Keywords: EGFP Enhanced Green Fluorescent Protein, FIGARO Fluid Interfaces Grazing Angles ReectOmeter, Hox homeodomain, ILL Institut Optical Density check during E, coli cell culture

ACMW air contrast matched water. AMP ampicillin. BAM brewster angle microscopy. BEs backscattered high-energy electrons. BSA bovine serum albumin. CLAMF chloramphenicol. DNA deoxyribonucleic acid. DTT DithioThreitol.

LIST OF FIGURES 2.1

Area of expression in embryo and adult life stages are shown.

Reproduced from reference [START_REF] Hughes | Hox genes and the evolution of the arthropod body plan[END_REF]. . . . . . . . . . . . . . . . . .

2.2

Hierarchical structures in brillar collagen showing a characteristic periodic structure forming various hierarchical orders of association: bers, brils and microbrils with three helical chains. Collagen ber diameter is from 1 to 20 µm. . . . . . .

3.2

Phospholipid of the plasma membrane with a hydrophilic head and two hydrophobic tails. . . . . . . . . . . . . . . . . . . . .

3.3

Scheme of Wilhelmy plate partially immersed in a water subphase where t is the plate thickness, θ is the contact angle, w is the plate width, h is the immersed depth and l is the plate length. In the inset there's the scheme of the forces acting on subphase molecules at the air-water interface. . . . . . . . . . 3.12 Interaction volume of an electron beam with a thick sample. .

Scattering length for dierent elements. The solid squares

indicate values for isotopes.Reproduced from reference [START_REF] Price | Introduction to Neutron Scattering[END_REF]. .

3.14 (a) -Reection of an incident beam from an ideally at interface. ki and kr are the incident and scattered wave vectors, with angles θ i = θ r = θ in the incidence plane; q is the wave vector transfer; and SLD (scattering length density) is the scattering length density of the semi-innite substrate. (b) -Calculated neutron reectivity prole for a beam incident on a silicon substrate where the surface is perfectly smooth. Reproduced from reference [START_REF] Russell | X-ray and neutron reectivity for the investigation of polymers[END_REF].k z,0 is equal to the magnitude of the incident (and reected) wavevector in air. . . . . . . . .

INTRODUCTION

Protein-based materials are currently the subject of intense research interest since they have an extended range of potential applications, such as improved bio-membrane biocompatibility for implanted medical devices and the creation of platform materials for novel biosensors and tissue engineering. In order to achieve these ob jectives, it is important to develop materials with a broad range of structural, mechanical, and functional properties optimized for each application. Self-assembly biopolymers are often used to generate the desired biomaterials with specic structure and application [START_REF] Huang | Functionalization and patterning of protein-based materials using active ultrabithorax chimeras[END_REF][START_REF] Nilebäck | Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism[END_REF][START_REF] Zhao | On the distribution of protein refractive index increments[END_REF]. For example, spider silk has a tensile strength comparable to the structural steel one, elastin-based materials are extremely extensible and with collage it is possible to produce 3D gels [START_REF] Li | Molecular basis for the extensibility of elastin[END_REF][START_REF] Wang | Eects of Hydrogel Stiness and Extracellular Compositions on Modulating Cartilage Regeneration by Mixed Populations of Stem Cells and Chondrocytes in Vivo[END_REF][START_REF] Koebley | Toughness-enhancing metastructure in the recluse spider's looped ribbon silk[END_REF]. A peculiar property of protein-based material, is the potential to incorporate other proteins with their several properties, into the material with a simple molecular engineering [START_REF] Huang | Functionalization and patterning of protein-based materials using active ultrabithorax chimeras[END_REF][START_REF] Jansson | Recombinant spider silk genetically functionalized with anity domains[END_REF][START_REF] Girotti | Recombinant Technology in the Development of Materials and Systems for Soft-Tissue Repair[END_REF].

A subset of proteins naturally self-assembles into useful materials. Common examples include the already cited silks and collagen but the list of molecular proteins is in constant expansion creating novel proteins with the property to form nano-scale scaolds. In the present dissertation we will briey discuss the properties of silk and collagen materials. However, among all of them Ultrabithorax-based material will be used in the present work, due to its properties. Ubx protein is a Hox transcription factor of Drosophila melanogaster fruit y that functions by binding DNA (deoxyribonucleic acid) to activate or repress transcription processes [START_REF] Liu | Multiple intrinsically disordered sequences alter DNA binding by the homeodomain of the Drosophila Hox protein ultrabithorax[END_REF][START_REF] Liu | Internal Regulatory Interactions Determine DNA Binding Specicity by a Hox Transcription Factor[END_REF]. In addition to its in vivo function, monomers from Ubx are known to spontaneously self-assemble at an air-water interface to form a monolayer, which has then been used as a basis for forming biopolymeric bers and lms. During this spontaneous self-assembly, nanoscopic aggregates of Ubx are formed that gradually assemble into protobrils [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF]. Lateral association among protobrils generates a surface lm that can be pulled into microscale bers or lifted o as lms [START_REF] Huang | The eect of genetically engineered spider silk-dentin matrix protein 1 chimeric protein on hydroxyapatite nucleation[END_REF]. The potential applications of this biopolymer have been enhanced by using an E. coli expression system to obtain puried fusion proteins that also self-assemble into biopolymeric membranes. Such fusion proteins utilize the Ubx portion for material formation while the biomolecule remains in an active conformation in the membranes. Functionalised Ubx-based materials have potential impact in numerous biomedical applications including wound healing, biocompatibility coatings and environmental sensing [START_REF] Rosenbloom | Extracellular matrix 4: the elastic ber[END_REF][START_REF] Wagenseil | Vascular Extracellular Matrix and Arterial Mechanics[END_REF].

In Following previous works on other types of protein lms [START_REF] Crawford | Serum albumin in 2D: A Langmuir monolayer approach[END_REF], critical parameters for this aggregation include the concentration of the Ubx protein and the solution in which the protein is suspended. The composition of the solution impacts the formation of the surface lm of Ubx monomers and hence the properties of resulting monolayer biopolymeric membrane. Indeed, the monomer must have a determined conguration in order to aggregate [START_REF] Bondos | Detection and prevention of protein aggregation before, during, and after purication[END_REF].

Moreover, the monomers must be at the air-water interface in order to aggregate with other monomers. Hence, conditions that promote aggregation at the surface of the water are required. Our hypothesis is that the waiting time for aggregation is an essential parameter to control to achieve an ordered structure of the biopolymeric membrane. That is, the monomers need time to change conformation, rise to the surface, then begin to aggregate and order themselves. We used the technique to investigate Ubx monomer aggregation and formation of the biopolymeric membrane by comparing the surface pressurearea (π-A) isotherms obtained under dierent conditions [START_REF] Langmuir | The constitution and fundamental properties of solids and liquids. II. Liquids[END_REF][START_REF] Langmuir | Activities of Urease and Pepsin Monolayers[END_REF][START_REF] Blodgett | Films Built by Depositing Successive Monomolecular Layers on a Solid Surface[END_REF]. The shape of the surface pressurearea (π-A) isotherm depends on the nature of amphiphiles, the compression speed, the spreading con- Finally, we decided to go a bit further in the lm formation analysis. After some parameter optimization done with ellipsometry, I moved to neutron reectometry. In this case, we decided to study the lm formation dependence on waiting time parameter at two contrasts ACMW (air contrast matched water) and D 2 O. With this method, it was able to estimate the lm thick- ness and to corroborate the observations made previously with Langmuir technique.

ULTRABITHORAX PROTEIN

Protein-based biomaterials are next generation technology due to their extended range of potential applications, such as drug delivery, tissue engineering, photonics, implanted medical devices and the creation of platform materials for novel biosensors [START_REF] Howell | Mechanically-Tunable, Protein-Based Materials Can be Functionalized with Other Proteins and with DNA[END_REF][START_REF] Jo | Biomaterial-based delivery systems of nucleic acid for regenerative research and regenerative therapy[END_REF]. This work will be focused on the study of lm formation of Ubx (Ultrabithorax) protein-based material. Ubx is a Hox transcription factor in the fruit y Drosophila melanogaster, that spontaneously self-assembles at an air-water interface to form a monolayer, which has then been used as a basis for biomaterial uses.

Homeodomain Transcription Factors

TR (transcription factor) are proteins that control the rate of transcription of genetic information from DNA to m-RNA (messenger ribonucleic acid) by binding to a specic DNA sequence [START_REF] Scott | The structure and function of the homeodomain[END_REF][START_REF] Prince | Construction, Cloning, and Expression of Synthetic Genes Encoding Spider Dragline Silk[END_REF]. In other words, their role is to guarantee that genes are expressed in the right cell, at the right time and in the right amount. Some TFs are characterized by the presence of Hox (homeodomain), 60 amino helix-turn-helix DNA-binding domain [START_REF] Prince | Construction, Cloning, and Expression of Synthetic Genes Encoding Spider Dragline Silk[END_REF][START_REF] Hsiao | The intrinsically disordered regions of the Drosophila melanogaster hox protein ultrabithorax select interacting proteins based on partner topology[END_REF][START_REF] Mcginnis | A homologous protein-coding sequence in drosophila homeotic genes and its conservation in other metazoans[END_REF].

A homeobox is a DNA sequence that encodes the homeodomain and Hox genes are homeobox-containing genes. Homeodomain Transcription Factors, known also as Hox proteins, operate during the embryo growth of bilaterally symmetric animals, to generate tissues, organs and appendages specifying the fate of structures in every tissue layer [START_REF] Hsiao | The intrinsically disordered regions of the Drosophila melanogaster hox protein ultrabithorax select interacting proteins based on partner topology[END_REF]. A given Hox protein can be involved in the specication of multiple body structures, that is it can control the expression of a variety of target genes, which may number into the hundreds [START_REF] Hughes | Hox genes and the evolution of the arthropod body plan[END_REF]. For example, Drosophila melanogaster Hox protein Ubx species the posterior-most legs, the halteres (which are organs that take the place of the second pair of wings and regulate the balance during ight) and the posterior aorta, portions of the midgut, ectoderm, musculature and central and peripheral nervous systems [START_REF] Rogulja-Ortmann | Antagonistic roles for Ultrabithorax and Antennapedia in regulating segmentspecic apoptosis of dierentiated motoneurons in the Drosophila embryonic central nervous system[END_REF][START_REF] Bienz | Homeotic genes and positional signalling in the Drosophila viscera[END_REF][START_REF] Hsiao | The intrinsically disordered regions of the Drosophila melanogaster hox protein ultrabithorax select interacting proteins based on partner topology[END_REF][START_REF] Hughes | Hox genes and the evolution of the arthropod body plan[END_REF], as shown in NOEs are an indication of disorder [START_REF] Aviles | The Conformation of Histone H5: Isolation and Characterisation of the Globular Segment[END_REF][START_REF] Fletcher | 4E binding proteins inhibit the translation factor eIF4E without folded structure[END_REF][START_REF] Daughdrill | The C-terminal half of the anti-sigma factor, FlgM, becomes structured when bound to its target, σ28[END_REF][START_REF] Muchmore | X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death[END_REF][START_REF] Romero | Sequence complexity of disordered protein[END_REF]. Ubx is also the only Hox protein in which predicted intrinsically disordered regions have been experimentally validated [START_REF] Hsiao | The intrinsically disordered regions of the Drosophila melanogaster hox protein ultrabithorax select interacting proteins based on partner topology[END_REF][START_REF] Liu | Multiple intrinsically disordered sequences alter DNA binding by the homeodomain of the Drosophila Hox protein ultrabithorax[END_REF] In addition to its in vivo function as a transcription factor, Ubx also self assembles into biomaterials through intermolecular dityrosine bonds [START_REF] Howell | Identication of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax[END_REF]. Bondos's lab in Texas (Texas A&M University) has characterized the materials and explored potential uses in a variety of areas [START_REF] Bondos | Detection and prevention of protein aggregation before, during, and after purication[END_REF][START_REF] Bondos | Flexibility and disorder in gene regulation: LacI/GalR and Hox proteins[END_REF][START_REF] Bondos | Context-dependent HOX transcription factor function in health and disease[END_REF][START_REF] Howell | Identication of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax[END_REF][START_REF] Howell | Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity[END_REF]. My work focuses on an in-depth study of Ubx lm-formation.

Protein-Based Materials

In the last four decades, following the pioneering work of Stanley Cohen and

Herbert Boyer [START_REF] Cohen | Construction of biologically functional bacterial plasmids in vitro[END_REF] the design and production of protein-based materials has been highly studied thanks to their versatility in medicine and biomedical devices in combination with good biocompatibility and biodegradability. [START_REF] Wen | Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons[END_REF][START_REF] Ayoub | Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes[END_REF][START_REF] Teulé | A protocol for the production of recombinant spider silk-like proteins for articial ber spinning[END_REF]. In fact, protein-based materials have dierent applications ranging from drug delivery, tissue engineering, biosensors and cell culture studies [START_REF] Howell | Mechanically-Tunable, Protein-Based Materials Can be Functionalized with Other Proteins and with DNA[END_REF][START_REF] Costa | Fibrous protein-based biomaterials (silk, keratin, elastin, and resilin proteins) for tissue regeneration and repair[END_REF]. Several proteins have the ability to self-assemble at air-water interface, forming a lm that can be re-arranged into other shapes, such as bers and 3D gels [START_REF] Åstrand | Assembly of FN-silk with laminin-521 to integrate hPSCs into a three-dimensional culture for neural dierentiation[END_REF][START_REF] Nilebäck | Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism[END_REF][START_REF] Bowen | Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk[END_REF][START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF][START_REF] Patterson | Materials composed of the Drosophila Hox protein ultrabithorax are biocompatible and nonimmunogenic[END_REF].

In the following 3 sections brief outlines of the properties of Ubx and 2 other commonly-used protein materials (silk and collagen) will be given.

Silk material

Silk proteins are produced by animals of the phylum of arthropods among which silkworms and spiders are the most prominent silk producers. Two of the most studied silk proteins are the broins produced by the domesticated silkworm Bombyx mori, used during the metamorphosis from larvae to moth, and the MaSp (major ampullate spidroin) produced by spiders and used for dierent purposes such as web building, prey swathing and reproduction [START_REF] Andersson | Silk spinning in silkworms and spiders[END_REF][START_REF] Nilebäck | Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism[END_REF]. Even though spiders and silkworms are distantly related, there are some features in common in spinning silk. Both of them produce and store in glands, highly repetitive, soluble and large silk proteins (> 300 kDa). When these proteins are pultrused through a narrowing conduct, which changes conformation in response to pH variations, they assemble forming silk bers under ambient conditions [START_REF] Andersson | Silk spinning in silkworms and spiders[END_REF][START_REF] Nilebäck | Interfacial Behavior of Recombinant Spider Silk Protein Parts Reveals Cues on the Silk Assembly Mechanism[END_REF]. The pH decrease along the conduct is a well known factor that regulates ber formation, bringing the protein conformation to change from disordered and partly helical into densely stacked β-sheet crystals [START_REF] Dicko | Spider silk protein refolding is controlled by changing pH[END_REF][START_REF] Domigan | Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands[END_REF][START_REF] Andersson | Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Eects on the Terminal Domains[END_REF]. An important role in silk assemble is played also by ion concentration gradients, dehydration of the protein dope and hydrodynamic forces along the conduct [START_REF] Tillinghast | Water extraction by the ma jor ampullate duct during silk formation in the spider, Argiope aurantia Lucas[END_REF][START_REF] Knight | Changes in element composition along the spinning duct in a Nephila spider[END_REF][START_REF] Spiess | Recombinant spider silk proteins for applications in biomaterials[END_REF].

Silk broin extracted from Bombyx mori assemble at the air-liquid interface [START_REF] Valluzzi | Orientation of silk III at the air-water interface[END_REF][START_REF] Valluzzi | The crystal structure of bombyx mori silk broin at the air-water interface[END_REF]. However, it is dicult to extract sucient quantities from spiders to meet the demands. Hence recombinant technology has been used to produce spidroins in heterologous hosts (host organisms, such as bacteria, yeasts, plants, and animals, which do not naturally have the expressing gene or one fragment) ensuring constant quality and biological safety as well as self-assembly property [START_REF] Bowen | Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk[END_REF]. However, recombinant silks are dicult to produce because of genetic instability of long and highly repetitive DNA sequences, translation inhibition by complex mRNA secondary structures, a high demand for glycine and alanine transfer RNAs, and overall metabolic burden caused by spidroin overexpression. Moreover, protein functionalization with other functional proteins or tags can aect self-assembly property [START_REF] Bowen | Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk[END_REF][START_REF] Saric | Engineering of silk proteins for materials applications[END_REF]. To solve this problem and produce native-sized silk proteins with high yields,the silk production process is engineered. For example, high molecular weight silk proteins are produced by splitting intein-mediated ligation via hosts like E. coli [START_REF] Bowen | Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk[END_REF] or plants [START_REF] Weichert | Seed-specic expression of spider silk protein multimers causes long-term stability[END_REF].

Collagen material

Collagen is one of the most abundant proteins in the extracellular matrices of many mammals connective tissues, comprising about 2535% of the dry weight of the body [START_REF] Deshmukh | Enigmatic insight into collagen[END_REF][START_REF] Bazrafshan | Spinnability of collagen as a biomimetic material: A review[END_REF]. Collagen is typically found in tendons, skin, ligaments, cartilages, bones, corneas, gut and blood vessels [START_REF] Lee | Biomedical applications of collagen[END_REF]. There are 29 of other materials, when they are processed as composite bers, sponge or lm [START_REF] Bazrafshan | Spinnability of collagen as a biomimetic material: A review[END_REF]. For this reason, over the past decade, researcher have been working

towards the possibility of mimicking the structure of collagen brils in native tissues, especially with the spinning technique.

Ultrabithorax material

As already mentioned, Ultrabithorax proteins are Hox transcription factors.

It has been proved that Ubx monomers spontaneously self-assemble under mild, aqueous conditions, forming biocompatible ordered materials at an airwater interface [START_REF] Bondos | Flexibility and disorder in gene regulation: LacI/GalR and Hox proteins[END_REF] (see Figure 2.3).

