44 research outputs found
The biology and evolution of the Dilp8-Lgr3 pathway: A relaxin-like pathway coupling tissue growth and developmental timing control
No full textMany insects, like cockroaches, moths, and flies, can regenerate tissues by extending the growth-competent phases of their life cycle. The molecular and cellular players mediating this coordination between tissue growth and developmental timing have been recently discovered in Drosophila. The insulin/relaxin-like peptide, Dilp8, was identified as a factor communicating abnormal growth status of Drosophila larval imaginal discs to the neuroendocrine centers that control the timing of the onset of metamorphosis. Dilp8 requires a neuronal relaxin receptor for this function, the Leucine rich repeat containing G protein coupled receptor, Lgr3. A review of current data supports a model where imaginal disc-derived Dilp8 acts on four central nervous system Lgr3-positive neurons to activate cyclic-AMP signaling in an Lgr3-dependent manner. This causes a reduction in ecdysone hormone production by the larval endocrine prothoracic gland, which leads to a delay in the onset of metamorphosis and a simultaneous slowing down in the growth rates of healthy imaginal tissues, promoting the generation of proportionate individuals. We discuss reports indicating that the Dilp8-Lgr3 pathway might have other functions at different life history stages, which remain to be elucidated, and review molecular evolution data on invertebrate genes related to the relaxin-pathway. The strong conservation of the relaxin pathway throughout animal evolution contrasts with instances of its complete loss in some clades, such as lepidopterans, which must coordinate growth and developmental timing using another mechanism. Research into these areas should generate exciting new insights into the biology of growth coordination, the evolution of the relaxin signaling pathway, and likely reveal unforeseen functions in other developmental stages.Fil: Gontijo, Alisson M.. Universidade Nova de Lisboa; PortugalFil: Garelli, Andres. Universidade Nova de Lisboa; Portugal. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentin
Retrotransposition causes age-dependent neuronal function decline
No full textTese de mestrado, Engenharia Biomédica e Biofísica, Universidade de Lisboa, Faculdade de Ciências, 2022Retrotransposable elements (RE) are DNA sequences that have the ability to change position within
the genome. They are present in most organisms and often comprise a large part of their genome. Because of their mobilization, REs are an intrinsic source of DNA damage and mutations that can alter
gene activity. Even though some of these mutations can eventually drive evolutionary novelty at the
population and species scales, more often, the RE-driven mutations are deleterious and lead to genomic
instability and disease at the individual scale. It comes as no surprise, therefore, that hosts have evolved
transcriptional and post-transcriptional mechanisms that repress REs activity.
Recent studies have correlated increased expression of REs with age and age-related diseases, such
as neurodegenerative disorders, which is suspected to be due to the tendency of RE silencing mechanisms
to fail over time. However, it remains unclear whether RE de-repression is a cause or a consequence of
the age-dependent cellular loss of function.
The goal of this project is to determine if retrotransposition of long interspersed nuclear element-1
(LINE1 or L1) can be a cause of neurodegeneration and neural function decline if it is not repressed
by the organism. To evaluate the effects of continuous expression of L1 in the nervous system in such a
context, we developed a heterologous system that allows tissue-specific expression, using the GAL4-UAS
system, of an L1 of the mosquito Anopheles gambiae (AgL1) in the evolutionary-distant fly Drosophila
melanogaster. Mosquitos and flies have evolved separately for about 250 million years, so it is expected
that their anti-RE systems no longer recognize each other.
We confirmed the expression of AgL1 transcripts in the adult fly nervous system using reverse
transcription-quantitative real-time polymerase chain reaction (RT-qPCR), and of the AgL1-encoded
proteins, ORF1p and ORF2p, by immunohistochemistry using antibodies against V5 and HA epitopes,
which respectively tag each protein in our construct. Further immunohistochemistry assays using an
anti-phosphorylated histone H2Av antibody, which indicates the presence of DNA double strand breaks
(DSBs), showed that AgL1-expressing flies had higher DSB levels in neural cells than their controls.
However, immunostaining using anti-activated caspase, a programmed-cell death indicator, revealed similar levels of neuronal death in both conditions.
Neuronal performance of ageing flies was analyzed using negative geotaxis (climbing) assays 2, 20,
30, and 40 days after eclosion. We observed a statistically significant decrease in climbing ability in
flies expressing AgL1 in the neurons, compared to their age-matched controls. Expression of AgL1 in
glial cells, in contrast, had no significant effect on climbing ability, suggesting that neurons are particularly sensitive to AgL1 activity. Feeding stavudine - a known retrotransposition inhibitor - to ageing
flies expressing AgL1 in neurons partially rescued their decreased climbing ability, suggesting AgL1
retrotransposition or its attempts had a causal role in the locomotor function decrease.
