions, we have produced reliable topology models for the majority of all membrane proteins in the model organisms E. coli and S. cerevisiae. The results were further expanded to ~15,000 homologous proteins in 38 fully sequenced eukaryotic genomes. This large set of reliable topology models should be useful, in particular as the structural data for eukaryotic membrane proteins is very limited.
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Melén, Karin. "Topology prediction of membrane proteins : why, how and when? /". Stockholm : Department of biochemistry and biophysics, Stockholm university, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-6875.
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Morén, Björn. "Caveolae associated proteins and how they effect caveolae dynamics". Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-92500.
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Caveolae are a type of invaginated membrane domain that has been shown to be involved in several disease states, including lipodystrophy, muscular dystrophies and cancer. Several of these diseases are caused by the lack of caveolae or caveolae-related signaling deficiencies in the tissues in which the caveolar domain are abundant such as lung, adipose, muscle and their related endothelial cells. Caveolae are formed through the assembly of the membrane inserted protein caveolin, cholesterol and the recently described family of cavin proteins, which together form the caveolae coat. The work in this thesis focuses on understanding the protein components and mechanisms that control the biogenesis and dynamics of caveolae. We have found that the protein EHD2 is an important regulator and stabilizer of the caveolar domain at the cell membrane. EHD2 is a dimeric ATPase known to oligomerize into ring-like structures around lipid membranes to control their shape. We have characterized the domain interactions involved in the specific targeting and assembly of this protein at caveolae. We propose a stringent regulatory mechanism for the assembly of EHD2 involving ATP binding and switching of the EH domain position to release the N-terminus and facilitate oligomerization in the presence of membrane species. We show that loss of EHD2 in cells results in hyper- dynamic caveolae and that caveolae stability at the membrane can be restored by reintroducing EHD2 into these cells. In a study of the protein cavin-3, which is known to be an integral component of the caveolar coat, we showed that this protein is targeted to caveolae via direct binding to the caveolar core protein caveolin1. Furthermore, we show that cavin-3 is enriched at deeply invaginated caveolae and regulate the duration time of caveolae at the cell surface. In combination with a biochemical and cellbiological approach, the advanced fluorescence microscopy techniques, like Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection microscopy (TIRF), combined with correlative Atomic Force Microscopy (AFM) have allowed us to characterize distinct caveolae-associated proteins and their respective functions at caveolae.
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Pérez, Culubret Adrià 1993. "Learning how to simulate : Applying machine learning methods to improve molecular dynamics simulations". Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2022. http://hdl.handle.net/10803/673392.
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Caracteritzar la dinàmica de les proteïnes és essencial per tal d'entendre la connexió entre seqüència i funció. La simulació de dinàmiques moleculars és una de les tècniques principals per a estudiar la dinàmica de proteïnes per la seva capacitat de capturar processos dinàmics computacionals en diferents escales temporals amb resolució atòmica. Tanmateix, hi ha limitacions que impedeixen que la simulació de dinàmiques moleculars es converteixi en un model substitutiu de les dinàmiques reals de proteïnes, principalment per limitacions de mostreig i la inexactitud dels camps de força utilitzats.
En aquesta tesi doctoral tractem aquestes limitacions mitjançant els últims avenços en aprenentatge automàtic. En la primera part de la tesi, desenvoluparem un nou algoritme de mostreig adaptatiu inspirat en mètodes d'aprenentatge reforçat, que aplicarem per a reconstruir la unió entre una proteïna desordenada i la seva parella d'unió. En la segona part de la tesi, desenvoluparem TorchMD, una llibreria d'aprenentatge profund per a simulacions de dinàmica molecular, que aplicarem per a aprendre un potencial "coarse-grained" per a simulacions de plegament de proteïnes.
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Chauvet, Sophie. "Fonctions des proteines hox : determinants intrinseques de specificite et genes effecteurs chez drosophila melanogaster". Aix-Marseille 2, 1999. http://www.theses.fr/1999AIX22052.
