Дисертації з теми "CLN6"
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1
Martin, Yella. "Investigation of the Batten Disease protein CLN6." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1444803/.
Повний текст джерелаАнотація:
The neuronal ceroid lipofuscinoses (NCLs, Batten Disease) are a group of lysosomal storage disorders caused by mutations in known and unknown proteins with different cellular locations. Mutations in the CLN6 gene cause a type of variant late infantile NCL (vLINCL). The CLN6 protein is located in the ER and how mutations in CLN6 cause accumulation of storage material in the lysosome is unclear. The aims of this thesis were to establish the tools and techniques necessary to analyse the CLN6 protein and to utilise these to investigate its subcellular location, to identify proteins that interact with CLN6 and to elucidate the early cellular responses to loss of functional vLINCL proteins, CLN5, CLN6 and CLN8. It was shown that CLN6 can be detected by Western blotting, indirect immunofluorescence and immunoprecipitation. A construct of CLN6 fused to horseradish peroxidase was cloned for localisation studies by indirect immunofluorescence and electron microscopy. CLN6 protein and mRNA levels could be depleted using RNA interference and this depletion assessed by Western blotting, indirect immunofluorescence and Q-PCR. It was not possible to establish whether CLN6 is located within a sub-domain of the ER using indirect immunofluorescence or electron microscopy. There was no effect on the localisation or stabilisation of wild-type or mutant CLN6 when proteasomal or lysosomal inhibitors were used, indicating that CLN6 does not traffic to the lysosome and that wild-type and mutant CLN6 were not degraded by ER-associated degradation. Endogenous CLN6 was identified in co-immunoprecipitation experiments, confirming that it may bind to itself. No other proteins were identified that bind to CLN6. Depletion of CLN5, CLN6 and CLN8 resulted in an increase in the size and a redistribution to perinuclear areas of late endosomes and lysosomes. An increase in long cis-Go g structures was also observed in response to siRNA against CLN6 and CLN8 but not CLN5.
2
Mohd, Ismail Izmira Farhana. "Identification of a novel mutation in the CLN6 gene (CLN6) in South Hampshire sheep affected with Neuronal Ceroid Lipofuscinosis." Thesis, The University of Sydney, 2014. http://hdl.handle.net/2123/14579.
Повний текст джерелаАнотація:
Neuronal ceroid lipofuscinoses (NCL/Batten disease) are a group of fatal inherited neurodegenerative diseases that occur in many species including humans, sheep, dogs and cattle. Typical NCL symptoms include progressive loss of vision, regression of mental and motor development, epileptic seizures and premature death. Currently there is no effective treatment or cure for NCL, with the underlying disease mechanisms still poorly understood. Advances in molecular genetics in recent years have allowed the characterisation of hundreds of causative mutations and polymorphisms in at least 17 disease-causing genes across all species. For some species, research colonies have been established for studies relevant to the corresponding human NCL variants. Best characterised of all animal models for NCL is the New Zealand South Hampshire (SH) sheep which is a model for the human variant late-infantile form of NCL (vLINCL). Past studies have revealed the ovine CLN6 gene (CLN6) as a strong candidate gene for this disease in South Hampshire sheep however no disease-causing mutation was identified. The main objective of the present thesis is the identification and characterisation of the mutation responsible for NCL in the South Hampshire sheep. It was proposed that the mutation lies in the non-coding regions within or flanking the gene and that this mutation affects gene regulation. Bioinformatic tools were initially used to identify conserved non-coding sequences (CNCS) which are deemed potential regions of interest for regulatory mutations. Due to the limited ovine genome resource available when the study was commenced in 2006, CLN6 orthologous sequences from other species were initially used for identification of highly conserved regions. Of the five identified CNCS (5’ UTR, 3’UTR and introns 1, 2 and 6) the region upstream of CLN6 and intron 1 were considered priorities for sequencing. Given that the Sanger sequencing method was laborious and time-consuming, and that there was rapid development of technology; the Sanger sequencing approach was abandoned and Next-generation sequencing (NGS) methods utilised for the following studies. The 454 Pyrosequencing NGS technology was used to sequence the complete ovine Bacterial artificial chromosome (BAC) to generate an ovine reference sequence for mutation screening approaches. The first mutation screening approach, sequence capture and targeted sequencing approach failed; however, the second approach involving sequencing of long-range PCR (LR-PCR) products successfully identified the disease-causing mutation. LR-PCR amplification of 14 regions within the ovine genome region spanning the CLN6 and flanking sequences followed by SOLID sequencing-by-ligation NGS method identified the disease-associated mutation as a 402bp deletion and 1bp insertion in ovine CLN6, namely g.-251_+150del and g.+150_151insC. The mutation is predicted to lead to the deletion of the whole of exon 1 and the ATG start codon as well as flanking non-coding sequence. Identifying the disease-causing mutation for NCL in SH sheep provides the long-awaited confirmatory evidence that ovine CLN6 is the causative gene for NCL in SH sheep. Future research in this large animal model will allow for more effective strategies for developing therapeutic approaches for NCL in humans and further strengthens the invaluable role of this animal model for NCL studies.
3
Cramer, Thomas [Verfasser]. "Generierung monoklonaler Antikörper gegen das Protein CLN6 / Thomas Cramer." Halle, 2017. http://d-nb.info/118038783X/34.
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4
Alves, Mariana Isabel Quaresma da Rocha. "Ceroido-Lipofuscinose Neuronal. Estudos de localização celular da proteína CLN6." Master's thesis, Faculdade de Medicina da Universidade do Porto, 2007. http://hdl.handle.net/10216/22151.
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5
Alves, Mariana Isabel Quaresma da Rocha. "Ceroido-Lipofuscinose Neuronal. Estudos de localização celular da proteína CLN6." Dissertação, Faculdade de Medicina da Universidade do Porto, 2007. http://hdl.handle.net/10216/22151.
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6
Dahlmann, Cordula [Verfasser]. "Retinaler Phänotyp dreier Mausmodelle für die neuronale Ceroidlipofuszinose : (CLN1-knockout Mausmodell, CLN3Δex7/8-knock-in Mausmodell und CLN6-knockout Mausmodell) / Cordula Dahlmann". Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2013. http://d-nb.info/1031097074/34.
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7
Lauronen, Leena. "Neuromagnetic studies on somatosensory functions in CLN3, CLN5 and CLN8 forms of neuronal ceroid lipofuscinoses." Helsinki : University of Helsinki, 2001. http://ethesis.helsinki.fi/julkaisut/laa/kliin/vk/lauronenle/.
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8
Trusso, Maria Allegra. "THE GENETICS OF BIPOLAR DISORDER AND THE ROLE OF HETEROZYGOSITY FOR NEURONAL CEROID LIPOFUSCINOSIS." Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1214195.
Повний текст джерелаАнотація:
Introduction. Bipolar Disorder (BD) is an heritable chronic mental disorder causing psychosocial
impairment, affecting patients with depressive/manic episodes. The familial transmission of BD does not
follow any of the simple Mendelian patterns of inheritance, demonstrating the involvement of multiple
susceptibility genes.
Materials and Method. Whole Exome Sequencing (WES) was performed in eight subjects of a large family
counting twelve BD affected people. We selected variants in common between the affected subjects, once
including and once excluding a “borderline” subject with moderate anxiety and traits of obsessive-
compulsive disorder.
Results. Results were in favour of a Digenic model of transmission, with a heterozygous missense variant in
CLN6 resulting in a “borderline” phenotype that if combined with a heterozygous missense variant in ZNF92
is responsible for the more severe BD phenotype. Both rare missense changes are predicted to disrupt the
protein function.
Conclusions. Loss of both alleles in CLN6 causes Neuronal Ceroid Lipofuscinosis, a severe progressive
neurological disorder of childhood. Our results indicate that heterozygous CLN6 carriers, previously reported
as healthy, may be susceptible to bipolar disorder late in life. Additional variants, such as that in ZNF92
reported here, may further worsen the phenotype in a setting of digenic disorder. Further investigation on a
larger cohort should be performed in order to better characterize the contribution of each gene.
9
Krzoska, Marta [Verfasser], and Thomas [Akademischer Betreuer] Braulke. "Untersuchungen zur Interaktion des krankheitsrelevanten CLN6-Proteins mit der Inositollipidphosphatase TPIP / Marta Krzoska. Betreuer: Thomas Braulke." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2011. http://d-nb.info/1020384301/34.
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10
Bessa, Carlos Jorge Pereira. "Molecular pathophysiology underlyng neuronal ceroid lipofuscinoses: CLN2 and CLN5." Doctoral thesis, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/24457.
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11
Bessa, Carlos Jorge Pereira. "Molecular pathophysiology underlyng neuronal ceroid lipofuscinoses: CLN2 and CLN5." Tese, Instituto de Ciências Biomédicas Abel Salazar, 2009. http://hdl.handle.net/10216/24457.
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12
De, Silva Weerakonda Arachchige Bhagya Nilukshi. "A study of neuronal ceroid lipofuscinosis proteins CLN5 and CLN8." Thesis, Kansas State University, 2015. http://hdl.handle.net/2097/35749.
Повний текст джерелаАнотація:
Master of ScienceBiochemistry and Molecular Biophysics Interdepartmental Program
Stella Yu-Chien Lee
Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative lysosomal storage disorders which is the most frequent group of inherited neurodegenerative disorders that affect children leading to severe pathological conditions such as progressive loss of motor neuron functions, loss of vision, mental retardation, epilepsy, ataxia and atrophy in cerebral, cerebella cortex and retina and eventually premature death. Among the many genes that cause NCL, mutations in CLN5 leads to different forms of NCL (infantile, late infantile, juvenile and adult) and mutations in CLN8 leads to progressive epilepsy with mental retardation (EPMR) and a variant late infantile form of NCL. The function(s) of both CLN5 and CLN8 proteins remain elusive. CLN5 is a glycosylated soluble protein that resides in the lysosome. We observed that endogenous CLN5 protein exist in two forms and identified a previously unknown C-terminal proteolytic processing event of CLN5. Using a cycloheximide chase experiment we demonstrated that the proteolytic processing of CLN5 is a post-translational modification. Furthermore treatment with chloroquine showed the processing occurs in low pH cellular compartments. After treatment with different protease inhibitors our results suggested the protease involved in the processing of CLN5 could be a cysteine protease. Using two glycosylation mutants of CLN5, retained in the endoplasmic reticulum (ER) or the Golgi we showed the proteolytic processing occurs in an organelle beyond the ER. This study contributes to understanding the characteristics of the CLN5 protein. CLN8 is an ER resident transmembrane protein that shuttles between the ER and the ER-Golgi intermediate compartment (ERGIC). In our study we identified a potential interaction between CLN8 and a PP2A holoenzyme complex consisting regulatory subunit A α isoform and regulatory subunit B α isoform. Using two CLN8 patient derived fibroblast cell lines we were able to show that the phosphorylated levels of PP2A target kinase Akt was reduced at both of its regulatory sites Ser473 and Thr308 and the activity of PP2A was increased. A delay of ceramide transport from ER to Golgi in CLN8 deficient patient cell lines was observed using BODIPY FL C5-Ceramide staining. Our results provide evidence for CLN8 protein being involved in the regulation of PP2A activity and trafficking of ceramide from ER to Golgi.
