17 research outputs found
Propietats aterogèniques de l'LDL electronegativa : inducció de l'expressió de citoquines, activitats enzimà tiques associades i unió a proteoglicans /
Descripció del recurs: el 28 de Gener de 2010L'LDL electronegativa (LDL(-)) és una fracció modificada d'LDL present a la circulació. Hi ha varies evidències que suggereixen el seu paper aterogènic ja que es troba augmentada en patologies d'alt risc cardiovascular, com en la hipercolesterolèmia familiar o en la diabetis mellitus. A més, presenta propietats inflamatòries, una major susceptibilitat a l'agregació i afinitat disminuïda pel receptor d'LDL. En la present tesi s'han estudiat algunes propietats aterogèniques d'aquesta partÃcula modificada, en concret s'han avaluat els següents aspectes: 1) Propietats inflamatòries. L'LDL(-) va induir una major expressió de citoquines (IL-6), quimioquines (IL-8, MCP-1 i GRO) i factors de creixement (GM-CSF) en cèl·lules endotelials de vena i d'artèria, i monòcits i limfòcits. La resposta de les cèl·lules endotelials arterials va ser major a nivell de producció total de citoquines i, a més, també van alliberar PDGF-B. No obstant, els monòcits i limfòcits, en resposta a l'LDL(-) també van expressar la citoquina anti-inflamatòria IL-10 la qual va disminuir la secreció de les altres citoquines pro-inflamatòries, modulant la resposta inflamatòria. 2) Activitat fosfolipolÃtica tipus fosfolipasa C associada. S'han descrit noves activitats enzimà tiques presents en l'LDL(-) que degraden eficientment fosfolÃpids amb colina. S'ha relacionat aquesta activitat fosfolipolÃtica amb la major susceptibilitat a l'agregació de l'LDL(-). Es desconeix l'origen d'aquesta activitat enzimà tica, tot i que s'ha descartat que sigui deguda al contingut augmentat en apoE, apoC-III o PAF-AH, i s'ha suggerit que en podria ser responsable una proteïna encara no identificada o la pròpia apoB-100. 3) Unió a proteoglicans de la matriu extracel·lular. Una subfracció de partÃcules d'LDL(-) van presentar una major unió a proteoglicans aïllats d'artèria humana. Aquesta subfracció de partÃcules estaven més agregades i tenien una elevada activitat fosfolipolÃtica. Fets que es van relacionar i, en conseqüència, es va hipotetitzar que la major activitat fosfolipolÃtica podria provocar l'agregació de les partÃcules i, al mateix temps, que aquestes s'unissin amb major afinitat als proteoglicans. En conclusió, l'LDL(-) sembla una partÃcula potencialment aterogènica degut a que té la capacitat d'induir l'alliberament de molècules inflamatòries en diferents tipus cel·lulars, i presenta una major agregació i afinitat pels proteoglicans, fent-la propensa a la retenció subendotelial.Electronegative LDL (LDL(-)) is a modified fraction of LDL present in circulation. Growing evidence suggests an atherogenic role. LDL(-) proportion is increased in diseases with high cardiovascular risk, as familial hypercholesterolemia or diabetes mellitus. Furthermore, it presents inflammatory properties, high susceptibility to aggregation and decreased LDL receptor affinity. In the current thesis some atherogenic properties of this modified particle have been studied, specifically, the following points have been evaluated: 1) Inflammatory properties. LDL(-) induced increased expression of cytokines (IL-6), chemokines (IL-8, MCP-1 and GRO) and grown factors (GM-CSF) on venous and arterial endothelial cells, monocytes and lymphocytes. Arterial endothelial cells produced a high amount of cytokines and, moreover, they also stimulated PDGF-B. On the other hand, monocytes and lymphocytes also expressed the anti-inflammatory cytokine IL-10, who decreased the secretion of the other pro-inflammatory cytokines, thereby counteracting the inflammatory response. 2) Phospholipase C-like phospholipolytic activity associated to LDL(-). Novel enzymatic activities present on LDL(-) that hydrolyze choline-containing phospholipids have been described. These activities have been involved in increased susceptibility to aggregation. A role for apoE, apoC-III and PAF-AH has been ruled out and our results suggest that apoB-100 could be involved. However, the exact origin remains unknown. 3) Binding to proteoglycans from the extracellular matrix. A subfraction of LDL(-) presented increased binding to proteoglycans isolated from human artery. This subfraction has increased aggregation and phospholipolytic activity. Both properties are related, suggesting that phospholipolytic activity promotes aggregation and favours binding to proteoglycans
Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes
The product of the Plasmodium falciparum genes clag3.1 and
clag3.2 plays a fundamental role in malaria parasite biology by
determining solute transport into infected erythrocytes.