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Les genes hox, tres conserves au cours de l'evolution, specifient l'identite des differentes parties du corps. Ils codent pour des facteurs de transcription a homeodomaine (hd) et sont exprimes de facon differentielle dans l'organisme le long de l'axe antero-posterieur. Un des challenges des trente dernieres annees a ete de comprendre comment les genes hox permettent de transformer une information de position en un mouvement de morphogenese. Il est necessaire pour cela de bien connaitre l'organisation des complexes hox, la regulation de leur expression, mais surtout, de comprendre les mecanismes de leur specificite d'action in vivo et de caracteriser leurs genes cibles. Dans une partie de ce memoire, je presente l'analyse de plusieurs genes cibles potentiels chez drosophila melanogaster. Ces genes ont ete identifies a partir d'une banque enrichie en fragments d'adn genomiques associes in vivo a la proteine hox ultrabithorax qui a ete construite dans l'equipe. Plus precisement, une etude systematique des clones de cette banque a permis l'analyse de quatre genes, regules par ubx, dont les sequences ont ete conservees au cours de l'evolution, nessy, d-larp et charybde et scylla (deux genes dupliques). Le clonage de ces genes, l'etude de leur regulation et les approches pour analyser leur fonction en aval des genes hox sont decrits dans cette these. De plus, dans le cas de d-larp, nous avons aussi etudie son orthologue murin : m-larp. En effet, alors qu'une grande partie des reseaux d'interactions impliquant les genes hox a ete conservee au cours de l'evolution, on dispose de peu de donnees sur la conservation des phenomenes se produisant en aval des genes hox. Nous avons clone m-larp afin d'etudier son profil d'expression et avons ainsi montre qu'il n'avait vraisemblablement pas conserve son statut de gene cible d'hox chez mus musculus. Dans une deuxieme partie de mon travail, je me suis interessee a la question de la specificite tissulaire des proteines hox. Une meme proteine hox, exprimee dans des domaines larges et dans differents tissus, n'active en general ses genes cibles que dans une sous partie de son domaine d'expression ; ce qui peut constituer une activite tissu specifique. S'il est maintenant clairement etabli que l'information de specificite d'action des proteines hox peut etre portee non seulement par l'hd mais aussi par d'autres regions de la proteine, aucun domaine implique dans la tissu-specificite n'avait ete decrit. Dans cette deuxieme partie de mon travail, je montre que les proteines hox possedent des determinants intrinseques de specificite tissulaire, certains de ces determinants sont situes en dehors de l'hd.
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Daniel, Sheril. "Molecular characterization of the Hsp70/Hsp90 organizing protein (Hop) phosphorylation, subcellular localization and interaction with Hsp90". Thesis, Rhodes University, 2008. http://hdl.handle.net/10962/d1004056.
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Hop (Hsp70-Hsp90 Organizing Protein) is a co-chaperone of two major molecular chaperones, Hsp70 and Hsp90, and acts by transferring substrates from Hsp70 to Hsp90. Although under normal conditions Hop is predominantly localized within the cytosol, Hop has been detected in the nucleus under certain conditions including cell cycle arrest. A putative nuclear localization signal (NLS) has been identified within Hop, which overlaps with the TPR2A domain (previously shown to be critical for Hop-Hsp90 interactions). Hop is phosphorylated in vitro by two cell cycle kinases, namely, casein kinase II (CKII) at S189 and cdc2-kinase at T198; both residues are found upstream of the putative NLS and TPR2A domain. Mimicking phosphorylation at either phosphorylation site appeared to affect the subcellular localization of Hop. The aim of this study was to characterize Hop with respect to its phosphorylation status in vivo, as well as its subcellular localization pattern under heat stress and determine how these properties affected its interaction with Hsp90 as a co-chaperone. Dephosphorylation of proteins under normal and heat shock conditions changed the isoform composition of Hop, providing strong evidence that Hop was phosphorylated in vivo. Surface plasmon resonance (SPR) and glutatione-S-transferase (GST) co-precipitation studies showed that a cdc2-kinase phosphorylated mimic of Hop disrupted Hop-Hsp90 binding. A full length Hop-EGFP construct, as well as substitution mutants of the predicted NLS residues within the Hop-EGFP construct, were transfected into baby hamster kidney (BHK)-21 cells in order to establish the subcellular localization of Hop under heat stress and to test whether predicted residues were critical for nuclear localization of Hop. Under normal conditions, both Hop-EGFP and the NLS mutants were predominantly cytosolic, but when the cells were subjected to heat stress, Hop and its NLS-mutants were localized to both the cytosol and the nucleus. SPR and GST co-precipitation studies showed that substitution of the residues within the major arm of the putative NLS abrogated Hop-Hsp90 interactions. The data obtained from this study, showed for the first time, that Hop was phosphorylated in vivo and suggested that phosphorylation of Hop by cdc2-kinase could inhibit Hop-Hsp90 interactions. Moreover, these results suggested that the subcellular localization of Hop was dependent on stress levels of the cell, particularly heat stress. We propose that the nuclear localization of Hop may be primarily regulated by stress and secondarily by cell cycle arrest. The major arm of the putative NLS did not affect the localization of Hop directly, but was shown to be critical for Hop-Hsp90 binding in vitro. The results of this study suggested that binding of Hop to Hsp90 sequestered Hop within the cytosol and that Hsp90 acted as a cytosolic retention factor for Hop. Both phosphorylation of Hop, and its subcellular localization, appeared to be intimately related to its interaction with Hsp90 as a co-chaperone.
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Cambronero, Christoffer. "Multiplex protein data and how to handle it". Thesis, Uppsala universitet, Statistiska institutionen, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-297390.
