Turbulence in partly vegetated channels: Experiments with complex morphology vegetation and rigid cylinders

Vegetation is a fundamental feature of riverine ecosystems, playing a variety of valuable ecological and biological roles. Concurrently, the presence of vegetation and its interaction with the flow alter the mean and turbulent flow field, with implications on flow resistance, water conveyance and transport of mass and energy. The proper understanding of these vegetation-influenced processes is essential for solving the existing and future river management challenges, concerning both societal needs and ecosystem requirements. The objective of this thesis is to provide new insight on the flow-vegetation hydrodynamic interaction with a specific focus on partly vegetated channels, a configuration representative of natural settings. Indeed, in natural watercourses, vegetation is generally found along river margins, partly obstructing the river cross-section and laterally interacting with the flow. Riparian vegetation presents a complex morphology and, owing to its flexibility, exhibits a dynamic and reconfiguring behavior under the flow forcing. In the analysis of flow in partly vegetated channels, these flow-influencing characteristics have been generally neglected, simulating vegetation with rigid cylinders. In the current study, two main experimental campaigns were performed to investigate the turbulent structure of the flow in partly vegetated channels, simulating vegetation with natural-like plant stands (PN) and with rigid cylinders (PR). The PN tests aimed at investigating the effects of plant morphology, reconfiguration and dynamic motions on the turbulent flow field. Furthermore, the effects of seasonal variability of plants on flow structure were explored. Results showed that plant morphology and reconfiguration play a key role in the vegetated shear layer dynamics, significantly affecting the exchange processes across the vegetated interface. The PR test series was performed to investigate the effects of vegetation density on the turbulent flow structure. The results showed that, for rigid vegetation, the density directly affects the shear layer features, governing the onset of large-scale coherent structures. Finally, the impacts of embedding natural plant features in the simulation of partly vegetated flows were explored by comparing the shear layers induced by complex morphology vegetation (PN) and by rigid cylinders (PR). In addition, an existing model for velocity prediction was tested against the experimental results, showing the need to improve existing models for taking into account the peculiar hydrodynamic behavior of natural vegetation

Impact of reconfiguration on the flow downstream of a flexible foliated plant

This paper explores the impacts of reconfiguration and leaf morphology on the flow downstream of a flexible foliated plant. 3D acoustic Doppler velocimetry and particle image velocimetry were used to experimentally investigate the hydrodynamic interaction between a foliated plant and the flow, testing two plants with different leaves morphology under different bulk flow velocities. The model vegetation was representative of riparian vegetation species in terms of plants hydrodynamic behavior and leaf to stem area ratio. To explore the effects of the seasonal variability of vegetation on the flow structure, leafless conditions were tested. Reconfiguration resulted in a decrease of the frontal projected area of the plants up to the 80% relative to the undeformed value. Such changes in plant frontal area markedly affected the spatial distributions of mean velocity and turbulence intensities, altering the local exchanges of momentum. At increasing reconfiguration, the different plant morphology influenced the mean and turbulent wake width. The leafless stem exhibited a rigid behavior, with the flow in the wake being comparable to that downstream of a rigid cylinder. The study revealed that the flexibility-induced reconfiguration of plants can markedly affect the local distribution of flow properties in the wake, potentially affecting transport processes at the scale of the plant and its subparts

The 40s Omega-loop plays a critical role in the stability and the alkaline conformational transition of cytochrome c