Expression of the two clag3 genes is mutually exclusive, such
that a single parasite expresses only one of the two genes at a
time. Here we investigated the properties and mechanisms of
clag3 mutual exclusion using transgenic parasite lines with
extra copies of clag3 promoters located either in stable
episomes or integrated in the parasite genome. We found that the
additional clag3 promoters in these transgenic lines are
silenced by default, but under strong selective pressure
parasites with more than one clag3 promoter simultaneously
active are observed, demonstrating that clag3 mutual exclusion
is strongly favored but it is not strict. We show that silencing
of clag3 genes is associated with the repressive histone mark
H3K9me3 even in parasites with unusual clag3 expression
patterns, and we provide direct evidence for heterochromatin
spreading in P. falciparum. We also found that expression of a
neighbor ncRNA correlates with clag3.1 expression. Altogether,
our results reveal a scenario where fitness costs and
non-deterministic molecular processes that favor mutual
exclusion shape the expression patterns of this important gene
family
Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum
Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt
within hosts Plasmodium falciparum undergoes drastic switches in
gene expression. To identify regulatory regions in the parasite
genome, we performed genome-wide profiling of chromatin
accessibility in two culture-adapted isogenic subclones at four
developmental stages during the intraerythrocytic cycle by using
the Assay for Transposase-Accessible Chromatin by sequencing
(ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs)
localize preferentially at transcriptional start sites (TSSs).
Chromatin accessibility by ATAC-seq is predictive of active
transcription and of the levels of histone marks H3K9ac and
H3K4me3. Our assay allows the identification of novel regulatory
regions including TSS and enhancer-like elements. We show that
the dynamics in the accessible chromatin profile matches
temporal transcription during development. Motif analysis of
stage-specific ATAC-seq sites predicts the in vivo binding sites
and function of multiple ApiAP2 transcription factors. At last,
the alternative expression states of some clonally variant genes
(CVGs), including eba, phist, var and clag genes, associate with
a differential ATAC-seq signal at their promoters. Altogether,
this study identifies genome-wide regulatory regions likely to
play an essential function in the developmental transitions and
in CVG expression in P. falciparum
Revisiting the initial steps of sexual development in the malaria parasite Plasmodium falciparum
Human to vector transmission of malaria requires that
some blood-stage parasites abandon asexual growth and convert
into non-replicating sexual forms called gametocytes. The
initial steps of gametocytogenesis remain largely
uncharacterized. Here, we study this part of the malaria life
cycle in Plasmodium falciparum using PfAP2-G, the master
regulator of sexual conversion, as a marker of commitment. We
demonstrate the existence of PfAP2-G-positive sexually committed
parasite stages that precede the previously known committed
schizont stage. We also found that sexual conversion can occur
by two different routes: the previously described route in which
PfAP2-G-expressing parasites complete a replicative cycle as
committed forms before converting into gametocytes upon
re-invasion, or a direct route with conversion within the same
cycle as initial PfAP2-G expression. The latter route is linked
to early PfAP2-G expression in ring stages. Reanalysis of
published single-cell RNA-sequencing (RNA-seq) data confirmed
the presence of both routes. Consistent with these results,
using plaque assays we observed that, in contrast to the
prevailing model, many schizonts produced mixed plaques
containing both asexual parasites and gametocytes. Altogether,
our results reveal unexpected features of the initial steps of
sexual development and extend the current view of this part of
the malaria life cycle
HDL and electronegative LDL exchange anti- and pro-inflammatory properties
