Biology subjects

Fonseca, J. P.

Publications and source records attributed to Fonseca, J. P..

5 recordsLinked to original sources

bioRxiv2021

Ribosomal DNA revealed an extensive role of allopolyploidy in the radiation of Ulex L.

We studied the phylogeny of Ulex L., a genus of spiny legumes, which its center of diversity in the Iberian Peninsula, using ribosomal DNA markers (rDNA), namely ETS, 5.8S and ITS (45S), and 5S intergenic spacer regions. One of the main findings was the presence of very different haplotypes in 5S-IGS genes and, to a less extent, in ETS and ITS, in seven polyploid taxa. We interpreted these results as an indication of hybrid origins and proposed allopolyploidy for U. argenteus ssp. subsericeus, U. australis ssp. australis, U. australis ssp. welwitschianus, U. densus, U. europaeus ssp. europaeus, U. europaeus ssp. latebracteatus, and U. jussiaei. These results reinforce an early hypothesis which stated that the radiation of Ulex occurred mainly by polyplodization. Phylograms showed two main clades, one grouping the hydrophilic U. gallii, U. breoganii and U. minor, and the other grouping the southern, xerophytic, taxa. The putative allopolyploids showed haplotypes, which grouped in both clades, indicating that allopolyploidy, occurred through hybridization from these hydrophilic and xerophytic lineages. The phylogenetic position of U. micranthus is not certain and it is discussed. The 5S-IGS showed to retain more polymorphisms than ETS gene or ITS markers. This result is compatible with the hypothesis that 5S rDNA region is less vulnerable to inter-loci concerted evolution than 45S, providing a more suitable marker for reconstructing histories of allopolyploid species. We discuss the taxonomic consequences of these results.

molecular biology↗

bioRxiv2020

TuNR: Orthogonal Control of Mean and Variability of Endogenous Genes in a Human Cell Line

Stochastic fluctuations at the transcriptional level contribute to isogenic cell-to-cell heterogeneity in mammalian cell populations. However, we still have no clear understanding of the repercussions of this heterogeneity, given the lack of tools to independently control mean expression and variability of a gene. Here, we engineered a synthetic circuit to independently modulate mean expression and heterogeneity of transgenes and endogenous human genes. The circuit, a Tunable Noise Rheostat (TuNR), consists of a transcriptional cascade of two inducible transcriptional activators, where the output mean and variance can be modulated by two orthogonal small molecule inputs. In this fashion, different combinations of the inputs can achieve the same mean but with different population variability. With TuNR, we achieve low basal expression, over 1000-fold expression of a transgene product, and up to 7-fold induction of the endogenous gene NGFR. Importantly, for the same mean expression level, we are able to establish varying degrees of heterogeneity in expression within an isogenic population, thereby decoupling gene expression noise from its mean. TuNR is therefore a modular tool that can be used in mammalian cells to enable direct interrogation of the implications of cell-to-cell variability.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2020

PDE-constrained optimization for estimating population dynamics over cell cycle from static single cell measurements

MotivationUnderstanding how cell cycle responds and adapts dynamically to a broad range of stresses and changes in the cellular environment is crucial for the treatment of various pathologies, including cancer. However, measuring changes in cell cycle progression is experimentally challenging, and model inference computationally expensive. ResultsHere, we introduce a computational framework that allows the inference of changes in cell cycle progression from static single-cell measurements. We modeled population dynamics with partial differential equations (PDE), and derive parameter gradients to estimate time- and cell cycle position-dependent progression changes efficiently. Additionally, we show that computing parameter sensitivities for the optimization problem by solving a system of PDEs is computationally feasible and allows efficient and exact estimation of parameters. We showcase our framework by estimating the changes in cell cycle progression in K562 cells treated with Nocodazole and identify an arrest in M-phase transition that matches the expected behavior of microtubule polymerization inhibition. ConclusionsOur results have two major implications: First, this framework can be scaled to high-throughput compound screens, providing a fast, stable, and efficient protocol to generate new insights into changes in cell cycle progression. Second, knowledge of the cell cycle stage- and time-dependent progression function allows transformation from pseudotime to real-time thereby enabling real-time analysis of molecular rates in response to treatments. AvailabilityMAPiT toolbox (Karsten Kuritz 2020) is available at github: https://github.com/karstenkuritz/MAPiT.

systems biology↗

bioRxiv2019

Biphasic Response of Protein Kinase A to Cyclic Adenosine Monophosphate Triggers Distinct Epithelial Phenotypes

Despite the large diversity of the proteins involved in cellular signaling, many intracellular signaling pathways converge onto one of only dozens of small molecule second messengers. Cyclic adenosine monophosphate (cAMP), one of these second messengers, is known to regulate activity of both Protein Kinase A (PKA) and the Extracellular Regulated Kinase (ERK), among other signaling pathways. In its role as an important cellular signaling hub, intracellular cAMP concentration has long been assumed to monotonically regulate its known effectors. Using an optogenetictool that can introduce precise amounts of cAMP in MDCKI cells, we identify genes whose expression changes biphasically with monotonically increasing cAMP levels. By examining the behavior of PKA and ERK1/2 in the same dose regime, we find that these kinases also respond biphasically to increasing cAMP levels, with opposite phases. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge to inhibit PKA. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, influencing the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational modeling, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.

systems biology↗

bioRxiv2018

A Toolkit for Rapid Modular Construction of Biological Circuits in Mammalian Cells

The ability to rapidly assemble and prototype cellular circuits is vital for biological research and its applications in biotechnology and medicine. Current methods that permit the assembly of DNA circuits in mammalian cells are laborious, slow, expensive and mostly not permissive of rapid prototyping of constructs. Here we present the Mammalian ToolKit (MTK), a Golden Gate-based cloning toolkit for fast, reproducible and versatile assembly of large DNA vectors and their implementation in mammalian models. The MTK consists of a curated library of characterized, modular parts that can be easily mixed and matched to combinatorially assemble one transcriptional unit with different characteristics, or a hierarchy of transcriptional units weaved into complex circuits. MTK renders many cell engineering operations facile, as showcased by our ability to use the toolkit to generate single-integration landing pads, to create and deliver libraries of protein variants and sgRNAs, and to iterate through Cas9-based prototype circuits. As a biological proof of concept, we used the MTK to successfully design and rapidly construct in mammalian cells a challenging multicistronic circuit encoding the Ebola virus (EBOV) replication complex. This construct provides a non-infectious biosafety level 2 (BSL2) cellular assay for exploring the transcription and replication steps of the EBOV viral life cycle in its host. Its construction also demonstrates how the MTK can enable important and time sensitive applications such as the rapid testing of pharmacological inhibitors of emerging BSL4 viruses that pose a major threat to human health.

synthetic biology↗