AgL1 retrotransposition capability was further evaluated using an established exogenous cell culture retrotransposition assay for human cells and a custom-modified retrotransposition assay for fly cells. We were able to demonstrate AgL1 retrotransposition in human HeLa cells, but not in Drosophila
melanogaster DL2 cells. This suggests that the decrease in climbing ability of AgL1-expressing flies is
the result of DNA damage caused by L1 retrotransposition attempts, rather than full retrotransposition
events
Retrotransposition causes age-dependent neuronal function decline
No full textTese de mestrado, Engenharia Biomédica e Biofísica, Universidade de Lisboa, Faculdade de Ciências, 2022Retrotransposable elements (RE) are DNA sequences that have the ability to change position within
the genome. They are present in most organisms and often comprise a large part of their genome. Because of their mobilization, REs are an intrinsic source of DNA damage and mutations that can alter
gene activity. Even though some of these mutations can eventually drive evolutionary novelty at the
population and species scales, more often, the RE-driven mutations are deleterious and lead to genomic
instability and disease at the individual scale. It comes as no surprise, therefore, that hosts have evolved
transcriptional and post-transcriptional mechanisms that repress REs activity.
Recent studies have correlated increased expression of REs with age and age-related diseases, such
as neurodegenerative disorders, which is suspected to be due to the tendency of RE silencing mechanisms
to fail over time. However, it remains unclear whether RE de-repression is a cause or a consequence of
the age-dependent cellular loss of function.
The goal of this project is to determine if retrotransposition of long interspersed nuclear element-1
(LINE1 or L1) can be a cause of neurodegeneration and neural function decline if it is not repressed
by the organism. To evaluate the effects of continuous expression of L1 in the nervous system in such a
context, we developed a heterologous system that allows tissue-specific expression, using the GAL4-UAS
system, of an L1 of the mosquito Anopheles gambiae (AgL1) in the evolutionary-distant fly Drosophila
melanogaster. Mosquitos and flies have evolved separately for about 250 million years, so it is expected
that their anti-RE systems no longer recognize each other.
We confirmed the expression of AgL1 transcripts in the adult fly nervous system using reverse
transcription-quantitative real-time polymerase chain reaction (RT-qPCR), and of the AgL1-encoded
proteins, ORF1p and ORF2p, by immunohistochemistry using antibodies against V5 and HA epitopes,
which respectively tag each protein in our construct. Further immunohistochemistry assays using an
anti-phosphorylated histone H2Av antibody, which indicates the presence of DNA double strand breaks
(DSBs), showed that AgL1-expressing flies had higher DSB levels in neural cells than their controls.
However, immunostaining using anti-activated caspase, a programmed-cell death indicator, revealed similar levels of neuronal death in both conditions.
Neuronal performance of ageing flies was analyzed using negative geotaxis (climbing) assays 2, 20,
30, and 40 days after eclosion. We observed a statistically significant decrease in climbing ability in
flies expressing AgL1 in the neurons, compared to their age-matched controls. Expression of AgL1 in
glial cells, in contrast, had no significant effect on climbing ability, suggesting that neurons are particularly sensitive to AgL1 activity. Feeding stavudine - a known retrotransposition inhibitor - to ageing
flies expressing AgL1 in neurons partially rescued their decreased climbing ability, suggesting AgL1
retrotransposition or its attempts had a causal role in the locomotor function decrease.