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Objective: To nd which methods and techniques that work well when working with multiplex proteindata. A short analysis of a dataset to show the methods is done at the end of the thesis. Material and methods: A study containing 171 individuals is used to test if there are any dierencesin protein values based on obesity, sugar level and type of obesity(5 subgroups). To investigate this OlinksProseek multiplex is used to generate 92 dierent protein values for each individual in the dataset. MainlyWelch's t-test and ANOVA are used as the comparison method for multiplex protein data. Since multipletests are performed the p-values are adjusted to avoid multiple type I error. Dierent ways to visualize thedata are also presented. Results: When comparing the obese and non-obese groups 25 proteins are shown to be signicantly dierent(p-value <0:05). Low and high sugar have no proteins that are signicantly dierent(p-value <0:05) betweenthe two groups and for the dierent types of obesity 18 proteins are signicant dierent(p-value <0:05). Conclusion: Both the tests for obesity and types of obesity show dierences between the groups while thesugar level do not have any proteins that signicantly dier between the groups. However when performinga multivariate techniques of low and high sugar the two can be separated indicating some dierences inprotein values.
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Zhang, Jingji. "Accuracy of mRNA Translation in Bacterial Protein Synthesis". Doctoral thesis, Uppsala universitet, Struktur- och molekylärbiologi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262901.
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Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch). Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides. We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.
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Page, Suzannah Jayne. "The expression of Hex, fli1 and Tal1 proteins in Xenopus embryos". Thesis, University of Portsmouth, 2011. https://researchportal.port.ac.uk/portal/en/theses/the-expression-of-hex-fli1-and-tal1-proteins-in-xenopus-embryos(0b7d6bcd-076c-4d5f-a6f7-cc6420cc4846).html.
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Understanding the gene regulatory networks driving the differentiation of the distinct cell types in embryos is a key component of understanding development. Amongst the first types of cell to differentiate during vertebrate development are blood cells, and haematopoiesis is tightly regulated by proteins known as haematopoietic transcription factors (HTFs). Although the mRNA expression patterns of many HTFs are now well characterised, little is known about their protein expression patterns or how their transcription regulation activity is controlled during early development. Knowledge of their spatial and temporal expression patterns and their post-translational regulation would greatly enhance our understanding of the genetic regulatory networks that produce blood. Antibodies have been successfully raised and characterised that recognise Xenopus Hex, fli1 and Tal1. Western blot experiments have shown that Hex and fli1 are expressed in oocytes and expression continues throughout development. This maternal expression is unexpected but is reminiscent of another embryonic HTF, Gata2. At stage VI of oogenesis the majority of fli1 and all Hex protein was localised within the cytoplasm, suggesting an alternative second function for each of these proteins. An intensively sampled developmental time-course carried out for fli1 between stages 24 and 31, revealed striking post-transcriptional control over fli1 expression. Neither Hex or fli1 proteins have been shown to be absent from the regions containing sites of haematopoiesis and both proteins were eluted in the same fractions following size exclusion chromatography using Xenopus extract, suggesting the possibility that they may be in complex together. Finally, either Tal1 protein is undetectable during early Xenopus embryogenesis, or is heavily modified. Since, at least for fli1, knockdown of the zygotic protein specifically affects blood development a number of questions are raised. What is different about the maternal and zygotic proteins, which means that the zygotic form is needed for haematopoiesis? What is the function of the maternal proteins – are they even capable of transcription regulation?
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Stutchbury, Benjamin. "Understanding how focal adhesion proteins sense and respond to mechanical signals". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/understanding-how-focal-adhesion-proteins-sense-and-respond-to-mechanical-signals(f46e2121-1642-41b8-98f6-e0c168d6f01e).html.
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The mechanical properties of the tissue vary widely around the body, from the soft brain to the rigid bone. Tissue cells are able to sense mechanical signals from their environment, which influence many aspects of cell behaviour such as migration, proliferation and differentiation. Focal adhesions (FAs) are large protein complexes that form the bridge between the extracellular matrix (ECM)-binding integrins and the contractile actin cytoskeleton. Here, they sense the rigidity of the local environment and translate this information into a cellular response, a process known as mechanotransduction. However, the FA proteins required for mechanotransduction, and the molecular mechanisms involved in this fundamental process, remain to be elucidated. Talin, vinculin, FAK and paxillin are four core FA-associated proteins that are thought to be involved in mechanotransduction. These proteins associate and dissociate from the complex in a constant state of flux. Using a live-cell imaging approach, I found that the rate of dynamic exchange of an FA protein correlates to its function. The FA appears to have a modular organisation; the slowest proteins have a structural role, such as talin and vinculin, responsible for directly linking integrin to actin and sensing the ECM stiffness. The signalling proteins are turned over more rapidly, including FAK and paxillin, and are responsible for directing the cellular response to force-generated signals from the ECM.The second results chapter focused on the force-dependent interactions between talin, vinculin and actin. The talin domains R2R3 were identified as the key mechanosensitive vinculin-binding sites, which are exposed upon the application of force across the talin rod. Vinculin binding to R2R3 led to actin associating with the central actin-binding site in the talin rod (ABS2), which is required for the transmission of actomyosin tension onto the underlying substrate as cellular traction force. Finally, the protein turnover data were incorporated into two mathematical models, describing talin and vinculin turnover, which were able to simulate the dynamic exchange of various talin and vinculin mutants in response to changing ECM stiffness. Using these models, the talin ABS2-actin and vinculin tail-actin interactions were found to be extremely important for sensing the stiffness of the ECM. These findings significantly increase our knowledge of the molecular mechanisms underpinning cellular mechanotransduction. Increased understanding of how mechanical signals are sensed and interpreted by the cell could lead to a number of novel therapies for a wide range of associated diseases, such as atherosclerosis, muscular dystrophy and cancer.