Ultrabithorax fusions

On of the most important advantage of protein-based materials is the possibility to incorporate novel functions to them by producing a fusion protein capable of both self-assembly and the desired new activity that can be engineered for many applications.

Although there are dierent techniques to incorporate one protein with another such as physical adsorption [START_REF] Beachley | Fabrication of nanober reinforced protein structures for tissue engineering[END_REF], cross-linking [START_REF] Murphy | Biomedical applications of chemicallymodied silk broin[END_REF][START_REF] Koh | Enhancement of neurite outgrowth using nano-structured scaolds coupled with laminin[END_REF], direct mixing with polymer solutions [START_REF] Kyle | Production of self-assembling biomaterials for tissue engineering[END_REF] and co-axial electrospinning [START_REF] Neal | Laminin nanober meshes that mimic morphological properties and bioactivity of basement membranes[END_REF][START_REF] Schneider | Biofunctionalized electrospun silk mats as a topical bioactive dressing for accelerated wound healing[END_REF], the gene fusion approach is used to produce Ubx fu- the resulting gene si placed in a living cell for protein production, the cell will produce a single polypeptide composed of the amino acid sequence of both proteins. The single polypeptide is called protein chimera or protein fusion. During the lm formation process, the functional protein is automatically embedded throughout the material maintaining its original activity [START_REF] Huang | Functionalization and patterning of protein-based materials using active ultrabithorax chimeras[END_REF].

There are several advantages using this technique. First of all, the two proteins don't have to be produced separately, reducing costs and time. Second, the resulting material is fully functionalised and uniform, that is each Ubx protein is attached to one functional protein and vice versa. Third, there is just one attachment between the two proteins (the exible linker in Figure 2.4) leading to a uniform orientation of the resulting protein. Finally, the lm formation brings a stabilization of functional proteins preserving their activities even in harsh conditions (e.g. heat, acid and other denaturating conditions) [START_REF] Howell | Identication of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax[END_REF].

METHODS

Langmuir trough Technique

Langmuir trough technique is a a well-established and sophisticated method to control interfacial molecular orientation creating a lms at air-water interface called Langmuir lm [START_REF] Ulman | An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly[END_REF]. Langmuir lm study seems to date back to the 18th century BC in Babylon, but it's with the American statesman Benjamin Franklin that the history of Langmuir lms began. In 1774 he made some simple quantitative calculations to nd out that a few-nanometresthick layer of oil had calming inuence on the water in the Clapham ponds [START_REF] Franklin | XLIV. Of the stilling of waves by means of oil. Extracted from sundry letters between Benjamin Franklin[END_REF]. In the 19th century Angela Pockels set the fundamentals for the airwater-interface monolayers quantitative characterisation. She studied the contamination of water surface as a function of the area using a rudimentary surface balance [START_REF] Pockels | Relations between the surface-tension and relative contamination of water surfaces[END_REF]. At the same time, Lord Rayleigh hypothesized the maximum extension of an oil lm represents a monolayer. It was about 25 years later that Irving Langmuir performed a systematic study of monolayers at air-water interface [START_REF] Langmuir | The constitution and fundamental properties of solids and liquids. II. Liquids[END_REF]. These studies led to him being awarded the Nobel Prize in 1932. Although Langmuir reported lm transfer onto solid substrate, the rst detailed study of sequential monolayer transfer was carried out by Blodgett [START_REF] Blodgett | Films Built by Depositing Successive Monomolecular Layers on a Solid Surface[END_REF][START_REF] Blodgett | Properties of built-up lms of barium stearate[END_REF].

In order to prepare a Langmuir lm, a given molecule, for this work EGFP-Ubx will be used, is dissolved in a water-insoluble solvent and then carefully spread onto the surface of a water solution. As the time passes and the solvent evaporates, a monolayer forms at the surface. When the available area, where the molecules are spread, is too large compared to the monolayer dimensions, the distance between adjacent molecules is so huge that their interaction is too weak. In this case we can treat the system as a two-dimensional gas. On the other hand, if the surface area is reduced, the molecules start to interact with each other and form a stronger lm and the system can be treated as The hydrophobic tail preferentially resides in the air and the hydrophilic head is preferentially immersed in the subphase. Since the amphiphiles have one hydrophobic tail and one hydrophilic head, they like to locate at the interfaces (e.g. air-water or oil-water). For this reason they are also called surfactants (surface active).

Once one o more droplet of a dilute solution of an amphiphile molecule is spread at the subphase surface, the barriers move and compress the molecules while the pressure and the area are recorded. The surface-pressure is described as:

π = σ 0 -σ (3.1)
Where π is the surface pressure, in mN/m, σ 0 is the surface tension of pure subphase and σ is the surface tension of the all system (subphase plus monolayer). The surface tension is a water property that measures the dierence in energy of molecules between the bulk and the surface. All the molecules t is the plate thickness, θ is the contact angle, w is the plate width, h is the immersed depth and l is the plate length. In the inset there's the scheme of the forces acting on subphase molecules at the air-water interface.

With the Wilhelmy plate-method, we can calculate the force due to surface tension on a partially immersed plate in the subphase, Figure 3.3. There are three forces acting on the plate: in the downward there are the gravity and surface tension force , while in the upward direction there is the force caused by the displaced water. The net downward force for a l × w × t Wilhelmy plate, with a material density ρ p , immersed to a depth h in a liquid whose density is ρ l is:

F = glwtρ p + 2γ(tw)cosθ -ρ l gtwh (3.2)
where γ is the liquid surface tension, θ is the contact angle and g is the gravitational constant. To determine the surface pressure it is just necessary to measure the force changes for a stationary plate between a clean surface and the same surface with an amphiphile monolayer. When the plate is fully wetted, cosθ = 1 and the surface pressure is:

π = -∆γ = - ∆F 2(t + w) (3.3) π = - ∆F 2w , if w >> t (3.4)
The force is determined by measuring the changing of plate mass, which is directly coupled to a sensitive electro-balance. The measure of surface pressure as a function of area at a constant temperature is also known as surface-pressure isotherm, or π-A and is an important indicator of the monolayer properties such as stability, reorientation of molecules, phase transitions and conformational transformations, Figure 3.4. Discontinuities in the π-A isotherms identify these changes indicating transition to dierent phases.

The phase behaviour is a consequence of several factors such as physical and chemical properties of the amphiphile and the temperature or composition of the subphase. For this reason, in many cases phase transitions are not observed.

When the surface pressure is low, the area between molecules is large and ideally there are no interaction. In this case, the molecules have a negligible size compared to the interface area, and thus will obey the equation πA = κT ,where T is the absolute temperature and κ is the Boltzmann constant. This is a two dimensional ideal gas equation, for this reason, this phase is called "gas phase". As the barriers move, the molecules are compressed in a smaller area and the intermolecular distance is decreased and consequently the surface pressure increases. A phase transition is observed in the isotherm: from gas phase molecules rearrange into a liquid phase, where the monolayer is coherent even though the molecules have a large degree of freedom. When the lm is further compressed, a second phase transition occurs, from the liquid to the solid state. In this condensed phase the molecules are packed and well oriented. If the monolayer compressed more and more, it collapses due to mechanical instability and a sharp pressure decrease is recorded [START_REF] Milner | Buckling of langmuir monolayers[END_REF]. For a Fresnel interface (an interface where the refractive index changes steeply from n 1 , to n 2 ) and for a p-polarization light (electric eld is parallel to the incidence plane), the reectivity, R p , is null at Brewster angle, θ b . Figure 3.6b shows the plot of R s and R p versus θ at air-glass interface, or rather when n 1 = 1 and n 2 = 2. As θ varies from 0°to 90°(from normal incidence to grazing), R s increases monotonically reaching the maximum value 1 at θ = 90. On the other hand, R p decreases initially, reaches the minimum value R p = 0 at θ = θ b and then increases up to R p = 1 at θ = 90. Thus, R s > R p for all angles θ and there's no reection for parallel polarized waves at the Brewster's angle. The reectivity Fresnel coecient for non-magnetic media is:

R p = n 2 cos θ i -n 1 cos θ t n 2 cos θ i + n 1 cos θ t (3.5)
where θ i is the incidence angle and θ t is the transmitted one. The Brewster angle can be calculated from Equation(3.5

) letting R p = at θ i = θ b : n 1 cos θ t = n 2 cos θ b (3.6)
with the use of Snell's law

n 2 sin θ t = n 1 sin θ b (3.7)
it is possible to eliminating θ t from Equation(3.6), and the Brewster angle is:

θ b = arctan n 2 n 1 (3.8)
In With this technique, both the shape of the condensed phase domains and their inner texture can be visualized. In fact, anisotropy plays an important role in the BAM images as inner textures and can be used to obtain structural analysis [START_REF] Clifton | Design and use of model membranes to study biomolecular interactions using complementary surface-sensitive techniques[END_REF][START_REF] Roldán-Carmona | Revisiting the Brewster Angle Microscopy: The relevance of the polar headgroup[END_REF]. The Reectivity, in this case, is described by the following equation:

R p = πd λ 2 1 1 -n 2 2 n 2 1 -n 2 2 -1 + n 2 2 n 2 1 2 (3.9)
where λ is the wavelength and d is thin lm thickness.

Ellipsometry

Even though at the beginning of nineteenth century, Augustin Jean Fresnel Light with p-and p-polarization remains linearly polarized upon reection.

Incident and reected bean can hence be described by their corresponding Jones vectors:

⃗ E inc =   |E i p |e iδ i p |E i s |e iδ i s   ⃗ E ref =   |E r p |e iδ r p |E r s |e iδ r s   (3.10)
To describe the changes in the state of polarization, it is possible to use two quantities:

∆ = (δ r p -δ r s ) -(δ i p -δ i s ) (3.11)
Fig. 3.9: Schema of ellipsometric measure.

tan Ψ = |E r p |/|E i p | |E r s |/|E i s | (3.12)
The reectivity properties of a sample within a given experiment are given by the corresponding reection coecients r p and r s :

r p = |E r p | |E i p | e iδ r p -δ i p r s = |E r s | |E i s | e iδ r s -δ i s (3.13)
If we assume that there is no coupling between the orthogonal p-and spolarizations (e.g. isotropic samples), the changes in the attenuation ψ and phase ∆ can be described with the following equation [START_REF] Magnusson | Mueller matrix ellipsometry studies of nanostructured materials[END_REF]:

tan ψe j∆ = r p r s = ρ (3.14)

Ellipsometry applied to ultra-thin lms

In the presence of an organic non-absorbing monolayer (refractive index n ∼ 1.3 -1.6) with a thickness below 2.5nm the sensitivity of ψ is negligible compared to the Delta one, so it is possible to consider just the phase shift.

For a single homogenous lm with refractive index n 1 = √ ϵ 1 in between two innite media with n 0 = √ ϵ 0 and n 2 = √ ϵ 2 (see Figure 3.9), the reectivity coecients can be written as:

∆ = arctan Im{ rp rs } Re{ rp rs } (3.15)
The reectivity coecients r p and r s can be written using the Fresnel equations which describe the reection at the surfaces n 0 -n 1 and n 1 -n 2 [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF]:

r p = |r p |e iδ r p = r 0,1,p + r 1,2,p e -i2β 1 + r 0,1,p r 1,2,p e -i2β r s = |r s |e iδ r s = r 0,1,s + r 1,2,s e -i2β 1 + r 0,1,s r 1,2,s e -i2β (3.16)
where β = 2π h λ n 2 1 -n 2 0 sin 2 θ, λ is the wavelength of incident light, h is the layer thickness and θ is the incident angle. In the thin lm limit (h << λ), it is possible to expand the complex reectivity coecients as a power series in terms of h/λ [START_REF] Motschmann | Ellipsometry in interface science[END_REF][START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF]:

∆ ∼ 4 √ ϵ 0 ϵ 2 π cos θ sin 2 θ (ϵ 0 -ϵ 2 )[(ϵ 0 + ϵ 2 cos 2 θ) -ϵ 0 ] • (ϵ 1 -ϵ 0 )(ϵ 2 -ϵ 1 ) ϵ 1 • h λ (3.17)
∆ is linearly proportionally to the ellipsometric thickness, η:

∆ = g(θ) λ η (3.18)
g(θ) is a function depending on bulk properties (n = √ ϵ) and incident angle:

g(θ) = 4πn 0 n 2 2 cos θ sin 2 θ (n 2 0 -n 2 2 )[(n 2 0 + n 2 2 cos 2 θ) -n 2 0 ] (3.19)
In our case n 2 = n water = 1.33, n 0 = n air = 1, θ = 50 and g(θ) = 75.70.

η = (n 2 1 -n 2 0 )(n 2 2 -n 2 1 ) n 2 1 h (3.20)
Generally, the material density in adsorbed organic layers decreases with increasing distance from the interface, so the refractive index of the layer varies over its height and η is calculated as an integral across the interface [START_REF] Drude | Ueber die Reexion und Brechung ebener Lichtwellen beim Durchgang durch eine mit Oberächenschichten behaftete planparallele Platte[END_REF][START_REF] Motschmann | Ellipsometry in interface science[END_REF][START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF][START_REF] De Feijter | Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the airwater interface[END_REF]:

η = (n 2 1 (z) -n 2 0 )(n 2 1 (z) -n 2 2 ) n 2 1 (z) dz (3.21)
Using the Gibbs convention, it is possible to introduce the parameter Γ, surface absorption per unit of surface area, as [START_REF] De Feijter | Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the airwater interface[END_REF]:

Γ = ∆c 1 (z)dz (3.22)
where ∆c 1 (z) = c 1 (z) -c 2 is the excess concentration of the solute in the adsorbed layer, c 1 (z) is the absolute concentration in the layer, and c 2 is the solute concentration in the bulk solution. De Feijter's equation can be used to derive a relation between the surface excess Γ and ∆ [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF][START_REF] De Feijter | Ellipsometry as a tool to study the adsorption behavior of synthetic and biopolymers at the airwater interface[END_REF]:

Γ = n 2 1 (n 1 -n 2 ) (n 2 1 -n 2 0 )(n 2 1 -n 2 2 )
• λ dn dc g(θ)

• ∆

(3.23)
where dn dc

is the refractive index increment. In this work the thin lm studied with ellipsometry is protein-based so the refractive index increment can be estimated from the protein compositional prediction [82,[START_REF] Zhao | On the distribution of protein refractive index increments[END_REF][START_REF] Khago | Protein refractive index increment is determined by conformation as well as composition[END_REF]. The refraction per gram R p of the protein is calculated as the weight average of the contributions from the individual amino acids R i :

R p = i R i M i i M i (3.24)
where M i is the residue molecular mass. In the same way, it is possible to calculate the protein partial specic volume, vp as:

vp = i vi M i i M i (3.25)
using the Lorentz-Lorenz formula [START_REF] Zhao | On the distribution of protein refractive index increments[END_REF][START_REF] Khago | Protein refractive index increment is determined by conformation as well as composition[END_REF], R = v(n 2 -1/(n 2 + 2)), the refractive index of the protein n 1 , is:

n 1 = 2Rp + vp vp -R p (3.26)
Assuming volume additivity, the refractive index of protein in solution can be calculated from the Wiener equation [START_REF] Wiener | Zur theorie der refraktionskonstanten[END_REF][START_REF] Zhao | On the distribution of protein refractive index increments[END_REF][START_REF] Khago | Protein refractive index increment is determined by conformation as well as composition[END_REF]:

dn dc = 3 2 vp n 2 n 2 1 -n 2 2 n 2 1 + 2n 2 2 (3.27)
where n 2 is the refractive index of the solvent, see Figure 3.15. Corrections for the wavelength were implemented [START_REF] Zhao | On the distribution of protein refractive index increments[END_REF][START_REF] Khago | Protein refractive index increment is determined by conformation as well as composition[END_REF]:

dn dc λ = dn dc 578nm 0.94 + 20000nm 2 λ 2 (3.28)

Scanning Electron Microscopy

The common optical microscope is really useful to image biological samples.

However, its resolution is physically limited by Abbe limit [START_REF] Abbe | Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung: I. Die Construction von Mikroskopen auf Grund der Theorie[END_REF]:

d min = λ 2nsin(θ) = λ 2N A (3.29)
where λ is the wavelength used, n is the refractive index of the medium, θ is the incident angle and NA is the numerical aperture. So, the smallest resolvable distance between two points is half the wavelength of the imaging light divided by NA. Since visible light (400nm < λ < 700nm) is used as source in the optical microscope, only samples whose characteristic dimension is bigger than 0,2 µm can be imaged with this technique. To overcome Abbe limit an electron source can be used, in fact electrons have shorter wavelength than photons (e.g. λ = 2.74 pm at E = 200 keV) Electron microscopy history is correlated to the development of electron optics. In 1926, Busch laid the basis of geometrical electron optics with the study of charged particles tra jectories in axially symmetric electric and magnetic elds, showing that such elds could act as particle lenses [START_REF] Bogner | A history of scanning electron microscopy developments: Towards "wet-STEM" imaging[END_REF][START_REF] Oatley | The early history of the scanning electron microscope[END_REF]. De Broglie, simultaneously, introduced the concept of wave-particle duality, that is every particle exhibits a wave-like behaviour . Hence, a wavelength was associated with charged particles [START_REF] Hawkes | Recent advances in electron optics and electron microscopy[END_REF][START_REF] Bogner | A history of scanning electron microscopy developments: Towards "wet-STEM" imaging[END_REF]. With these two discoveries, the idea of an electron microscope began to take shape. Several years later, in 1930s, Ruska made the rst attempt with an electron microscope prototype, facing many technical limits to overcome [START_REF] Ruska | über Fortschritte bei der Abbildung elektronenbestrahlter OberÄchen[END_REF][START_REF] Ruska | The Development of the Electron Microscope and of Electron Microscopy (Nobel Lecture)[END_REF]. Ruska and Knoll tried to implement Busch's lens formula experimentally constructing the rst transmission electron microscope (TEM) in 1931, with a magnication of 16 [START_REF] Haguenau | Key Events in the History of Electron Microscopy[END_REF]. The rst scanning electron microscope was built in 1935 by Knoll and his group. However, the resolution was low (∼ 100µm) due to the missing demagnication that would have produced a ner probe. Since then, many scientists worked to improve SEM (scanning electron microscopy) to the instrument we know and use nowadays.