13
Thelen, Melanie [Verfasser], and Thomas [Akademischer Betreuer] Braulke. "Expression und Proteinwechselwirkungen von murinem Cln6 und seine Rolle in der Pathogenese der Neuronalen Ceroid Lipofuszinose / Melanie Thelen. Betreuer: Thomas Braulke." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1069376795/34.
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14
Larkin, Heidi. "Rôle de Calnuc dans le triage endosomial des récepteurs lysosomiaux et implication potentielle dans les maladies du lysosome." Thèse, Université de Sherbrooke, 2016. http://hdl.handle.net/11143/8204.
Повний текст джерелаАнотація:
Résumé : Calnuc est une protéine ubiquitaire qui lie le calcium et qui est présente au réseau trans-golgien (TGN) ainsi qu'aux endosomes. Notre groupe a précédemment mis en évidence le rôle de Calnuc dans le transport de Low density lipoprotein receptor-related protein 9 (LRP9), un récepteur aux lipoprotéines de faible densité qui cycle entre le TGN et les endosomes. Les récepteurs lysosomiaux au mannose-6-phosphate (MPR) et Sortiline sont bien caractérisés et empruntent également cette voie. À l'image de LRP9, nous avons montré que Calnuc prévient leur dégradation aux lysosomes en participant à leur recyclage à partir des endosomes vers le TGN. En fait, Calnuc est importante pour l'activation et l'association membranaire de Rab7, une petite protéine G qui recrute ensuite le complexe Rétromère responsable du transport rétrograde des récepteurs. La glycoprotéine lysosomiale Ceroid lipofuscinosis neuronal 5 (CLN5) est également impliquée dans ce processus. La structure et la fonction de cette dernière n'étant pas clairement définies, nous avons établi qu'elle est synthétisée sous forme d’une glycoprotéine transmembranaire de type II, mais son domaine N-terminal cytoplasmique et son segment transmembranaire sont rapidement éliminés suivant le clivage du peptide signal de manière à former une protéine CLN5 mature fortement associée à la membrane par une hélice amphipathique (AH). La compréhension des propriétés de base de CLN5 est particulièrement pertinente puisque la protéine est impliquée dans certaines variantes de céroïdes-lipofuscinoses neuronales (NCL), une maladie neurodégénérative rare causée par une surcharge des lysosomes. D'ailleurs, nos données indiquent que les mutants pathologiques de CLN5 dépourvus de cette AH perdent leur association membranaire, sont retenus au réticulum endoplasmique et sont rapidement dégradés. En raison de la similitude des fonctions de Calnuc et de CLN5 au niveau du triage endosomial, nous avons exploré le lien entre les deux protéines. Calnuc cytosolique et CLN5 luminale semblent former un complexe, par l'intermédiaire de la protéine transmembranaire CLN3, de façon à influencer l'activité de Rab7. CLN3 étant aussi associée aux NCL, nous avons finalement exploré la potentielle implication de Calnuc dans la maladie. L'absence de Calnuc entraîne des phénotypes cellulaires typiques des NCL comme un engorgement des lysosomes, une accumulation de matériel autofluorescent et une augmentation de l'autophagie. Les niveaux protéiques de Calnuc sont diminués dans toutes les lignées de fibroblastes de patients atteints de NCL disponibles ce qui indique que Calnuc pourrait être impliquée dans certains types de NCL. La présente thèse couvre donc la découverte de la fonction de Calnuc dans le transport intracellulaire, jusqu'à son implication potentielle dans les NCL, de même qu'une étude topologique de CLN5.Abstract : Calnuc is a ubiquitous Ca2+-binding protein present on the trans-Golgi network (TGN) and endosomes. We previously highlighted the role of Calnuc in the transport of Low density lipoprotein receptor-related protein 9 (LRP9), a low density lipoprotein (LDL) receptor that cycles between the TGN and endosomes. Lysosomal receptors mannose-6-phosphate receptor (MPR) and Sortilin are well-characterized and also use the TGN-to-endosome trafficking pathway. Similarly to LPR9, we showed that Calnuc prevent their degradation in lysosomes by acting in their recycling from endosomes to the TGN. In fact, Calnuc is a important for the activation and the membrane association of Rab7, a small G protein which then recruit the Retromer complex known to be responsible for the retrograde transport of receptors. Lysosomal glycoprotein Ceroid lipofuscinosis neuronal 5 (CLN5) is also involved in this process. Because its structure and function have not yet been clearly defined, we established that it is synthesized as a type II transmembrane (TM) glycoprotein, but its cytoplasmic N-terminus and TM segment are rapidly removed following signal-peptide cleavage to generate mature CLN5 which is tightly associated to membrane through an amphipathic helix (AH). The understanding of the basic properties of CLN5 is particularly important given that CLN5 is involved in some variants of neuronal ceroid lipofuscinosis (NCL), a rare neurodegenerative disease caused by lysosomal overload. Moreover, our data indicate that CLN5 pathological mutants deprived of AH lose their membrane association, are retained in the endoplasmic reticulum, and are rapidly degraded. Based on the similarity featured by Calnuc and CLN5 in endosomal sorting, we explored the link between these two proteins. Cytosolic Calnuc and luminal CLN5 seem to form a complex, through the transmembrane protein CLN3, in order to influence the activity of Rab7. As CLN3 is also associated with NCL, we finally explored the potential involvement of Calnuc in this disease. Canuc depletion leads to typical NCL phenotypes such as lysosome enlargement, accumulation of autofluorescent material and of an increased of autophagy induction. Canuc's levels are decreased in all fibroblasts cell lines of NCL patients available indicating that Calnuc could be involved in some types of NCL. This thesis thus covers the discovery of the function of Calnuc in intracellular transport up to its potential involvement in the NCL, as well as a topological study CLN5.
15
Heine, Claudia. "Untersuchung zur Pathogenese der neuronalen Ceroid-Lipofuszinosen am Menschen und an den Tiermodellen Ovis aries (LINNEAUS 1758) und Mus musculus (LINNEAUS 1758) CLN6 und Cathepsin-D-Defizienz /." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=967749301.
Повний текст джерела
16
Isosomppi, Juha. "Molecular and cell biology of infantile (CLN1) and varaint late infantile (CLN5) neuronal ceroid lipofuscinoses." Helsinki : University of Helsinki, 2003. http://ethesis.helsinki.fi/julkaisut/laa/haart/vk/isosomppi/.
Повний текст джерела
17
Sharifi, Azita. "Biologie moléculaire et cellulaire de trois protéines menmbranaires du lysosome impliquées dans les maladies neurologiques : La sialine, CLN3 et CLN7." Paris 11, 2010. http://www.theses.fr/2010PA11T033.
Повний текст джерела
18
O'Hare, Megan Beatrice. "Examining CLN7 function in Drosophila." Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/examining-cln7-function-in-drosophila(8281ab94-be3e-43f9-ab7b-4d0474f1de5d).html.
Повний текст джерелаАнотація:
My thesis aims to establish the normal functional role of the transmembrane protein CLN7; mutations in Cln7 cause a debilitating neurodegenerative disorder in infants known as Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). NCL are a collection of early-onset inherited neurodegenerative disorders characterised by abnormal accumulations of autofluorescent storage material within lysosomal organelles. Mutations in thirteen genes have so far been identified in patients across all forms of NCL and disease models established, however no model has been generated for late infantile CLN7 disease. I propose to generate a knock out mutant in order to establish Drosophila as a model to study the cellular role of the hitherto functionally unknown CLN7. Initially I determined a potential shared conservational functionality between Drosophila CLN7 homologue, CG8596, and human MFSD8/CLN7 by establishing a similar subcellular localisation pattern within the endo-lysosomal pathway in vitro. In order to study the functional role of CLN7 I designed an imprecise P-element excision screen, which yielded three genetically null mutants for Cln7. Characterisation of the mutants revealed loss of Cln7 in Drosophila did not recapitulate the hallmark intralysosomal storage material accumulation whilst also causing a profound lifespan extension. However, studying the developing synapse in late stage larvae revealed loss of Cln7 to have a significant effect on a specific motoneuron innervation at the NMJ. Behavioural assays showed Cln7-/- larvae displayed altered larval locomotion; mutant animals were unable to maintain consistent movement and showed impaired complex movement. Defects in tonic motoneuron innervation were observed in Cln7 loss of function animals suggesting their intrinsic properties make them more vulnerable to loss of Cln7 compared to the unaffected phasic motoneurons. As a consequence of reduced synaptic input from tonic motoneurons, physiological studies revealed Cln7-/- animals display characteristic phasic motor input in the form of synaptic depression and larger recorded EPSPs. Examination of model systems and patient autopsy tissue for other NCL forms have revealed severe CNS regional-specific neuronal death underpins the disorder, whilst cellular studies reveal a multitude of mechanisms of pathology including cellular pathways such as autophagy and oxidative stress. I carried out genetic interaction studies focussing on the NMJ, these suggest CLN7 functions downstream of autophagy but upstream of mTORC2 to regulate synapse development.
19
Brooks, Helen Rachel Elisabeth. "Modelling late infantile CLN2 disease." Thesis, King's College London (University of London), 2015. http://kclpure.kcl.ac.uk/portal/en/theses/modelling-late-infantile-cln2-disease(f776762d-3d0c-4713-80ae-0eaa56addb5b).html.
