Like most organisms, Drosophila melanogaster can synchronize its physiological and behavioural processes by possessing internal circadian clock that regulates. Naturally fluctuating timing cues, like light and temperature (also known as Zeitgebers), synchronize these endogenous and self‐sustained clocks with external time. In Drosophila, synchronization of the circadian clock by light has been studied in detail, but much less is known about the molecular mechanisms underlying temperature entrainment. Previous data from our lab shows that Nocte, a Chordotonal organ (Ch organ) located protein, is required for normal temperature entrainment in Drosophila. However, neither the function of Nocte in temperature entrainment nor the molecular underlying mechanisms are clear. To address these issues, a proteomics strategy of combing co‐immunoprecipitation and MS/MS sequencing was applied to isolate potential interactors of Nocte. IR25a was one of the most promising candidates, which was later confirmed by behavioural tests using RNA interference: Reducing IR25a expression in Chorgan resulted in abnormal behaviour during temperature cycles, similar to what had been described for Nocte mutant. To further confirm the interaction between Nocte and IR25a, I showed that IR25a physically interacts with Nocte in vivo. Moreover, using an IR25a‐gal4 line, I was able to show that IR25a is expressed in subsets of chordotonal organs (Ch organ) including Johnston's Organs (JO), where Nocte is also highly expressed. These results, along with the behavioural data mentioned above are consistent with the proteomics results and suggest that Nocte and IR25a physically and functionally interact. IR25a mutants were employed to further investigate the function of IR25a in temperature entrainment. First of all, I found that both central and peripheral clocks in wild type flies can be synchronized to temperature cycles with only two degree differences (12h: 12h, 27 °C: 25 °C). In contrast, synchronization of locomotor activity rhythms in the IR25a null mutants to the same temperature cycles and other TC's with 2°C amplitude was eliminated. Under the same conditions, the oscillations of the core clock proteins TIMLESS (TIM) and PERIOD (PER) that normally occur in fly heads were completely abolished inIR25a null mutants, suggesting that IR25a is required for temperature entrainment of peripheral clocks. In the central brain pacemaker neurons, the oscillations of TIM in dorsal and lateral neurons were also affected by the IR25a mutants. On the contrary, IR25a is not required for light entrainment and temperature compensation, suggesting that IR25a is specifically involved in temperature synchronization. Moreover, temperature entrainment of the IR25a null mutants can be partially restored by applying larger temperature intervals (29°C: 25°C) indicating that IR25amay function as amplitude detector independent of absolute temperature values. Finally, neuronal activity in IR25a+ neurons is crucial for the synchronization of circadian clocks to low amplitude temperature cycles. Re‐constitution of functional olfactory receptors required the assembly of IR25a with IR76a and IR76b. Interestingly, IR76a and IR76b are neither required for temperature entrainment at the behavioural level nor expressed in the Ch organs. To check if other potential IRs interacting with IR25a exist, I screened the expression pattern of most divergent IRs using IR‐gal4/UAS‐GFP flies. IR56a was isolated as a potential partner of IR25a because it is also expressed in the femur chordotonal organs. To investigate the function of IR56a in temperature entrainment, I generated a null mutant of IR56a. Surprisingly, this gene is not required for synchronizing clocks to a temperature cycle (27°C: 25°C) at the behavioural level. However, the behavioural and molecular phenotypes of IR56a mutant under different temperature cycles need to be further characterized.