In Figure 3.10 there's a schematic diagram of a SEM. In electron microscopy, a high-energy electron beam is focused on a specimen, and scanned along a pattern of parallel lines. A thermionic gun emits electrons, called PEs (primary electrons), when a very high electric eld applied. PEs are formed into a nely focused beam passing trough magnetic lenses and sent to the sample.The beam probe interacts with specimen surface and as a result signals are generated, collected and analysed to form an image. Interactions produce mainly SEs (secondary electrons), with lower energies compared to the primary ones, BEs (backscattered high-energy electrons) and characteristic X-rays [START_REF] Bogner | A history of scanning electron microscopy developments: Towards "wet-STEM" imaging[END_REF][START_REF] Stokes | Principles and Practice of Variable Pressure / Environmental Scanning[END_REF].

To produce an electron beam, a high voltage is applied to a lament, which can be either a thermionic emitter or a eld emission source. The source brightness, β(A/m 2 sr), is given by the current density per unit of solid angle: where j b is the current density, i b is the emission current, α is the convergence semi-angle an d 0 is the crossover beam diameter. In conclusion, the brightness increases linearly with the accelerating voltage V 0 [START_REF] Stokes | Principles and Practice of Variable Pressure / Environmental Scanning[END_REF].

β = j b π(α) 2 = 4i b (π) 2 d 0 (α) 2
There are two types of electron gun in general use, thermionic emitters and Field emission sources. Thermionic emitters are usually V-shape tungsten laments or pyramidal lanthanum hexaboride, LaB 6 , single crystal. They are heated, by passing a current through them, to over 25000°C to produce thermal emission of electrons from the tip. In fact, their work function Φ (the minimum thermodynamic energy needed to remove an electron from a solid,), is low: Φ tungsten = 4.5e.V. and Φ LaB 6 ∼ 2.5e.V.. Since Φ LaB 6 < Φ tungsten , the LaB 6 laments provide larger maximum beam current which means a brighter beam. Field emission sources, know also as eld emission guns or cold cathode emitters, do not require a heating but an high negative potential is applied until quantum mechanic tunnelling eect occur and electrons escape the surface work function barrier. They are brighter and more stable than thermionic emitters, with very small deviation in electronic energy [START_REF] Stokes | Principles and Practice of Variable Pressure / Environmental Scanning[END_REF][START_REF] Henini | Scanning electron microscopy: An introduction[END_REF]. Moreover, they exhibit a much narrower energy spread, and thus a larger fraction of emitter electrons are used. Since the electron extraction process is unconditioned by thenal beam voltage, eld emission sources are perfect for low-voltage imaging SEM. Compared to thermionic sources, eld emission ones oer higher resolution (∼ 1nm), excellent performances over a wide range of accelerating voltages (0.5 -30 kV) and higher resolution at low voltages. However, a better vacuum is required in this case to preserve the tip ( 10-10 instead of 10 -6 for thermionic sources). Once the electronic beam is emitted, it passes through an anode to be accelerated and then two or three magnetic lenses demagnify the beam into a ne probe which is nally scanned across the specimen, for example we pass from a d 0 ∼ 50µm after the thermionic electron gun emission to a nal spot size d 0 ∼ 10nm. Even thou electrons can be focused by both electrostatic and electromagnetic elds, the aberration given by the latter are smaller which brings to their extensive application in SEM.

⃗ F = e( ⃗ E + ⃗ v ⊗ ⃗ B) (3.31)
where ⃗ v is the electron velocity in the beam. As electrons pass through the lens, they feel fringing magnetic eld lines that extend across the lens gap Fig. 3.12: Interaction volume of an electron beam with a thick sample.

between the polepieces and o-axis electrons begin to spiral toward the optic axis [START_REF] Goldstein | The SEM and Its Modes of Operation[END_REF]. This is schematically shown in Figure 3.11b. When the current intensity is increased, the magnetic eld force increases and the crossover point of the beam moves upwards. At the contrary, when the current decreases, the crossover point moves downwards.

As already mentioned, when the electron beam is scanned across the sample surface, electrons interact and penetrate into the specimen in a tear-drop shape3.12. The penetration dimensions depend on several factors including incidence angle, atomic masses of the elements in the specimen and primary electrons energy. The penetration depth increases the higher the beam energy, incident angle and the lighter the atomic mass (e.g. for a GaAs specimen with E = 20 keV at normal incidence, electrons penetrate ∼ 1µm into the sample) [START_REF] Henini | Scanning electron microscopy: An introduction[END_REF].

SEM imaging technique is based on SEs detection, which are usually electrons from or near the sample surface whose energy is typically below 50 eV [START_REF] Henini | Scanning electron microscopy: An introduction[END_REF].

Since SEs escape from a very shallow specimen area whose dimension is comparable to the beam size, they provide the highest spatial resolution images.

Topographic information are obtained but, thanks to some BEs collected from the secondary electron detector, some compositional contrast is also present. BEs are those electrons that approach the atom nuclei suciently close to be scattered through a large angle and re-emerge from the surface.

Their energy is higher compared to SEs one, but they are not as numerous. Moreover, since they come from a sample area bigger than beam size, their spatial resolution is lower. However, their have a key role in compositional information (elements of higher atomic mass give brighter signals) and in crystallographic information (because electron channelling occurs).Other secondary eects due to incident PEs energy loss can be produced such as X-rays, Auger electrons and visible light.

Neutron Reectivity

Although all the techniques previously described are useful to analyse samples at air-water interface, the spatial resolution is not suitable to study deeply the mechanisms of lm formation or to measure sample thickness or the chemical composition. Thanks to wavelengths comparable to interatomic distances (typical λ = 2-30Å), NR (neutron reectometry) is a powerful tool for the surface behaviour investigation providing a spatial resolution below ∼ 1.0nm with penetration depth over ∼ 100nm. First observed in 1946 by

Fermi and Zinn [START_REF] Fermi | Reection of Neutrons on Mirrors[END_REF] after the experimental discover made by James Chadwick in 1932 [START_REF] Chadwick | Possible existence of a neutron[END_REF], NR (neutron reectometry) has specic properties that make this technique useful and interesting. In the present work the nomenclature of Russell [START_REF] Russell | X-ray and neutron reectivity for the investigation of polymers[END_REF][START_REF] Russell | On the reectivity of polymers: Neutrons and X-rays[END_REF] will be used, for further details it is possible to consult its works.

Neutrons are subatomic particles with neutral charge, their electric dipole moment is either zero or too small to be measured and their mass is comparable to the proton mass. The absence of charge allows the neutrons to avoid any interaction with the electron cloud of an atom and interact, instead, with the atom core, in other words there's no coulombic interaction with matter. Since nuclei are several orders of magnitude smaller than atoms and nuclear forces are very short range (on the order of a few femtometers), neu-

trons
SLD = j b j ρ j (3.33)
where b j is the coherent scattering length of a nucleus and ρ j is the atom density of the respective nucleus. SLD can be either positive or negative depending the sign of b j .

Reectivity from a single layer

In the present work, we'll discuss specular reection, where the incident angle of the neutron beam is equal to the reected angle, (Figure 3.14 θ i = θ r = θ).

Dening the ⊥ direction as the direction normal to the surface, for specular reection the scattering vector has only a ⊥ component. This is the only scattering that occurs for samples or systems without any structure in the and kr are the incident and scattered wave vectors, with angles θ i = θ r = θ in the incidence plane; q is the wave vector transfer; and SLD is the scattering length density of the semi-innite substrate. (b) -Calculated neutron reectivity prole for a beam incident on a silicon substrate where the surface is perfectly smooth. Reproduced from reference [START_REF] Russell | X-ray and neutron reectivity for the investigation of polymers[END_REF].k z,0 is equal to the magnitude of the incident (and reected) wavevector in air.

plane of surface (lateral structure).

Neutron reectivity is usually described with neutron momentum transfer vector, q, since the scattering is elastic |q incident | = |q ref lected |:

q ⊥0 = 4 π λ sin θ (3.34) q ⊥0 = 2k sin θ, k = 2π λ (3.35)
where θ is the angle of incidence of the neutron beam with the surface and λ is the associated neutron wavelength, calculated from de Broglie equation:

λ = h m n v (3.36)
where h is the Planck constant (h = 6.626 • 10 -34 J • Hz -1 ), m n is the neutron mass and v is the neutron velocity. When the neutron beam coming from air impinges on a semi-innite medium that consists of a single type of atom with a scattering length b, the neutron wave interacts with a constant potential V:

V = 2πh 2 m n N b (3.37)
where N b is the scattering length density. The neutron kinetic energy is:

E = h2 k 2 2m n (3.38)
If the kinetic energy associated to the perpendicular component of k is smaller than the potential V(i.e. E ⊥ < V ), the neutron energy is not enough to overcome the potential barrier and the neutron beam is totally reected from the surface. It is possible to calculate the critical angle, θ c , at which the total reection occurs:

h2 k ⊥ 2 2m n = 2πh 2 m n N b (3.39) 2h 2 π 2 m n λ 2 sin 2 θ c = 2πh 2 m n N b (3.40) sin θ c = λ 2 N b π (3.41)
When the SLD has negative values, no total reection can occur. If E ⊥ > V , neutron transmission occurs. The transmitted beam energy corresponds to the energy of the incident beam minus the potential:

h2 k ⊥,t 2 2m n = h2 k ⊥,i 2 2m n - 2πh 2 m n N b (3.42) which gives k ⊥,t 2 = k ⊥,i 2 -4πN b = k ⊥,i 2 -k c 2 (3.43)
where k c is the critical value of k ⊥,i .

If we consider the system from a classical optics point of view, incident radiation on a surface can be refracted and reected due to the dierence of refractive indices at the interface. In general, the refractive index of a specimen is slightly less than 1 and is given by [START_REF] Cubitt | Chapter 2.8.3 NEUTRON REFLECTION: PRINCIPLES AND EXAMPLES OF APPLICATIONS SCATTER-ING IN MICROSCOPIC PHYSICS AND CHEMICAL PHYSICS Neutron Scattering Contents[END_REF]:

n 2 = k 2 t k 2 i = 1 - λ 2 N b π (3.44)
For most materials, N b << 1, so for λ < 60 Åit is possible to approximate n as:

n ≈ 1 - λ 2 N b 2π (3.45)
It is the change in the refractive index from one medium to another that cause neutron reection. This will occur at the interface between two dissimilar materials.

Reectivity from multiple layers

In a system containing multiple layers the reection coecient at an interface between the ith and (i + 1)th layer is:

r i,i+1 = k ⊥,i -k ⊥,i+1 k ⊥,i + k ⊥,i+1 (3.46)
In the case of air-substrate interface the reection coecient, r 0,1 , is simply:

r 0,1 = k ⊥,0 -k ⊥,1 k ⊥,0 + k ⊥,1 (3.47)
The reectivity, R (which corresponds to the ratio of reected to incident neutrons in layer 0.), is given by

R = r 0,1 r 0,1 * (3.48)
where the asterisk denotes the complex conjugate. From (3.47),(

(3.48), the reectivity for the air-substrate case is:

R(k ⊥,0 ) = 1 -[1 -(k c,1 /k ⊥,0 ) 2 ] 1/2 1 + [1 -(k c,1 /k ⊥,0 ) 2 ] 1/2 2 (3.49)
remembering (3.35) the reectivity can be written also as:

R(q) = q -[q 2 -q 2 c ] 1/2 q + [q 2 -q 2 c ] 1/2 2 (3.50)
When q >> q c it is possible to approximate the above expression as

R(q) ≈ 16π 2 q 4 N 2 b (3.51)
Therefore, for an innitely sharp interface, the reectivity will vary with q -4 at high values of q. In Figure 3.14a the calculated neutron reectivity prole for a silicon substrate with a perfectly at surface is shown. Total external reection occurs below k ⊥,0 = k c,1 [START_REF] Russell | X-ray and neutron reectivity for the investigation of polymers[END_REF]. The scattering length density can fall o even more rapidly than q -4 in presence of diuse interfaces or roughness at the air interface. These deviations are directly related to the width of the interface [START_REF] Russell | X-ray and neutron reectivity for the investigation of polymers[END_REF]. Reproduced from reference [START_REF] Russell | X-ray and neutron reectivity for the investigation of polymers[END_REF].

then has its own ('Fresnel') reection coecient, given by equation (3.46), at the air/lm and lm/substrate interfaces, separated by a distance d, as

shown in Figure 3.15. The total reection coecient (reectance) from the sample is given by summing the amplitudes of all the waves that emerge from the 0-1 interface following repeated reection and refraction at the two interfaces. This summation results in a total reection coecient, r, given by:

r = r 0,1 + r 1,2 exp(2ik ⊥,1 d) 1 + r 0,1 r 1,2 exp(21k ⊥,1 d) (3.52)
From (3.48), the reectivity is

R = |r 0,1 | 2 + |r 1,2 | 2 + 2Re{r 0,1 r 1,2 exp(2ik ⊥,1 d)} 1 + |r 0,1 r 1,2 | 2 + 2Re{r 0,1 r 1,2 exp(2ik ⊥,1 d)} (3.53)
in the case where the reectances are real, (3.53) reduces to

R = r 2 0,1 + r 2 1,2 + 2r 0,1 r 1,2 cos(2k ⊥,1 d) 1 + r 2 0,1 r 2 1,2 + r 0,1 r 1,2 cos(2k ⊥,1 d) (3.54)
This reectivity shows an oscillation behaviour due to the cosine, which is directly related to the thickness d of the layer by:

d ∝ π ∆k ⊥,0 = 2π ∆q (3.55)
where ∆q is the 'distance' in q space between adjacent minima in a plot of R(q). In Figure 3.14b a calculated reectivity prole for a lm, 500 Åin thickness, on a substrate where the interfaces between the lm and the air from the (n+1)th and nth layers respectively:

r n,n+1 = k ⊥,n -k ⊥,n+1 k ⊥,n + k ⊥,n+1 (3.56)
The reectance by the (n-1)th and nth layer can then be written as:

r n-1,n = r ′ n-1,n + r ′ n,n+1 exp(2id n k n ) 1 + r ′ n-1,n r ′ n,n+1 exp(2id n k n ) (3.57)
where the prime indicates the reectance given by equation(3.56), r n-1,n includes reections at the (n-1)-n and n-(n+1) interfaces.Using equation (3.57) the reectance at the (n-2)/(n-1) interface is calculated as:

r n-2,n-1 = r ′ n-2,n-1 + r n-1,n exp(2id n-1 k n-1 ) 1 + r n-2,n-1 r n-1,n exp(2id n-1 k n-1 ) (3.58)
This recursion is then continued until the air/specimen interface is reached.

Many methods have been established to solve the problem for a multilayerstratied medium on top of an innitely thick substrate, based on Parratt's recursive algorithm [START_REF] Parratt | Surface studies of solids by total reection of x-rays[END_REF] and Abèle's optical matrix method [START_REF] Heavens | Optical Properties of Thin Solid Films[END_REF]. In the optical matrix method described by Born and Wolf [START_REF] Born | Principles of Optics[END_REF], a characteristic matrix M for reection and transmission is dened for each interface:

M i = cos(k i d i ) -sin(k i d i )/k i k i sin(k i d i ) cos(k i d i ) (3.59)
Multiplying these matrices for each layer in a system leads to the the total reectivity curve for a series of layers with known properties. Using this method, dierent combinations of layers can lead to the same reectivity prole, which is an eect of the well known phase problem caused by the fact that only intensities can be experimentally accessed, but not the phase.

The reectivity can be calculated exactly for perfect layered structures, with sharp interfaces between layers. However, for most systems of interest the interfaces are not perfectly sharp [START_REF] Cubitt | Chapter 2.8.3 NEUTRON REFLECTION: PRINCIPLES AND EXAMPLES OF APPLICATIONS SCATTER-ING IN MICROSCOPIC PHYSICS AND CHEMICAL PHYSICS Neutron Scattering Contents[END_REF], as a result of either material diusion between interfaces (see Figure 3.17 a) or roughness with peaks and troughs over a large range of length scales (see Figure 3.17 b) [START_REF] Cubitt | Chapter 2.8.3 NEUTRON REFLECTION: PRINCIPLES AND EXAMPLES OF APPLICATIONS SCATTER-ING IN MICROSCOPIC PHYSICS AND CHEMICAL PHYSICS Neutron Scattering Contents[END_REF].

As a consequence, both the rough and diuse cases cause a faster decline of reectivity with momentum transfer than the q -4 dependency described in Equation 3.51. There are several approaches to mathematically model roughness. The method used in the Motot software (used in the present work to analyse the data) is to treat roughness as an error function erf ( z √ σ ) [START_REF] Névot | Caractérisation des surfaces par réexion rasante de rayons X. Application à l'étude du polissage de quelques verres silicates[END_REF], where σ is the roughness measure, which corresponds to a multiplication of the reectivity with a Debye Waller factor [START_REF] Cubitt | Chapter 2.8.3 NEUTRON REFLECTION: PRINCIPLES AND EXAMPLES OF APPLICATIONS SCATTER-ING IN MICROSCOPIC PHYSICS AND CHEMICAL PHYSICS Neutron Scattering Contents[END_REF]:

R(q) ≈ 16π 2 q 4 N 2 b • e -q 2 σ2
(3.60) Fig. 3.17: Three surface proles: on the left sharp interface, at the centre a rough surface, on the right a diuse surface. The rough and diuse surfaces can result in identical specular reectivities.

FIGARO

In NR experiments, the specular reected intensity is measured as a function of momentum transfer q: R R i (q)

(3.61)
Remembering that the perpendicular component of the momentum transfer vector is given by

q ⊥ = 2k ⊥ sin θ = 4π λ sin θ (3.62)
It is possible to vary q ⊥ by varying either the wavelength λ or the incident angle θ leading to two dierent methods of measurements: monochromatic reectometry or TOF (time of ight) reectometry.

In the rst case the wavelength is xed and the incidence angle is varied during the experiments.