Повний текст джерелаАнотація:
The neuronal ceroid lipofuscinoses (NCLs/Batten disease) are a group of fatal autosomal recessive lysosomal storage disorders (LSDs). There are no effective treatments available for any form of NCL. This thesis focuses on modelling late infantile CLN2 disease (LINCL), which is caused by mutations in the CLN2 gene, resulting in deficiency of the lysosomal enzyme tripeptidyl peptidase I (TPP1). Potential treatments of this disorder are based upon delivering this missing enzyme to the brain but in order to judge the efficacy of future therapies, pre-clinical studies must be carried out in disease models. A mouse model of CLN2 disease exists, which is deficient in TPP1 and in this thesis I characterised the disease progression of this Tpp1-/- mouse. This analysis revealed a pattern of disease progression that is unique in the NCLs to these Tpp1-/- mice, where neuronal cell loss was initiated in the thalamus and preceded microglial and astrocytic activation. Subsequently, neuronal cell loss spread to the cortical target regions and was accompanied by glial activation. In addition, the effects of the adaptive immune system on CLN2 disease progression were explored by examining the same Tpp1-/- mice on an immunodeficient background. These immunocompromised mice had a more pronounced disease phenotype in terms of atrophy of regional brain structures and neuronal cell loss in the somatosensory cortex and specific thalamic nuclei than the equivalent TPP1-deficient mice with an intact immune system. The TPP1-deficient mice which lacked the adaptive immune responses lived longer, however this extended lifespan allowed a more severe neurodegerative phenotype to develop. This thesis also presents the process of developing a human neural progenitor cell (hNPC) model of late infantile CLN2 disease by introducing a CLN2 disease-causing mutation into a hNPC line using genome editing. Transcription activator-like effector nucleases (TALENs) are genome-editing tools that fuse naturally occurring DNAbinding proteins that can be designed and arranged according to a simple code to target a specific DNA sequence, to a DNA cleavage domain. These TALENs can be used to edit genomes by inducing double strand breaks. In this thesis I present my attempts to produce TALENs to target the CLN2 gene and to produce a clonal hNPC cell line that harbours a CLN2 disease mutation. I show that a culture of hNPCs was isolated that contained largely wild-type hNPCs and a smaller population of hNPCs containing the desired CLN2 disease-causing mutation. I also describe an optimised method for transfection of hNPCs that is now being used by other researchers wishing to carry out genome editing in this hNPC line.
20
Lensink, Ingrid. "Characterisation of the Batten disease gene, CLN3 /." Title page, abstract and contents only, 2000. http://web4.library.adelaide.edu.au/theses/09PH/09phl573.pdf.
Повний текст джерелаАнотація:
Thesis (Ph.D.)-- University of Adelaide, Dept. of Cytogenetics and Molecular Genetics, 2000.Copies of author's previously published articles inserted. Errata sheets pasted onto front end papers. Bibliography: leaves 156-182.
21
Jones, Kiera Megan. "The photodissociation of ClNO : a computational approach." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/7652/.
Повний текст джерелаАнотація:
The detailed photodissociation dynamics of ClNO on the 2 ¹ A' and 1 ³A'' states have been investigated using computational methods. Chapter 3 concerns the photodissociation of the 2 ¹A' state. New, purely ab initio potential energy surfaces are described, calculated using the MRCI method. Transition dipole moments and non-adiabatic coupling matrix elements have been calculated across the extent of the surface. To investigate the dissociation dynamics, wavepacket propagations using the MCTDH method have been performed, yielding NO product state distributions which match extremely well with experimental results. In addition, classical trajectory calculations incorporating surface hopping have modelled transitions through a conical intersection in the asymptotic region of the surfaces, and have allowed anisotropy parameters to be obtained. In chapter 4 the photodissociation dynamics of the 1 ³A'' state are described. A new potential energy surface and set of transition dipole moments have been calculated, and these have been used in quantum and classical dynamics calculations. In this case, whilst the quantum results agree well with experiment, the classical results do not. In chapter 5, coherent control on the 1 ³A'' state is discussed. Using the MCTDH method, both transform limited and shaped ultrashort laser pulses at a series of energies have been used to influence the dynamics. The NO vibrational and rotational state distributions can be changed considerably using femtosecond pulse pairs separated by a variable delay. Finally, chapter 6 examines the effect of spin-orbit coupling on the states of ClNO and surfaces taking this into account have been obtained at the CASSCF level. These surfaces are used to qualitatively explain experimental product state distributions. Further to this, dissociation on the spin-orbit equivalent of the 2 ¹A' state has been studied using a preliminary wavepacket propagation; the predicted spin-orbit product state distributions agree well with experiment.
22
Redaelli, F. "Caratterizzazione funzionale e ruolo di CLN8 nei processi neurodegenerativi." Doctoral thesis, Università degli Studi di Milano, 2009. http://hdl.handle.net/2434/63207.
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23
Brandenstein, Laura Isabel [Verfasser], and Stefan [Akademischer Betreuer] Hoth. "Analysen des Adaptorprotein-abhängigen Transports des lysosomalen Membranproteins CLN7 und von Mausmodellen (Mus musculus) der CLN7-Erkrankung / Laura Isabel Brandenstein ; Betreuer: Stefan Hoth." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1162621796/34.
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24
Lange, Jenny. "Neuron-glia interactions in infantile neuronal ceroid lipofuscinosis (CLN1 disease)." Thesis, King's College London (University of London), 2016. https://kclpure.kcl.ac.uk/portal/en/theses/neuronglia-interactions-in-infantile-neuronal-ceroid-lipofuscinosis-cln1-disease(ceed9c8f-f40d-4796-be7b-e799fe88b431).html.
Повний текст джерелаАнотація:
The neuronal ceroid lipofuscinoses (NCLs) are the most common cause of childhood dementia and are invariably fatal. Early, localised glial activation occurs in all forms of NCL and has been shown to be an accurate predictor of areas in which neuronal loss is most pronounced. Indeed, in a tissue culture model of juvenile NCL, glial cells have been shown to actively contribute to neuronal dysfunction and death. So far the role of glial cells has not been established in other forms of NCL and in order to assess glial function in infantile NCL (INCL, CLN1 disease), a series of different tissue cultures were generated from Ppt1 deficient mice (Ppt1-/- ). These studies revealed that both Ppt1-/-astrocytes and microglia exhibit a more activated phenotype under basal conditions, as well as alterations to their protein expression profile following pharmacological stimulation. Ppt1-/- astrocytes displayed moderately reduced glutamate uptake, as well as changes in lactate release. Perhaps as a consequence of these changes, Ppt1- /-astrocyte survival was severely impaired. In addition the morphological phenotypes of Ppt1-/-neurons were explored, revealing decreased neurite outgrowth, complexity and a reduction in cell body size. Ppt1-/-neuronal cultures contained significantly fewer inhibitory neurons and displayed decreased cell survival after prolonged time in culture. Most importantly, using a co-culture system, the presence of Ppt1-/- glial cells appeared to increase cell death in both WT and Ppt1-/- cultures. Notably, Ppt1-/- microglia appeared to trigger increased Ppt1-/- neuronal death, whereas Ppt1-/- astrocytes also exhibited increased cell death. Ppt1-/- glial cells also affected both wild type and Ppt1-/- neuronal morphology, including further reduced neurite outgrowth. In contrast, wild type glial cells ameliorated some of the morphological defects observed in Ppt1-/- neurons, and this was most apparent when wild type astrocytes where grown in co-cultures with Ppt1-/- neurons. Taken together, these finding present novel evidence for compromised glial function in Cln1 disease, demonstrating that both Ppt1-/- microglia and astrocytes may potentially contribute to the neurodegeneration observed in CLN1 disease. These data highlight the importance of targeting glial cells in the development of therapeutic interventions for CLN1 disease. Furthermore, although sharing some similarities Ppt1-/-glial phenotypes were broadly different from those observed in the tissue culture model of juvenile NCL, suggesting that the role of glia may differ between forms of NCL.
25
Budden, Theodore. "CLN5 deficiency results in alterations in the activation of autophagy." Thesis, Kansas State University, 2014. http://hdl.handle.net/2097/20473.
Повний текст джерелаАнотація:
Master of ScienceDepartment of Biology
Stella Y. Lee
CLN5 is one of several proteins that when mutated result in the lysosomal storage disorder (LSD) Neuronal Ceroid Lipofuscinosis (NCL). CLN5 is a soluble lysosomal protein that has no known function at this time. Previously we showed that eight asparagine residues in CLN5 are N-glycosylated, and that this modification is important for the protein’s transport and function. Now, we have identified a link between the activation of autophagy and CLN5 deficiency. The autophagy-lysosomal protein degradation system is one of the major pathways the cell uses to degrade intracellular material and recycle cellular building blocks. It was recently shown that other CLN proteins affect the relative level of autophagy, indicating a potential link between the autophagy pathway and the NCLs. By knocking down endogenous CLN5 in HeLa we showed that, upon stress induction, cells responded with higher levels of autophagy activation. Consistent with these knockdown experiments, there is a higher level of the autophagy marker protein, LC3-II, in CLN5 patient cells that are naturally deficient for the CLN5 protein. Pharmaceutical induction of autophagy through different means also showed higher LC3-II levels compared to control, though patterns differed in the type of autophagy induced. In summary, we discovered that the autophagy pathway is altered in CLN5 deficient cells, indicating a potential role for CLN5 in autophagy. Further analyses of the autophagy pathway will shed light on where CLN5 is acting and the mechanism by which defective CLN5 causes NCL.
26
Nelvagal, Hemanth Ramesh. "Spinal cord pathology in CLN1 disease : a novel therapeutic target." Thesis, King's College London (University of London), 2017. https://kclpure.kcl.ac.uk/portal/en/theses/spinal-cord-pathology-in-cln1-disease(8b1dc3ed-dfd9-442d-a427-43ded0d82a6a).html.