AgL1 retrotransposition capability was further evaluated using an established exogenous cell culture retrotransposition assay for human cells and a custom-modified retrotransposition assay for fly cells. We were able to demonstrate AgL1 retrotransposition in human HeLa cells, but not in Drosophila
melanogaster DL2 cells. This suggests that the decrease in climbing ability of AgL1-expressing flies is
the result of DNA damage caused by L1 retrotransposition attempts, rather than full retrotransposition
events
Dilp8 requires the neuronal relaxin receptor Lgr3 to couple growth to developmental timing
No full textHow different organs in the body sense growth perturbations in distant tissues to coordinatetheir size during development is poorly understood. Here we mutate an invertebrate orphanrelaxin receptor gene, the Drosophila Leucine-rich repeat-containing G protein-coupled receptor 3(Lgr3), and find body asymmetries similar to those found in insulin-like peptide 8 (dilp8)mutants, which fail to coordinate growth with developmental timing. Indeed, mutation or RNAintereference (RNAi) against Lgr3 suppresses the delay in pupariation induced by imaginaldisc growth perturbation or ectopic Dilp8 expression. By tagging endogenous Lgr3 andperforming cell type-specific RNAi, we map this Lgr3 activity to a new subset of CNS neurons,four of which are a pair of bilateral pars intercerebralis Lgr3-positive (PIL) neurons that respondspecifically to ectopic Dilp8 by increasing cAMP-dependent signalling. Our work sheds newlight on the function and evolution of relaxin receptors and reveals a novel neuroendocrinecircuit responsive to growth aberrations.Fil: Garelli, Andres. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnol.conicet - Bahia Blanca. Instituto de Invest.bioquimicas Bahia Blanca (i); ArgentinaFil: Heredia, Fabiana. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; PortugalFil: Casimiro, Andreia P.. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; PortugalFil: Macedo, Andre. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; PortugalFil: Nunes, Catarina. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; PortugalFil: Garcez, Marcia. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; PortugalFil: Mantas Dias, Angela R.. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; PortugalFil: Volonté, Yanel Andrea. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnol.conicet - Bahia Blanca. Instituto de Invest.bioquimicas Bahia Blanca (i); ArgentinaFil: Uhlmann, Thomas. Dualsystems Biotech Ag; SuizaFil: Caparros, Esther. Universidad Miguel Hernández. Facultad de Medicina; EspañaFil: Koyama, Takashi. Instituto Gulbenkian de Ciência; PortugalFil: Gontijo, Alisson M.. Nova University of Lisbon. CEDOC-Chronic Diseases Research Center; Portuga
The intersectional genetics landscape for humans
No full textBACKGROUND: The human body is made up of hundreds-perhaps thousands-of cell types and states, most of which are currently inaccessible genetically. Intersectional genetic approaches can increase the number of genetically accessible cells, but the scope and safety of these approaches have not been systematically assessed. A typical intersectional method acts like an "AND" logic gate by converting the input of 2 or more active, yet unspecific, regulatory elements (REs) into a single cell type specific synthetic output. RESULTS: Here, we systematically assessed the intersectional genetics landscape of the human genome using a subset of cells from a large RE usage atlas (Functional ANnoTation Of the Mammalian genome 5 consortium, FANTOM5) obtained by cap analysis of gene expression sequencing (CAGE-seq). We developed the heuristics and algorithms to retrieve and quality-rank "AND" gate intersections. Of the 154 primary cell types surveyed, >90% can be distinguished from each other with as few as 3 to 4 active REs, with quantifiable safety and robustness. We call these minimal intersections of active REs with cell-type diagnostic potential "versatile entry codes" (VEnCodes). Each of the 158 cancer cell types surveyed could also be distinguished from the healthy primary cell types with small VEnCodes, most of which were robust to intra- and interindividual variation. Methods for the cross-validation of CAGE-seq-derived VEnCodes and for the extraction of VEnCodes from pooled single-cell sequencing data are also presented. CONCLUSIONS: Our work provides a systematic view of the intersectional genetics landscape in humans and demonstrates the potential of these approaches for future gene delivery technologies.publishersversionpublishe
Influência da época de semeadura e da população de plantas na produtividade de cultivares de sorgo granífero no Sudoeste Goiano.
No full text
Mutations in genes involved in nonsense mediated decay ameliorate the phenotype of <it>sel-12 </it>mutants with amber stop mutations in <it>Caenorhabditis elegans</it>