The structural and redox properties of a non-covalent complex reconstituted upon mixing two non-contiguous fragments of horse cytochrome c, the residues 1 - 38 heme-containing N-fragment with the residues 57 - 104 C-fragment, have been investigated. With respect to native cyt c, the complex lacks a segment of 18 residues, corresponding, in the native protein, to an omega ( W)loop region. The fragment complex shows compact structure, native-like alpha-helix content but a less rigid atomic packing and reduced stability with respect to the native protein. Structural heterogeneity is observed at pH 7.0, involving formation of an axially misligated low-spin species and consequent partial displacement of Met80 from the sixth coordination position of the heme-iron. Spectroscopic data suggest that a lysine ( located in the Met80-containing loop, namely Lys72, Lys73, or Lys79) replaces the methionine residue. The residues 1 - 38/57 - 104 fragment complex shows an unusual biphasic alkaline titration characterized by a low (pK(a1)= 6.72) and a high pK(a)-associated state transition (pK(a2)= 8.56); this behavior differs from that of native cyt c, which shows a monophasic alkaline transition ( pK(a)= 8.9). The data indicate that the 40s Omega-loop plays an important role in the stability of cyt c and in ensuring a correct alkaline conformational transition of the protein

A comprehensive framework tool for performance assessment of NBS for hydro-meteorological risk management

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Evaluating the Impact of Nature-Based Solutions: A Handbook for Practitioners

The Handbook aims to provide decision-makers with a comprehensive NBS impact assessment framework, and a robust set of indicators and methodologies to assess impacts of nature-based solutions across 12 societal challenge areas: Climate Resilience; Water Management; Natural and Climate Hazards; Green Space Management; Biodiversity; Air Quality; Place Regeneration; Knowledge and Social Capacity Building for Sustainable Urban Transformation; Participatory Planning and Governance; Social Justice and Social Cohesion; Health and Well-being; New Economic Opportunities and Green Jobs. Indicators have been developed collaboratively by representatives of 17 individual EU-funded NBS projects and collaborating institutions such as the EEA and JRC, as part of the European Taskforce for NBS Impact Assessment, with the four-fold objective of: serving as a reference for relevant EU policies and activities; orient urban practitioners in developing robust impact evaluation frameworks for nature-based solutions at different scales; expand upon the pioneering work of the EKLIPSE framework by providing a comprehensive set of indicators and methodologies; and build the European evidence base regarding NBS impacts. They reflect the state of the art in current scientific research on impacts of nature-based solutions and valid and standardized methods of assessment, as well as the state of play in urban implementation of evaluation frameworks

L’equity crowdfunding: i fattori critici di successo

Questa tesi si basa su una raccolta e una successiva elaborazione di dati effettuata su Crowdcube, la piattaforma di crowdfunding di investimento leader a livello mondiale, con lo scopo di analizzare il fenomeno dell’equity-crowdfunding in tutte le sue diverse caratteristiche. Attraverso gli strumenti grafici e computazionali, si studierà un campione di circa 100 progetti, con l’obiettivo finale di individuare e studiare “i fattori critici di successo”, derivanti dalle correlazioni tra caratteristiche dei progetti presentati e successo ottenuto da questi ultimi nell’ottica di finanziamenti ricevuti. L’importanza di questo studio è data dalla necessità dei progettisti che presentano le campagne di crowdfunding di capire come poter rassicurare e convincere gli investitori, in modo da avere una raccolta fondi di successo. L’analisi sarà arricchita da tabelle, istogrammi e grafici, con lo scopo di supportare l’esplicazione dei vari concetti e delineare eventuali trend