Повний текст джерелаАнотація:
The neuronal ceroid lipofuscinoses (NCLs) are a group of up to 14 inherited progressive neurodegenerative lysosomal storage disorders affecting children and young adults. Together, they are the most common pediatric neurodegenerative storage disorders. Symptoms include loss of vision, epileptic seizures and the loss of cognitive and motor function. A lack of any effective therapies means that all forms are fatal. CLN1 disease or Infantile NCL is one of the most rapidly progressing forms of the disease and is caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase – 1 (PPT1). Ppt1 deficient (Ppt1-/-) mice recapitulate features of the human disease and have proved to be a useful tool in characterizing disease progression and pathology in the brain. However, these pathological changes fail to fully explain the sensorimotor deficits seen in this mouse model as well as in human CLN1 disease. Along with the limited success of various brain directed therapies, this led us to analyze the spinal cord. Our analysis revealed unexpectedly profound and rapidly progressing disease pathology in the spinal cords of these mice, which occurs earlier than similar events in the brain. This included regional atrophy, neuroinflammation, and significant neuron loss at all levels of the cord as well as novel phenotypes indicating a postnatal developmental delay and significant white matter pathology. Automated gait analysis also showed novel early phenotypes in these mice including an increased dependence on the forelimbs for locomotion. Similar spinal cord pathology was also demonstrated in human INCL autopsy samples as well as in mouse models of the other major forms of NCL. Targeting the spinal cords of Ppt1-/-mice with enzyme replacement therapy (ERT) and gene therapy significantly improved disease pathology, motor function and lifespan in these mice, demonstrating the clinical significance of spinal cord pathology in these mice. Furthermore, combining intracranial and intrathecal gene therapy showed a synergistic effect, showing the greatest improvements for any CLN1 disease therapy to date. Taken together, these findings highlight the spinal cord as not only being significantly affected in CLN1 disease, but also as a novel and effective therapeutic target.
27
GARSUAULT, SOPHIE. "Degradation et fonction de cln1 identification de sequences en cis responsables de la degradation et de la fonction de cln1, une cycline g1 de saccharomyces cerevisiae." Paris 6, 1998. http://www.theses.fr/1998PA066496.
Повний текст джерелаАнотація:
Ces travaux ont consiste a identifier des signaux en cis necessaires a la degradation et a la fonction de cln1, une cycline g1 de s. Cerevisiae. Les cyclines g1 sont au coeur de la regulation du cycle cellulaire eucaryote, controlant la transition entre proliferation et differenciation. Chez la levure saccharomyces cerevisiae, cln1 et cln2 s'accumulent dans la cellule en phase g1, declenchent l'engagement irreversible dans un nouveau cycle en activant la kinase cdc28, puis disparaissent brutalement en raison de leur haute instabilite. L'association a cdc28 se fait par l'intermediaire d'une region tres conservee chez toutes les cyclines, la cyclin-box, situee dans la region n-terminale, et correspondant au premier domaine de repliement defini par cristallographie (cyclin-fold). Nous montrons que la derniere helice du second cyclin-fold joue un role determinant dans la fonction de cln1. Peu d'exemples de sequences situees hors de la cyclin-box et contribuant a la fonctionnalite de la cycline ont ete rapportes jusqu'a maintenant. Des deletions carboxy-terminales progressives ont permis de montrer que les determinants de la degradation sont repartis dans la region c-terminale de cln1, et sont similaires a ceux identifies chez cln2 et cln3. Les sites de phosphorylation semblent etre les determinants majeurs, mais la region c-terminale contient d'autres signaux non identifies, necessaires pour une degradation rapide. Les determinants de proteolyse contenus dans la region carboxy-terminale de cln1 ne fonctionnent pas comme des signaux autonomes de degradation, contrairement a ce qui a ete observe chez cln2 et cln3. La mutation ponctuelle dans la cyclin-box d'un residu hautement conserve, l'arginine 72, est suffisante pour abolir la fixation a cdc28, et a stabiliser significativement la cycline. Ainsi, la fixation de la cycline a la kinase est un pre-requis a sa degradation rapide. Nous montrons par ailleurs que les cyclines libres - non fixees a cdc28 - sont reconnues par une voie de degradation operant plus lentement, ne reconnaissant probablement pas les memes signaux que la voie de degradation des cyclines associees a cdc28.
28
Choi, Young-Jun 1967. "Biochemical and molecular aspects of an esterase from Lactobacillus casei CL96." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36892.
Повний текст джерелаАнотація:
In order to establish the potential of an esterase from Lactobacillus casei for application in the dairy industry, this study was designed with the following objectives: (1) to determine the optimal cultural conditions for growth and enzyme production, (2) to construct a positive selection vector with low copy number for gene cloning, (3) to clone and sequence the gene encoding for esterase, (4) to biochemically characterize the purified recombinant esterase, and (5) to over-express an esterase of L. casei into Escherichia coli as well as into a methylotrophic yeast, Pichia pastoris using strong promoters.Maximal growth and enzyme production in L. casei were obtained after 20 h in basal MRS medium containing 1% (w/v) lactose at pH 7.0, 30°C. Among various substrates (C2--C16) tested, the highest activity was towards C6 and C8. Although the enzyme was produced constitutively, tributyrin induced the enzyme production by a 2.5 fold.
A new E. coli and lactic acid bacteria shuttle vector with low copy and positive selection termed pCWL70 was constructed. High transformation efficiency and significant vector stability of pCWL70 were found in L. casei and Lactococcus lactis.
An esterase gene (estI) of L. casei CL96 was localized in a 3.3 kb Bam HI DNA fragment that contains an open reading frame of 1,800 bp. The open reading frame estI was isolated by PCR and subcloned into the E. coli expression vector pET29a, and Pichia expression vector pPICZ B, that allows the inducible expression under the control of the T7 promoter and an alcohol oxidase promoter (AOX1), respectively. E. coli BL21(DE3)pLysS containing estI expressed a novel 67.5 kDa protein corresponding to the EstI in N-terminal fusion with the S · tag peptide.
An esterase of L. casei CL96 was successfully over-expressed in E. coli up to 500 folds (about 25% of total cellular protein) as well as in the P. pastoris. In high cell density fed-batch fermentation, the recombinant Pichia strain containing linearized pCESTc was grown at pH 6.5 and 30°C, and the cell density peaked at about 180 h with 468 g wet cell weight per liter. The final yield was 3.7 x 106 units/ml, which is 980-folds higher than that of native L. casei cells.
The amino acid sequence of EstI indicated that the esterase is a member of a novel GHSMG family of lipolytic enzymes and S-formylglutathione hydrolases (FGH). The putative catalytic triad of EstI consists of residues Ser325, Asp516 and His558 as demonstrated by amino acid sequence alignments. (Abstract shortened by UMI.)
29
Moharir, Akshay. "Role of N-glycosylation in trafficking and stability of human CLN5." Thesis, Kansas State University, 2012. http://hdl.handle.net/2097/14143.
Повний текст джерелаАнотація:
Master of ScienceDivision of Biology
Stella Y Lee
Neuronal Ceroid Lipofuscinoses (NCLs) are a group of lysosomal storage diseases that are characterized by accumulating autofluorescent lipopigments in cells. NCLs are a form of progressive neurodegenerative diseases with symptoms ranging from blindness, loss of speech and motor activities to ataxia and seizures. Patients do not live to adulthood in most cases, making it prevalent in children. Among the many genes that cause NCL, CLN5 leads to different forms of NCL (infantile, late infantile, juvenile, and adult). CLN5 protein resides in the lysosomes but its function has not been established. It is predicted to contain eight N-glycosylation sites, but the role of N-glycosylation on its function and trafficking has not been assessed. We analyzed the role of N-glycosylation on the transport and stability of human CLN5. We created N-glycosylation mutants of each site by changing the Asn to Gln and our analysis of these mutants show that all the eight N-glycosylation sites are used in vivo. We also report effects of abolishing individual N-glycosylation sites on the trafficking of CLN5. While the lack of glycosylation at some sites results in CLN5 being retained in the ER or Golgi, others do not affect CLN5 trafficking. Cycloheximide chase experiments show that one of the mutants (N401Q) in CLN5 leads to lower protein levels in cell pellets with an increased secretion compared to CLN5 wild type, while other mutations show differential stability in cell pellets. These results demonstrate that each N-glycosylation site plays a different role(s) in the stability, transport and/or function of CLN5.
30
Jimenez, Rondan Felix Ruben. "The Biology of Claudin 6 (Cldn6) in the Developing Mouse Lung." BYU ScholarsArchive, 2015. https://scholarsarchive.byu.edu/etd/4414.
Повний текст джерелаАнотація:
The tight junctions (TJ), which are located in the apical region between epithelial and endothelial cells, regulate the paracellular diffusion of ions and small molecules and play an important role in maintaining cell polarity, cell-cell integrity, and permeability. In the lung, epithelial cells are attached by TJ structures. They provide a permeable barrier and cell communication. The loss of barrier integrity, which is maintained by the expression of claudins (Cldn), results in cellular permibilization and leads to paracellular diffusion of solutes and harmful molecules. There are 27 known Cldn homologous members in mice and human. Cldn6 is mostly expressed in embryonic stem cells and associated with the programing of epithelial cells during embryo development and lung morphogenesis. In order to test the hypothesis that Cldn6 expression affects lung morphogenesis, we analyzed the expression pattern of Cldn6 during lung ontogenesis to examine cell-specific expression pattern of Cldn6 during each embryonic period in the mouse lung. Also, we assessed transcriptional regulators and control mechanisms that precisely influence Cldn6 expression in pulmonary cells. We discovered that Cldn6 is an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. We found that normal down-regulation of Cldn6 as development proceeds influences differentiation associated with the transition between the embryonic to the alveolar stage. Conditional gain-of-function and loss-of-function experiments in animal models prove to be the most beneficial tool in deciphering the impact of Cldn in organ formation and maintenance. We generated a conditional transgenic mouse that provides the opportunity to genetically up-regulate Cldn6 in distal lung. Our transgenic mouse showed a delay in lung development and down-regulation of transcriptional factors. Cldn6 is both temporally and spatially controlled in the developing lung and its regulation is maintained by critical transcriptional control networks managed by TTF-1. In lung diseases, altered Cldn expression leads to diseases such as COPD, asthma, and ARDS. The tight junctional proteins are differentially regulated by tobacco smoke exposure and Cldn6 is potentially involved as neighboring epithelial cells respond to tobacco smoke. We exposed adult mice to controlled doses of second hand smoke during four days and A-549 cells to 10% CSE for 6 hours. We discovered that mice lungs respond by down-regulating Cldn6 basal levels and impair barrier function. These results reveal that midgestational up-regulation of Cldn6 and its marked down-regulation as development proceeds illustrate the notion that Cldn6 function is important during early programming stages of lung morphogenesis.