One advantage of this technique is the possibility to chose the wavelength that maximizes the ux from the neutron source and therefore the resolution is the most ecient. Although this is a simple and very useful conguration for solid samples, where the incidence angle is varied by varying the sample position, for liquid samples θ can not be easily varied. In this cases, and many others, it is usually used the TOF reectometry: the incident angle is kept is produced by nuclear ssion), they travel with dierent velocities depending on the associated energy and thus wavelength:

E = 1 2 m n v 2 and E = 2π 2 h2 m n λ 2 (3.63) then v = 2πh m n λ (3.64)
Since the distance between the source and the detector is known and all the neutrons start at the same time (choppers are used to cut a continuous beam into multiple pulses, thus a common starting time is determined), it is possible to measure the time at which each neutron arrives at the detector and calculate the associated wavelength. Therefore, neutrons are sorted according to wavelengths and, following that, momentum transfers upon interaction with the sample. In this way, it is possible to measure the reectivity prole R(q) from neutrons reected at the same incidence angle. In order to increase the q range, multiple angles are generally chosen for an experiment.

In principle, the wavelength resolution is xed at:

∆λ λ = ∆t t (3.65)
However, the pulses duration is one of the limitations for resolution, as well as divergence beam and scattering background. In general, the background is subtracted from each measurement using the area detector. The range of q covered for a xed angle θ is inversely proportional to the λ range, for the cold neutrons used at the ILL λ ∼ 1 -30 Å. For soft matter, the TOF technique is much more applicable than the monochromatic one, and data can be recorded much faster (hence more eciently). The intensity at a given wavelength is less, but the eciency is gained by measuring a broad wavelength range simultaneously, which can be helpful for samples that may change with time.

As already mentioned, neutrons are delivered to FIGARO by a nuclear reactor producing an intense continuous neutron ux (1.5 × 10 15 neutrons per second per cm 2 , with a thermal power of 58.3 MW). Since the energy of neutrons is too high to be used in large scale, it is important to direct the neutron beam to a moderator tank (e.g. D 2 at 25K) where neutrons have multiple scatterings losing energy during each elastic collision. In this way, neutrons reach the thermal equilibrium and their energy is of the order of k B T , where T is the temperature of the water moderator, and k B is the Boltzmann coecient.

FIGARO is equipped with a 2D 500X150 mm 2 3 He tubular aluminium monoblock detector. The resolution used for the experiments was 7% ∆λ/λ. Each sample was measured at two dierent incident angles of 0.62 • and 3.8 • , with a wavelength range of ∈ [2Å, 30 Å] and thus the q-range ∈ [0.009Å -1 ,0.2Å -1 ].

PROTEIN PRODUCTION RESULTS

Production in Swansea laboratories

After a two-week period of training in Texas A&M University at Bondos' laboratories and following published protocols [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF][START_REF] Hessinger | The Drosophila Hox gene Ultrabithorax acts in both muscles and motoneurons to orchestrate formation of specic neuromuscular connections[END_REF][START_REF] Patterson | Materials composed of the Drosophila Hox protein ultrabithorax are biocompatible and nonimmunogenic[END_REF] it was possible to start Ubx protein production in Swansea laboratories for the rst time. Producing a protein in new laboratories is not easy as the conditions change. To do so, it was necessary to adapt the protocols to our necessity.

The overall process, to obtain pure proteins, requires 5 days and is divided in two steps: bacteria growth and protein purication. The loaded proteins were: the standard, the pellet (the solid part discarded after the centrifugation before the column step), the crude, the unbound proteins collected during the column step when the supernatant was loaded, fractions E0, E4 and E5, the Ni-NTA column matrix. In the crude, EGFP-Ubx was present, as expected since it was the liquid collected before the column purication, but in the unbound the Ubx band was missing, meaning that the majority of Ubx proteins was trapped by the matrix. At the same time, Ni-NTA matrix did not show the EGFP-Ubx presence because after the column wash and the elution process all the proteins were released.

However, in the pellet there was still some EGFP-Ubx, so the protein extraction was not totally ecient. Unfortunately this passage could not be improved because one possible consequence would be the protein degradation. In this case, it was preferable to lose proteins rather than have higher concentrations with degraded EGFP-Ubx.

At the end of the process, it was possible to conclude that the proteins were pure with a ratio over 95%, which was a great result especially because it was the rst protein production done in Swansea laboratories.

A = e • c • L (4.1)
where A is absorbance (maximum value of absolute spectrum), e is the molar extinction coecient of the absorbing species, c is the concentration of protein (our case EGFP) and L is the path length of light. Assuming that EGFP extinction coecient is e ∼ 56mM -1 sm -1 at 488 nm [START_REF] Tsien | The green uorescent protein[END_REF][START_REF] Nielsen | Absorption spectrum of the green uorescent protein chromophore anion in vacuo[END_REF], and the cuvette light path is 1 cm, we could measure the absolute spectra and calculated the concentration. Absolute spectra measured for all the fractions, collected after the rst protein purication, are shown in Figure 4.6.

The dotted black line helps to indicate the maximum value at λ = 490 nm.

Once the maximum value of absolute spectra is measured, it is possible to calculate protein concentration as:

c = A • 1/dilution e (4.2)
For example, in the case described in Figure 4.6, the maximum value for the fraction E1 is A = 0.033, so the concentration will be calculated as c = 0.033•20/56 = 0.0119 mM = 11.9 µM. In Table4. Comparing the concentrations listed in Table 4.2 and in Table 4.3 it is possible to see that the two methods agree for small concentrations, but for higher values, such as for fractions E4 and E5, the two methods do not. This is due to the fact that the linear range of the assay for BSA in between 0.2 to 0.9 mg/mL. In our case, the concentration is higher so the values are not reliable. For this reason, we prefer to use the rst method, when possible.

Demonstration of industrial-scale production of Ubx

In anticipation of a future increasing demand of Ubx proteins, it was decided to start the process of protein production in large volumes. In this regard, the entire process was reviewed and adjusted for the use of a fermenter. This is a container used to grow bacteria and fungi in large amounts, some fermenters have the capacity to hold thousands of litres. In the present work, a 15-L fermenter was used.

The transformation process was done as described in section 4.1.1, as this step can not be changed.

Once the bacteria colonies were ready, it was possible to start the small growth. Instead of 8 small pots, 1L of autoclaved LB media was inoculated with some colonies picked from the LA agar plate, after have added AMP and CLAMP (1 mL of each). Bacteria were let grow overnight at 37

• C shaking at 250 rpm. The following day, 6 L of LB media were prepared following the receipt and were put inside the fermenter where it was sterilised prior to using it. Once the media was autoclaved, it was let cool down to 37

• C. Then 6 mL of each antibiotics and 7 mL of cultured LB media were added. In this case only ∼ 1% of the nal volume of bacteria cell culture was added because the fermenter was designed to optimize bacteria and fungi growth, facilitating the oxygenation and agitation and resulting in a faster cell growth. For the purpose of this work, however, it was important to not reach mid-log too quickly or E. coli would not be vigorous enough to produce proteins. Moreover, 10 mL PEG were added to avoid the foam formation. The parameters set were:

• temperature T = 37 • C, yellow line in Figure 4.8;

• Air = fault(4), pink line in Figure 4.8;

• Agitation = 350 rpm;

• pH = 7.00, red line in Figure 4.8.

Once the inoculation was started, the OD was checked, see Table4.4.

Tab. 4.4: Optical Density check during E. coli cell culture. • C, as it is shown in Figure 4.8. Then, it was possible to add IPTG to induce the EGFP-Ubx gene transcription and the temperature was set to 26

• C for the overnight growth. The following day it was possible to harvest the cells. Unfortunately, the laboratory was not equipped with a large centrifuge and the harvesting process was done with a bench centrifuge that could process only 6 falcon tubes of 40 mL each, so the maximum volume used was 240 mL. To spin down 6 L of bacteria cells it took 3 hours. However the media was left on the bench for 3 hours, so the pellets weren't good and were thrown away.

Although the rst attempt was unsuccessful, an other PhD student in our group was able to produce a large batch of EGFP-Ubx protein following the described procedure.

Production in Grenoble laboratories

In August 2020, during the Covid-19 pandemic, a re had broken out at Swansea University's campus and the laboratory, where EGFP-Ubx proteins were produced and stored, was destroyed. For this reason, the protein production was moved to Grenoble to produce at least one batch used to nish the experiments. The protocol used was the same described earlier and just few changes were made.

The transformation was done using an old plasmid that survived the re in Swansea. The following day, 200 mL of LB media were inoculated with one single colony scratched from the plate and let grow overnight in the incubator at 37

• C. Then 2 baed asks, each containing 1 L of media, were inoculated and placed in the shaker at 37

• C. Since the LB media used was a bit richer with nutrients to what the normal protocol suggested (15 g tryptone instead of 10 g), and in the shaker the asks were producing too much foam, the speed was lowered to 60 rpm. From this point the OD was checked until the mid-log was reached, see Table4.5.

Tab. 4.5: Optical Density check during E. coli cell culture. • C overnight. The day after cells were harvested and frozen at -20 • C until use.

For this purication, the lysing buer was prepared using 4 aliquots of DNase I at 20 u/µL, 10 mM of manganese and magnesium, 2 pellets of complete, 0.5 mg of Pefabloc (instead of PMSF) and two spatula tips of lysozime. Once the pelled was lysed, it was centrifuged and the supernatant was loaded in the column. Unfortunately during the purication process the matrix was disturbed a bit and the proteins had been eluted during the last wash (G80 buer) instead of during the elution step (G300 buer). This brought a problem of pH, because G80 buer had pH = 8.0 while G300 buer had pH = 7.0, and a problem of salt concentration.

Soon after the purication, EGFP-Ubx concentration was calculated with BSA method because a spectrometer was not available, see Figure 4.9.

Fig. 4.9: Absorbance-concentration relationship for BSA proteins.

We decided to verify protein concentration with SDS-PAGE gel. The protocol used was the same used previously and the result is shown in Figure 4.10. In the gel it was decided to load the crude, the unbound, wash number 1, wash number 2, wash number 3, elution 1, elution 2, BSA (whose molecular weight is equal to EGFP-Ubx one) at concentration 1 mg/mL; 2 mg/mL and 3 mg/mL and the standard. As visible, there are no proteins in the three washes, as expected. The three columns with BSA indicated respectively one big band at around 70 kDa, which was the molecular weight expected. In elution 2, E2, it seemed there were no proteins while in elution 1, E1, there was a big band indicating a high concentration of proteins. However, the band indicated the presence of proteins with molecular weight ∼ 25 kDa.

This meant that all the proteins produced were degraded and could not be used for the planned experiments and I was forced to use stored proteins.

Fig. 4.10: Gel containing the crude, the unbound proteins, three washes W1, W2, W3, fractions E1, E2, BSA at three concentrations 1 mg/mL, 2 mg/mL, 3 mg/mL and the standard.

MACROSCOPIC RESULTS

As already mentioned, Ubx monomers spontaneously self-assemble ex vivo at air-water interface forming a biomaterial that can be reshaped (into microscale bers or lms) and used in many applications. Although both Ubx monomers and Ubx lms have been highly studied and characterized (e.g. tissue engineering scaolds [START_REF] Howell | Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity[END_REF]), lm formation step has not been fully studied leading to a lack of understanding the mechanisms which bring monomers to aggregate into a lm. This missing knowledge can allow us to optimize materials assembly and yield. In fact, mechanical properties can be tuned by altering the Ubx, eliminating tyrosines in the protein structure [START_REF] Howell | Identication of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax[END_REF]. Moreover, the nature of these early stages can impact on lm structure and behaviour of the resulting lm. The concept Ubx study can be applied to all self-assemble proteins. In the present work, EGFP-Ubx fusion has been used. The goal of this work is to understand the early mechanisms of lm formation and the rst step toward this direction is the study of inuence of external parameters upon lm formation. This is the reason why the Langmuir technique has been used, since the lm formation process used by Bondos' group is similar to the sample preparation used in Langmuir technique. This method enables us to measure the surface tension as a function of surface area at constant temperature, recording a π -A isotherm.

Langmuir results and discussion

Using the Langmuir trough prevented me from following the protocol developed by Bondos' group, because the amount of proteins used is too high and the G0 buer contains a high concentration of salts. As a result of the high amount of both EGFP-Ubx and salts, a saturation of π-A measurements at early stages is visible. It was important to avoid this condition as in this case it could not be easy to compare isotherms coming from dierent parameter changes. For this reason, we drastically reduced both the amount of proteins utilized and the concentration of salts and glucose in the buer. Considering that no Langmuir studies have been conducted with Ubx protein or one of its fusion, I referred to previous works done with Bovine Serum Albumin (BSA) [START_REF] Trillo | Monolayers of human serum albumin -II. Eect of pH and ionic strength of substrate[END_REF][START_REF] Crawford | Serum albumin in 2D: A Langmuir monolayer approach[END_REF][START_REF] Gálvez-Ruiz | Dierent approaches to study protein lms at air/water interface[END_REF], whose molecular weight is comparable to that of EGFP-Ubx, 69.07 kDa.

The measurements of π -A isotherms were carried out on a KSV NIMA Langmuir trough Large KN2003 (PTFE; total area 841 cm 2 ) equipped with a dipping well and a dipping mechanism. The Langmuir trough was cleaned prior to use with warm water and acetone to remove any trace of contaminants. The stock spreading solution was prepared by diluting EGFP-Ubx puried proteins with Millipore water in order to reach a 10 µM concentration. The amount of added proteins in each experiment diers depending on the parameter-eect study. Fresh protein solution was dripped directly and symmetrically on the whole trough interface using a 1 mL pipette, three droplets per side, two droplets are placed near the moving barriers and one is placed near the Wilhelmy plate, see Figure 5.2. The droplets were carefully put in contact to the surface, avoiding any kind of disturbance, with the barriers fully opened. The spreading process plays an important role in lm formation [START_REF] Campbell | Polyelectrolyte/surfactant lms spread from neutral aggregates[END_REF]. In this phenomenon inertia, gravity and surface tension are the driven forces, balanced by viscous one [START_REF] Gorter | On the spreading of proteins[END_REF][START_REF] Chebbi | Dynamics of unidirectional gravity-inertial spreading of oil on water[END_REF][START_REF] Berg | Marangoni-driven spreading along liquid-liquid interfaces[END_REF]. Initially, inertia and gravity dominate the spreading process and then, as the proteins expand at air-water interface, surface tension gradients act as dominant force [START_REF] Chebbi | Dynamics of unidirectional gravity-inertial spreading of oil on water[END_REF][START_REF] Berg | Marangoni-driven spreading along liquid-liquid interfaces[END_REF]. The phenomenon of uid ow caused by surface tension gradient is called Marangoni spreading [START_REF] Gorter | On the spreading of proteins[END_REF][START_REF] Berg | Marangoni-driven spreading along liquid-liquid interfaces[END_REF][START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF][START_REF] Campbell | Polyelectrolyte/surfactant lms spread from neutral aggregates[END_REF]. When this phenomenon occurs, the resulting protein lm at air-water interface remains stable for many hours (the duration of measurements) [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF][START_REF] Campbell | Polyelectrolyte/surfactant lms spread from neutral aggregates[END_REF] and EGFP-Ubx monomers eventually dissolve into the subphase and can adsorb slowly to the interface [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF][START_REF] Campbell | Polyelectrolyte/surfactant lms spread from neutral aggregates[END_REF].

Once the proteins were loaded, they were allowed to equilibrate for a variable time (chosen following Majithia's work [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF]), called waiting time, before the compression was started. Films were obtained at a barrier compression rate of 25 cm 2 /min at pH ∼ 8.0 and room temperature (∼ 25 • C). Since the protein is partially soluble in water, we chose to use cm 2 as unity of measure for the Area, instead of nm 2 /molecule.

At the beginning I focused on nding the perfect conditions to obtain π -A isotherms with all the signicant features (e.g. gas phase and liquid phase [START_REF] Crawford | Serum albumin in 2D: A Langmuir monolayer approach[END_REF],

as described in section 3. As can be seen, the isotherms collected using plain water as subphase are very similar to each other in shape and values for the four dierent EGFP-Ubx concentrations (even though for the 1 nmol concentration the Surface Pressure values are really small). They present the required features of gas and liquid phase, however, no lm formed at the air-water interface. The absence of lm formation could be explained in two ways: the concentration of proteins is too low and monomers interact forming small islands of proteins that can not laterally associate; plain water can not prevent the monomers precipitation leading to the formation of aggregates that disperse in the bulk reducing the amount of available proteins at air-water interface. In order to distinguish which of the two hypothesis is most relevant, I repeated the same experiment using, instead, a common aqueous solution for Langmuir technique, 0.1 M NaCl, and the same EGFP-Ubx monomer concentrations, Figure 5.3b. NaCl, in fact, stabilises the protein conformation helping, in this way, lm formation [START_REF] Bondos | Detection and prevention of protein aggregation before, during, and after purication[END_REF]. Thus, if no lm formed at air-water interface, the problem was denitely the presence of inadequate number of proteins. The very rst dierence between the previous case is that π -A values are higher in this case (we go from 15 to 25 mN/m), even though at 1 nmol in 0.1M

NaCl the curve is comparable to the one obtained for 2 nmol in water. For this reason, in the case of 1 nmol of proteins spread in NaCl subphase, the hypothesis that the concentration is too low is the most reasonable. Moreover, at this concentration, it is impossible to wrap bers or lms and a monolayer was not visible to the naked eye. Thus, the formed lm is not strong and compact enough. On the other hand, an increase in protein concentration results in higher surface pressure values and bers could be drawn from the compressed lms for the 2, 3, and 4 nmol concentrations. However the 4 nmol concentration creates a very strong material that can be easily pulled but it is is too close to the saturating the Langmuir trough system and the measurement cannot be stabilized (e.g. a lm can be detected at the very beginning with all the barriers fully open, a further compression would lead to a more compact lm). Thus, this concentration could not be used to study parameter eects since small variations would not be measurable due to saturation.. As the 2 nmol and 3 nmol concentrations behave very similarly, the 2 nmol EGFP-Ubx concentration was chosen to enable stable lm formation while allowing maximum use of the dynamic range of Langmuir while varying other parameters.

The most relevant and important result from this rst experiment was the ability to pull bers after waiting only 10 minutes from the spreading of such low concentration of EGFP-Ubx. This result had not been achieved by Bondos group identifying a new phase of material formation that has never been studied. This upends everything we knew about the lm formation process [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF].