Hybrid zones are regions where genetically differentiated forms come together and exchange genes through hybrid offspring. The study of characters gradually changing across such zones (clines) can give insight into evolutionary processes, providing exceptionally sensitive estimates of the intensity of selection, and allowing the detection of loci that might be involved in reproductive isolation and speciation. The Alpine grasshopper Podisma pedestris has a hybrid zone in Southern France where two populations meet. They differ in their sex chromosome system, and strong selection against hybrids is observed. These distinct populations likely have split and re-joined several times during the Quaternary glacial cycles. A model explaining the selection observed against hybrids postulates hundreds of loci of small effect spread over two differentiated genomes meeting in secondary contact. Yet, over 50 years of study to-date non have been discovered. However, so far the study of P. pedestris has not made use of high-throughput sequencing data which provides an unprecedented resolution of molecular markers. I am aiming to close the gap with this thesis. I assemble the grasshopper's mitochondrial genome sequence and infer what proportion of its genome is made up by mitochondrial inserts (Numts). Using transcriptome data from two individuals, I then go on to fit demographic models, finding the populations split approximately 400 000 years ago and that the current-day population sizes are considerably smaller than the ancestral one. The final data chapter explores the genetic architecture of the hybrid zone using data from a targeted sequence capture of hundreds of loci covering some 10 000 polymorphic sites. Only two loci under selection are identified, which is surprising given the power of the analysis. Both loci are located on the X chromosome and are subject to weak selection (0.3% and 0.03%). This shows the power of hybrid zone analysis to infer targets of selection. The results are discussed in light of a theoretical chapter on the 'inexorable spread' phenomenon and lead to the proposal for further research into the causes of the reproductive isolation observed between the grasshopper populations.

The barn swallow (Hirundo rustica) is represented by six subspecies and has two well-studied ornamental traits, tail length and ventral plumage colour, which vary geographically among subspecies. Sexual selection on these traits has been suggested to drive speciation. The European subspecies rustica has pale ventral feathers and long tail streamers, and females prefer males with longer tail streamers. The North American erythrogaster has shorter tails and red ventral plumage and their females use redness of ventral plumage as a mate choice cue. In the Middle East, the subspecies transitiva bears long tail streamers and red ventral feathers, both of which have been suggested to show male attractiveness. The Asian subspecies gutturalis has a pale belly with short but dimorphic tails. Studies in Japanese populations have suggested that the white spot on the tail feathers and throat patch are sexually selected in males, but this explanation leaves the dimorphism of tail streamers unexplained. To further investigate the sexually selected traits of gutturalis, especially the role tail streamers might play, I studied a population of the barn swallow gutturalis in China between 2013 and 2015, and conducted a partial cross-fostering experiment in 2015. My data indicate that male tail streamers are sexually selected in the gutturalis population in China. Longer-tailed males (with deeper fork tails) were cuckolded less frequently and had a higher reproductive success, they also had mates that invested more in parental care and had a higher total peak body mass of offspring. Both male and female body condition (body mass and tarsus length) influenced offspring growth. Males with larger body mass initiated breeding earlier and their offspring had a larger body mass on day seven after hatching and grew faster as determined by a cross-fostering experiment. Larger females reared offspring that grew faster and reached a higher peak body mass both in original nests and nests with cross-fostered nestlings. Based on feeding rate observations and the cross-fostering experiment, it seems that females obtain indirect benefits rather than direct benefits from mating with more attractive males.