31
Quilis, Bayarri Inma. "Mecanismes d'especificitat funcional dels complexes CDK-Ciclines CLN." Doctoral thesis, Universitat de València, 2011. http://hdl.handle.net/10803/81537.
Повний текст джерелаАнотація:
La progressió del cicle cel.lular en les cèl.lules eucariotes es deu a l'activació seqüencial
de diferents complexos CDK-ciclina. En Saccharomyces cerevisiae una sola CDK,
Cdc28, s'associa amb nou ciclines diferents i és la ciclina la que determina la funció del
complexe al llarg de cada fase del cicle cel.lular. Les ciclines Cln1 i Cln2 controlen la
transició G1 / S. Aquestes ciclines havien estat considerades equivalents per la seva
homologia de seqüència, regulació i funció, però una diferència funcional entre elles va
ser descrita establint a Cln2 com l'efector principal dels processos morfogenètics durant
la transició G1 / S. En aquest treball s´han tractat de descriure les bases moleculars de
l´especificitat funcional de Cln2. S'ha realitzat un anàlisi funcional utilitzant ciclines
quimèriques que contenen diferents fragments de Cln1 i Cln2 per tal d'identificar les
regions responsables de la funcionalitat específica de Cln2 en comparació amb Cln1.
Els nostres resultats ens porten a concloure que la regió entre els aminoàcids 225 i 299
del Cln2 és necessària i suficient per a la seva funció específica. També hem estudiat la
localització de Cln2 i Cln1. Ambdues ciclines es distribueixen entre el nucli i el
citoplasma, però Cln1 mostra una major acumulació nuclear. La via clàssica
d´importació participa en l´entrada de les dues ciclines al nucli, però només Cln2 és
exportada fora del nucli per la carioferina Msn5. Hem identificat els senyals de
localització de les ciclines. El senyal de localització nuclear no es correspon amb cap
NLS clàssic descrit i es troba en la regió entre els aminoàcids del 130 al 226 d´ambdues
ciclines. Sorprenentment, l´exportació de Cln2 depén de la regió entre els aminoàcids
225 i 299. Aquesta correlació entre la seqüència d´exportació i la regió responsable de
la funció específica de Cln2 dóna suport a que siga l'exportació al citoplasma el
mecanisme que confereix les seves funcions específiques a Cln2 durant la transició G1/
S. Quan dita regió s´introdueix en Cln1 la ciclina resultant presenta una distribució
menys nuclear i és capaç de realitzar les funcions citosòliques pròpies de Cln2. Per tant,
sembla ser la seua regulació espacial la que determina la funcionalitat diferencial de
Cln1 i Cln2.
Paralel.lament s´ha estudiat la degradació de les ciclines i s´ha observat que Cln1 i Cln2
presenten la mateixa estabilitat. Tanmateix, mentre que Cln2 és degradada per SCFGrr1 i
SCFCdc4, Cln1 és degradada preferentment per SCFGrr1. La seqüència N-terminal de la
ciclina determina el seu patró de degradació: la seqüència de Cln2 és necessària per a
que la proteïna siga degradada per SCFCdc4. En conclusió, la degradació no determina la
funcionalitat de les ciclines però estableix un nivell més de regulació diferencial entre
Cln1 i Cln2 relacionat amb l´anterior, ja que l´estabilitat de les ciclines depén de la
localització.
Per una altra part s´ha analitzat el paper de Cdc24 en la funció específica de Cln2
concluint que si bé Cln2 sembla més eficient que Cln1 en facilitar l´eixida del nucli de
Cdc24, no és aquesta eixida sinò altres funcions en el procés de gemmació les que Cln2
realitza de forma específica. S´han identificat les carioferines responsables del transport
de Cdc24: és importada al nucli per la via clàssica d´importació i exportada per Xpo1.
S´han identificat tres possibles senyals de localització diferents, un NES i dos NLS,
però els anàlisis de mutants en residus d´aquestes regions no han permés demostrar la
dependència de la localització de la proteïna Cdc24 d´aquests senyals.
Finalment es va realitzar una caracterització dels mutants en les diferents carioferines de
la família de la importina β, la cual va mostrar que molts dels mutants podien
relacionar-se amb el cicle cel.lular en processos com la regulació d´Start, la replicació o
la morfogènesi cel.lular.Cell cycle progression in eukaryotic cells is driven by sequential activation of different CDK-cyclin complexes. In Saccharomyces cerevisiae a single CDK, Cdc28, associates with nine different cyclins and it is the cyclin who determines the complex function along each cell cycle stage. Cln1 and Cln2 cyclins control the G1/S transition. These cyclins had been considered equivalent due to their sequence homology, regulation and function, but a functional difference between them was described establishing Cln2 as the main effector of the morphogenetic processes during G1/S transition. In this work we are trying to describe the molecular basis of Cln2 functional specificity. Functional analysis have been performed using chimeric cyclins containing different fragments of Cln1 and Cln2 in order to identify specific functional regions of Cln2 compared to Cln1. Our results lead us to conclude that the region between amino acids 225 and 299 of Cln2 is necessary and sufficient for its specific function. We have also studied Cln2 and Cln1 localization. Both cyclins are distributed between the nucleus and the cytoplasm but Cln1 shows stronger nuclear accumulation. Karyopherin Kap95 imports both cyclins into the nucleus but only Cln2 is exported out of the nucleus by karyopherin Msn5. We have detected localization signals in cyclins. Strikingly, Cln2 export depends on the region between amino acids 225 and 299. This strongly supports that the export to cytoplasm confers its specific functions to Cln2 during G1/S transition.
32
Grand, Teddy. "Analyse fonctionnelle de mutations du gène CLN5 impliquées dans la maladie de Dent." Paris 6, 2010. http://www.theses.fr/2010PA066180.
Повний текст джерелаАнотація:
La maladie de Dent est une maladie rénale héréditaire liée au chromosome X. Elle apparait dès l’enfance ou à l’âge adulte et se traduit par une protéinurie de bas poids moléculaire accompagnée le plus souvent d’une hypercalciurie, d’une hyperphosphaturie et d’une néphrocalcinose. Cette atteinte du tubule contourné proximal conduit régulièrement à une insuffisance rénale terminale, elle est due à la mutation du gène CLCN5 codant le transporteur ClC-5. L’ensemble du travail a permis de classer les mutants ClC-5 en trois types. Les ClC-5 mutants de type 1 (16% des mutations) présentent une maturation N-glycosidique et une localisation subcellulaire normale mais une conduction altérée voire abolie. Les mutants de type 2, les plus courants (75% des mutations), démontrent une altération de leur maturation N-glycosidique, ainsi qu’une absence d’adressage à la membrane plasmique et aux endosomes précoces en raison de leur rétention dans le réticulum endoplasmique par les systèmes contrôle-qualité. Enfin, les mutants de type 3 voient la stabilité de leur forme mature diminuer par rapport à la protéine sauvage, provoquant ainsi une réduction de leur expression à la membrane plasmique. En revanche, leur adressage aux endosomes précoces n’est pas altéré. En conclusion, ce travail de thèse aura permis de mieux comprendre l’impact fonctionnel de mutations dans le gène CLCN5 en vue d’une éventuelle intervention pharmacologique adaptée en fonction du type de défaut observé. Il aura aussi permis d’identifier des zones clés de la protéine pour la conduction ionique et servira de point de départ à d’autres études plus poussées sur les conductances de H+ et de Cl- des mutants de type 1.
33
Carmody, Carol Ann. "CLND and in-source CID ESMS : a route to a truly quantitative HPLC detector?" Thesis, University of Southampton, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401831.
Повний текст джерела
34
Ariunbat, Khandsuren [Verfasser]. "Untersuchungen zu molekularen Grundlagen lysosomaler Dysfunktion an Zellmodellen der neurodegenerativen CLN7-Erkankung / Khandsuren Ariunbat." Hamburg : Staats- und Universitätsbibliothek Hamburg Carl von Ossietzky, 2021. http://d-nb.info/1230988106/34.
Повний текст джерела
35
Nemer, Kathleen. "The role of punicic acid (c9t11c13-CLNA) in lipid and energy metabolism of mice." Connect to resource, 2009. http://hdl.handle.net/1811/37220.
Повний текст джерела
36
Kaleem, Abuzar [Verfasser], and Guido [Akademischer Betreuer] Hermey. "Functional characterization of Ceroid Lipofuscinosis Neuronal 3 (CLN3) interactions / Abuzar Kaleem ; Betreuer: Guido Hermey." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1216629544/34.
Повний текст джерела
37
Schultz, Mark. "Cell biological defects in juvenile neuronal ceroid lipofuscinosis." Diss., University of Iowa, 2013. https://ir.uiowa.edu/etd/6498.
Повний текст джерелаАнотація:
Mutations in the CLN3 gene cause Juvenile Neuronal Ceroid Lipofuscinosis (JNCL), a form of Batten disease that is grouped within the broad class of lysosomal storage diseases. JNCL displays a primary central nervous system phenotype characterized by rapid onset blindness, wide spread brain atrophy and reversal of learned abilities with death occurring 10-20 years after symptom onset. The mechanisms underlying these phenotypes are not known. CLN3 encodes CLN3, a protein with no known molecular function. CLN3 is expressed at very low levels natively in most cells, and is highly hydrophobic.
Similar to other lysosomal storage diseases, it is difficult to ascertain the primary versus the secondary defects when the protein functions along the endosomal-lysosomal pathway. In JNCL one common finding among several labs, in various cellular systems, is a fluid-phase endocytotic defect. I took this commonality as a key to CLN3 function, and pursued cell biological pathways required for fluid-phase endocytosis. Fluid-phase endocytosis is regulated by cycling of the small GTPase Cdc42 and I discovered increased Cdc42-GTP in CLN3-null mouse brain endothelial cells. In mouse brain endothelial cells enhanced Cdc42-GTP increased Cdc42 dependent signaling, filopodial formation, and retarded cell migration. I also found reduced plasma membrane association of ARHGAP21, a known negative regulator of Cdc42. My data supports a model where loss of CLN3 reduces ARHGAP21 plasma membrane recruitment, and causes aberrant Cdc42 activation. Thus irregular Cdc42 activation underlies the commonly reported fluid-phase endocytic defects in JNCL.