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Guimarães, Larissa Oliveira. "Caracterização de subpopulações de Leucemia Mielóide Aguda portadora do rearranjo MLL quanto à resposta diferencial ao tratamento em longo prazo com Citarabina". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-07012016-115206/.
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A natureza heterogênea da Leucemia Mielóide Aguda (LMA) tornou-se um desafio para o sucesso da quimioterapia convencional com o agente Citarabina (Ara-C), especialmente em leucemias com prognóstico desfavorável, como aquelas portadoras do rearranjo MLL. Visto que as células de LMA-MLL são consideradas sensíveis ao Ara-C quando comparadas às leucemias que não apresentam o rearranjo, mas a recaída à doença é frequente, a presente tese propôs estudar a relação entre características biológicas relacionadas às bases da resistêmcia ao Ara-C em LMA-MLL. A abordagem proposta foi a seleção de subpopulações de linhagens celulares portadoras do rearranjo MLL submetidas ao tratamento em longo prazo com Ara-C, comparando-as com as linhagens não expostas à droga. As células foram caracterizadas quanto: 1) ao potencial proliferativo na presença ou ausência de Ara-C; 2) a distribuição das células no ciclo celular; 3) a distribuição de marcadores clássicos de superfície de células-tronco hematopoiéticas, CD34 e CD38; e 4) o perfil de expressão global dos RNAs transcritos. O tratamento em longo prazo selecionou células mais resistentes ao Ara-C que as células parentais. Além disso, quanto ao ciclo celular, as células selecionadas com Ara-C apresentaram apoptose reduzida (fase sub-G1), acúmulo na fase de síntese (fase S) e aumento da capacidade proliferativa após reexposição à droga (fase G2-M). Quanto à análise de marcadores de células-tronco hematopoiéticas, observou-se que após o tratamento em longo com Ara-C, uma das linhagens celulares apresentou distribuição bimodal do marcador CD38. Quando separadas por sorting em citometria de fluxo, observou-se que as subpopulações com níveis distintos de expressão de CD38, denominadas MV-4-11 CD38High e MV-4-11 CD38Low apresentaram resposta distinta ao tratamento com Ara-C. Quando avaliadas quanto ao perfil global de expressão gênica, constatou-se que MV-4-11 CD38High eram mais semelhantes às células parentais, e que MV-4-11 CD38Low formavam um grupo isolado, distinto das outras duas populações celulares. A análise de ontologia gênica (GO) evidenciou que entre as categorias mais representativas de processos biológicos estavam atividades associadas à capacidade proliferativa, ao desenvolvimento e a resposta a estímulos. As análises de agrupamentos hierárquicos mostraram que: 1) o cluster de genes do desenvolvimento HOXA estava mais expresso nas células MV-4-11 CD38Low do que em MV-4-11 CD38High, que apresentaram expressão mais elevada do cluster HOXB; 2) o gene HOX mais diferencialmente expresso foi HOXA13, associado na literatura com prognóstico desfavorável em outros tipos de câncer; 3) dos genes associados a resposta a estímulos, o único relacionado à via de metabolização do Ara-C diferencialmente expresso entre as linhagens foi NME1; 4) aqueles que participam das vias de reparo de pareamento incorreto, reparo por excisão de bases e por excisão de nucleotídeos encontraram-se mais expressos nas células MV-4-11 CD38High que em MV-4-11 CD38Low. Além disso, diversas quinases dependentes de ciclinas (CDKs) também estiveram diferencialmente expressas entre MV-4-11 CD38High e MV-4-11 CD38Low. Sugere-se por fim, que o modelo in vitro proposto neste estudo para simular a situação de resistência ao Ara-C em subpopulações de LMA-MLL, demonstrou que os mecanismos de resposta à Citarabina nesta doença, vão além de alterações na detoxificação e metabolização da droga, e parecem mais associados a vantagens proliferativas e do desenvolvimento das células leucêmicas. Estas vias devem ser exploradas como alvos potenciais na terapia combinada ao Ara-C.The heterogeneity of Acute Myeloid Leukemia (AML) became a challenge for the success of the conventional chemotherapy agent Cytarabine (Ara-C), especially in leukemias with poor prognosis, as those harboring MLL rearrangement. Since AML-MLL cells are considered sensitive to Ara-C when compared with leukemias that do not carry the rearrangement, but relapse is frequent, the present dissertation proposed to study the relationship between biological characteristics related to the basis of chemoresistance to Ara-C in AML-MLL. We proposed an approach based on the selection of