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Cetartiodactyls include terrestrial and marine species, all generally endowed with a comparatively lateral position of their eyes and a relatively limited binocular field of vision. To this day, our understanding of the visual system in mammals beyond the few studied animal models remains limited. In the present study, we examined the primary visual cortex of Cetartiodactyls that live on land (sheep, Père David deer, giraffe); in the sea (bottlenose dolphin, Risso’s dolphin, long-finned pilot whale, Cuvier’s beaked whale, sperm whale and fin whale); or in an amphibious environment (hippopotamus). We also sampled and studied the visual cortex of the horse (a closely related perissodactyl) and two primates (chimpanzee and pig-tailed macaque) for comparison. Our histochemical and immunohistochemical results indicate that the visual cortex of Cetartiodactyls is characterized by a peculiar organization, structure, and complexity of the cortical column. We noted a general lesser lamination compared to simians, with diminished density, and an apparent simplification of the intra- and extra-columnar connections. The presence and distribution of calcium-binding proteins indicated a notable absence of parvalbumin in water species and a strong reduction of layer 4, usually enlarged in the striated cortex, seemingly replaced by a more diffuse distribution in neighboring layers. Consequently, thalamo-cortical inputs are apparently directed to the higher layers of the column. Computer analyses and statistical evaluation of the data confirmed the results and indicated a substantial correlation between eye placement and cortical structure, with a markedly segregated pattern in cetaceans compared to other mammals. Furthermore, cetacean species showed several types of cortical lamination which may reflect differences in function, possibly related to depth of foraging and consequent progressive disappearance of light, and increased importance of echolocation.

Arylalkylamine N-acetyltransferase (AANAT) plays a crucial role in synchronizing internal biological functions to circadian and circannual changes. Generally speaking, only one copy of AANAT gene has been found in mammals, however, three independent duplications of this gene were detected in several cetartiodactyl lineages (i.e., Suidae, Hippopotamidae, and Pecora), which originated in the middle Eocene, a geological period characterized with the increased climate seasonality. Lineage-specific expansions of AANAT and the associated functional enhancement in these lineages strongly suggest an improvement in regulating photoperiodic response to adapt to seasonal climate changes. In contrast, independent inactivating mutations or deletions of the AANAT locus were identified in the four pineal-deficient clades (cetaceans, sirenians, xenarthrans, and pangolins). Loss of AANAT function in cetaceans and sirenians could disrupt the sleep-promoting effects of pineal melatonin, which might contribute to increasing wakefulness, adapting these clades to underwater sleep. The absence of AANAT and pineal glands in xenarthrans and pangolins may be associated with their body temperature maintenance. The present work demonstrates a far more complex and intriguing evolutionary pattern and functional diversity of mammalian AANAT genes than previously thought and provides further evidence for understanding AANAT evolution as driven by rhythmic adaptations in mammals.

Cathelicidins are a family of innate immunity effectors present in most vertebrates. Cathelicidin-derived antimicrobial peptides play an essential role in host defence, with a direct antimicrobial action as part of the innate immune responses and the capacity to modulate cellular immunity, affecting both innate and adaptive responses. Some can also have protective roles connected with wound healing, angiogenesis or sepsis. Cathelicidins are characterized by a conserved pro-region, known as 'cathelin-like' domain, carrying a highly variable, C-terminal antimicrobial domain. Many mammalian species express only one cathelicidin, but artiodactyles are known to express up to a dozen, bearing structurally and functionally very diverse AMPs. These including small disulfide-stabilized cyclic peptides, extended linear peptides rich in specific residues (e.g. proline, arginine, phenylalanine, tryptophan) and helical peptides of different sizes. Cetaceans are phylogenetic with artiodactyls, so are also expected to express this wide repertoire of cathelicidin-derived AMPs, whose functional characteristics may however have been shaped by the particular pathogenic microbiota they face in their aquatic lifestyle. With the known artiodactyl peptides, these might provide interesting leads for biomedical application. My Ph.D. followed to two parallel lines of research: i) the study of cathelicidin AMPs identified in the dolphin Tursiops truncatus (bottlenose dolphin), identified by searching genomic databases, to define their antimicrobial activities and obtain information on the modes-of-action, using different types of functional assays. These studies were carried out in parallel with selected orthologues from other artiodactyls, such as cow, sheep or pig, to screen for differences among them, and revealed both convergences and some interesting differences, especially relating to a proline-rich (PR-AMP) and an -helical one. These studies established a potent antimicrobial activity for the helical peptide, with a remarkable capacity too inhibit bacterial growth also at sub-lethal concentrations. The PR-AMP instead showed an increased internalization capacity, due to its apparent efficient use of alternative transporters. These data provided interesting insights into specific aspects of Cetaceans cathelicidins in relation to those of other mammals, and lay the groundwork for the possible development of novel antimicrobial agents. ii) the possible application of the bovine cathelicidin-derived PR-AMP Bac7(1-35) as a vehicle for delivering antibiotic cargo into susceptible bacterial cells. This peptide acts intracellularly, entering cells using specific transport systems. The aim was to conjugate it to the antibiotic tobramycin and expand its capacity to penetrate into Gram-negative pathogens, possibly overcoming resistance mechanisms. I began to develop synthetic strategies for modifying the antibiotic and then linking it to the peptide vehicle. Functional assays showed that one type of construct had a broader spectrum of activity than the individual components, and in some cases was active against resistant strains. This new strategy may potentially be applied to other types or classes of currently available antibiotics, as long as they can be modified for conjugation without affecting activity.

Conserved noncoding sequences (CNSs) of vertebrates are considered to be closely linked with protein-coding gene regulatory functions. We examined the abundance and genomic distribution of CNSs in four mammalian orders: primates, rodents, carnivores, and cetartiodactyls. We defined the two thresholds for CNS using conservation level of coding genes; using all the three coding positions and using only first and second codon positions. The abundance of CNSs varied among lineages, with primates and rodents having highest and lowest number of CNSs, respectively, whereas carnivores and cetartiodactyls had intermediate values. These CNSs cover 1.3–5.5% of the mammalian genomes and have signatures of selective constraints that are stronger in more ancestral than the recent ones. Evolution of new CNSs as well as retention of ancestral CNSs contribute to the differences in abundance. The genomic distribution of CNSs is dynamic with higher proportions of rodent and primate CNSs located in the introns compared with carnivores and cetartiodactyls. In fact, 19% of orthologous single-copy CNSs between human and dog are located in different genomic regions. If CNSs can be considered as candidates of gene expression regulatory sequences, heterogeneity of CNSs among the four mammalian orders may have played an important role in creating the order-specific phenotypes. Fewer CNSs in rodents suggest that rodent diversity is related to lower regulatory conservation. With CNSs shown to cluster around genes involved in nervous systems and the higher number of primate CNSs, our result suggests that CNSs may be involved in the higher complexity of the primate nervous system.

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: ( i ) Odontocetes are monophyletic; ( ii ) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; ( iii ) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and ( iv ) the rapid radiation of extant cetacean lineages occurred some 28–33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.