Therapeutic development for JNCL has been hampered in part from the varying phenotypes ascribed to CLN3 deficiency. My discovery that the fluid-phase endocytic defects result from Cdc42 pathway aberrations, which in turn contributed to multiple downstream phenotypes, opened the door to novel JNCL therapeutics. Here I present work showing that a Cdc42 inhibitor corrects the Cdc42 dependent defects in vitro and multiple defects in a JNCL mouse model.
38
Menoyo, Molins Alexandra. "Nutrient availability regulates cell cycle through a Pho85 CDK-dependent control of Cln3 cyclin stability." Doctoral thesis, Universitat Internacional de Catalunya, 2012. http://hdl.handle.net/10803/101414.
Повний текст джерелаАнотація:
Cell cycle control by trophic factors has a key role in regulation of cell proliferation in all organisms. Nutrients are one of these important factors needed by cells to reproduce, so very well regulated mechanisms must exist that connect nutrient availability to cell cycle. Hence the importance on studying how exactly nutrient-dependent signaling pathways work.
Cln3, the most upstream G1 cyclin in Saccharomyces cerevisiae, is one well demonstrated common effector of multiple nutrient-dependent signaling pathways. Moreover, its role in cell cycle is crucial. So it is a good candidate to regulate cell cycle progression in response to nutrient availability.
One important question is to find the protein that could directly modulate Cln3 levels in response to nutrient availability. This protein could play as a nutrient sensor and as a cell cycle regulator at the same time.
In the present thesis, Pho85 is founded to be the protein that could run these two highly different tasks, because of its well-characterized properties on sensing phosphate availability and the well-known functions on modulating cell cycle as CDK.
The results of the present work clearly demonstrate that when phosphate is present, Pho85 regulates Cln3 levels by increasing the stability of the cyclin through specific phosphorylations, promoting cell cycle progression. Contrary, under phosphate depletion conditions, Pho85 become inactive and Cln3 is rapidly degraded, leading to a cell cycle arrest in order to maintain cell chronological lifespan.El control del cicle cel•lular per factors tròfics té un paper important en la proliferació cel•lular de tots els organismes. Els nutrients són uns d’aquests factors importants requerits per les cèl•lules per reproduir-se, per tant deuen existir mecanismes molt ben regulats que connecten la disponibilitat de nutrients amb el cicle cel•lular. Per això, l’estudi de com funciona la senyalització cel•lular de nutrients i com afecta a la progressió del cicle és altament rellevant. Cln3, la ciclina de G1 més primerenca a Saccharomyces cerevisiae, és un efector comú de múltiples vies de senyalització de nutrients. A més, el seu paper en el cicle cel•lular és crucial. Per tant aquesta proteïna és una bona candidata per regular la progressió del cicle cel•lular en resposta a la disponibilitat de nutrients. Una qüestió important a resoldre és trobar la proteïna que podria modular directament els nivells de Cln3 depenent de la presència de nutrients. Aquesta proteïna actuaria com a sensor de nutrients i com a reguladora del cicle cel•lular alhora. A la present tesi, es mostra a Pho85 com la proteïna que pot fer aquestes dues tasques, tant per les seves propietats ben conegudes en la detecció de fosfat, com per les seves funcions de CDK modulant el cicle cel•lular. Els resultats d’aquesta tesi demostren clarament que quan el fosfat és present, Pho85 modula els nivells de Cln3 incrementant l’estabilitat de la ciclina mitjançant fosforilacions específiques, promovent la progressió del cicle cel•lular. Per altra banda, sota condicions de manca de fosfat, Pho85 esdevé inactiva i Cln3 és degradada ràpidament, conduint a un arrest del cicle cel•lular per mantenir la longevitat de la cèl•lula.
El control del ciclo celular por factores tróficos tiene un papel importante en la proliferación celular de todos los organismos. Los nutrientes son uno de estos factores importantes requeridos por las células para reproducirse, por lo tanto deben existir mecanismos muy bien regulados que conecten la disponibilidad de nutrientes con el ciclo celular. Por ello, el estudio de cómo funciona la señalización celular de nutrientes y cómo afecta a la progresión del cicle es altamente relevante. Cln3, la ciclina de G1 más temprana en Saccharomyces cerevisia, es un efector común de múltiples vías de señalización de nutrientes. Además, su papel en el ciclo celular es crucial. Por lo tanto esta proteína es una buena candidata para regular la progresión del ciclo celular en respuesta a la disponibilidad de nutrientes. Un tema importante a resolver es encontrar la proteína que podría modular directamente los niveles de Cln3 dependiendo de la presencia de nutrientes. Esta proteína actuaría como sensor de nutrientes y como reguladora del ciclo celular. En la presente tesis, se muestra a Pho85 como la proteína que puede hacer estas dos tareas, tanto por sus propiedades bien conocidas en la detección de fosfato, como por sus funciones de CDK modulando el ciclo celular. Los resultados de esta tesis demuestran claramente que cuando el fosfato está presente, Pho85 modula los niveles de Cln3 incrementando la estabilidad de la ciclina mediante fosforilaciones específicas, promoviendo la progresión del ciclo celular. Por otro lado, bajo condiciones de ausencia de fosfato, Pho85 es inactivada y Cln3 se degrada rápidamente, conduciendo a una parada del ciclo celular para mantener la longevidad de la célula.
39
Steenhuis, Pieter [Verfasser], and Stephan [Akademischer Betreuer] Storch. "Intracellular Sorting and Biochemical Analysis of the Lysosomal Membrane Protein CLN7 / Pieter Steenhuis. Betreuer: Stephan Storch." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1069376825/34.
Повний текст джерела
40
Galal, Jana [Verfasser], and Stephan [Akademischer Betreuer] Storch. "Generation and analysis of a cell-based model of CLN7 disease / Jana Galal. Betreuer: Stephan Storch." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2015. http://d-nb.info/1071370111/34.
Повний текст джерела
41
Seifert, Anja Carolin Verfasser], and Robert [Akademischer Betreuer] [Bähring. "Funktion von Kv4.2/KChIP3-Kanalkomplexen in Gegenwart des lysosomalen Proteins CLN3 / Anja Carolin Seifert ; Betreuer: Robert Bähring." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1205491554/34.
Повний текст джерела
42
Oetjen, Sandra [Verfasser], and Guido [Akademischer Betreuer] Hermey. "Characterization of interactions and trafficking of the Neuronal Ceroid Lipofuscinosis protein CLN3 / Sandra Oetjen ; Betreuer: Guido Hermey." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://d-nb.info/1116604981/34.
Повний текст джерела
43
Seifert, Anja Carolin [Verfasser], and Robert [Akademischer Betreuer] Bähring. "Funktion von Kv4.2/KChIP3-Kanalkomplexen in Gegenwart des lysosomalen Proteins CLN3 / Anja Carolin Seifert ; Betreuer: Robert Bähring." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1205491554/34.
Повний текст джерела
44
Salinas, Silvia Adriana. "HPLC separation of amines with a zirconia-based column coupled to a gas- phase chemiluminescence nitrogen specific detector (CLND)." Texas A&M University, 2003. http://hdl.handle.net/1969.1/241.
Повний текст джерелаАнотація:
Gas phase chemiluminescence nitrogen specific detector (CLND)is used for the direct analysis of underivatized nitrogen-containing components such as alkylamines that can not be detected by the so called universal HPLC detector, the UV detector. However, alkali metal hydroxides can not be used as mobile phase constituents with the CLND because they form non volatile particulate combustion products that foul the detector. Therefore, trimethylsulfonium hydroxide (TMSOH) has been selected as a strong base for use with the CLND, because its combustion products, CO2, H2O and SxOy are volatile. An alkali-stable zirconia-based column was used and coupled to the CLND. Zirconia-based columns are mechanically and hydrolytically more stable than silica-based columns, which have a working pH range from 3 to 8 only. Zirconia-based columns can be used at a pH from 1 to 14 and can be used at temperatures up to 200˚C.
The separation of amines was carried out at high pH values where the amino groups were deprotonated. Primary, secondary, tertiary and quaternary amines were separated using a pH=13.7 mobile phase that contained only TMSOH, methanol and water. Good peak shapes were observed for all, except n-alkylamines and samples that contained both amino groups and alcohol groups.
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Han, Bong Kwan. "The G1 cyclin Cln3p regulates vacuole homeostasis through phosphorylation of a scaffold protein, Bem1p, in Saccharomyces cerevisiae." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4795.
Повний текст джерелаАнотація:
How proliferating cells maintain the copy number and overall size of their organelles is
not clear. In the budding yeast Saccharomyces cerevisiae the G1 cyclins Cln1,2,3p
control initiation of cell division by regulating the activity of the cyclin-dependent
kinase (Cdk) Cdc28p. We show that Cln3p controls vacuolar (lysosomal) biogenesis and
segregation. First, loss of Cln3p, but not Cln1p or Cln2p, resulted in vacuolar
fragmentation. Although the vacuoles of cln3ÃÂ cells were fragmented, together they
occupied a large space, which accounted for a significant fraction of the overall cell size
increase in cln3ÃÂ cells. Second, cytosol prepared from cells lacking Cln3p had reduced
vacuolar homotypic fusion activity in cell-free assays. Third, vacuolar segregation was
perturbed in cln3ÃÂ cells. Our findings reveal a novel role for a eukaryotic G1 cyclin in
cytoplasmic organelle biogenesis and segregation.
Furthermore we show that the scaffold protein Bem1p, a critical regulator of
Cdc42p activity, is a downstream effector of Cln3p/Cdc28p complex. The Cdc42p
GTPase is known to be required for vacuole fusion. Our results suggest that Ser72 on
Bem1p is phosphorylated by Cdc28p in a Cln3p-dependent manner to promote vacuole fusion. Replacing Ser72 with Asp, to mimic phosphorylation at an optimal Cdkconsensus
site located in the first SH3 domain of Bem1p, suppressed vacuolar
fragmentation in cells lacking Cln3p. Using in vivo and in vitro assays, we found that
Cln3p was unable to promote vacuole fusion in the absence of Bem1p or in the presence
of a non-phosphorylatable Bem1p-Ser72Ala mutant. Furthermore, activation of Cdc42p
also suppressed vacuolar fragmentation in the absence of Cln3p. Our results provide a
mechanism that links cyclin-dependent kinase activity with vacuole fusion through
Bem1p and the Cdc42p GTPase cycle.