subpopulations of cell lines bearing MLL rearrangement submitted to the long-term treatment with Ara-C, comparing them with the cell lines that were not previously exposed to the drug. The cells were characterized according to: 1) the proliferative potential in the presence and absence of Ara-C; 2) the distribution of the cells in the cell cycle; 3) distribution of hematopoietic stem cell classic surface markers, CD34 and CD38; and, 4) global expression profile of transcribed RNAs. The long-term treatment selected cells that are more resistant to Ara-C than the cells that were not previously treated (parental cells). Besides, according to cell cycle, the cells selected by Ara-C treatment present decreased apoptosis (sub-G1 phase), accumulation in the synthesis phase (S-phase) and increase in the proliferative capability after re-exposition to the drug (G2-M phase). Regarding the hematopoietic stem cell markers, we observed that after Ara-C long-term treatment, one of the cell lines exhibited a bimodal distribution of the CD38 marker. When sorted by flow cytometry, we observed that both subpopulations with distinct levels of CD38 expression, called MV-4-11 CD38High and MV-4-11 CD38Low also showed distinct response to Ara-C. When evaluated regarding to their global gene expression profiles, we verified that MV-4-11 CD38High were more closely related to the parental cells, and MV-4-11 CD38Low made up an isolated group, distinct of the other cell populations. Gene ontology (GO) analysis revealed that among the most representative categories of biological processes, activities associated with proliferative capability, development and response to stimuli were included. The hierarchical clustering analysis showed that: 1) the cluster HOXA of genes of development was more expressed in the MV-4-11 CD38Low than in the MV-4-11 CD38High cells, that presented increased expression of HOXB cluster; 2) the most differentially expressed HOX gene was HOXA13, which according to the literature is associated with poor prognosis in other types of cancer; 3) among the genes associated with response to stimuli, the only one related to Ara-C-metabolizing pathway that was differentially expressed between the cell lines was NME1; 4) those genes that take part in the mismatch repair, base excision repair and nucleotide excision repair pathways were more expressed in the MV-4-11 CD38High than in the MV-4-11 CD38Low cells. Additionally, several cyclin-dependent kinases (CDKs) were also differentially expressed between MV-4-11 CD38High and MV-4-11 CD38Low. Finally, we suggest that the in vitro model proposed in this study to mimic the situation of chemoresistance to Ara-C in subpopulations of AML-MLL, showed that the mechanisms of Ara-C response in this disease, go beyond changes in drug detoxification and metabolization, and seem more associated to proliferative and development advantages of the leukemic cells. These pathways should be explored as potential targets to Ara-C combination therapies.
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Sun, An-Qiang. "How Do Enzymes Wear Out? Effects of Posttranslational Modifications on Structure and Stability of Proteins; The Triosephosphate Isomerase Model". Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc798116/.
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Triosephosphate isomerase (EC 5.3.1.1., TPI) undergoes specific posttranslational modifications (deamidation and oxidation) which are believed to initiate protein turnover by destabilization of the dimer. The crystal structures, amino acid sequences, and aging related changes of TPI from various species have been independently characterized by several laboratories. TPI has thus become the prototype enzyme for examining the initial steps in protein turnover. The binding of substrate enhances the specific deamidation of the mammalian enzyme, and a general mechanism of 'molecular wear and tear' [Gracy, R. W., Yiiksel, K. 0., Chapman, M. L., and Dimitrijevich, S. D. (1990) in Isozymes-Structure, Function and Use in Biology and Medicine (Ogita, Z-I., and Markert, C. L., Eds) pp. 787-817, Wiley-Liss, New York] has been proposed to explain how enzymes may wear out.
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Lin, Zhen St George Clinical School UNSW. "Molecular mechanism of cancer related to urokinase receptor: DNAzyme-mediated inhibition and Novel protein interactors of urokinase receptor". Awarded by:University of New South Wales. St George Clinical School, 2007. http://handle.unsw.edu.au/1959.4/31893.