Once the protein concentration has been xed, the next step was the study of inuence on lm formation at dierent salt concentrations trying to reach the same NaCl concentration (0.5 M) used by Bondos group with G0 buer.

Although I could not use 0.5 M NaCl buer because at 0.4 M the system could not been stabilized and the measure was saturating, three dilutions have been explored using 2 nmol of EGFP-Ubx: 0.1 M NaCl, 0.2 M NaCl and 0.3 M NaCl, Figure 5.4a. In this case we can see a similar trend seen in Figure 5.3b with increasing π -A values as the salt concentration increases. We decided anyway to proceed in my experiments with 0.1 M NaCl concentration because increasing the amount of salt does not bring major advantages and we preferred to follow previous works in protein lm-formation at air-water interface [START_REF] Trillo | Monolayers of human serum albumin -II. Eect of pH and ionic strength of substrate[END_REF]110,[START_REF] Crawford | Serum albumin in 2D: A Langmuir monolayer approach[END_REF]. conditions would lead to alteration in protein structure and the consequent appearance of aggregates and precipitates. This phenomenon is one of the cause of properties loss, in our case self-assembly. To prevent this problem, it is important to study the eects of cosolvents upon protein solubility and aggregation. In order to chose the cosolvent to be studied, I referred to Bondos' article [START_REF] Bondos | Detection and prevention of protein aggregation before, during, and after purication[END_REF] 5.4b can be done on the appearance of sawtooth during gas phase for all subphases, except water. We hypothesised that they were caused by EGFP-Ubx islands sliding over each other during the barrier closure, they rotated away from it. So, the increase in π-A values didn't indicate a lm formation rather a collision of protein-base islands.

After having explored the eects of cosolvents, we decided to proceed the study using two aqueous solutions: 0.1 M NaCl and 0.1 M NaCl + 0.05 M N aH 2 P O 4 because the rst one gives lm that can be pulled even with small concentration of salts and the last one because it is the combination of salts that gives the strongest and more compact lm.

The last set of experiments done with Langmuir technique was focused on the exploration of waiting time dependence, Figure 5.5a and 5.5b. This parameter has never been taken in account as until now the proteins has been left

to form lm for hours in order to obtain an organised, compact and strong lm. At the beginning I explored the lm formation using 2 nmol of proteins dissolved in 0.1 M NaCl solution. Following Majithia's work [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF], the lm have been left to equilibrate for 10 minutes, 60 minutes and 120 minutes, In this case the waiting times chosen are not enough as at 10 and 60 minutes the π -A isotherms are comparable, loosing the decreasing eect. After two hours, instead, the surface pressure values are higher as expected. So, the non-monotonic eect disappears if we consider only these three measures.

For this reason, I decided to explore lm-formation at 25 minutes too. From the graph it is visible that at the latter waiting time we nd again the nonmonotonic eect. Thus, the time frame in which the descending-ascending behaviour is altered.

Our hypothesis is that at the early stages of lm formation (e.g. minutes)

the lm is partially disorganised as a lot of islands of brils are formed at air-water interface all with dierent orientations in 2D space, as observed by Majithia et all [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF]. When the trough barriers are closed, they have not got enough time to interact and orientate and, being forced to stay in a small area, they form a lm that can be anyway pulled into bers even without a proper periodicity. As the time passes, monomers interact also with the buer and cosolvent and amount of material adsorbed at the air-water interface decreases over time (e.g. 30 to 60 minutes, depending on cosolvents chosen). In this time frame the ability to pull bers is reduced and no sam-ples can be collected because there are no bers or because the bers are too thin to be collected. For longer waiting times (e.g. 120 minutes), the amount of monomers at the surface increases and the lm structure results well organised as the brils islands have enough time to interact and to rotate so that the bril result orientate with each other before the lm is compressed [START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF]. For this reason, π -A isotherms indicate a more compact lm. LA values have been calculated for the dierent waiting times and the two aqueous solutions described above, as listed in Table5.1. In general, it is possible to say that for both aqueous solutions LA follows the non monotonic eect seen for π -A isotherms.

Limiting Area analysis

Fiber collection

With Langmuir technique it is possible to obtain macroscopical information about the resulting lm by comparing dierent π -A isotherms and to make some hypothesis regarding the lm organization and structure. However, in order to have a complete comparison between the results obtained with

Langmuir trough and what have been fully characterized by Bondos' group, it was important to collect bers from each Langmuir experiment, in particular for the waiting-time comparison.

Following standard procedures, using a 10µL sterile plastic inoculation loop, EGFP-Ubx bers have been collected at the end of lm compression so that all the lms had in common the same available surface area [START_REF] Mendes | Generating Novel Materials Using the Intrinsically Disordered Protein Ubx[END_REF]. By gently touching of the plastic loop on the air-water interface, it is possible to pull bers that can be wrapped around it. The wrapped bers were left dehydrate slowly in a sterile plastic Petri dish sealed with paralm. The plastic loop was also xed at the surface of the container. In this way any violent shaking was prevented (that could break the bers) and at the same time any air dust contamination was avoided. The Petri dish was stored at room temperature until use. First of all, we took bers using 2 nmol of EGFP-Ubx proteins spread in 0.1 M NaCl subphase using the Langmuir trough at dierent waiting times. We were able to collect bers at 10 and 120 minutes waiting time, but none at 60 minutes. Fibers were analysed using SEM (1 kV voltage and 5 µA current with a 2.5k magnication) to check the ber structure (see Figure 5.7). Although the two diameters are comparable (see Table 5.2), the rst visible dierence we can see in the ber structure is the wrinkles alignment: when the waiting time is high, 120 minutes, the ber presents a well aligned structure with uniform parallel wrinkles (Figure 5.7 (b)). The latter have also comparable dimensions. However, when the waiting time is drastically reduced, 10 minutes, bers present fewer wrinkles, if compared with the previous case, with dierent dimensions (see Figure 5.7 (a)). It is clear that the dierent waiting times have an impact on ber structures and, in general, on lm organization. For this reason, in order to analyse the dierences in lm formation when varying the waiting time, measurements using the G0 buer were carried out using the Langmuir trough. In this way, thanks to the high density and viscosity of the buer, bers could be collected also in the descendent portion of Langmuir data (using the other types of buer would not have gave us the opportunity to). Moreover, using G0 buer enables the comparison with earlier results from the Bondos group [START_REF] Huang | Size dictates mechanical properties for protein bers self-assembled by the drosophila hox transcription factor ultrabithorax[END_REF]. As already mentioned, the π -A isotherms cannot be used since the instrument was saturated, but the Langmuir trough was used for controlled compression and not to get measurements. Using G0 buer was not only en- and the buer (G0) were the same, the material formed at air water interface is dierent meaning that the waiting-time parameter extensively aects the protein organization.

The second visible dierence in all three images is the wrinkle alignment.

Matlab data analysis

Once these studies had been concluded, we decided to go forward and analyse samples taken using 0.1 M NaCl and 0.01 M NaCl + 0.05 M N aH 2 P O 4

solutions with an in-house developed MATLAB code. In this way it was possible to obtain quantitative structural information from an image. The goal of our analysis was to measure the alignment and orientation of wrinkle structure in the collected bers. The concept on which the analysis is based is the axial autocorrelation and the full width at half maximum (FWHM) height, see Figure 5.10.

Once the ber image is loaded in MATLAB and the wrinkle orientation in dened (xing the y-axis), a threshold is set based on the background to create a binary mask of the ber. The lter removes small areas captured by the threshold because considered noise. On the other hand, in order to remove the background noise, all the intensities outside the mask area on the original image are set to zero. At this point it was essential to use an image attening algorithm to separate and dierentiate the brils (Figure 5.9). Assuming a cylindrical shape for UBX ber, the ber visible part is considered to be a semicircle with the diameter equal to the width of the ber. The centre point of the bre was dened as the midpoint between the two widest points on the bre. From that centre point, pixel intensity values are expanded horizontally as if the cylinder was being unrolled into a 2D where θ is the angle between the virtual centreline of the bre and the pixel of interest (Figure 5.10b) and d is the ber diameter. In order to dene the width per pixel, we used the image resolution, 20 nm/pixel. The width per pixel parameter is important for the calculation of the arc length between a pixel and its neighbour, based on Equation 5.1. This arc length is converted back into pixels and rounded to the nearest whole value. The image intensity value at the original pixel is then repeated as many times as necessary to match the length of the arc. This was repeated for each horizontal line of pixels. When the radius of the ber is much larger than the radius of the wrinkle, we can cut o the portion of the image that extends beyond a 60 Since for some waiting times it was not possible to collect bers because of the the inability to produce them, and for some waiting times it was not possible to wrap samples, due to the break caused by the small diameter (e.g. d ≤ 2µm), we dierentiate the ability to collect small bers from the inability to produce them. The symbol '-' is used if it was impossible to see any, '+' if it was possible to see some which was too weak to be collected.

The vertical autocorrelation values corroborate the qualitative observations seen in Figure 5.8. There is a signicant increase in the structural linearity and continuity of the bers pulled after 120 minutes of waiting time as compared to both the 10-and 60-minute wait time groups. As already mentioned, our main hypothesis explains the dierent wrinkle structure with the brils islands orientation. When the lm is formed and squeezed in minuteslong period of time, the island are not parallel and when bers are pulled, the applied mechanical force pulls a straight ber, but the lm structure is not well organized and the wrinkles are non-uniform. On the other hand, in hours-long waiting time, the island interact with each other and rotate so that they are parallel. When the lm is compressed and bers are pulled structure is more organized. For this reason, we can conclude that the island along the ber where they are structured, Figure 5.12(a). After a 60-minutes waiting period, there is no consistent size or structure in the image (Figure 5.12(b)), reecting the fact that wrinkles are disordered. Finally, at 120minutes waiting time, the structure of the wrinkles is uniform and organized and their size increases in dimension up to ∼1 µm.

Metal Disk coating

During the Langmuir experiments, we decided to study the interaction between Ultrabithorax protein-based lms and titanium substrates. The goal of these observations was to understand the coating durability on titanium surface and the possibility to use Ubx-based material for cell growth applications. In fact, the titanium substrates used, have great applications in cell biology and previous works done by Bondos group, showed that Ubx monomers fused with vascular endothelial growth factor (VEGF) proteins generate bers able to help tissue regeneration [START_REF] Howell | Functionalization of Ultrabithorax Materials with Vascular Endothelial Growth Factor Enhances Angiogenic Activity[END_REF]. However, no studies have been done with Ubx lms, so I started with EGFP-Ubx material.So I performed some preliminary test using sterile titanium discs. The rst step was to clean the surface in a sonicator with some IPA for 15 minutes to re-move any dirtiness from the surface and then do a plasma cleaning. Since the goal of this step was to sterilize the plates before the coating and not to remove T iO 2 from the surface, I opted for Sulfur hexauoride (S F 6 ) and Argon (Ar) plasma cleaning instead of HF, which is more aggressive and dangerous.

As I had never done plasma cleaning and I was not sure where to start from, I have tried with dierent parameters. The rst set of parameters used was: Then, I used 100 sccm Ar, Pressure = 70 mT, Power = 50 W and time = 2 minutes. However, the disc seemed to be as bright as it was initially (Figure 5.13b), so I tried using the same parameters used previously except the power which was increased from 50 W to 200 W. However, the disc was again as bright as it was previously (Figure 5.13c). At this point I decided to proceed with Ubx lm coating with the rst and the last cleaned discs, so that I could compare the resulting lm.

For the metal coating I decided to use Langmuir trough in order to have controlled process. I used buer G0 with 5% w/v Glucose and I doubled the quantity of N aH 2 P O 4 so that it was possible to obtain the strongest lm possible with a 2-hours waiting time parameter instead of 16-hours one. For this purpose, I increased also the protein concentration using 10 nmol instead of 2 nmol. With the aid of a dipping arm equipped with Langmuir trough, it was possible to coat the discs uniformly in both sides of the piece even if only one surface was cleaned. Once coated, the discs were put in a plastic petri dish, sealed with paralm, and let rest to dehydrate. After a week the discs have been re-hydrated with some PBS buer and the lm was checked with a uorescent microscope, using a ×4 objective lens and a λ = 488 nm as excitation source.

Antimicrobial peptide coating

The EGFP-Ubx lm coating work was particularly useful to test two new Ubx fusions and their antimicrobial action.

Our group in Swansea designed two expression constructs to create fusion proteins positioning antimicrobial peptides at the N-terminus of Ubx, using a exible linker and SUMO tag to ensure protected expression and precise excision of the peptides. The aim was to use the Ubx lm-formation ability combined with the peculiar ability to kill bacteria and fungi of specic peptides. In this way it could be possible to coat surfaces (e.g. door handle) with a thin lm of these Ubx fusions and inhibit the growth of bacteria, stains, odours and product degradation. Cathelicidin LL-37 (LL37)

and Bovine myeloid antimicrobial peptide-18 (BMAP) were chosen for their well-studied antimicrobial properties [START_REF] Costa | Protein-Based Films Functionalized with a Truncated Antimicrobial Peptide Sequence Display Broad Antimicrobial Activity[END_REF][START_REF] Bucki | Cathelicidin LL-37: A multitask antimicrobial peptide[END_REF]. In Figure 5.16 a model of the BMAP-Ubx fusion protein is shown illustrating the dityrosine bond forming residues thought to be responsible for material formation. To measure Fig. 5.16: Model of the BMAP fusion protein illustrating the dityrosine bond forming residues thought to be responsible for material formation.

the antimicrobial eect of coated surfaces with Ubx, a quantitative kinetic antimicrobial screening assay was used. This is based on ISO 22196 standards and is appropriate for use with protein covered surfaces. The assay is based on the resazurin microplate method, using kinetic analysis of growth of Bacillus subtilis, a commonly found non-pathogenic fast-growing grampositive bacteria, as the initial test organism. Resazurin reduction, resulting from cell metabolism, produces a highly uorescent dye which was quantied in a 96 well format using a Biotek 96 well plate reader, excitation wavelength λ ex = 520 nm and emission wavelength λ em = 590 nm. Three substrates were chosen for this experiment:

• titanium Grade 2 discs (diameter ∼6 mm), chosen due to its high resistance to corrosion and biocompatibility, as a stable and an inert oxide layer spontaneously forms when its surface is exposed to oxidising media;

• titanium Grade 2 discs (diameter ∼6 mm) that were previously coated with EGFP-Ubx lms and etched with piranha solution (a mixture of sulfuric acid,H 2 SO 4 , water and hydrogen peroxide, H 2 O 2 ) to remove Ubx lm.

This substrate was chosen with a view to a possible recycling of the materials;

• silicon wafer squares (side = 6 mm), chosen because it is a suitable support for growing and/or mounting cells.

The experiments were set up aseptically in a Hepaltered ow hood. The surfaces to be tested were initially sterilised by immersion in 60% ethanol for 10 mins followed by rinsing 4 times in sterile PBS. Then, they were coated with Ubx lm, dried, placed in a sterile Petri dish and aligned on a labelled perforated sterile rack for ease of manipulation. The Petri dish was situated As can be seen, Ubx coating in general do not inhibit bacterial growth, on the contrary it seems to slightly improve it. At the same time, compared to other two samples, non-etched titanium substrates are inhibiting a bit more the growth. This eect is removed when the surface it cleaned with piranha solution, probably due to chemical change at the surface. In general, bacteria grew better on etched and Ubx coated titanium surfaces.

In conclusion, it is better to use titanium grade 2 disks coated with Ubx rather 5.2 BAM results and discussion The rst experiment was done with plain water, shown in Figure 5.19. As soon as I spread proteins, the ROI/sig immediately arose and the luminous intensity was linearly increasing, meaning that more and more proteins were collecting at air-water interface. However, in general during the Langmuir cycle there is not a clear structure variation even though the surface pressure indicates the presence of a lm. Probably a really thin lm formed at air-water interface but it was not strong enough and no bers were visible at the end of the process.

The further experiment was done using 0.1 M KCl subphase. In this case as soon as the proteins were spread, thick protein-based islands were visible.

Moreover, during the compression-expansion cycle, it was possible to see two types of structures: a single compact lm oating on non-compact material that shows a sort of lateral structure that is not complete, as can be seen in picture number 4 and 5 in Figure 5.20. During the equilibration time, it was possible to monitor lm formation at air-water interface. Interestingly, lateral association between a big thick island and a small agglomerate od EGFP-Ubx brils, as can be seen in Figure 5.21.

Fig. 5.21: Lateral association between big thick island and a small protein agglomerate.

6. MOLECULAR RESULTS

Ellipsometry Data

Ellipsometry results were used to help the planning and the interpretation of NR experiments, as they provide complementary informations about the surface excess during the formation of protein lms.

Optimization of spread lms

To simplify the calculation of surface excess Γ from ellipsometry data [mg/m 2 ],

following the calculations presented in the supporting information of reference [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF], it is possible to chose one model for the considered system. In this case, there are two possible models :

• The model changes in the surface coverage as changes to the layer thickness at constant density (`oil-like' behaviour). As layer density is constant, there is no solvent in the surface layer, so n surf is a constant, hence the relation between the Γ and ∆ surf is linear [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF]. In the EGFP-Ubx protein case:

Γ = 0.495∆ surf (6.1)
• The model changes at constant thickness (`particle-like' behaviour). In this case, n surf is a variable related to the density or coverage of protein in the layer, hence the relation between Γ and ∆ surf is quadratic. An empirical t to simulated data gives Γ(∆ s urf ) is quadratic [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF] and for the protein studied we have:

Γ = -0.0374∆ 2 surf + 0.6126∆ surf (6.2)
where ∆ surf = ∆ -∆ 0 is the change in ∆ from the specimen at the interface, ∆ is the measured value, and ∆ 0 is the calibration value for pure subphase.

For protein-based lms, it is recommended to use the 'particle like' model as they tend to form monolayers with increasing area coverage rather than increasing thickness.