46
Imler, Elliot, and Elliot Imler. "A Drosophila Model of Autosomal Dominant Adult-Onset Neuronal Ceroid Lipofuscinosis (ANCL/CLN4) Links Toxicity to CSP Activity." Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621895.
Повний текст джерелаАнотація:
Autosomal dominant adult onset neuronal ceroid lipofuscinoses (ANCL/CLN4) is a rare neurodegenerative disorder caused by mutations in the human gene DNAJC5 which encodes cysteine string protein alpha (CSPα). ANCL is characterized by the appearance of aberrant lysosomal storage material in the post-mortem brains of patients, who usually die from widespread neuronal loss within 10 years from the onset of symptoms. CSPα is a neuroprotective co-chaperone specifically localized to synaptic vesicles (SVs) and is evolutionarily conserved in all animals. CSPα forms a chaperone complex with HSC70 to properly fold a limited number of synaptic proteins. Complete loss of CSP leads to neurodegeneration and reduced lifespans in flies and mice. However, the mechanism of degeneration induced by ANCL mutations is currently unknown and there are no available animal models to study the dysfunctional proteins in situ. In this thesis, I describe the generation and subsequent characterization of the first animal model of ANCL, using the fruit fly Drosophila melanogaster. First, I show that human CSPα (hCSPα) is conserved functionally from humans to flies. Wildtype hCSPα expressed in flies localizes properly to SVs and is able to rescue lifespan defects in CSP null mutant flies. Overexpression of hCSPα proteins with the ANCL causing L115R and L116Δ mutations recapitulates numerous phenotypes consistent with human disease pathology. This includes the appearance of high molecular weight (HMW) SDS-resistant aggregates on western blots, accumulation of aberrant osmophilic membrane structures observed via electron microscopy, and a dose-dependent reduction in adult viability. Mutant hCSPα is mislocalized from SVs to enlarged abnormal endosomes, which accumulate in neuronal axons and somata. These endosomes strongly co-localize with the endosomal sorting required for transport (ESCRT) complex protein HRS, contain large amounts of ubiquitinated proteins, and lack markers of lysosomal maturation. This suggests that the ANCL causing mutations may cause disruptions in endo-lysosomal trafficking via an ESCRT related mechanism. To probe the genetic nature of the mutant alleles I expressed the mutant hCSPα transgenes with various doses of endogenous Drosophila CSP (dCSP). I show that loss of dCSP suppresses toxicity, as well as the aberrant endosomal accumulations and HMW aggregates induced by overexpression of mutant hCSPα. Additionally, expression of a combination of the wildtype and mutant hCSPα showed a super-additive effect on viability and HMW aggregates. This suggests that the disease-causing mutations may act as hypermorphic gain of function alleles, contrary to existing models, which suggest a dominant-negative mechanism. I also performed an F1 candidate screen for genetic modifiers of toxicity, using a robust and easy-to-score adult eye morphology and pigmentation phenotype. Using this approach, I discovered several strong interactors, both enhancers and suppressors, including member of the ESCRT trafficking pathway and other known CSP-interacting proteins. Of particular interest was the CSP co-chaperone Hsc70, which had several loss of function alleles among the strongest observed suppressors. Loss of Hsc70 also greatly reduces toxicity and endosomal accumulations of overexpressed mutant hCSPα but interestingly does not have a significant effect on the levels of HMW CSPα aggregates. This further supports the model that ANCL mutations act as hypermorphs, with a toxic mechanism involving CSP’s endogenous interactions with HSC70. Finally, I discuss the implications of these findings in relation to previous studies of the ANCL causing mutations and endogenous CSPα/HSC70 function and propose a novel mechanistic disease model. This model postulates that mutant CSP is properly trafficked to synapses but, after a brief lifespan as a properly functioning HSC70 co-chaperone, is then ubiquitinated and localized onto endosomes. Ubiquitinated mutant CSP is then clustered by HRS but is unable to mature properly through an ESCRT dependent degradation pathway. These endosomes are retrogradely trafficked through the axon to the soma where they fuse, accumulate, and persist, eventually leading to cellular toxicity via an unknown mechanism. The hypermorphic nature of the mutants can be explained by the novel observation that normal endogenous CSP also traffics through a retrograde ESCRT dependent pathway, where it intersects and co-accumulates with mutant CSP, potentially contributing to toxicity.
47
Shi, Heng. "Supercritical Fluid Chromatography with Chemiluminescent Nitrogen and Sulfur Detection." Diss., Virginia Tech, 1997. http://hdl.handle.net/10919/30546.
Повний текст джерелаАнотація:
The need for sensitive and selective detectors in supercritical fluid chromatography (SFC) is particularly evident since SFC can be used to analyze classes of compounds that are not readily amenable to either gas chromatography (GC) or liquid chromatography (LC). These compounds include species that are nonvolatile or thermally labile and , in addition, contain no chromophore that can be used for spectrophoto detection. The objective of this research is therefore to interface selective chemilumninescent detectors with SFC in the sensitive detection of nitrogen- and/or sulfur containing compounds.
The chemiluminecent nitrogen detector (CLND), a gas-phase detector which is specific for nitrogen-containing compounds, was first evaluated as a detector for use with capillary SFC. The potential use of the CLND for food flavor and petroleum samples was demonstrated. In addition to equimolar nitrogen response, the CLND showed good sensitivity and large linear dynamic range. Minimum detectable quantity (MDQ) was 60 pg of nitrogen with a linear range of over 3 orders of magnitude. Nitrogen to carbon selectivity of 105 was obtained. Capillary SFC with simultaneous flame ionization and chemiluminescent detection was also demonstrated.
The second portion of the research investigated the CLND for packed column SFC with methanol modified CO2. The only modification made in the CLND for packed column SFC is the pyrolysis furnace. The CLND and UV were used to interface with SFC via a post-column split. Methanol-modified CO2 was also demonstrated to be compatible with the CLND even with a high mobile phase flow rate. The use of pressure and modifier programs appears to be feasible as is evidenced by the baseline studies which have been performed, as well as by the applications demonstrated.
The last portion of the research focused on the evaluation of a new generation sulfur chemiluminescent detector (SCLD), which is also a gas-phase detector, with packed column SFC using both pure and methanol modified CO2. The minimum detectable quantities were determined to be 2.6 pg or 14 pg sulfur for mobile phase employing pure CO2 or 8% methanol modified CO2 respectively. The evaluation study also showed excellent selectivity and linearity, as well as day-to-day repeatability. The capabilities of the SFC-SCLD system for sulfur speciation and detection of thermally labile pesticides and polar sulfonamides, as well as petrochemical samples were presented.Ph. D.
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Palou, Marín Gloria. "El mecanisme de vigilància de la fase S estabilitza els nivells de la ciclina Clb6 en resposta a estrès genotòxic." Doctoral thesis, Universitat Autònoma de Barcelona, 2009. http://hdl.handle.net/10803/3621.
Повний текст джерелаАнотація:
Les cèl·lules eucariotes estan constantment exposades a dany al DNA i a estrès replicatiu (estrès genotòxic), font d'inestabilitat genòmica. L'anomenat mecanisme de vigilància (checkpoint) de la fase S detecta la presència d'estrès genotòxic i genera una resposta que inclou l'aturada preventiva de la replicació i del cicle cel·lular, per tal de donar temps a la superació del problema, i la protecció de la replicació dels cromosomes. Quan els elements centrals d'aquest mecanisme de control estan mutants, les cèl·lules esdevenen genèticament inestables i, en humans, predisposa al càncer. Malgrat el seu paper crític en la preservació de la integritat del genoma, es desconeix la manera com el mecanisme de vigilància de la fase S executa bona part de la seva resposta. Els elements i vies que constitueixen el checkpoint estan conservats evolutivament. El nostre treball s'ha centrat en el llevat de gemmació Saccharomyces cerevisiae. En aquest organisme la proteïna quinasa efectora principal és Rad53. Amb l'objectiu de descobrir dianes a través de les quals Rad53 regula la fase S, s'han assajat en una reacció quinasa in vitro diferents proteïnes candidates a ser substrat de Rad53. Això ha permès la identificació de la ciclina de fase S Clb6 com a substrat de Rad53 in vitro.
Les ciclines són les subunitats activadores de la quinasa dependent de ciclina Cdc28/Cdk1, que promou la progressió del cicle cel·lular. Cdc28/Cdk1 és activada per diferents ciclines específiques de les diferents fases del cicle cel·lular, que li confereixen especificitat de substrat. Clb6 és una de les dues ciclines de fase S (junt amb Clb5), que promouen la replicació del DNA. Mentre que Clb5 és present des del final de la fase G1 fins a anafase, Clb6 és eliminada durant la primera meitat de la fase S.
Per tal d'estudiar si Clb6 és diana de Rad53 in vivo, s'ha estudiat el seu comportament en resposta a estrès genotòxic. Interessantment, en una fase S compromesa els nivells de Clb6 es mantenen estables, i aquesta estabilització és dependent de Rad53.
Experiments amb l'inhibidor de la traducció proteica cicloheximida mostren que l'estabilització de Clb6 requereix síntesi de novo. Donat que en un cicle cel·lular no pertorbat Clb6 és expressada exclusivament en fase G1, sota el control del factor de transcripció MBF. Els nostres resultats indiquen que MBF es reactiva en una fase S compromesa: la subunitat transactivadora Swi6, exclosa del nucli en una fase S no pertorbada, és nuclear en una fase S compromesa; per altra banda, la sobreexpressió d'una forma hiperestable del repressor d'MBF Nrm1 dóna lloc a la desaparició de Clb6 malgrat la presència continuada d'estrès genotòxic i una resposta checkpoint funcional. Fins ara MBF era considerat un factor de transcripció exclusiu de la fase G1.
