21 research outputs found
Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA
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Anàlisi genètica i funcional de la migranya hemiplègica i la migranya comuna
No full text[cat] Aquesta tesi es centra en la genètica de la migranya. La migranya comuna és un trastorn neurològic caracteritzat per episodis recurrents de mal de cap. Els criteris de la IHS (International Headache Society) subclasifiquen la malaltia en migranya amb aura (MA) i migranya sense aura (MO). L'aura són símptomes neurològics transitoris que poden acompanyar el mal de cap. Les aures més freqüents són les aures visuals, tot i que també existeixen les aures sensorials essent l'aura hemiplègica la seva forma severa. La nostra investigació es va dividir en dues areas d'acord amb la base genètica dels trastorns, d'una banda, s'ha estudiat la genètica complexa de la migranya comuna, d'altra banda s'ha estudiat una forma rara de la migranya que presenta una herència mendeliana anomenada migranya hemiplègica familiar (FHM).
Per entendre més la base genètica de la migranya comuna es va utilitzar un estudi d'associació tipus cas-control amb gens candidats. Amb aquesta finalitat es van seleccionar al voltant de 550 pacients amb migranya (MA i MO) i el seu corresponent grup de control. Per tal d'analitzar la seva implicació en la susceptibilitat genètica a la migranya, es van triar gens que codifiquen per als canals de la superfamília heterogeni de potencial receptor transitori (Transient Receptor Potential- TRP) que se sap que estan implicats en les vies nociceptives. Aquesta feina ha donat lloc a una publicació (Carreño et al. SNP variants within the vanilloid TRPV1 and TRPV3 receptor genes are associated with migraine in the Spanish population. Am J Med Genet B Neuropsychiatr Genet. 2012).
En el cas particular de les formes monogèniques de FHM es coneixen tres gens involucrats en la malatia (CACNA1A, ATP1A2 i SCN1A), les proteïnes codificades per aquests gens tenen un paper rellevant en la neurotransmissió del glutamat. L'anàlisi funcional de les mutacions que causen FHM han mostrat en última instància un augment de l'alliberament de la neurotransmissió. En el cas de mutacions al CACNA1A s'ha vist un efecte de guany de funció, a diferència de les mutacions al ATP1A2 que presenten un efecte de pèrdua de funció. En aquest treball s'ha fet un screening mutacional per identificar mutacions en pacients per seqüenciació directa. Quan les mutacions eren suficientment interessants s'han generat construccions en vectors d'expressió per subseqüents estudis funcionals en cèl·lules eucariotes. Aquesta feina ha donat lloc a tres publicacions. A la primera (Serra et al. A mutation in the first intracellular loop of CACNA1A prevents P/Q channel modulation by SNARE proteins and lowers exocytosis. Proc Natl Acad Sci. 2010) es va identificar un canvi que modula la funció del canal de CACNA1A. Aquest estudi ajuda a explicar la contribució genètica en la heterogeneïtat clínica d'una família i a entendre millor el mecanisme molecular dels canals de calci tipus P/Q. El segon (Carreño et al. Acute striatal necrosis in hemiplegic migraine with the novo CACNA1A mutation. Headache. 2011) és un informe d'un pacient que presenta una necrosi aguda stratial. Té una rellevància clínica a causa de l'aparició primerenca dels símptomes neurològics previs als atacs hemiplègics. El tercer i últim treball (Carreño et al. Screening of the ATP1A2 and CACNA1A genes in patients with hemiplegic migraine: clinical, genetic and functional studies. [work in progress]) recull l'screening mutacional al gens ATP1A2 i CACNA1A en 19 pacients amb FHM. Es van identificar 5 mutacions prèviament descrites i dues mutacions noves.[eng] This Thesis is focused in migraine genetics, migraine is a prevalent neurological disorder characterized by recurrent episodes of headache. This research was divided in two areas according to the genetic basis of the disorders; on the one hand we studied the common migraine with a complex genetics, on the other hand we studied the rare mendelian forms of familial hemiplegic migraine (FHM).
To understand more the genetic basis of the common migraine a case-control association study approach was used with candidate genes. For that purpose, around 550 patients with migraine and their corresponding control group were selected. In order to analyze their involvement in the genetic susceptibility to migraine, we chose genes encoding for channels of the heterogeneous superfamily of Transient Receptor Potential (TRP) which are known to be involved in the nociceptive pathway.