Ruolo delle istidine nella struttura e nella stabilità del citocromo C

L’attività funzionale biologica di una proteina dipende essenzialmente dalla sua abilità ad adottare una struttura specifica. In condizioni fisiologiche, la macromolecola si ripiega nel lo stato nativo (biologicamente attia) partendo da uno stato non ripiegato. Capire il meccanismo attraverso cui la catena polipeptidica si ripiega ad assumere la sua struttura tridimensionale nativa (folding) è certamente una delle sfide più importanti della scienza del XXI secolo.Le proteine possono “collassare” in stati parzialmente ripiegati in condizioni di equilibrio o di non equilibrio.Tali stati possono potenzialmente rappresentare degli ottimi modelli di intermedi del processo di folding; studiarne le proprietà strutturali, è determinante per capire più a fondo il meccanismo di folding.Tali intermedi giocano un ruolo cruciale sulle funzioni cellulari e sulla stabilità ; ciò indica che il processo di ripiegamento proteico è di tipo cooperativo.Studi di cinetica rapida sono spesso utilizzati per caratterizzare intermedi transienti,dei quali il più importante è certamente il molten globule, uno stato compatto,caratterizzato da regioni a struttura secondaria simili allo stato nativo, ma con struttura terziaria fluttuante; esso manca infatti di molte delle interazioni terziarie specifiche presenti nella struttura nativa. Dagli studi strutturali, si osserva che nel molten globule la catena laterale adotta una grande varietà di conformazioni rispetto alla proteina nativa, in accordo con una sua aumentata flessibilità (rispetto alla proteina nativa).Lo studio del folding del citocromo c è stato effettuato utilizzando una vasta gamma di metodiche spettroscopiche; tra queste, il dicroismo circolare , la trasformata di Fourier , la spettroscopia Raman e NMR, che hanno permesso di osservare cambiamenti strutturali di alcune regioni distinte della macromolecola.Il citocromo c è costituito da una singola catena polipeptidica di 104 aminoacidi, organizzata in cinque regioni a-elica e tre regioni ad ansa(Ω loops). Gli Ω-loosp sono segmenti proteici privi di struttura secondaria, localizzati sulla superficie della proteina, costituiti principalmente da amminoacidi polari, e caratterizzati da elevata flessibilità. Il citocromo c contiene il gruppo prostatico (l’eme) legato covalentemente alla matrice proteica tramite due legami tioetere con le cisteine Cys14 e Cys17. Il Fe-eme è inoltre coordinato in posizione prossimale all’His18 e in posizione distale alla Met 80. Lo stato A (i.e. il molten globule ) del citocromo c di cavallo, è stato studiato approfonditamente. A pH 2.2 e a bassa forza ionica, il ferri-citocromo è sostanzialmente non ripiegato (“unfolded”); tuttavia, l’aggiunta di sali, induce la proteina a ripiegarsi in una struttura compatta, lo stato A, stabilizzato dal legame degli anioni ai gruppi carichi positivi situati sulla superficie della proteina. Lo stato A ha struttura α-elicaoidale comparabile a quella dello stato nativo, ma la sua conformazione terziaria è fluttuante. In particolare, nello stato A rimane conservato il “core idrofobico”, costituito dalle eliche N- e C terminali e dal gruppo eme, è stabilizzato da interazioni non covalenti; tuttavia le regioni a cappio ( loops) sono fluttuanti e parzialmente disordinate. Dei due ligandi assiali del ferro-eme, solo l’istidina 18 rimane coordinata al metallo, mentre il legame assiale con la metionina 80 risulta modulato dal tipo e dalle dimensioni degli anioni presenti in soluzione..Scopo di questa tesi è definire il ruolo del legame idrogeno formato nella proteina nativa, tra l’anello imidazolico dell’istidina in posizione 26 (nell’Ω-loop 20s) e il gruppo carbonilico del residuo in posizione 44 (nell’Ω-loop 40s), per la stabilità e le proprietà strutturali e funzionali del citocromo c. Allo stesso tempo, la sostituzione della H26 per mutagenesi, permetterà di stabilire le cause per cui tale residuo è invariante nella sequenza aminoacidica della proteina ( sia nei vertebrati che nelle piante superiori). L’ipotesi più accreditata è che la rottura del legame idrogeno, dovuta alla mutazione di H26, liberi i due loops determinando così un aumento della flessibilità della proteina e il probabile spiazzamento della Met80 dalla sesta posizione di coordinazione al ferro eme. Questa mutazione, se dovesse stabilizzare uno stato intermedio compatto, potrebbe produrre un valido modello di intermedio di folding del citocromo c. Il citocromo del iso-1 di