Per últim hem explorat el paper de l'estabilització de Clb6 en una fase S compromesa. El mutant nul clb6 no presenta cap fenotip detectable, suggerint que Clb6 pot operar en forma redundant dins de la resposta a estrès genotòxic. Per aquest motiu hem optat pel plantejament complementari: estudiar l'efecte de mantenir la presència de Clb6 en una fase S no pertorbada (i per tant en absència de la resta de la resposta checkpoint). En aquestes condicions les cèl·lules repliquen el seu DNA amb aparent normalitat, però queden aturades en un estadi previ a anafase. Aquests resultats suggereixen que Clb6 pot ser un element efector de la branca S-M del mecanisme de vigilància de la fase S, que estaria constituïda per diversos punts de control solapat. Aquest paper justificaria que Clb6 hagi de ser eliminada de forma avançada respecte a Clb5.
Eukaryotic cells are permanently exposed to DNA damage and to replication stress (genotoxic stress), a source of genomic instability. A surveillance mechanism, the S phase checkpoint, detects and responds to genotoxic stress, and elicits a response that includes the arrest of cell cycle progression -to give time to overcome the insult- and the protection of chromosome replication. When the checkpoint central elements are mutated cells become genetically unstable, which in humans results in a high frequency of cancer.
Despite such critical role to preserve genomic integrity, much remains unknown about the way how the S phase checkpoint executes its response. Because the checkpoint elements and pathways are conserved, our work has been carried out in the budding yeast Saccharomyces cerevisiae. In this organism Rad53 is the S phase checkpoint effector kinase. To identify novel Rad53 targets, we carried out an in vitro kinase assay with different potential substrate proteins involved in cell cycle control. This approach identified the S phase cyclin Clb6 as an in vitro Rad53 substrate.
Cyclins are the activatory subunits for Cdc28/Cdk1, the cyclin dependent kinase that drives cell cycle progression in budding yeast. Cdc28/Cdk1 is activated by different cell cycle phase specific cyclins, that also confer substrate specificity to the kinase. Clb6 and Clb5 are the S phase cyclins, promoting the onset of DNA replication. Interestingly, whereas the presence of Clb5 spans from late G1 to anaphase, Clb6 is eliminated earlier, during S phase.
To study whether Clb6 is a target of Rad53 in vivo, we explored the effect of genotoxic stress on the cyclin. When cells are exposed to replication stress or to DNA damage during S phase, the presence of Clb6 is stabilized, and such stabilization depends on Rad53.
Experiments with the inhibitor of protein translation cycloheximide show that the stabilization of Clb6 requires de novo synthesis. In an unperturbed cell cycle Clb6 is expressed only during G1 phase, under the control of the transcription factor MBF. Our results show that MBF is reactivated in a compromised S phase: the transactivatory subunit Swi6, excluded from the nucleus in an unperturbed S phase, shows nuclear localization during a compromised S phase; in addition, the overexpression of a hyperstable form of the MBF repressor Nrm1 results in Clb6 elimination, despite the continued presence of genotoxic stress and a functional checkpoint response. To date, MBF was considered a transcription factor operating solely and characteristically during G1 phase.
Finally we have explored the role of Clb6 stabilization in response to genotoxic stress. The clb6 null mutant does not present a detectable phenotype, which is compatible with a redundant role in the response to genotoxic stress. We therefore chose to address the question the other way round, and study the effect of keeping the levels of Clb6 stable during an otherwise unperturbed S phase (and therefore in the absence of the vast checkpoint response that could mask the specific role of Clb6). Under such conditions cells replicate normally, but arrest in a stage previous to anaphase. These results suggest that Clb6 may be an effector element in the S-M branch of the S phase checkpoint. One such role would explain why Clb6 must be eliminated in S phase, ahead of Clb5 during mitosis.
49
Khan, Kamron N., Mohammed E. El-Asrag, Cristy A. Ku, Graham E. Holder, Martin McKibbin, Gavin Arno, James A. Poulter, et al. "Specific Alleles of CLN7/MFSD8, a Protein That Localizes to Photoreceptor Synaptic Terminals, Cause a Spectrum of Nonsyndromic Retinal Dystrophy." ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2017. http://hdl.handle.net/10150/624955.
Повний текст джерелаАнотація:
PURPOSE. Recessive mutations in CLN7/MFSD8 usually cause variant late-infantile onset neuronal ceroid lipofuscinosis (vLINCL), a poorly understood neurodegenerative condition, though mutations may also cause nonsyndromic maculopathy. A series of 12 patients with nonsyndromic retinopathy due to novel CLN7/MFSD8 mutation combinations were investigated in this study. METHODS. Affected patients and their family members were recruited in ophthalmic clinics at each center where they were examined by retinal imaging and detailed electrophysiology. Whole exome or genome next generation sequencing was performed on genomic DNA from at least one affected family member. Immunofluorescence confocal microscopy of murine retina cross-sections were used to localize the protein. RESULTS. Compound heterozygous alleles were identified in six cases, one of which was always p.Glu336Gln. Such combinations resulted in isolated macular disease. Six further cases were homozygous for the variant p.Met454Thr, identified as a founder mutation of South Asian origin. Those patients had widespread generalized retinal disease, characterized by electroretinography as a rod-cone dystrophy with severe macular involvement. In addition, the photopic single flash electroretinograms demonstrated a reduced b- to a-wave amplitude ratio, suggesting dysfunction occurring after phototransduction. Immunohistology identified MFSD8 in the outer plexiform layer of the retina, a site rich in photoreceptor synapses. CONCLUSIONS. This study highlights a hierarchy of MFSD8 variant severity, predicting three consequences of mutation: (1) nonsyndromic localized maculopathy, (2) nonsyndromic widespread retinopathy, or (3) syndromic neurological disease. The data also shed light on the underlying pathogenesis by implicating the photoreceptor synaptic terminals as the major site of retinal disease.
50
Martínez, Láinez Joan Marc. "Regulación del ciclo celular por ploidía en Saccharomyces cerevisiae." Doctoral thesis, Universitat Internacional de Catalunya, 2017. http://hdl.handle.net/10803/461358.
Повний текст джерелаАнотація:
Una de les característiques cel·lulars amb més impacte sobre la seva fisiologia cel·lular és la mida, regulada per multitud de factors extrínsecs i intrínsecs, i una característica de vital rellevància ja que afecta al volum de diferents orgànuls i a la seva proporció, l’arquitectura interna de la cèl·lula i té capacitat de adaptar-se al contingut de DNA. Respecte a aquest últim, i gràcies a estudis que es remunten a un segle fins l’actualitat, es postula que existeix un mecanisme pel que la cèl·lula es capaç de regular la seva mida cel·lular mitjançant la ploïdia. Això passa al llarg de tot l’arbre de la vida, existint evidències d’una correlació lineal entre la mida i el nombre de cromosomes que contenen las cèl·lules. No obstant, aquest mecanisme continua esquiu i es desconeix quins elements participen.
Aquest estudi, basat en un model eucariota como és S. cerevisiae, es va iniciar gràcies a la observació prèvia de que vectors llançadora centromèrics, YCp, produeixen un increment de mida. En aquest treball se ha determinat que el centròmer és el principal element inductor d’aquest fenotip, descartant altres opcions com la quantitat de DNA o la recuperació de vies metabòliques aportada per aquests vectors, el que es va confirmar mitjançant cromosomes artificials, YAK, i la integració de nous centròmers condicionals en els cromosomes de llevat. A més, s’ha desenvolupat un sistema per quantificar el número de centròmers aportant a la cèl·lula mitjançant fluorescència, protocol que ha permès relacionar de forma molt precisa la dosis centromèrica a la mida cel·lular. Pel que es refereix al mecanisme molecular implicat, es va observar que un nombre elevat de centròmers augmenta la degradació de la ciclina Cln3 a través de elements del SCF presents en el nucli, el que produeix un clar retràs a la fase G1 i, com a conseqüència, un increment en la mida cel·lular. En aquest mecanisme es revela la participació de proteïnes senyalitzadores del centròmer, com són Mad3, Mad2, i Bub3, així com les interacciones in vivo entre Mad3 i Cln3 o Cdc4. Aquestes dades apunten la existència d’un nou mecanisme molecular per la regulació de la mida cel·lular per ploïdia.Una de las características celulares con más impacto sobre su fisiología celular es el tamaño, regulada por multitud de factores extrínseco e intrínsecos, y una característica de vital relevancia ya que afecta el volumen de diferentes orgánulos y su proporción, la arquitectura interna de la célula y tiene capacidad de adaptarse al contenido de DNA. Respecto a este último, y gracias a estudios que se remontan desde hace un siglo hasta la actualidad, se postula que existe un mecanismo por el cual la célula es capaz de regular su tamaño celular mediante ploidía. Esto ocurre a lo largo de todo el árbol de la vida, existiendo evidencias de una correlación lineal entre el tamaño y el número de cromosomas que contienen las células. No obstante, este mecanismo permanece esquivo y se desconoce que elementos participan. Este estudio, basado en un modelo eucariota como es S. cerevisiae, se inició gracias a la observación previa que vectores lanzadera centroméricos, YCp, producen un incremento de tamaño. En este trabajo hemos determinado que el centrómero es el principal elemento inductor de este fenotipo, descartando otras opciones como la cantidad de DNA o la recuperación de vías metabólicas aportada por estos vectores, lo que se confirmó mediante cromosomas artificiales, YAK, y la integración de nuevos centrómeros condicionales en los cromosomas de levadura. Además, se ha desarrollado un sistema para cuantificar el número de centrómeros aportado a la célula mediante fluorescencia, protocolo que ha permitido relacionar de forma muy precisa la dosis centromérica al tamaño celular. Por lo que se refiere al mecanismo molecular implicado, hemos observado que un número elevado de centrómeros aumenta la degradación de la ciclina Cln3 a través de elementos del SCF presentes en el núcleo, lo que produce un claro retraso en la fase G1 y, en consecuencia, un incremento en el tamaño celular. En este mecanismo hemos desvelado la participación de proteinas señalizadoras del centrómero, como son Mad3, Mad2, y Bub3, así como las interacciones in vivo entre Mad3 y Cln3 o Cdc4. Estos datos apuntan a la existencia de un nuevo mecanismo molecular para la regulación del tamaño celular por ploidía.