The ellipsometry data were recorded using a phase modulated Picometer Light ellipsometer using a HeNe laser with λ = 632 nm at θ = 50 • . Measurements were made using a static trough, a glass Petri dish with a diameter of Moreover, considering that the surface excess for both cases tend immediately to plateau, it is possible to state that EGFP-Ubx proteins are spread as stable lms at air-water interface, despite their solubility in aqueous solutions [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF]. The EGFP-Ubx protein-based lm remains at the air-water interface for the duration of the measurements. In fact, protein lms can be spread from concentrated droplets [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF]. This can be the reason why, using BAM, it was not possible to image clearly the lm formation during the waiting time or during the barrier compression process. Instead, a bright solid protein-based island was visible from the very beginning. However, remembering Langmuir trough data, it is clear that this surface-loading process depends on protein concentration. In fact, if there are not enough proteins, they dissolve into the bulk and then slowly adsorb at air-water interface again. The solution was poured into the glass Petri dish and the surface was aspirated for 3 seconds. The surface excess was then monitored for 4 and 12 hours (Figure 6.3). From the graph it is visible that, except an initial uc-Fig. 6.3: Time-resolved surface excess of adsorption technique. In the gure frame the same data are displayed on a t 1/2 scale. The dotted black lines help to visualise the linear component.

tuation due to the surface aspiration, the two measures are consistent. This means that the kinetics of EGFP-Ubx adsorption from a premixed solution at a clean air-water interface is stable and not sub jected to external parameter inuence (e.g. temperature or humidity). In both cases, after an initial rapid increase, the surface excess seems to stabilize and reaches a plateau.

The temporal uctuations visible at the plateau may be related to the fusion of protein islands as they form and grow laterally at the air/water interface, passing in and out of the laser beam [START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF]. However, the quantity of subphase is so small that after 3 hours it evaporates creating a laser misalignment that can contribute to signal oscillation. For this reason, for our study the experiments won't be longer than 2 hours.

In Figure 6.3 it is interesting to notice that the surface excess tends to values close to the ones seen in the spreading technique, Γ∼ 1.79 mg/m 2 . Thus, it is possible to conclude that EGFP-Ubx lm formed at air-water interface after the adsorption from a premixed solution tends to be similar to the one obtained when proteins are spread at the surface. However the amount of time required to the two lms to be similar is dierent: 2 hours against 12 hours.

Following Ramsden work [START_REF] Ramsden | Concentration scaling of protein deposition kinetics[END_REF], the surface excess was plot also on a t It is possible to conclude that the lm formed at air-water interface by adsorption from premixed solution resembles the lm formed by spreading.

However, since in the adsorption case the time-frame required is 6 times longer compared to the spreading case, in the present work only the latter case will be used for lm formation. might contribute to protein degradation. From Figure 6.4 it is possible to verify that the protein behaviour is consistent, meaning that no important degradation occurred over time and the tyrosine sites were preserved. After having veried the ability to pull bers, it was decided to proceed with the experiments using the old proteins.

At the beginning it was important to check the consistency of old proteins spread in the subphases used for neutron reectivity. Thus, experiments using 0.1 M NaCl solutions were repeated three times using both H 2 O (Figure 6.5a) and D 2 O (Figure 6.5b) as solvents.

As can be seen in the two diagrams, the time-resolved surface excess is consistent over time, in the limit of ellipsometer resolution. This means that EGFP-Ubx monomers self-assemble at air-water interface with a consistent lateral association. However, when EGFP-Ubx proteins are spread in H 2 O solution, the curves dier in shape at initial times and then converge for With all these results it was possible to proceed with neutron reectivity experiments.

c 2 = Γ D 2 O Γ H 2 O • c U bx

Neutron reectometry data

As already mentioned in the chapter 3.5, during NR experiment a neutron beam was directed toward the air-water interface where the proteins were spread and the intensity of reected beam was recorded in specular set up.

The reectivity prole, R(Q z ), was then calculated as ratio between the reected neutron beam and the incident one as a function of momentum transfer perpendicular to the surface Q z = (4π / λ) • sin(θ), where λ is the neutron beam wavelength and θ is the incidence angle. The data were reduced using COMSOS [START_REF] Nelson | Co-renement of multiple-contrast neutron/X-ray reectivity data using MOTOFIT[END_REF]. R(Q z ) is a function depending on the prole along the z direction of the scattering length density (SLD),ρ. The data were analysed with the software Motot [START_REF] Nelson | Co-renement of multiple-contrast neutron/X-ray reectivity data using MOTOFIT[END_REF]. The system was modelled as a single layer of proteins, characterized by and average ρ, a thickness d and a roughness σ, oating on a semi innite liquid. The parameters found with this model, were then used to calculate a reectivity prole that could be compared to the measured one. The tting process was done using a 5% ∆χ 2 /χ 2 . A common problem in neutron reectometry is the absence of a biunivocal relationship between the reectivity proles and the length density proles. More than one scattering length density proles can be related to a reectivity prole. To solve this issue more experiment are performed on the same system having a dierent isotopical composition. This method, referred as contrast variation, is based on the hypothesis that upon isotopic substitution of one or more components of the experimental system, the main nanostructural features does not change signicantly. As we will see in the following sections, this hypotesis is not strictly fullled in the systems that we are studying here. Hence, in the case of Ubx lms dierent technical expedients are required to use. The mixtures used in the following experiments are D 2 O and air contrast matched water (ACMW). The latter solvent is a mixture of 8.13% v/v D 2 O + 91.87% v/v H 2 O, and its SLD resulted equal to zero being therefore equivalent to air. A bare ACMW-air interface does not reects neutrons,ensuring that the only scattering comes from the EGFP-Ubx. Hence, this contrast is particularly sensitive to the properties of the protein layer. To both ACMW and D 2 O dierent salts were added in order to obtain comparable results with what was previously done. The rst visible result was the dierence between the reectivity for ACMW at A 1 = 300 cm 2 and A 2 = 110 cm 2 . When the barriers were fully open, there was a noisy signal coming from neutrons at low Q, that would become even noisier for higher values of Q (for this reason we did not measure it). On the other side, after the lm compression the resulting signal was less noisy and stronger. This result was coherent with surface-pressure (π-A) values: at the beginning, the Ubx-monomers interactions were minimal, and the monomers started to aggregate forming small islands. As the barriers moved closer, the interactions became stronger and stronger because of the decrease of area per molecule. This resulted in an increase of π-A values and consequently in a more compact lm structure. So at this protein concentration a lm was able to form at the surface.

Dierences can be seen also when the proteins were spread in D 2 O. In this case, the signal is higher mainly due to the fact that the contrast between D 2 O-air is higher (ACMW-air contrast is zero), so the intensity of the re- ection is high. Moreover, we know that with D 2 O the lms are thicker, because this subphase promotes the monomers interaction, hence a lm formation could be seen from the very beginning. The compression enhanced the interactions and we could observe a variation in reectivity which meant a thickness variation maintaining the other parameters constant.

In conclusion, we were able to detect the presence of EGFP-Ubx lm at air-liquid interface. A lm was measured even without compression, which perfectly agreed with the observations made with BAM. Moreover, we were able to set the parameters that let us measure neutron reectivity.

Experiments

After that optimum conditions for sample preparations were achieved, it was possible to to better plan a neutron reectometry experiment to investigate the lm formation. For this purpose, a new beam time was obtained in February 2021. From preliminary investigations using laboratory-based techniques, it seemed that the key dierences with waiting-time parameter corresponded to either sub-micron lateral structural developments, or structural changes normal to the interface. Thus, the goal of this new set of experiment was the probe of these changes.

NR experiments were performed at FIGARO reectometer equipped with the same Langmuir trough used previously (trough area at barriers fully open equal to 256 cm 2 and area at full compression equal to 127 cm 2 ). The charac- terisation of the protein adsorption at the air-water interface was done using two subphases, D 2 O and ACMW (140 mL), to maximise the contrast between the liquid and the protein. D 2 O is a contrast that generally provides a good reectivity signal, while ACMW is a contrast that maximise the sensitivity to the structural aspects of the protein lms, although it has generally a lower reectivity signal. In order to match the Langmuir experiments, previously described, we decided to investigate the three waiting-times responsible for the non monotonic eect (10, 60 and 120 minutes) of π-A values, previously described using subphases with 0.1 M NaCl. For simplicity, in this work we won't specify the salt concentration as it will remain the same for all the results shown. After the preliminary experiments with ellipsometer, we noticed that EGFP-Ubx protein lm had two dierent behaviours in ACMW and D 2 O. That is, by using the same concentration of protein in the solution, two dierent lms were forming at the air-water interface (in ACMW was less thick than the one in D 2 O). Therefore, we chose a higher concentration in ACMW that would result in a protein lm at the air/water interface that had similar features (thickness, hydration), at least as a result of the ellipsometry experiments. In order to be able to compare NR data and Langmuir results, The rst set of experiments done was with EGFP-Ubx concentration equal to c 1 in ACMW. In Figure 6.9 on the top, the reectivity and SLD are shown, while the bottom part shows the model parameters used to t the data. As can be seen, the three reectivity curves, which were shifted vertically for clarity, present similar shapes, but the dierences between them are suciently large to obtain signicant dierence in results of the data tting, as it can be seen from the SLD graph reported in Figure 6. This behaviour can be explained with lm organization structure. When the brils composing the protein-based material are well aligned to each other, the lm resulted to be thin and the bril structure avoid to incorporate solvent. On the other hand, a disordered material present a thick lm in which there is a higher quantity of solvent. As discussed in the previous chapter, at 120 minutes the lm presented a highly ordered pattern resulting in a thin thickness, while, on the other hand, after 60 minutes of equilibration, the material structure presented disordered pattern where brils are non-continuous and winding and the thickness was higher compared to the other cases. In the middle of these two extremes, at 10 minutes waiting time, lm present is not perfectly aligned and its thickness is more similar to the 120-minutes It is possible to conclude that with this experiment we were able to corroborate the observations done with experiments made starting from the same protein concentrations in the liquid phase. We could ask if the lateral morphologies determined using BAM and described in the previous chapter 5.2 might aect the interpretation of the NR data. It is known that the presence of lateral morphologies on a surface aect the neutron reection in two dierent ways wheatear these morphologies extend on a length scale larger or smaller of the coherent length of the neutrons. In the case where two dierent morphologies are present on the surface of water whose lateral extension is larger than the coherent length of the neutrons (e.g. macroscopical regions with and without protein lms), the resulting reectivity prole would be given by a linear combination of the reectivities of the two regions weighted by the relative fraction of the areas occupied by the two morphologies. However, that was not the case in our NR experiments as the reectivity proles could be well tted with one single reectivity proles. We can then conclude that our protein lms at the stage in which the measurement were conducted, presented a homogeneous composition at least on the scale of the coherent length of the neutrons.

The second set of experiment was done spreading a protein concentration c 2 in ACMW and c 1 in D 2 O and a co-renement of the two measures have been done. The use of two dierent concentrations of EGFP-Ubx at the two contrasts was dictated by the observation that the thickness of the protein lm changes signicantly if we use D 2 O or ACMW. We then adapted the concen- tration of the protein in ACMW to match the elliposmetric thickness of the protein lm in D2O. In Figure 6.10 reectivity and SLD data of EGFP-Ubx monolayers spread in ACMW and D 2 O for dierent waiting times are shown.

In the table, the structural parameters obtained from the co-renement of NR curves are listed. In this case, it is possible to see that the reectivity curves present really small changes. Moreover, the non monotonic eect seen previously disappears in this case, for both ACMW and D 2 O subphase, returning in the regime studied by Bondos' group. The lm thickness, calculated with co-renement, decreases while the waiting time increases too, going from 41.4±1.0Å to 30.0±0.8Å and nally 22.3±1.4Å The same trend is followed by SLD values and roughness. The non-monotonic eect disappears also for the two experiments in ACMW and D 2 O separately, demonstrating that this phenomenon is not caused by co-renement. This clearly indicates that as time passed, the monomers rst and the brils then, had enough time to organise and form a smooth structured material, with a decreasing content solvent. This result shows that the concentration of the EGFP-Ubx, used to deposit the lm, has a key role in the nal properties of the lm.

The explanation of this phenomenon can be attributed to dierent causes to the two subphases used, ACMW and D 2 O. In the rst case, in fact, probably the protein concentration used (c 2 ) was too high for the amount of liquid (140 mL) in which it was spread. The non-monotonic behaviour was not fully explored at dierent protein concentrations. In the case of D 2 O sub- phase, instead, the loss of non-monotonic behaviour can be explained with the stronger hydrophobic bonds compared to the one present in H 2 O. Since Ubx proteins present hydrophobic aminoacids at the outside, their structure is aected by the change of subphase hydrophobic bonds and the stability of the monomers increased. Moreover, D 2 O has a higher density compared to H 2 O which is a key parameter in Ubx lm-formation. In fact, G0 buer used for lm formation contains 5% w/v D-Glucose that increases the density of the liquid and helps to stabilise the protein structure. So, in this experiment, D 2 O eects are similar to D-Glucose ones. In conclusion, this second set of experiments shows a key information concerning the use of the contrast variation on EGFP-Ubx lms at the air water interface that might be relevant for other protein systems at the air water interface. Since the properties of the resulting lm strongly depend on the isotopic composition of the liquid subphase, care must be taken in the choice of the the reectivity curves that are used to perform co-renement.

Further studies are needed on this topic. For example, it would be useful to repeat the reectometry experiments at lower concentration in D 2 O to check if similar trends with waiting time can be obtained as in the concentration c 1 .

CONCLUSIONS & FUTURE WORKS

Ultrabithorax (Ubx) protein is a Hox transcription factor that regulates the transcription rate of genetic information inside fruit y cells. Interestingly, it was found that this protein presents a unique property in addition to its in vivo function as a transcription factor: Ubx self-assembles into biomaterials through intermolecular dityrosine bonds at air-water interfaces [START_REF] Howell | Identication of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax[END_REF]. Ubx protein-based materials are non-immunogenic, biodegradable, biocompatible, and have stabilizing eects on incorporated proteins [START_REF] Patterson | Materials composed of the Drosophila melanogaster protein ultrabithorax are cytocompatible[END_REF][START_REF] Patterson | Materials composed of the Drosophila Hox protein ultrabithorax are biocompatible and nonimmunogenic[END_REF][START_REF] Hsiao | The intrinsically disordered regions of the Drosophila melanogaster hox protein ultrabithorax select interacting proteins based on partner topology[END_REF][START_REF] Huang | Functionalization and patterning of protein-based materials using active ultrabithorax chimeras[END_REF]. These unique properties and the ability to incorporate functional proteins make the materials ideal for use in a variety of elds. In this dissertation, I explored EGFP-Ubx protein-based material in order to understand which parameters inuence the lm formation and, consequently, the resulting lm structure.

These results have a great impact in lm customization and optimization for dierent applications.

The rst and principal result was the ability to produce EGFP-Ubx proteins in Swansea laboratories with high purity, even though in small batches.

Without this outcome it could not be possible to achieve the results described.

Once the protein production was established, it was possible to proceed with

2 . 3

 23 TEM images showing the hierarchical lm formation of EGFP (Enhanced Green Fluorescent Protein)-Ubx (Ultrabithorax) at the air water interface. (a)-Ubx aggregation at 15 minutes after dilution; (b)-protobrils formation after 1h in dilution; (c)-parallel alignment of protobrils after 1-2h; (d)-bril formation of 50 nm diameter after 2h; (e)-lateral association of brils into lm; (f )-Ubx lm drawn into ber. Reproduced from reference [80]. . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Schematic gene sequence of Ubx fusion proteins used to create functionalised materials. The functional protein sequence (green, purple and red) are shown linked to exible linker, in yellow, followed by the self-assembling protein Ubx in brown. . 3.1 Langmuir trough scheme. A, bath where the subphase is poured; B moving barriers to control the surface area dimension; C surface-pressure balance with Wilhelmy plate measuring the surface pressure. . . . . . . . . . . . . . . . . . . . . .

3 . 4

 34 Scheme of surface pressure (π) vs. area (A) and orientation of surfactants in dierent phases. . . . . . . . . . . . . . . . . . . plane-view and end-view representation of wave nature of light . b -Light beam incident upon and reected from a surface showing the plane of incidence and the s-direction and p-direction. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 a -Optical setup of BAM. b -Reection coecients vs. incidence angle for s-and p-polarized light at air-glass interface. n a = 1 and n g = 2. . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Brewster Angle Microscopy physical functioning. . . . . . . . . 3.8 Example of BAM image . . . . . . . . . . . . . . . . . . . . . 3.9 Schema of ellipsometric measure. . . . . . . . . . . . . . . . . 3.10 Schematic diagram of a scanning electron microscope. . . . . . 3.11 a -Schematic diagram of an electromagnetic lens. The coil windings are inside the iron shroud and the eld is produced across the lens gap between polepieces. b(i) -Diagram showing how electrons are focussed pass trough an electromagnetic lens. b(ii) -Conventional optical ray diagram for comparison.