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The urokinase receptor (uPAR) plays a central role in metastatic process. It???s evident uPAR is overexpressed across a variety of tumour cells and leads to the increased aggressiveness and poor prognosis of cancer. Inhibition of uPAR expression can block metastatic potential in many tumours. In addition, besides uPA, there are several other proteins which have been confirmed to interact with uPAR, such as vitronectin and integrins. These interactions also contribute to signal transduction and the functions of uPAR complex. Therefore, downregulation of uPAR expression by targeting uPAR mRNA or protein, or by regulating the uPAR partners would be potential therapeutic strategies for prevention of cancer metastasis. There are two main aspects contained in this thesis. Firstly, three specific DNAzymes targeting uPAR mRNA were designed to downregulate uPAR expression in vitro and their effects to decrease cancer cell invasion studied in a human osteosarcoma cell line Saos-2. The results showed that two of them (Dz483 and Dz720) cleaved uPAR transcript in vitro with high efficacy and specificity and the Dz720 inhibited uPAR protein levels by 55% in Saos-2 cells. Besides, the Dz720 significantly suppressed Saos-2 cell invasion using an in vitro matrigel assay. Secondly, two potential uPAR partners from yeast two-hybrid screening, a heat shock protein MRJ and an anti-apoptosis protein HAX-1, were characterised and their functions binding with uPAR investigated. The interactions were confirmed by co-immunoprecipitation, GST-pull down assay and confocal microscopy in cancer cells. In addition, there was a 50% increase in cell adhesion after transfection with MRJ. This increase in adhesion is dependent on the uPAR/full length MRJ interaction as cells transfected with the mutant construct containing only N-terminal region or C-terminal region of MRJ had no increase in cell adhesion. The observed increase in adhesion to vitronectin by MRJ was also blocked by an anti-uPAR domain I antibody suggesting that the induced adhesion is at least in part contributed by uPAR on the cell surface. Together, the identification of both MRJ and HAX-1 as uPAR interactors provides further insight into the intricate relationship between uPAR and other proteins which may develop potential approaches for cancer therapy.
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Lacerda, Tonielli Cristina Sousa de. "Papel do complexo PrPc-HOP e vesículas extracelulares em câncer colorretal". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-20092016-101001/.
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O câncer colorretal (CCR) é o terceiro tipo de câncer mais comum no mundo. Apesar dos avanços nos tratamentos convencionais, aproximadamente dois terços dos pacientes com CCR são submetidos à cirurgia potencialmente curativa. Entretanto, grande parte desses pacientes evolui mal, apresentando recidivas e/ou metástases. A busca de novos alvos moleculares para a terapia do CCR revelou a proteína celular Prion (PrPC) como um possível candidato. Trabalhos recentes sugerem participação direta ou indireta de PrPC no crescimento de tumores, na formação de metástases, na composição de complexos multiproteicos e na indução de vias de sinalização envolvidas em diversos processos biológicos, como proliferação. Além disso, PrPC foi descrito como um importante modulador do crescimento de tumor colorretal. Resultados prévios mostraram que a interação da proteína PrPC com a proteína HSP70/HSP90 Organizing Protein (HOP) induz proliferação em glioblastomas. HOP é uma proteína predominantemente citoplasmática, podendo também ser secretada associada às vesículas extracelulares. Assim, o presente estudo objetivou avaliar o papel do complexo PrPC-HOP e das vesículas extracelulares no desenvolvimento e progressão dos tumores colorretais. Os nossos resultados mostram que HOP induziu migração e invasão em linhagens de CCR de maneira dependente de PrPC, uma vez que o uso do peptídeo sem atividade que compete pelo sítio ligação de HOP a PrPC inibiu estes processos. Além disso, nossos dados apontaram que o aumento de migração e invasão das células de CCR induzida pela interação PrPC-HOP é mediada pela ativação da via ERK1/2. Os achados in vitro estimularam a avaliação do perfil de expressão de PrPC e HOP por imuno-histoquímica em tecidos de pacientes com diferentes tipos de tumores colorretais. Nossos resultados sugeriram que essas proteínas são importantes no início do desenvolvimento tumoral e na transição de adenomas para adenocarcinomas, não havendo correlação entre a presença de HOP e/ou PrPC com metástase, linfonodos acometidos, estadiamento, sobrevida ou região tumoral versus tecido normal. Em relação ao papel das vesículas extracelulares na progressão dos tumores colorretais, nossos resultados mostraram que linhagens celulares que apresentam padrões parecidos de agressividade tumoral podem ter perfis de secreção de proteínas e vesículas extracelulares bastante diferentes, induzindo, portanto, processos biológicos com intensidades distintas. O meio condicionado e as vesículas extracelulares da linhagem