Targeting the heart:extracellular vesicles and beyond

Extracellular vesicles are cell-derived tiny particles that naturally transport different molecules including RNA between cells, inducing a response in the receiving cell. This thesis focused on the use of extracellular vesicles for cardiac applications by manipulating their content and favouring their accumulation in the heart. This research showed that these vesicles can be enriched with long non-coding RNAs, molecules known to play important roles in the heart. They were further shown to transport them to different cells that are usually present in the heart. However, once intravenously injected in animals, these vesicles do not accumulate in the heart, limiting their use for cardiac therapies. Finally, we proposed the use of a transporter that would degrade in response to an external stimulus and locally release the vesicles loaded on it. With this non-invasive system we were able to double the amount of vesicles that reach and accumulate in the heart, paving the way for improved vesicle-based treatments

Complete mitochondrial genome of the Florida manatee (Trichechus manatus latirotris, Sirenia)

The Florida manatee (Trichechus manatus latirostris) is an endangered subspecies of the West Indian manatee (T. manatus), which inhabits inland and marine waters of southeastern United States. In this study, we assembled the mitochondrial genome (mtDNA) of the Florida manatee from whole genome shotgun reads. As a result, we show that the currently annotated T. manatus mtDNA belongs to a different species, the Amazonian manatee (T. inunguis). The newly assembled Florida manatee mtDNA is 16,881 bp in length, with 13 protein-coding genes, two ribosomal RNAs (rRNAs), 22 transfer RNAs (tRNAs) and one non-coding control region (D-loop). Phylogenetic analysis based on the control region indicates the newly assembled mtDNA is haplotype A01, characteristic of T. m. latirostris, while the current mtDNA associated with the Florida manatee genome assembly has a Ti02 haplotype that is found in Amazonian manatees and hybrids

High prevalence of subclinical frog virus 3 infection in freshwater turtles of Ontario, Canada

Ranaviruses have been associated with chelonian mortality. In Canada, the first two cases of ranavirus were detected in turtles in 2018 in Ontario, although a subsequent survey of its prevalence failed to detect additional positive cases. To confirm the prevalence of ranavirus in turtles in Ontario, we used a more sensitive method to investigate if lower level persistent infection was present in the population. Here we report results via a combination of qPCR, PCR, Sanger sequencing and genome sequencing from turtles from across Ontario, with no clinical signs of illness. We found 2 positives with high viral load and 5 positives with low viral load. Histopathology found subtle histological changes. DNA sequences identified two types of frog virus 3 (FV3), and genome sequencing identified a ranavirus similar to wild-type FV3. Our results show that the virus has been present in Ontario's turtles as subclinical infections

Population-specific signatures of intra-individual mitochondrial DNA heteroplasmy and their potential evolutionary advantages

Heteroplasmy is the existence of more than one mitochondrial DNA (mtDNA) variant within a cell. The evolutionary mechanisms of heteroplasmy are not fully understood, despite being a very common phenomenon. Here we combined heteroplasmy measurements using high throughput sequencing on green turtles (Chelonia mydas) with simulations to understand how heteroplasmy modulates population diversity across generations and under different demographic scenarios. We found heteroplasmy to be widespread in all individuals analysed, with consistent signal in individuals across time and tissue. Significant shifts in haplotype composition were found from mother to offspring, signalling the effect of the cellular bottleneck during oogenesis as included in the model. Our model of mtDNA inheritance indicated that heteroplasmy favoured the increase of population diversity through time and buffered against population bottlenecks, thus indicating the importance of this phenomenon in species with reduced population sizes and frequent population bottlenecks like marine turtles. Individuals with recent haplotypes showed higher levels of heteroplasmy than the individuals with ancient haplotypes, suggesting a potential advantage of maintaining established copies when new mutations arise. We recommend using heteroplasmy through high throughput sequencing in marine turtles, as well as other wildlife populations, for diversity assessment, population genetics, and mixed stock analysis

Spatial and temporal distribution of mitochondrial lineages in the European wild boar