In the particular case of FHM, a monogenic form of the disorder, there are three genes known to be involved in the FHM (CACNA1A, ATP1A2 and SCN1A), whose encoded proteins are playing a relevant role in the neurotransmission of the glutamate. Functional analysis of the mutations causing FHM have shown ultimately an increased neurotransmission release. CACNA1A previous studies reveled a gain-of-function effect from FHM mutations, unlike mutations on ATP1A2 that present a loss-of-function effect. Our work consisted on identifying mutations in patients by direct sequencing. If the mutations were interesting enough vector constructions were generated for functional studies in eukaryotic cells. This work gave rise to three publications: First; the identification of a change that modulates the function of the CACNA1A channel. This study contributes to explain the genetic contribution in the clinical heterogeneity of one family and to know more about the molecular mechanism of the P/Q calcium channel. Second; a report of a patient that presents an acute stratial necrosis that had clinical relevance because of the early onset of the neurological symptoms previous to the hemiplegic attacks. Third; a mutational screening of ATP1A2 and CACNA1A genes in 19 patients with FHM. 5 previously described mutations and two new mutations were found. Functional studies were carried out for the newly mutations
Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography–tandem mass spectrometry
No full textA sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.1–50 and 0.5–100 nM, respectively. Fully 13C isotope-labeled and deuterated internal standards were used to achieve accurate quantification. The applicability of the method to analyze serum, urine, and cell culture supernatants was demonstrated by recovery experiments and the evaluation of matrix effects. Precision for the analysis of serum, urine, and cell culture supernatants ranged between 1.3% and 16.0%, 1.5% and 13.5%, and 1.0% and 17.4%, respectively. The method was applied to analyze changes in tryptophan metabolism in cell culture supernatants from IFN-?-treated monocytes and immature or mature dendritic cells.BiotechnologyApplied Science
L1 retrotransposition in neurons is modulated by MeCP2
No full textRetrotransposition in neuronsL1 retrotransposons are dynamically regulated and active genomic elements that affect gene expression and neuronal function throughout brain development. According to a new study by Alysson Muotri and colleagues, the absence of MeCP2, a modulator of DNA methylation implicated in several neurodevelopmental disorders, increases L1 retrotransposon activity in rodent models. This increase in susceptibility to L1 retrotransposition is duplicated in iPS cells derived from patients with Rett syndrome. These data correlations suggest that disease-related genetic mutations may influence L1 retrotransposon activity, adding another layer of complexity to our understanding of molecular neurological disorders
The role of the Bmi1-GSK3β pathway in glioblastoma
No full textMalignant gliomas remain one of the deadliest of all cancers despite maximal
therapy. They present unique challenges to therapy with a median survival of 12 months.
Simultaneous activation of several growth promoting and anti-apoptotic pathways
represents the basis for the failure of monotherapies against this disease. In order to
efficiently block growth of glioblastoma (GBM) cells, we have applied several
combinatorial approaches. We have found that combination of histone deactylase
inhibitors along with the glycolytic inhibitor 2-deoxyglucose (2DG) efficiently induced
apoptosis in GBM cells. Furthermore, combination of the microtubule inhibitor
patupilone and AEE788 –an inhibitor of EGFR, which is frequently activated in gliomas,
induced apoptosis in GBM cells at doses that as single drugs were not effective. In GBM
and other cancers, subpopulations of tumor cells with stem cell properties that are
believed to constitute a tumor cell reservoir, have been identified. GBM cells frequently
express the progenitor cell markers Nestin and Sox2 and low levels of the differentiation
markers CNPase, GFAP and !-tubulin III. Bmi1 and Glycogen synthase kinase 3 (GSK3)
has been implicated in stem cell maintenance, but how Bmi1 regulates differentiation is
still unknown. We have identified a link between Bmi1 and GSK3 and showed that
blocking GSK3 may be instrumental to reduce the GBM cancer stem cell pool. We found
that the GSK3 inhibitors SB216763 as well as Lithium chloride depleted the cancer stem
cell population in GBM cells and induced tumor cell differentiation, irrespective of the
CD133 status. Cell proliferation and colony formation were markedly reduced in a dosedependent
manner.