Electronegative LDL [LDL(–)] is a minor modified LDL subfraction present in blood with inflammatory effects. One of the antiatherogenic properties of HDL is the inhibition of the deleterious effects of in vitro modified LDL. However, the effect of HDL on the inflammatory activity of LDL(–) isolated from plasma is unknown. We aimed to assess the putative protective role of HDL against the cytokine released induced in monocytes by LDL(–). Our results showed that LDL(–) cytokine release was inhibited when LDL(–) was coincubated with HDL and human monocytes and also when LDL(–) was preincubated with HDL and reisolated prior to cell incubation. The addition of apoliprotein (apo)AI instead of HDL reproduced the protective behavior of HDL. HDL preincubated with LDL(–) promoted greater cytokine release than native HDL. Incubation of LDL(–) with HDL decreased the electronegative charge, phospholipase C-like activity, susceptibility to aggregation and nonesterified fatty acid (NEFA) content of LDL(–), whereas these properties increased in HDL. NEFA content in LDL appeared to be related to cytokine production because NEFA-enriched LDL induced cytokine release. HDL, at least in part through apoAI, inhibits phospholipase-C activity and cytokine release in monocytes, thereby counteracting the inflammatory effect of LDL(–). In turn, HDL acquires these properties and becomes inflammatory
Characterization of the accessible genome in the human malaria parasite Plasmodium falciparum
Human malaria is a devastating disease and a major cause of poverty in resource-limited countries. To develop and adapt
within hosts Plasmodium falciparum undergoes drastic switches in
gene expression. To identify regulatory regions in the parasite
genome, we performed genome-wide profiling of chromatin
accessibility in two culture-adapted isogenic subclones at four
developmental stages during the intraerythrocytic cycle by using
the Assay for Transposase-Accessible Chromatin by sequencing
(ATAC-seq). Tn5 transposase hypersensitivity sites (THSSs)
localize preferentially at transcriptional start sites (TSSs).
Chromatin accessibility by ATAC-seq is predictive of active
transcription and of the levels of histone marks H3K9ac and
H3K4me3. Our assay allows the identification of novel regulatory
regions including TSS and enhancer-like elements. We show that
the dynamics in the accessible chromatin profile matches
temporal transcription during development. Motif analysis of
stage-specific ATAC-seq sites predicts the in vivo binding sites
and function of multiple ApiAP2 transcription factors. At last,
the alternative expression states of some clonally variant genes
(CVGs), including eba, phist, var and clag genes, associate with
a differential ATAC-seq signal at their promoters. Altogether,
this study identifies genome-wide regulatory regions likely to
play an essential function in the developmental transitions and
in CVG expression in P. falciparum
Impaired Binding Affinity of Electronegative Low-Density Lipoprotein (LDL) to the LDL Receptor Is Related to Nonesterified Fatty Acids and Lysophosphatidylcholine Content â€
Deciphering the principles that govern mutually exclusive expression of Plasmodium falciparum clag3 genes
The product of the Plasmodium falciparum genes clag3.1 and
clag3.2 plays a fundamental role in malaria parasite biology by
determining solute transport into infected erythrocytes.
Expression of the two clag3 genes is mutually exclusive, such
that a single parasite expresses only one of the two genes at a
time. Here we investigated the properties and mechanisms of
clag3 mutual exclusion using transgenic parasite lines with
extra copies of clag3 promoters located either in stable
episomes or integrated in the parasite genome. We found that the
additional clag3 promoters in these transgenic lines are
silenced by default, but under strong selective pressure
parasites with more than one clag3 promoter simultaneously
active are observed, demonstrating that clag3 mutual exclusion
is strongly favored but it is not strict. We show that silencing
of clag3 genes is associated with the repressive histone mark
H3K9me3 even in parasites with unusual clag3 expression
patterns, and we provide direct evidence for heterochromatin
spreading in P. falciparum. We also found that expression of a
neighbor ncRNA correlates with clag3.1 expression. Altogether,
our results reveal a scenario where fitness costs and
non-deterministic molecular processes that favor mutual
exclusion shape the expression patterns of this important gene
family