3. 15 2 (Fermenter

 152 (a) -Reection of an incident beam from two ideally at interfaces, one is a thin layer of thickness d on a semi-innite substrate (with scattering length density N b 2 and N b 1 for the lm and substrate respectively). (b) -Calculated neutron reectivity prole for a 500 Åthick layer of perdeuterated poly(styrene), d-PS, on a silicon substrate. The oscillations are due to the thickness of the specimen. Reproduced from reference [108]. . . . . . . . . . . . . . . . . . . . . . . . . . . 3.16 Diagram of a n-layers specimen with variable thicknesses (d n ) on a substrate. The index n + 1 corresponds to the substrate, n to the layer immediately adjacent to the substrate, down to index 0 which corresponds to the vacuum/air. . . . . . . . . . ix 3.17 Three surface proles: on the left sharp interface, at the centre a rough surface, on the right a diuse surface. The rough and diuse surfaces can result in identical specular reectivities. . 3.18 Sketch of the reectometer FIGARO. . . . . . . . . . . . . . . 4.1 Transformation technique scheme: using the heat shock technique, the cell membrane is made permeable to plasmid DNA which can enter into E. coli cell. . . . . . . . . . . . . . . . . . 4.a) -E. coli colonies on LB agar plate. (b) -400 mL of autoclaved LB with 0.4 mL of 50µg/mL AMP and 0.4 mL of 30µg/mL CLAMF (c) -Harvested bacteria cells using 30 minutes of centrifugation at 3400 rpm at 4°C. . . . . . . . . . . . 4.3 Column for protein purication with 10 mL nickel-nitrilotriacetic acid resine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 SDS-PAGE of protein samples scheme. Proteins are loaded into gel walls and electrical current is applied. Proteins run through the gel and are separated by size. . . . . . . . . . . . 4.5 (a) gel containing standard, the crude and all the elution fractions (E0, E1, E2, E3, E4, E5, E6); (b) gel containing the standard, the pellet, the crude, the unbound proteins, fractions E0, E4 and E5 and the Ni-NTA column matrix. The red rectangles underline EGFP-Ubx presence or absence. . . . . . 4.6 Absolute spectra of 1/20 dilution measured for all the fractions collected during protein purication. . . . . . . . . . . . . . . parameters during E. coli cell growth. Yellow line indicates the temperature, the red one indicates the pH and the pink the air ow. . . . . . . . . . . . . . . . . . . . . . . . 4.9 Absorbance-concentration relationship for BSA proteins. . . . 4.10 Gel containing the crude, the unbound proteins, three washes W1, W2, W3, fractions E1, E2, BSA at three concentrations 1 mg/mL, 2 mg/mL, 3 mg/mL and the standard. . . . . . . . 5.1 Steps of EGFP-Ubx protein-based lm formation. (a) -EGFP-Ubx is diluted in a Teon-coated tray lled with G0 buer; (b) -after a 16 h incubation period under an aluminium pan, with a microcentrifuge tube rack EGFP-Ubx lm is gently squeezed reducing its area; (c) -the aluminium pan is placed again on top of the tray leaving the lm to set for an hour; (d) -a plastic inoculation loop is used to touch the surface and pull bers (e). . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 M 1 M

 11 droplet disposition schema. Droplets are dripped at the surface gently in a symmetric way: three droplets per side, two droplets are placed near the moving barriers and one is placed near the Wilhelmy plate. . . . . . . . . . . . . . . . . 5.3 Isothermal compression curve measured with 10-minute waiting time at dierent concentrations: 1nmol, 2nmol, 3nmol and 4nmol. The subphase used was plain water (a) and 0.1 M NaCl (b). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Isothermal compression curve measured with 10-minute waiting time spreading 2nmol EGFP-Ubx. (a) -the subphase used has and increasing salt concentration: water, 0.1 M, 0.2 M and 0.3 M NaCl; (b) -the subphase used has dierent salts diluted: 0.1 M NaCl, 0.1 M KCl, 0.01 M PBS and 0.1 M NaCl + 0.05 M N aH 2 P O 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Isothermal compression curve measured varying the waiting time parameter (10 minutes, 60 minutes and 120 minutes) and spreading 2nmol EGFP-Ubx with (a) -0.1 M NaCl; (b) -0.NaCl + 0.05 M N aH 2 P O 4 . . . . . . . . . . . . . . . . . . . 5.6 Representation of limiting area projection. . . . . . . . . . . . 5.7 Fibers collected using 2 nmol EGFP-Ubx proteins spread in 0.1 M NaCl buer using (a) -10 minutes waiting time and (b) -120 minutes waiting time. It was not possible to collect bers from 60 minutes waiting time. . . . . . . . . . . . . . . . 5.8 Fiber images obtained using Langmuir trough with 2 nmol Ubx in G0 buer and using (a) -10 minutes waiting time, (b) -60 minutes waiting time and (c) -120 minutes waiting time. All the images have been taken using a Scanning Electron Microscope with 1 kV voltage. . . . . . . . . . . . . . . . . . . 5.9 Fiber attening diagram. The view from the SEM (A) can be attened by assuming a cylindrical shape and calculating arc lengths. The blacked-out regions are outside of the θ = 60 • threshold. . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.10 Diagram vertical autocorrelation (A) along with the graph of the computed sum values (B). . . . . . . . . . . . . . . . . . . 5.11 Ideal case of perfectly aligned bers (a) and the corresponding autocorrelation (b) and wavelet analysis (c). . . . . . . . . . . 5.12 Wavelet analysis of the bers reveals lateral uniformity and wrinkle sizes for waiting times of 10 (a), 60 (b), and 120 minutes (c), using 2 nmol of EGFP-Ubx spread G0 buer. . . . . xi 5.13 Titanium discs cleaned for 15 minutes with IPA in a sonicator prior to do Plasma cleaning for 2 minutes with (a) SF 6 , with 30 sscn ow rate, p = 200 mT, P = 200 W; (b) Ar, with 100 sscn ow rate, p = 70 mT, P = 50 W; (c) Ar, with 100 sscn ow rate, p = 70 mT, P = 200 W. . . . . . . . . . . . . . . . 5.14 Fluorescent pictures taken with ×4 objective lens using an excitation wavelength λ = 488 nm. The disc was cleaned with SF 6 plasma, (a) and (b), and Ar plasma, (c) and (d), prior the dip coating with EGFP-Ubx. . . . . . . . . . . . . . . . . 5.15 Fluorescent pictures taken with ×4 objective lens using an excitation wavelength λ = 488 nm. The disc was cleaned with SF 6 plasma and checked (a) 1 week (b) 4 weeks (c) 7 weeks and (d) 11 weeks after the dip coating with EGFP-Ubx. . . . . 5.16 Model of the BMAP fusion protein illustrating the dityrosine bond forming residues thought to be responsible for material formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.17 Left part: scheme of experiment. Silica surfaces are indicated with S, etched titanium discs are indicated with O and nonetched ones with N. Ubx-coated surfaces are indicated with 'x', while control surfaces are indicated with 'c'. On the top of samples bacteria solutions, C and D, and plain PBS (indicated as PBX) were pipetted. Right part: results of overnight uorescence. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.18 A-U BAM time-lapse during barrier expansion. The protein lm was created using 2 nmols EGFP-Ubx proteins spread in 0.1 M NaCl solution and let rest 2 hours before the barrier closure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.19 π-A isotherm with BAM images taken during lm compressionexpansion in plain water. . . . . . . . . . . . . . . . . . . . . . 5.20 π-A isotherm with BAM images taken during lm compressionexpansion in 0.1 M KCl. . . . . . . . . . . . . . . . . . . . . . 5.21 Lateral association between big thick island and a small protein agglomerate. . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Dierent loading approaching (a) one single droplet was let to fall from the top at the centre of Petri dish; (b) one single droplet was spread by touching gently the surface at the centre of Petri dish; (c) adsorbed layers from premixed solutions. surface excess of spreading technique with adsorption (black), one droplet touching gently the surface (red), one droplet falling at the centre of the static trough (blue) . . surface excess of adsorption technique. In the gure frame the same data are displayed on a t 1/2 scale. The dotted black lines help to visualise the linear component. . . . 6.4 Time-resolved surface excess of old proteins tested in 2020 (red), old proteins tested in 2021 (blue) and new proteins tested in 2021 (black). . . . . . . . . . . . . . . . . . . . . . . 6.5 Time-resolved surface excess of old proteins spread in 0.1 M NaCl using H 2 O (a) and D 2 O (b) as subphase. . . . . . . . . . 6.6 Time-resolved surface excess of EGFP-Ubx proteins spread in dierent subphases: H 2 O, 0.1 M NaCl in H 2 O, D 2 O and 0.NaCl in D 2 O. . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7 Time-resolved surface excess of EGFP-Ubx proteins spread in D 2 O and H 2 O with two concentrations c 1 and c 2 . . . . . . . . 6.8 Measured reectivity proles at two contrasts obtained for two compression areas (300 and 110 cm 2 ). In the inset Correspond- ing SLD proles from the NR curves. . . . . . . . . . . . . . . 6.9 Top left: Reectivity prole the protein concentration c 1 spread in ACMW with barriers fully compressed. The curves are shifted vertically for clarity. Top right: Corresponding SLD proles from the NR curve for the protein concentration c 1 spread in ACMW with barriers fully compressed. Bottom:

  ditions and the temperature. Our experiments indicated that the waiting time between the introduction of Ubx monomers and the Langmuir barrier compression was an additional critical parameter in formation of biopolymeric membranes. Moreover, we developed a new image analysis technique based on autocorrelation of SEM (scanning electron microscopy) images of the biopolymeric membranes. This autocorrelation technique is applicable to any digital image, and in our experiments was used to quantify the linear structure of the Ubx biopolymeric membranes.

Figure 2 . 1 .Fig. 2 . 1 :

 2121 Fig. 2.1: Area of expression in embryo and adult life stages are shown. Reproduced from reference [62].

  recognized and categorized types of collagen with unique characteristics and functions. Collagen proteins have complex hierarchical structures containing dierent levels of structure. Firstly three amino-acid chains are wound together in a triple helical conguration forming a microbril. Microbrils are then organised into larger elongated brils. Finally, collagen bers are formed by bril self-assembly (see Figure 2.2).

Fig. 2 . 2 :

 22 Fig. 2.2: Hierarchical structures in brillar collagen showing a characteristic periodic structure forming various hierarchical orders of association: bers, brils and microbrils with three helical chains. Collagen ber diameter is from 1 to 20 µm.

Fig. 2 . 3 :

 23 Fig. 2.3: TEM images showing the hierarchical lm formation of EGFP-Ubx at the air water interface. (a)-Ubx aggregation at 15 minutes after dilution; (b)protobrils formation after 1h in dilution; (c)-parallel alignment of protobrils after 1-2h; (d)-bril formation of 50 nm diameter after 2h; (e)-lateral association of brils into lm; (f)-Ubx lm drawn into ber. Reproduced from reference [80].

Fig. 2 . 4 :

 24 Fig. 2.4: Schematic gene sequence of Ubx fusion proteins used to create functionalised materials. The functional protein sequence (green, purple and red) are shown linked to exible linker, in yellow, followed by the self-assembling protein Ubx in brown.

  a liquid. Since Pockels studies in 1935, scientists studied and developed a mechanised computer-controlled instrumentation for the lm manipulation at air-water interface. The instrumentation is called Langmuir trough, a schematic description is shown in Figure 3.1. In this scheme, A is a bath where the subphase is poured, B are the moving barriers and C is a balance that measures the surface pressure. Both bath and barriers are usually made of Teon, rst used by Fox and Zisman [41]. This material, in fact, enables scientists to clean properly the instrumentation using aggressive chemicals to ensure a complete removal of organic contaminants. The surface-pressure measuring balance used is the Wilhelmy plate[132].

Fig. 3 . 1 :

 31 Fig. 3.1: Langmuir trough scheme. A, bath where the subphase is poured; B moving barriers to control the surface area dimension; C surface-pressure balance with Wilhelmy plate measuring the surface pressure.

Fig. 3 . 2 :

 32 Fig. 3.2: Phospholipid of the plasma membrane with a hydrophilic head and two hydrophobic tails.

Fig. 3 . 3 :

 33 Fig. 3.3: Scheme of Wilhelmy plate partially immersed in a water subphase where

Fig. 3 . 4 :

 34 Fig. 3.4: Scheme of surface pressure (π) vs. area (A) and orientation of surfactants in dierent phases.

3. 2 Fig. 3 .Fig. 3 .

 233 Fig. 3.5: a -Schematic plane-view and end-view representation of wave nature of light . b -Light beam incident upon and reected from a surface showing the plane of incidence and the s-direction and p-direction.

  the case of air-water interface (n a = 1 and n w = 1.333), the Brewster angle is θ b = 53.13 at a wavelength λ = 532nm. Even though in reality the refractive index doesn't change steeply between two media, the reected light has a minimum at θ b . Spreading a thin molecular lm at the air-water interface leads to a change in the refractive index and thus, to a measurable change in reectivity[101, 124] (Figure 3.7).

Fig. 3 . 7 :

 37 Fig. 3.7: Brewster Angle Microscopy physical functioning.

Fig. 3 . 8 :

 38 Fig. 3.8: Example of BAM image

(

  1788 -1827) and Dominique François Arago (17861853) have mathematically studied and characterized the light polarization, the concept of partially polarized light arose only around the year 1852 when Sir George Gabriel Stokes demonstrated that it was possible to completely characterize any arbitrary polarization state from four experimental measurements [116, 126]. Thanks to Stokes' work, in 1946, Subrahmanyan Chandrasekhar formulated the equations for radioactive transfer of partially polarized light[27, 126]. In addition to Stokes theory, new developments made possible the polarization states measure as a function of wavelengths that brought to ellipsometry. Ellipsometry is a very sensitive technique which examines the optical properties of surfaces and thin lms. It is based on the use of polarized light. Along with frequency and wavelength, light polarization is one of fundamental parameter used to describe transverse waves and species the geometrical orientation of the oscillations(perpendicular to the direction of motion of the wave)[5], Figure 3.5a. Measurements are based on the change in polarization of light reected at a surface, where the relative amplitude and phase of the p-and s-polarized components change by dierent amounts[65, 21].

( 3 . 30 )Fig. 3 . 10 :

 330310 Fig. 3.10: Schematic diagram of a scanning electron microscope.

Fig. 3 .

 3 Fig. 3.11: a -Schematic diagram of an electromagnetic lens. The coil windings are inside the iron shroud and the eld is produced across the lens gap between polepieces. b(i) -Diagram showing how electrons are focussed pass trough an electromagnetic lens. b(ii) -Conventional optical ray diagram for comparison.

2 ,

 2 penetration depth is high and they can travel large distances through most materials without being scattered or absorbed. This allows the study of volume eects and bulky components. Moreover, since neutron scattering is possible only with nuclei, NR is sensitive to the isotopic composition of matter.Neutrons-nuclei scattering contains only s-wave components, which implies that the scattering is isotropic and can be described by the scattering length parameter, b (typically b ∼ 10 -12 cm.). It is a quantity that depends on the details of the interaction between the neutron and nucleus components. Both the sign and value of b change irregularly with Z (atomic number) and A (atomic weight), in contrast to x-ray scattering for which the atomic scattering length is a monotonically increasing function of Z[START_REF] Price | Introduction to Neutron Scattering[END_REF]. This phenomenon is shown in Figure3.13. A striking example is the case of hydrogen and deuterium which are two dierent isotopes of the same element whose scattering length values and even sign are completely dierent (b = -3.74 fm and 6.67 fm respectively). Moreover, b is dierent for the dierent spin states of the neutron-nucleus system,I ± 1 this dierence is most signicant for hydrogen nuclei, leading to incoherent (background) scattering from hydrogenated samples. With the scattering length it is possible to quantify the scattering

Fig. 3 .

 3 Fig. 3.13: Scattering length for dierent elements. The solid squares indicate values for isotopes.Reproduced from reference [98].

Fig. 3 .

 3 Fig. 3.14: (a) -Reection of an incident beam from an ideally at interface. ki

  Fig. 3.15: (a) -Reection of an incident beam from two ideally at interfaces, one is a thin layer of thickness d on a semi-innite substrate (with scattering length density N b 2 and N b 1 for the lm and substrate respectively). (b) -Calculated neutron reectivity prole for a 500 Åthick layer of perdeuterated poly(styrene), d-PS, on a silicon substrate. The oscillations are due to the thickness of the specimen. Reproduced from reference [108].

  Fig. 3.16: Diagram of a n-layers specimen with variable thicknesses (d n ) on a substrate. The index n + 1 corresponds to the substrate, n to the layer immediately adjacent to the substrate, down to index 0 which corresponds to the vacuum/air.

  Fig. 3.18: Sketch of the reectometer FIGARO.

4. 1 . 1 42 •Fig. 4 . 2 : 37 • 1 Tab. 4 . 1 :

 11424237141 Fig. 4.1: Transformation technique scheme: using the heat shock technique, the cell membrane is made permeable to plasmid DNA which can enter into E. coli cell.

Fig. 4 . 4 :Fig. 4 .

 444 Fig. 4.4: SDS-PAGE of protein samples scheme. Proteins are loaded into gel walls and electrical current is applied. Proteins run through the gel and are separated by size.

4. 1 . 4 Fig. 4 . 6 :

 1446 Fig. 4.6: Absolute spectra of 1/20 dilution measured for all the fractions collected during protein purication.

Fig. 4 . 7 :

 47 Fig. 4.7: Absorbance-concentration relationship for BSA proteins.

First

  of all, absorbance for known BSA concentration was measured at 590 nm and a linear absorbanceconcentration trend (y = mx + q) was calculated, see Figure 4.7. Then, OD was measured for EGFP-Ubx proteins and the concentration was calculated, see

Fig. 4 . 8 :

 48 Fig. 4.8: Fermenter parameters during E. coli cell growth. Yellow line indicates the temperature, the red one indicates the pH and the pink the air ow.

Fig. 5 . 1 :

 51 Fig. 5.1: Steps of EGFP-Ubx protein-based lm formation. (a) -EGFP-Ubx is diluted in a Teon-coated tray lled with G0 buer; (b) -after a 16 h incubation period under an aluminium pan, with a microcentrifuge tube rack EGFP-Ubx lm is gently squeezed reducing its area; (c) -the aluminium pan is placed again on top of the tray leaving the lm to set for an hour; (d) -a plastic inoculation loop is used to touch the surface and pull bers (e).

Fig. 5 . 2 :

 52 Fig. 5.2: EGFP-Ubx droplet disposition schema. Droplets are dripped at the surface gently in a symmetric way: three droplets per side, two droplets are placed near the moving barriers and one is placed near the Wilhelmy plate.

1 )Fig. 5 . 3 :

 153 Fig. 5.3: Isothermal compression curve measured with 10-minute waiting time at dierent concentrations: 1nmol, 2nmol, 3nmol and 4nmol. The subphase used was plain water (a) and 0.1 M NaCl (b).

Fig. 5 . 4 :

 54 Fig. 5.4: Isothermal compression curve measured with 10-minute waiting time spreading 2nmol EGFP-Ubx. (a) -the subphase used has and increasing salt concentration: water, 0.1 M, 0.2 M and 0.3 M NaCl; (b) -the subphase used has dierent salts diluted: 0.1 M NaCl, 0.1 M KCl, 0.01 M PBS and 0.1 M NaCl + 0.05 M N aH 2 P O 4 .