Previously it has been shown that Rsp5p, a member of Nedd4 ubiquitin ligases in yeast, is modified by the ubiquitin-like protein SUMO and that this modification is performed by Siz1p, a member of PIAS SUMO ligases that are in turn substrates of Rsp5p-dependent ubiquitylation, thus defining a previously unidentified system of crosstalk between the ubiquitin and SUMO systems in yeast. This project aims to identify whether similar crosstalk pattern exists in human cells. In vitro ubiquitylation assays showed that some of the human Nedd4 family members (Nedd4.1, Nedd4.2, WWP1) are capable of ubiquitylating the human SUMO ligase PIAS3, while in contrast, Smurf2 does not appear to be able to modify this protein. This modification is partially WW-PY-motif-dependent as ubiquitylation level of PIAS3 mutants with altered PY motifs conducted by Nedd4.1 or Nedd4.2 was reduced, but not completely disrupted. Interestingly, in vitro SUMOylation assay revealed that Nedd4.1 is SUMOylated even in the absence of SUMO E3 ligases and an apparent interaction between the SUMO E2 (Ubc9) and Nedd4.1 was observed both in vitro and in vivo. I show that auto- SUMOylation of Nedd4.1 is accompanied with the formation of thioester-linked conjugates between Nedd4.1 and SUMO, but these do not involve cysteine residues (C867, C778, and C627) within the HECT domain itself and is not occurring at a predicted SUMOylation consensus site (K357). Furthermore, I have shown that Nedd4.1 and SUMO1/2 colocalize in HeLa cells, and that overexpression of epitope tagged Nedd4 and SUMO1/2, followed by denaturing pull-downs demonstrates that both Nedd4.1 and Nedd4.2 can be SUMOylated in vivo. Meanwhile, I have generated a SUMO trap based on SUMO interacting motifs (SIMs) and confirmed its ability of capturing SUMOylated proteins both in vivo and in vitro. Its use reveals that Nedd4 SUMO conjugates could be captured by SUMO trap when Nedd4 and SUMO were co-expressed in HeLa cells, again confirming Nedd4.1 as a substrate for SUMO1 or SUMO2. In conclusion, I show that SUMOylation of Nedd4.1 does exist in HeLa cells, and on the other hand, some of Nedd4 family members are responsible for PIAS3 ubiquitylation in vitro, providing evidence of a crosstalk between Nedd4 family of ubiquitin ligases and PIAS family of SUMO ligases in mammals.

The controlled organization of individual molecules and nanostructures with nanoscale accuracy is of great importance in the investigation of single-molecule events in biological and chemical assays, as well as for the fabrication of the next generation optoelectronic devices. In this regard, the precise patterning of individual molecules into hierarchical structures has attracted substantial research interest in recent years. DNA has been shown to be an ideal structural material for this purpose, due to the specificity of its programmability and outstanding chemical flexibility. DNA origami can display a high degree of positional and precise binding sites, allowing for complex arrangements and the assembly of different nanoscale architectures. In this project, we present a novel platform based on the use of DNA scaffolds for the organization of individual nanomoieties (with nanoscale spatial control), and their selective immobilisation on surfaces for single-molecule investigations. In particular, semiconductor quantum dots (QDs), fluorescence molecules, linear small peptides, and structural proteins were tethered with single-molecule accuracy on DNA origami; their subsequent organization in array configuration on nanopatterned surfaces allowed us to fabricate and test different platforms for single-molecule studies. In particular, we developed a Focused Ion Beam (FIB) nanofabrication strategy and demonstrated its general applicability for the assembly of functionalised DNA nanostructures in highly uniform nanoarrays, with single-molecule control. In addition, we further explored this nanofabricated platform for biological investigations at the single-molecule level, from protein-DNA interactions to cancer cell adhesion studies with single-molecule control. Investigations have been carried out via fluorescence microscopy, scanning electron microscopy (SEM), Focused Ion Beam (FIB) and atomic force microscopy (AFM). By and large, combining the programming ability of DNA as a scaffolding material with a one-step lithographic process, we have developed a platform of general applicability for the fabrication of nanoscale chips that can be employed in a variety of single-molecule investigations.

Sexual selection is ubiquitous in all sexually reproducing species and a powerful evolutionary force. The effect of sexual selection on population fitness has caused wide debate and has been proposed to both enhance adaptation rates, but also possibly increase extinction risk. Using experimental evolution, the strength of sexual selection was altered by biasing adult sex ratios in replicated populations of the Indian meal moth, Plodia interpunctella. Under increasing temperatures strong sexual selection and polyandry was associated with increased population fitness. Suggesting sexual selection could provide a buffer against climate change by increasing adaptation rates. However, no effect on male mating success was observed. In contrast, under stable temperatures male mating success was increased by strong sexual selection, however, this did not translate to increased population fitness. These results indicate that female choice is a potentially strong selective pressure in P. interpunctella. Moreover, under stabilising and directional selection the costs and benefits of sexual selection change. In a field study, dung beetle species richness and abundance were compared across a gradient of habitat disturbance, ranging from old-growth rainforest to oil palm plantation. Species persistence within altered habitats was positively associated with expressing horns and relative horn lengths, suggesting male-male competition increases a species ability to persist within modified landscapes. There was not a strong effect of relative testes mass or sexual size dimorphism on the abundance of species. Additionally, by examining the expression of sexually selected traits within species, horn length and testes mass appear to be condition dependent, but only the expression of horns was effected by habitat change. Overall, from both laboratory and field studies it was found that sexual selection can increase adaptation rates and the persistence of species within altered and changing environments. Moreover, both sets of studies suggest pre-copulatory sexual selection to be an important aspect of sexual selection in driving this adaptation.