lievito rappresenta sistema ideale nello studio dei citocromi c di classe c; la sua struttura determinata a Raggi X è disponibile ed è stato sviluppato un sistema di mutagenesi capace di produrre mutanti stabili. Il nostro studio ha evidenziato il ruolo chiave del legame-H tra His26-Glu 44 nel conferire stabilità della proteina nativa, bloccando i Ω-loops 20s e il 40s , con conseguente aumento della rigidità della macromolecola. L’assenza di tale legame induce cambiamenti conformazionali nella struttura terziaria della macromolecola che riducono sensibilmente la stabilità dalla proteina. L’alta flessibilità mostrata dal mutante H26Y, indebolisce il legame assiale Fe(III)-Met80, e altera il comportamento della proteina stessa. Come risultato, la transizione conformazionale alcalina avviene a pH più basso (pKa = 7.5 anzichè 8.4) e, contrariamente a quanto si osserva nella proteina nativa, a pH neutro il mutante H26Y resulta essere composto da almeno due specie con diversa coordinazione assiale al metallo. I dati ottenuti indicano che di questo legame-H la rottura induce nella proteina la formazione di uno stato di “molten globule” a pH fisiologico, caratterizzato da due forme in equilibrio,secondo il seguente schema: His18-Fe(III)-Met80↔His18-Fe(III)-X dove X è un ligando endogeno,probabilmente una lisina secondo quanto suggerito dalle misure di Raman effettuate.Con lo scopo di determinare il ruolo svolto delle altre due istidine presenti nella sequenza aminoacida della proteina di lievito (H33 and H39), sono stati prodotti e caratterizzati i doppi mutanti H26YH33Y, H26YH39K, H33YH39K, che conservano ciascuno una sola istidina nella sequenza aminoacidica. In particolare, la H39 è stata mutata con una lisina presente nella sequenza del citocromo c equino. I dati ottenuti indicano che H33 e His39 non alterano le proprietà della proteina, evidenziando così il ruolo critico svolto dal residuo H26.Sono state studiate anche le proprietà del mutante H26Y del cyt c equino, dal momento che malgrado mostri una struttura simile a quella dell’iso-1-cyt c. del lievito (come rilevato dalla cristallografia a raggi X), ha però maggiore stabilità. Il citocromo c equino prodotto nel nostro laboratorio mostra proprietà spettroscopiche ed una stabilità praticamente identiche alla proteina nativa, (evidenziate da misure CD, assorbimento elettronico e Raman). A pH neutro il mutante H26Y equino non mostra cambiamenti significativi rispetto alla proteina wt (nativa) ma è caratterizzato da un’isomerizzazione alcalina con pk più basso rispetto alla forma wt (pKa: 8.3 anzichè 9.2), ma comunque più alto di quello del mutante di lievito. La variazione di pKa osservata fra il mutante e la forma wt ( ∆ (∆Go ) = 5.1 KJ/mol) indica l’effetto l’effetto della mutazione in posizione 26 sulla stabilità della proteina .E’ interessante notare che una una stessa variazione di ∆pKa viene osservata per il citocromo c di lievito; ciò suggerisce che la mutazione H26Y influisce sulla stabilità delle due proteine in modo simile, nonostante la maggiore stabilità del citocromo c equino a pH 7.0, ciò suggerisce che le proprietà del gruppo ionizzante, che controlla la transazione alcalina (i.e. “ trigger ”) sono ugualmente influenzate in entrambe le proteine; dunque, i gruppi responsabili per la maggiore stabilità del citocromo c equino a pH neutro non sono coinvolti nei cambiamenti conformazionali associati all’isomerizzazione alcalina del citocromo c.Protein function critically depends on the protein ability to adopt a specific structure. Remarkably, a protein can efficiently fold to the native state from the unfolded state(s) on physiological time scale. Understanding how unfolded polypeptides self-assemble into a correctly folded structure represents one of the great challenges of the 21st century’s science. Proteins can be induced to collapse into partially folded states, under equilibrium or non-equilibrium conditions. A variety of partially folded proteins were shown to represent suitable models for intermediates forming during protein folding; therefore, determining the structural properties of these partially folded states becomes crucial for a deeper understanding of the protein folding mechanism. Partially folded proteins also play a role for important cell functions and are thought to have a central role in protein stability. However, due to the cooperative nature of folding a minimal amount of intermediates is observed. Fast-kinetics is widely employed for the characterization of transiently