Figure 5 .Fig. 5 . 5 :

 555 Figure 5.5a. Interestingly it is possible to see a non-monotonic lm-growth varying the equilibration time. At a given trough area the surface pressure

Fig. 5 . 6 : 128 ±

 56128 Fig. 5.6: Representation of limiting area projection.

Fig. 5 . 7 :

 57 Fig. 5.7: Fibers collected using 2 nmol EGFP-Ubx proteins spread in 0.1 M NaCl buer using (a) -10 minutes waiting time and (b) -120 minutes waiting time. It was not possible to collect bers from 60 minutes waiting time.

Table 5 . 2 )

 52 , d: for 10 min d = 4.5µm ± 1µm; for the 60 minutes waiting time parameter its value is 42.8µm ± 2.8µm and 120 min-waiting-time diameter it is equal to 25.5µm ± 2µm. Although the amount of spread proteins (2nmol)

Fig. 5 . 9 :

 59 Fig. 5.9: Fiber attening diagram. The view from the SEM (A) can be attened by assuming a cylindrical shape and calculating arc lengths. The blacked-out regions are outside of the θ = 60 • threshold.

•Fig. 5 . 10 :Fig. 5 . 11 :

 510511 Fig. 5.10: Diagram vertical autocorrelation (A) along with the graph of the computed sum values (B).

Fig. 5 . 12 :

 512 Fig. 5.12: Wavelet analysis of the bers reveals lateral uniformity and wrinkle sizes for waiting times of 10 (a), 60 (b), and 120 minutes (c), using 2 nmol of EGFP-Ubx spread G0 buer.

Figure 5 . 8 (

 58 Figure 5.8 (2 nmol EGFP-Ubx spread in G0 buer). The average pixel intensity values used was 0.1µm. The 10 minutes-waiting-time ber presents consistently thin wrinkles (∼0.25 µm in dimensions) with at least 2 areas

SF 6 Fig. 5 . 13 :

 6513 Fig. 5.13: Titanium discs cleaned for 15 minutes with IPA in a sonicator prior to do Plasma cleaning for 2 minutes with (a) SF 6 , with 30 sscn ow rate, p = 200 mT, P = 200 W; (b) Ar, with 100 sscn ow rate, p = 70 mT, P = 50 W; (c) Ar, with 100 sscn ow rate, p = 70 mT, P = 200 W.

Fig. 5 . 14 :

 514 Fig. 5.14: Fluorescent pictures taken with ×4 objective lens using an excitation wavelength λ = 488 nm. The disc was cleaned with SF 6 plasma, (a) and (b), and Ar plasma, (c) and (d), prior the dip coating with EGFP-Ubx.

  scheme of experiment is depicted: silica surfaces are indicated with S, etched titanium discs are indicated with O and non-etched ones with N. Ubx-coated surfaces are indicated with 'x', while control surfaces are indicated with 'c'.On the top of samples bacteria solutions, C and D, and plain PBS (indicated as PBX) were pipetted. In Figure5.17 on the right part, the results of overnight uorescence are shown.

Fig. 5 . 17 :

 517 Fig. 5.17: Left part: scheme of experiment. Silica surfaces are indicated with S, etched titanium discs are indicated with O and non-etched ones with N. Ubxcoated surfaces are indicated with 'x', while control surfaces are indicated with 'c'. On the top of samples bacteria solutions, C and D, and plain PBS (indicated as PBX) were pipetted. Right part: results of overnight uorescence.

Fig. 5 .

 5 Fig. 5.18: A-U BAM time-lapse during barrier expansion. The protein lm was created using 2 nmols EGFP-Ubx proteins spread in 0.1 M NaCl solution and let rest 2 hours before the barrier closure.

Fig. 5 .

 5 Fig. 5.19: π-A isotherm with BAM images taken during lm compressionexpansion in plain water.

Fig. 5 .

 5 Fig. 5.20: π-A isotherm with BAM images taken during lm compressionexpansion in 0.1 M KCl.

6 cm and a liquid volume of 25 cm 3 .

 3 For the presented experiments we used 25 mL 0.1 M KCl solution and we added 0.591 nmol EGFP-Ubx proteins. The protein concentration chosen for the experiments, was calculated taking into account the subphase volume reduction from Langmuir trough used for the BAM (350 mL) to the Petri dish used (25 mL). The concentration was, thus, scaled from 1 to 0.6 nmol.

Fig. 6 . 1 :

 61 Fig. 6.1: Dierent loading approaching (a) one single droplet was let to fall from the top at the centre of Petri dish; (b) one single droplet was spread by touching gently the surface at the centre of Petri dish; (c) adsorbed layers from premixed solutions.

Fig. 6 . 2 :

 62 Fig. 6.2: Time-resolved surface excess of spreading technique with adsorption (black), one droplet touching gently the surface (red), one droplet falling at the centre of the static trough (blue)

6. 1 . 2

 12 Fig. 6.4: Time-resolved surface excess of old proteins tested in 2020 (red), old proteins tested in 2021 (blue) and new proteins tested in 2021 (black).

Fig. 6 . 5 :

 65 Fig. 6.5: Time-resolved surface excess of old proteins spread in 0.1 M NaCl using H 2 O (a) and D 2 O (b) as subphase.

Figure 6 .

 6 Figure 6.6 a comparison between dierent aqueous solutions is shown. First of all, it is interesting to notice that in both cases of plain D 2 O and H 2 O,

(6. 3 )

 3 where Γ D 2 O and Γ H 2 O are the corresponding surface excess values when the lm is spread in D 2 O and H 2 O, respectively; and c 1 =c U bx is the lower protein concentration, used until now. The results are shown in Figure6.7

Fig. 6 . 6 :

 66 Fig. 6.6: Time-resolved surface excess of EGFP-Ubx proteins spread in dierent subphases: H 2 O, 0.1 M NaCl in H 2 O, D 2 O and 0.1 M NaCl in D 2 O.

Fig. 6 . 7 :

 67 Fig. 6.7: Time-resolved surface excess of EGFP-Ubx proteins spread in D 2 O and H 2 O with two concentrations c 1 and c 2 .

6. 2 . 1 1 M

 211 Optimisation of the samples for NR experimentsIn January 2020, the rst NR experiment was performed with EGFP-Ubx.Experiments were performed at FIGARO reectometer (Institut Laue-Langevin, Grenoble) equipped with the Langmuir trough. The instrument was used in kinetic mode. Since no one has ever tried NR with the protein in question, the goal of this experiment was to explore and optimise the parameters to obtain good reectivity signal. Even if one day (out of two) was lost due to technical problems of the instrument, it was possible to investigate an initial structural characterisation of the protein adsorption at the air-water interface at before beginning the compression (trough area = 300 cm 2 ) and full compression (area 110 cm 2 ) using two contrasts D 2 O and ACMW with 0.KCl. For the experiments 140 mL of subphase were used and the amount of EGFP-Ubx proteins used, 50 µl of 10µM, was calculated to match ellipsometry results shown previously. The NR curves in Figure6.8 represented two dierent stages of lm formation and the ts corresponded to hydrated protein layers.

Fig. 6 . 8 :

 68 Fig. 6.8: Measured reectivity proles at two contrasts obtained for two compression areas (300 and 110 cm 2 ). In the inset Corresponding SLD proles from the NR curves.

  we decided to scale the EGFP-Ubx concentration based on surface-Area (at barriers fully open) reduction. So, since the trough area scales from 800 cm 2 to 256 cm 2 , the amount of proteins was scaled from 2 nmol to 0.6 nmol.For this reason, we chose to use c 1 = 33.5 µL of 17.76 µM stoking solution of EGFP-Ubx. Moreover, a second protein concentration, and c 2 = 50 µL of 17.76 µM, was used for ACMW subphase, to match what was observed with ellipsometry. Proteins were spread at the surface by gently touching the interface and the Langmuir trough barriers were closed after the selected equilibration time. Moreover, we hypothesized that there were no structural dierences between the lm obtained in ACMW and in D 2 O subphases due to the fact that dytirosine bonds, involved in lm lm-formation, are very specic. This was important in order to perform NR co-renement at two contrasts with two lms that in rst approximation were similar. Neutron reectivity was measured as a function of time during both the equilibration and the compression period. In this work, measures at full compression are shown.

10 - 6 Å - 2 10 - 6 Å - 2

 10621062 9 right. The black line represents the SLD values associated with 10-min waiting time, the blue one is associated to 60-min and the red to 120-min. As can be seen, the high-peak SLD values seem to follow a sort of non-monotonic eect seen previously for Langmuir curves: from SLD = 0.8•at 10 minutes, the value decreases to 0.6•for 60 min and the reaches 1•10 -6 Å -2 for 120 minutes waiting-time. The same trend can be seen for lm thickness, visible in the bottom part of Figure 6.9. The thickness of EGFP-Ubx lm formed after 10 minutes was 17.1±2.8Å with a hydration of 53.0±0.2 %. When the monomers equilibrated for 60 minutes before the barrier compression, the surface lm was 26.9±1.6 Å thick, with a solvent component equal to 70.3±1.8 %. Finally, for longer waiting time periods, 120 minutes, the thickness of the resulting lm was 11.6±1.0 Å with a hydration of 36.5±0.4%.

waiting-time than to the 60 Fig. 6 . 9 :

 6069 Fig. 6.9: Top left: Reectivity prole the protein concentration c 1 spread in ACMW with barriers fully compressed. The curves are shifted vertically for clarity. Top right: Corresponding SLD proles from the NR curve for the protein concentration c 1 spread in ACMW with barriers fully compressed. Bottom: Structural parameters obtained from the NR curves: d (thickness [Å]), and solvent content[%] for the protein concentration c 1 spread in ACMW with barriers fully compressed.

2 Fig. 6 .

 26 Fig. 6.10: Top: Reectivity and corresponding SLD proles at three waiting time (10, 60 and 120 minutes) spreading c 1 of EGFP in ACMW and c 2 of EGFP in D 2 O. Bottom: Structural parameters obtained from the NR curves: d (thickness [Å]), solvent content[%] and roughness (σ) [Å].

  the proposed analysis. The rst experiments involved the monitoring of lm formation in a LB trough where three critical parameters have been studied: the concentration of monomers used, the subphase in which the protein is spread and the waiting time needed for monomers to equilibrate before the area reduction through the barrier compression. At the end of these studies we discovered a new material formation regime depending on waiting time parameter. Moreover, a new ber-formation protocol was developed using 5times lower protein concentration in 100-times less time compared to all the works published until now. The ability to pull bers after just 10 minutes using such a low protein concentration was surprising and totally unexpected. This result had not been achieved by Bondos group identifying a new phase of material formation that has never been studied.We developed an in-house Matlab tool, based on autocorrelation and wavelet analysis, to analyse the structure of pulled bers at dierent conditions. With this code it was possible to make hypotheses on lm structure at air-water interface by quantifying the order of wrinkles present in the bers. This tool can be adapted to any digital image.With EGFP-Ubx I made some tests on surface coating analysing the degradation time frame, discovering that the lm remains stable for 4 weeks with almost no changes. The coating lm started to degrade after 7 weeks becoming more and more extensive after 11 weeks. With plain Ubx lm coating, instead, we tested the antimicrobial property of coated surfaces nding out that the best substrate to use is titanium grade 2 and cannot be cleaned to a future reuse with piranha solution because the etching would change chemically the surface causing an environment favourable to bacterial growth.The following step was to analyse lm formation more in deep. After some parameter optimization done with ellipsometry, I moved to neutron reectometry. I was able to estimate the lm thickness and to probe the time eect of EGFP-Ubx at two contrasts, corroborating the observations made with low protein concentration.At the end of the present work it is important to point some possible future works. First of all, it would be important to study deeper the isotopic eect on lm formation and repeat the reectometry experiments at lower concentration in D 2 O as mentioned in the previous chapter 6.2.2. Nonetheless, the shown results raised several questions concerning the specic inuences on the structure and properties of the materials assembled with the new protocol. A possible study in collaboration with Bondos group could be useful to determine whether similar interactions form in bers created with the two dierent methods.

  the present work, we use a Ubx fusion with EGFP (Enhanced Green

	Fluorescent Protein) to investigate the mechanisms of Ubx lm formation,
	particularly during early stages of Ubx monomer aggregation at the air-
	water interface. Fusion of EGFP does not aect the ability of Ubx to self-
	assemble. However, we discovered this fusion plays an important role in the
	lm-formation dynamics. In fact, Ubx monomers precipitate in water and,
	presumably due to the positive charges (predicted net charge of +11), they
	explode without any counter ions. The negative charges present in EGFP
	prevent the explosion of Ubx monomers.

However, at the state of art, the main problem is given by the lack of understanding of the physical and chemical mechanisms underlying material formation. This means that if we do not understand how the lm forms, it is not possible to fully control the process. In order to ll this gap, my work was focused on understanding the mentioned process with laboratory-based techniques.

Table 4 .

 4

			3.	With this method, the pro-
			tein concentration was calculated
			in	mg/mL,	in	order	to	switch
	Elution	OD	c[mg/mL]	c[µM]
	E0	0.46	0.44	06.7
	E1	0.59	0.68	10.3
	E2	0.62	0.74	11.2
	E3	0.70	0.88	13.3
	E4	0.83	1.10	16.6
	E5	0.77	1.00	15.2
	E6	0.44	0.42	06.4

to µM it is necessary to divide the concentration by molecular weight. Tab. 4.3: Table.

  in which a list of possible cosolvent for Ubx are listed. The eect of specic cosolvent can be seen by direct comparison with other co-aH 2 P O 4 ) is not classied as cosolvent but helps the thermal stability of the protein. As can be seen in the graph, the shape of the curves changes as well as the π -A values, meaning that fewer proteins aggregate and more proteins are able to reach the air-water interface to form a more compact lm. It is clear that potassium chloride can stabilize better solvent-solvent interactions with respect to sodium chloride, in fact surface tension is higher for the same area values. At the same time, PBS eects are comparable to KCl one although its concentration is smaller.

	solvents. In this work water (as a contrast), 0.1 M NaCl, 0.1 M KCl, 0.01 M
	PBS (which contains sodium phosphate, sodium chloride, potassium chloride
	and potassium phosphate) and 0.1M NaCl + 0.05 M N aH 2 P O 4 buers were
	used (see Figure 5.4b). The cosolvent chosen are acting as kosmotropes in
	a range between 0 -1 M for Ubx monomers and their fusions even though
	sodium phosphate monobasic (N

This can be described by the fact that PBS contains both NaCl and KCl plus their phosphate versions, thus we have a combined action of dierent co-solvents. The most unexpected result relates to the sodium chloride plus sodium phosphate buer. I chose the same concentration for N aH 2 P O 4 as in the G0 buer used for both EGFP-Ubx purication and lm-formation by Bondos' group. We can see that the lm is more compact and a lm is detected already at 600 cm 2 , as it is for PBS buer, but, contrary to what happens to latter, surface pressure does not drop to zero but monotonically increase. A last observation on Figure

  Tab. 5.2: Mean values of diameters, d,[µm] for the bers collected with Langmuir trough at dierent waiting times, t, and subphases, 0.1 M NaCl and G0 buer. Missing values are reported with the symbol `-`, if it was impossible to see any. ± 0.2 -5.2 ± 0.4 4.5 ± 1.0 42.8 ± 2.8 25.5 ± 2.0 At 120 minutes we can see a highly ordered pattern with large and well dened wrinkles (whose dimensions are in the range of 0.7-1.7µm). On the other hand, bers obtained after 60 minutes present a very disordered pattern where wrinkles are non-continuous and winding. In the middle of these two extremes, there's the 10 minutes waiting time case, whose bers present some wrinkle-formations that are not perfectly aligned. Comparing what we have obtained with Bondos' analysed bers[START_REF] Ma Jithia | On the design of composite protein-quantum dot biomaterials via self-assembly[END_REF], we can conclude that 2hours waiting-time bers are really similar to what already studied, but the remaining two bers are dierent in diameter and/or in wrinkle structure.

			0.1 M NaCl			G0 buer
	t [min]	10	60	120	10	60	120
	d [µm]	3.9

Table 5 . 3 .

 53 Tab. 5.3: Mean values of FWHM [µm]for the bers collected with Langmuir trough at dierent waiting time and solutions. Missing values are reported with the symbol `-`, if it was impossible to see any, with `+' if it was possible to see some which were too weak.

	Buer	0.1 M NaCl	0.1M NaCl+0.05M N aH 2 P O 4	G0 buer
	Time [min]	10	60	120	10	60	120	10	60	120
	FWHM [µm]	6.4	-	7.7	+	+	5.2	4.6	2.2	9.4

  1/2 scale, see box in Figure6.3. He described the initial protein adsorption as a function of diusion coecient, D 2/3 , bulk concentration, c, and time, t 1/2 .As can be seen in the box plot, both Γ curves show a linear behaviour with positive slope, the dotted black lines help to visualise the linear tendency, before the slope decreases. The change of the slope is explained with the subsequent jamming of molecules after a critical surface density[START_REF] Ramsden | Concentration scaling of protein deposition kinetics[END_REF][START_REF] Campbell | Spread Films of Human Serum Albumin at the Air-Water Interface: Optimization, Morphology, and Durability[END_REF].

  O are higher than the one for H 2 O (although Γ D 2 O ∼ Γ H 2 O+N aCl ). The main cause of this behaviour can be give by isotope-specic eects from the dierence in energy of H-bonding according to the isotopic composition of the subphase. Thus, it is clear that by using the same concentration of protein in the solution, it ends up with two dierent protein lms at the air-water interface: the lm in H 2 O being less thick than the one in D 2 O. This lm dierence brings to two dierent situations that can not be compared during neutron reectivity data analysis. Therefore, it was decided to use a higher protein concentration in H 2 O so that the resulting EGFP-Ubx lm at the air/water interface has similar features (thickness, hydration) to the one forming at D 2 O surface. The new protein concentration was calculated following the equation:

2 O and H 2 O, the addition of a kosmotrope, NaCl, helps to prevent proteins aggregation so that more EGFP-Ubx monomers are able to reach the air-water interface and participate in lm formation. The observations made with Langmuir technique are thus conrmed with ellipsometry. For this reason, it was decided to use 0.1 M NaCl solutions for further experiments. Moreover, it is possible to point out that Γ values for D 2