The clinical consequences of ionizing radiation exposure remain one of the leading causes of death in the United States. Much research has been carried out to discover a potential countermeasure for acute radiation syndrome (ARS) without success. The United States Food and Drug Administration (US FDA) has not accepted any effective and harmless ionizing radiation therapy agents (radioprotectors) for treating ARS. It has recently been discovered that g-tocotrienol (GT-3), one of the E vitamers chiefly present in palm oil, has radioprotective abilities in mice and nonhuman primate (NHP) models. Though GT-3 is one of the most promising countermeasures discovered, the separation and purification from other vitamers or its matrix is difficult. This has limited its characterization, derivatization, and biomedical application. We have therefore designed novel chromatographic methods to optimize separation and purification. Thin layer chromatography (TLC) was used to ascertain the best solvent system for column chromatography (CC). Exactly 8% ethyl acetate in hexane employed in TLC and CC resulted in good separation (Rf ≥ 0.3) and purification. Various fractions presumed to contain GT-3 were collected and analyzed to confirm the exact structure using 1H NMR, 13C NMR, DEPT, and GC-MS. Results obtained so far have revealed the exact structure of the compound. However, some traces of impurities have been indicated by the NMR outcomes; therefore, high-performance liquid chromatography (HPLC) will be used to maximize GT-3 purification. This present study will be instrumental in elucidating the biochemical structure of various complex plant bioactive components that are hard to isolate and analyze. It is envisioned that this work will help to erase the knowledge deficit in medicinal chemistry and assist in the development of new medications for ARS.

Atherosclerotic disease is characterized by the formation of lipid-ladden plaques in artery walls. During later stages of disease, these plaques become calcified by mechanisms involving the trans-differentiation of vascular smooth muscle cells (VSMC) to osteoblast-like cells. Although vascular calcification was thought to be a passive mechanism, evidence shows that this process is heavily modulated by various cell signaling mechanisms, including CB2 endocannabinoid receptors. Previous studies have shown that known CB2 antagonists accelerate VSMCs trans-differentiation to an osteoblast-like phenotype, indicating that this receptor serves an anti-calcification signal. The goal of this investigation is to determine if a series of 11-[2-arylmethylene)hydrazono]-PBD derivatives with established CB2 binding affinity function as CB2 antagonists or agonists in a cell culture model of VSMC osteoblastic trans-differentiation. MOVAS cells were grown in standard media or osteogenic media (to induce trans-differentiation) supplemented with and without the various PBD derivatives. Following the treatment period, the extent of osteoblast-like activity was evaluated by alizarin red staining for calcium deposition. To quantify the staining present, the dye was extracted using cetylpyridinium chloride hydrate solution and then analyzed via UV-Vis spectroscopy at 570 nm. The ability of the derivatives to modulation of osteoblastic transdifferentiation of MOVAS cells was further evaluated by performing Western blot analysis for expression of Runx2, an essential transactivator of osteoblast differentiation. Results of this work determined that some of the PBD derivatives increased the calcification compared to the control, indicating that they likely act as CB2 receptor antagonists, while others decreased calcification compared to the control, indicating that they likely act as CB2 receptor agonists. Not only do these results characterize the interactions of these compounds with CB2 receptors, they demonstrate that these PBD derivatives have biological activity. These results also further implicate CB2 receptors as a regulator of VSMC cell calcification, which could lead to novel drug therapies for the treatment of atherosclerotic plaques.