WiDr apresentaram maior potencial de indução de migração quando comparado com a linhagem HCT8. Além disso, a modulação negativa da proteína VPS4, uma das responsáveis pela formação dos corpos multivesiculares, mostrou-se uma abordagem interessante no estudo da secreção de vesículas por células de CCR, uma vez que o dominante negativo de VPS4 promoveu diminuição do cargo proteico e da secreção de vesículas extracelulares, redução da proliferação celular e do efeito indutor do processo de migração na linhagem WiDr. Assim, em conjunto, o presente trabalho indicou que o complexo PrPC-HOP pode ser um bom alvo terapêutico nos processos de migração e invasão em CCR. Ainda, essas proteínas se mostraram importantes nos estágios iniciais da formação dos tumores. A modulação da secreção de vesículas extracelulares pode contribuir para retardar a progressão dos tumores colorretais.Colorectal cancer (CRC) is the third most common type of cancer in the world. Despite improvements in conventional treatments, approximately two-thirds of CRC patients undergo potentially curative surgery. However, most of these patients evolve poorly, showing recurrence and/or metastasis. Search of new molecular targets for CRC therapy revealed the cellular protein Prion (PrPC) as a putative candidate. Recent studies have shown that PrPC exhibit direct or indirect participation in tumor growth, formation of metastasis, composition of multiprotein complexes and induction of signaling pathways involved in many biological processes such as proliferation. Moreover, PrPC has been described as an important modulator of colorectal tumor growth. Previous findings showed that the interaction between PrPC and its ligand HSP70/90 heat shock organizing protein (HOP) induces gliobastoma proliferation. It is well known that HOP localizes mainly in the cytoplasm but HOP is also secreted associated with extracellular vesicles. In this way, the present study sought to evaluate the role of PrPC-HOP complex and extracellular vesicles in the development and progression of CRC. We demonstrate that HOP induces the migration and invasion of CRC cell lines in a PrPC-dependent manner because the use of HOP peptide, which is able to bind to PrPC, blocking PrPC-HOP complex formation, inhibited the migration and invasion processes. In addition, our data showed that the enhancement of migration and invasion induced by PrPC-HOP interaction is mediated by ERK1/2 pathway activation. These in vitro results lead us to evaluate the PrPC and HOP expression by immunohistochemistry in tissues from patients with different tumor types. Our data showed that these proteins could be important for the initial steps of tumor development, represented by the transition from adenoma to adenocarcinoma. No correlation was found among HOP and/or PrPC expression and metastasis, lymph node involvement, staging, survival or tumor area versus normal tissue. Regarding the role of extracellular vesicles in the progression of colorectal tumors, our results showed that cell lines exhibiting similar aggressive tumor behavior can have a different protein secretion pattern and a distinct profile of extracellular vesicles release, which could induce biological process with different intensities. The conditioned medium and the extracellular vesicles derived from WiDr cell line showed a higher potential to induce migration than HCT8 cell line. Moreover, the negative modulation of VPS4, one of the proteins responsible for multivesicular body formation, showed to be an interesting approach in the study of extracellular vesicles secretion secreted by CRC cells; the negative dominant of VPS4 promoted in the WiDr cell line a reduction in the protein cargo and secretion of the extracellular vesicles, a decrease of cell proliferation and induction of migration process. Therefore, taken together, our data highlights that PrPC-HOP complex can be considered a new therapeutic target in migration and invasion processes of CRC. Moreover, these proteins appeared to be important at onset of tumor formation. The modulation of extracellular vesicles secretion may contribute for delaying the progression of colorectal tumors.
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Hawkins, Kenneth R. "Designing the diffusion immunoassay (DIA) : how properties of the analyte affect DIA performance /". Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/8076.
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NAPOLI, ILARIA. "How the fragile X mental retardation protein represses protein synthesis: a mechanism of translational control in dendrites". Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/765.
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La Sindrome dell'X Fragile è la forma di ritardo mentale ereditario più frequente nella popolazione con un'incidenza 1/4000 nei maschi e 1/6000 nelle femmine. La patologia è causata da mutazioni nel gene FMR1 il cui prodotto proteico, FMRP, è altamente espresso nei neuroni.
FMRP è una proteina che lega gli RNA messaggeri neuronali e si localizza lungo i dendriti e gli assoni dei neuroni dove controlla il trasporto e la sintesi proteica dei messaggeri associati.