In many terrestrial species, the geographic distribution of DNA lineages was heavily affected by the climatic fluctuations that occurred during the Quaternary, although the impact of human populations, especially on harvested species, might have confounded the pattern in more recent times. In game species such signals are expected to be overshadowed by the effects of more recent management (local extinctions, introductions, translocations). Past global climate changes had a profound impact on the evolutionary history of many species and left a genetic signature which is often still detectable nowadays. The wild boar is one the most widely distributed terrestrial animals, naturally occurring from Western Europe and Northern Africa to Japan. Previous studies in European animals suggested that Iberia, Balkans and Italy played a major role as refugia during the Last Glacial Maxima (LGM), and these three areas were the main responsible for the recolonization of European continent after the LGM. This PhD dissertation investigated the geography of the genetic diversity of the European wild boar (Sus scrofa). It aims to infer how past climate influenced the current observed pattern of genetic diversity distribution of the mitochondrial DNA. A comprehensive dataset of more than 700 sequences from the hypervariable region of the mitochondrial DNA from 74 populations, covering the whole European continent (from Portugal to Russia) was used. The results confirmed the existence of a ubiquitous mitochondrial clade in Europe (E1), a highly differentiated clade exclusive of Italy (E2) and East Asian haplotypes (A) at very low frequencies, presumably introgressed from commercial pig breeds. No Near Eastern haplotype was detected. A clear south-north decreasing gradient of diversity was observed, with maximum levels hosted in presumed glacial refugia. Seven structured groups were found, with distributions highly correlated to geography. Population size within groups was generally constant through time, with only slight recent changes. Sardinia was the most differentiated population, with 64% private haplotypes. A surprising similarity between western (Iberia) and eastern Europe was observed, while central populations showed a higher affinity to the Italian gene pool. Notably, a monophyletic subclade, was shared at high frequencies by Central European (France and Germany) and Italian wild boars. To predict the distribution of the wild boar during the LGM and its relationship with the current distribution of the genetic diversity a maximum entropy method of niche modeling was used. The climate models predicted that Iberia, Balkans and Italy had great habitat suitability during the LGM, serving as refugia. Most part of Europe north to 45°N was unsuitable to the species. Italy and Iberia showed greater suitability when compared to other regions of Europe, which can indicate that these two areas may have retained large population size even during cold periods. The suitability map and the current distribution of the genetic diversity exhibited similar patterns, with northern areas showing low suitability and lower genetic diversity, while southern regions had higher values of suitability and genetic diversity. The current distribution of genetic diversity was highly influenced by past climate events, especially related to the LGM. Post-LGM colonization of the continent from southern refugia can explain the current phylogeographic patterns in the European wild boar. More recent changes to population size and gene flow could have acted more at a local scale, without a great impact on the global framework

New Genetic Insights About Hybridization and Population Structure of Hawksbill and Loggerhead Turtles From Brazil

An extremely high incidence of hybridization among sea turtles is found along the Brazilian coast. This atypical phenomenon and its impact on sea turtle conservation can be elucidated through research focused on the evolutionary history of sea turtles. We assessed high-quality multilocus haplotypes of 143 samples of the 5 species of sea turtles that occur along the Brazilian coast to investigate the hybridization process and the population structure of hawksbill (Eretmochelys imbricata) and loggerhead turtles (Caretta caretta). The multilocus data were initially used to characterize interspecific hybrids. Introgression (F2 hybrids) was only confirmed in hatchlings of F1 hybrid females (hawksbill × loggerhead), indicating that introgression was either previously overestimated and F2 hybrids may not survive to adulthood, or the first-generation hybrid females nesting in Brazil were born as recent as few decades ago. Phylogenetic analyses using nuclear markers recovered the mtDNA-based Indo-Pacific and Atlantic lineages for hawksbill turtles, demonstrating a deep genetic divergence dating from the early Pliocene. In addition, loggerhead turtles that share a common feeding area and belong to distinct Indo-Pacific and Atlantic mtDNA clades present no clear genetic differentiation at the nuclear level. Finally, our results indicate that hawksbill and loggerhead rookeries along the Brazilian coast are likely connected by malemediated gene flow

Discovery and implementation of a novel pathway for n-butanol production via 2-oxoglutarate

Background: One of the European Union directives indicates that 10% of all fuels must be bio-synthesized by 2020. In this regard, biobutanol - natively produced by clostridial strains - poses as a promising alternative biofuel. One possible approach to overcome the difficulties of the industrial exploration of the native producers is the expression of more suitable pathways in robust microorganisms such as Escherichia coli. The enumeration of novel pathways is a powerful tool, allowing to identify non-obvious combinations of enzymes to produce a target compound. Results: This work describes the in silico driven design of E. coli strains able to produce butanol via 2-oxoglutarate by a novel pathway. This butanol pathway was generated by a hypergraph algorithm and selected from an initial set of 105,954 different routes by successively applying different filters, such as stoichiometric feasibility, size and novelty. The implementation of this pathway involved seven catalytic steps and required the insertion of nine heterologous genes from various sources in E. coli distributed in three plasmids. Expressing butanol genes in E. coli K12 and cultivation in High-Density Medium formulation seem to favor butanol accumulation via the 2-oxoglutarate pathway. The maximum butanol titer obtained was 85 \ub1 1 mg L-1 by cultivating the cells in bioreactors. Conclusions: In this work, we were able to successfully translate the computational analysis into in vivo applications, designing novel strains of E. coli able to produce n-butanol via an innovative pathway. Our results demonstrate that enumeration algorithms can broad the spectrum of butanol producing pathways. This validation encourages further research to other target compounds