Future work giving a deeper insight into the regulatory mechanisms of the
receptor tyrosine kinases and downstream effectors will help us to identify more specific
targets. Understanding the mechanisms why some targeted therapies work and others fail
will finally bring us to the level that efficient long-term treatment strategies can be
envisaged
Mitogenomes from Two Uncommon Haplogroups Mark Late Glacial/Postglacial Expansions from the Near East and Neolithic Dispersals within Europe
No full textThe current human mitochondrial (mtDNA) phylogeny does not equally represent all human populations but is biased in favour of representatives originally from north and central Europe. This especially affects the phylogeny of some uncommon West Eurasian haplogroups, including I and W, whose southern European and Near Eastern components are very poorly represented, suggesting that extensive hidden phylogenetic substructure remains to be uncovered. This study expanded and re-analysed the available datasets of I and W complete mtDNA genomes, reaching a comprehensive 419 mitogenomes, and searched for precise correlations between the ages and geographical distributions of their numerous newly identified subclades with events of human dispersal which contributed to the genetic formation of modern Europeans. Our results showed that haplogroups I (within N1a1b) and W originated in the Near East during the Last Glacial Maximum or pre-warming period (the period of gradual warming between the end of the LGM, ~19 ky ago, and the beginning of the first main warming phase, ~15 ky ago) and, like the much more common haplogroups J and T, may have been involved in Late Glacial expansions starting from the Near East. Thus our data contribute to a better definition of the Late and postglacial re-peopling of Europe, providing further evidence for the scenario that major population expansions started after the Last Glacial Maximum but before Neolithic times, but also evidencing traces of diffusion events in several I and W subclades dating to the European Neolithic and restricted to Europe
Author Correction: Rare genetic variants affecting urine metabolite levels link population variation to inborn errors of metabolism
No full textMaking Sense of the Census: Classifying and Counting Ethnicity in Oceania, 1965-2011
No full textAs the flagship government effort to count and classify its population, censuses are a key site for rendering and making visible group boundaries. Despite claims to objective rationality, however, census taking is a political and inherently subjective exercise. Censuses help shape the very categories they claim to capture: censuses do more than reflect social reality, they also participate in the social construction of this reality (Kertzer and Arel, 2002b, p. 2). While ethnicity – as a social construct – is imagined, its effects are far from imaginary, and census categorisations may have significant material consequences for the lives of citizens.
Although an increasing number of studies have examined how and why governments in particular times or places count their populations by ethnicity, studies that are both cross-national and longitudinal are rare. Attempting to in part bridge this gap, this thesis studies census questionnaires from 1965 to 2011 for 24 countries in Oceania. In doing so, it explores three general questions: 1) how ethnicity is conceptualised and categorised in Oceanic censuses over time; 2) the relationship between ethnic counting in territories to that of their metropoles; and 3) Oceanic approaches towards multiple ethnic identities. Spread over an area of thirty million square kilometres of the Pacific Ocean, Oceania provides an interesting context to study ethnic counting. The countries and territories which make up the region present an enormous diversity in physical geography and culture, languages and social organization, size and resource endowment. As the last region in the world to decolonise, Oceania includes a mix of dependencies and sovereign states.
The study finds that engagement with ethnic classification and counting is near-ubiquitous across the time period, with most countries having done so in all five cross-sectional census rounds. In general terms, in ethnic census questions ‘racial’ terminology of race and ancestry has been displaced over the focal period by ‘ethnic’ terminology of ethnicity and ethnic origin. Overall, the concept of ethnic origins predominates, although interestingly it is paired with race in the US territories, reflecting the ongoing social and political salience of race in the metropole. With respect to ethnic categories provided on census forms (and thus imbued with the legitimacy of explicit state recognition) the study finds a shift away from the imagined and flawed Melanesian/Micronesian/ Polynesian racial typology and other colonial impositions to more localised and self-identified Pacific identities. It is theorised that these shifts are emblematic of broader global changes in the impetuses for ethnic counting, from colonially-influenced ‘top down’ counting serving exclusionary ends to more inclusive, ‘bottom up’ approaches motivated by concerns for minority rights and inclusive policy-making