No full textAbstract Background Presenilin proteins are part of a complex of proteins that can cleave many type I transmembrane proteins, including Notch Receptors and the Amyloid Precursor Protein, in the middle of the transmembrane domain. Dominant mutations in the human presenilin genes PS1 and PS2 lead to Familial Alzheimer's disease. Mutations in the Caenorhabditis elegans sel-12 presenilin gene cause a highly penetrant egg-laying defect due to reduction of signalling through the lin-12/Notch receptor. Mutations in six spr genes (for suppressor of presenilin) are known to strongly suppress sel-12. Mutations in most strong spr genes suppress sel-12 by de-repressing the transcription of the largely functionally equivalent hop-1 presenilin gene. However, how mutations in the spr-2 gene suppress sel-12 is unknown. Results We show that spr-2 mutations increase the levels of sel-12 transcripts with Premature translation Termination Codons (PTCs) in embryos and L1 larvae. mRNA transcripts from sel-12 alleles with PTCs undergo degradation by a process known as Nonsense Mediated Decay (NMD). However, spr-2 mutations do not appear to affect NMD. Mutations in the smg genes, which are required for NMD, can restore sel-12(PTC) transcript levels and ameliorate the phenotype of sel-12 mutants with amber PTCs. However, the phenotypic suppression of sel-12 by smg genes is nowhere near as strong as the effect of previously characterized spr mutations including spr-2. Consistent with this, we have identified only two mutations in smg genes among the more than 100 spr mutations recovered in genetic screens. Conclusion spr-2 mutations do not suppress sel-12 by affecting NMD of sel-12(PTC) transcripts and appear to have a novel mechanism of suppression. The fact that mutations in smg genes can ameliorate the phenotype of sel-12 alleles with amber PTCs suggests that some read-through of sel-12(amber) alleles occurs in smg backgrounds.</p
Progression through pupariation behaviors requires dilp8-Lgr3 signaling between the cuticle epidermis and thoracic CNS neurons
No full textHigher dipterans undergo metamorphosis within a puparium, a protective capsule made up of the reshaped and hardened cuticle of the last larval instar. Puparium formation (pupariation) in Drosophila starts when wandering stage larvae reduce their locomotion and initiate increasingly strong whole body contractions that together with the internalization of the three anteriormost body segments remodel the body and cuticle, reducing their length/width ratio from ~5 to ~3. This behavior lasts 5-10 min and culminates with the extrusion of a proteinaceous mix (glue) produced by the salivary glands. The animal then slowly moves forward in a caterpillar-like fashion, traveling about half its length for ~1 min to its final pupariation site. We call this behavior glue-spreading behavior (GSB), as it helps to spread the glue over the ventral part of the animal, promoting its attachment to the underlying substrate. Following GSB, the final shape of the puparium is set, even though weak and periodic contractions occur over the next 40-50 min (post-GSB), the operculum becomes defined, and the cuticle (i.e., the future puparium) starts to gradually sclerotize and tan. Here, we show that proper progression through three pupariation behaviors (pre-GSB, GSB, and postGSB) requires the Dilp8-Lgr3 pathway, a relaxin-like pathway that has been previously implicated in controlling the timing of pupariation in animals carrying aberrantly growing imaginal discs. During pupariation, however, Dilp8-Lgr3 signaling is spatiallyand temporally-distinct: a strong, epidermis-derived dilp8 expression peak that starts at the pre-GSB phase of pupariation appears to signal via Lgr3 in a novel subpopulation of thoracic CNS neurons. While this signaling ensures partial progression through the preGSB phase, and total progression through GSB and post-GSB phases, it is not required for sclerotization and tanning. These results demonstrate a new transient epidermis to neuron signaling event that facilitates progression through the cascade of pupariationassociated behaviors.Fil: Garelli, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidade Nova de Lisboa; PortugalFil: Heredia, Fabiana. Universidade Nova de Lisboa; PortugalFil: Volonté, Yanel Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidade Nova de Lisboa; PortugalFil: Pereirinha, Joana. Universidade Nova de Lisboa; PortugalFil: Casimiro, Andrea. Universidade Nova de Lisboa; PortugalFil: Viegas, Filipe. Universidade Nova de Lisboa; PortugalFil: Belém, Claudia. Universidade Nova de Lisboa; PortugalFil: Tanaka, Kohtaro. Instituto Gulbenkian de Ciências; PortugalFil: Cardoso, Gisela. Universidade Nova de Lisboa; Portugal. Universidade de Sao Paulo; BrasilFil: Macedo, Andre. Universidade Nova de Lisboa; PortugalFil: Leal, Ana. Universidade Nova de Lisboa; PortugalFil: Kotowicz, Malwina. Universidade Nova de Lisboa; PortugalFil: Prado Spalm, Facundo Heber. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Mendes, César. Universidade Nova de Lisboa; PortugalFil: Gontijo, Alisson M.. Universidade Nova de Lisboa; PortugalInsect Hormones 2019KolymbariGreciaBashirullah, ArashLeopold, Pierr
Correlations between pre-and post-fasting growth in Nile tilapia
No full textWe tested the correlation between growth rate before and after a food deprivation phase in twelve single held Nile tilapias, Oreochromis niloticus. The experiment was divided into three phases: Before food deprivation (phase 1: 15 d), food deprivation (phase 2: 12 d) and refeeding (phase 3: 15 d). The specific growth rate - SGR, food conversion efficiency - FCE and feed ingestion increased significantly during phase 3. Positive and significant correlations were found either to SGR or to feed ingestion between Phase 1 and 3 but not for FCE. The SGR on phase 3, moreover, were positively correlated to FCE and feed ingestion, while on phase 1 SGR was positively correlated to FCE only. Thus, high pre-fasting SGR or feed ingestion reflects in likewise high post-fasting SGR or feed ingestion values. Moreover, since SGR and FCE are correlated to each other in both phase 1 and 3, but phase 3 SGR is also correlated to feed ingestion; We could suppose that hyperphagic behaviour could be the main compensatory mechanism. Accordingly, we suggest that a fish with an elevated growth performance shall display a proportionally raised post-fasting growth response in order to normalize its predetermined growth trajectory and resume its normal growth rate. © GSP.Peer Reviewe