Nel presente progetto abbiamo analizzato il meccanismo attraverso il quale FMRP regola la sintesi proteica alle sinapsi, caratterizzando il complesso ribonucleoproteico contenente FMRP che agisce bloccando la traduzione di specifici mRNAs. I nostri studi rivelano che la proteina CYFIP1/Sra1, un noto interattore di FMRP, può interagire con il fattore traduzionale eIF4E. Il complesso ribonucleoproteico CYFIP/FMRP contiene anche il piccolo RNA non codificante Brain Cytoplasmic RNA 1 (BC1) e il messaggero del gene Map1b. Stimolando i neuroni in coltura e i sinaptosomi con il fattore neurotrofico BDNF si osserva la parziale dissociazione del complesso prima descritto con successiva attivazione della traduzione. Questi dati contribuiscono ad accrescere la conoscenza sul meccanismo di azione della proteina FMRP, compromesso nei pazienti affetti dalla Sindrome dell'X Fragile.Regulated protein synthesis in neuronal dendrites is crucial for synaptic plasticity and brain development. The Fragile X mental retardation protein (FMRP) represses translation of specific mRNAs in neuronal dendrites; how it exerts this effect, however, is largely unknown. One protein that interacts with FMRP is CYFIP1/Sra1, a component of the WAVE complex involved in actin polymerization. Here, we show that CYFIP1/Sra1 also interacts with the cap-binding translation initiation factor eIF4E through a novel domain that is structurally related to that present in eIF4E-BPs; this doman in CYFIP1/Sra1 is necessary for translational repression. Furthermore, the Brain Cytoplasmic RNA 1 (BC1), increases the affinity of FMRP for the CYFIP1-eIF4E complex. Importantly, in the brain CYFIP1 is associated with BC1 RNA as well as with Map1b, an FMRP-target mRNA. Reduction of CYFIP1 in neurons leads to an increase of Map1B protein. BDNF, a developmental stimulus that triggers dendritic protein synthesis, causes CYFIP1/Sra1, FMRP and eIF4E to dissociate in dendrites and at synapses. CYFIP1/Sra1 thus mediates the translational repression of FMRP in the brain. In addition, CYFIP1/Sra1 links translational control with the actin skeleton and therefore potentially with mRNP transport from dendrites into spines. Finally, we dissect one of the molecular mechanisms through which FMRP regulates neuronal mRNA translation and unravel one of the pathways that maybe impaired in patients with the Fragile X Syndrome.
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Meyer, Tim [Verfasser]. "How Structural Details Influence the Result of pKa Calculations in Proteins / Tim Meyer". Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1072622351/34.
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Chun-Yue, Lee. "Exploring the biological functions of AlkB proteins and how they relate to AAG". Thesis, Massachusetts Institute of Technology, 2009. http://hdl.handle.net/1721.1/46598.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2009.Includes bibliographical references.
Our DNA is constantly under the assault of DNA damaging agents that are ubiquitous in nature and unavoidable. Fortunately, our cells have evolved DNA repair mechanisms to maintain genomic integrity against this constant attack. An important type of DNA damage is alkylation damage, which has been the focus of this thesis, the major goal of which is to explore the biological role of a set of alkylation repair proteins, the E. coli AlkB and two human AlkB homologs (ABH2 and ABH3), and how they relate to the 3methyladenine DNA glycosylase (AAG). AAG is a base excision repair (BER) protein that has been well-studied and is known to be involved in the repair of a wide variety of substrates. On the other hand, the direct reversal protein AlkB, and its human homologs, have not been so extensively characterized, but it is known that they can repair not only DNA, but also RNA. Although there are eight human AlkB homologs, attention was focused on ABH2 and ABH3 since they are the more well-characterized homologs and recently implicated in DNA repair.In order to investigate the role of the AlkB proteins, particularly in mammalian cells, I expressed ABH2 and ABH3 in established human cell lines and investigated whether their expression would enhance cell survival after alkylation treatment. However, no detectable phenotype was observed in the cell lines upon treatment with the alkylating agent methyl methanesulfonate (MMS). This is possibly due to endogenous ABH levels being sufficient for repair. We therefore turned to characterization of the Abh2 and Abh3 null mice, as compared to wildtype and to another alkylation repair deficient animal, Aag null mice. In addition to the primary substrates 1methyladenine and 3-methylcytosine, AlkB, ABH2, and ABH3 can also repair an important class of damage, the etheno base DNA lesions, which can also be repaired by AAG.
(cont.) Here we have shown in a mouse model that Abh2 and Abh3 overlap with Aag in protecting mice from sensitivity in response to chemically induced chronic inflammation, in which etheno base lesions are readily generated. In addition, we also employed a biochemical approach using a comprehensive library of lesion-containing DNA oligonucleotides to study the redundancy in repair activity between AAG and AlkB. In doing so, we have found new substrates for AAG and in particular, 1-methylguanine, is a new substrate shared between AAG and AlkB. Thus, although these two proteins employ different mechanisms for repair, our studies established further evidence of the interplay between these proteins and the different repair pathways they represent, underscoring the importance of alkylation damage repair for proper cell homeostasis.
by Chun-Yue Lee.
Ph.D.
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Meyer, Tim Friedrich [Verfasser]. "How Structural Details Influence the Result of pKa Calculations in Proteins / Tim Meyer". Berlin : Freie Universität Berlin, 2015. http://nbn-resolving.de/urn:nbn:de:kobv:188-fudissthesis000000099528-6.
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