populated intermediates; of these, the most important is the molten globule, a compact, partially folded state possessing native-like secondary structure but lacking the extensive, specific side-chain packing interactions of the native structure. As demonstrated by structural studies, in the molten globule the side-chains adopt a greater variety of conformations with respect to the native protein, and the protein is characterized by high flexibility (if compared to the native state). In order to understand what changes take place in the protein during the final stages of folding, spectroscopic techniques, as circular dichroism, Fourier transform infrared, Raman and NMR have have been successfully employed; these techniques provide precious information on changes occurring in distinct regions of the macromolecule. Cytochrome c is a single polypeptide chain organized in five α-helices and three omega loops. Ω-loops are protein segments devoided of secondary structure, localized mostly on protein surface, characterized by the presence of polar aminoacids and enhanced flexibility. Also, cytochrome c contains heme as prosthetic group, which is linked to the polypeptide chain by two tioether bonds formed by the cysteins in position 14 and 17. The heme-iron is also axially coordinated with the two residues His 18 and Met 80. The molten globule (A state) of ferric cytochrome c has been extensively characterized. At pH 2.2, ferric cytochrome c is substantially unfolded at low ionic strength; upon addition of salts, the protein cooperatively folds to a compact structure, the A state, stabilized by the binding of anions to the positively charged groups on the protein surface. The A state possesses α-helix structure comparable to that of the native form, but a fluctuating tertiary conformation. In particular the hydrophobic core containing the two major helices (the N and C-terminal helices) and the heme group is preserved in the A state, stabilized by non bonded interactions, while the loop regions are fluctuating and partially disordered. Of the two native axial ligands of the heme-iron, only His 18 is thought to remain coordinated to the heme iron while the Met80 axial bond is lost in the presence of large anions, and is recovered by the presence of small anions. With this in mind, in this thesis our intent is to define wich contribution is provided by the hydrogen bond linking the histidine in position 26 (Ω loop 20s) to the carbonyl group of the residue in position 44 (Ω-loop 40s), to stability, folding, and functional properties of cytochrome c. At the same time, we want to explore the possibility to generate a molten globule state of cytochrome c at neutral pH. The interest on His 26 lies in the fact that this residue, it is an invariant residue in vertebrate and higher plant cytochromes c . In yeast cytochrome the lack of the H26-E44 H-bond, due to mutation of H26, is expected to free the two loops with a consequent increase of the protein flexibility and the likely displacement of M80 from the axial coordination to the heme-iron; thus, such a mutation shoud stabilize a compact intermediate state acting as a suitable model for a kinetic folding compact intermediate of cyt c. Yeast iso-1-cytochrome c represents an ideal model-system for c-class cytochromes; a high-resolution X-ray structure of the protein is available and a system to generate mutants by direct mutagenesis has been developed. Our study highlights the crucial role played by the His26-Glu 44 Hbond, wich confers great stability to native iso-1-cit c by keeping the 20’s and the 40’s Ω-loops joined and sterically close, thereby enhancing the overall rigidity of the macromolecule. The absence of the such H-bond induces changes in the tertiary structure of the macromolecule associated to a dramatic decrease of protein stability. The flexibility of the H26Y variant weakens the native M80-Fe(III) axial bond strength, therefore altering the protein behaviour. As a result, the alkaline conformational transition occurs at lower pH (pKa = 7.5 rather than 8.4) and, contrary to the native protein, at neutral pH the H26Y variant results to be a mixture of (at least) two species with different axial coordination to the metal. These observations indicate that this H-bond is particularly important for protein stabilization; its rupture induces in the protein the formation of a “molten globule” state under physiological-like conditions. This state is an equilibrium mixture of two forms, one characterized by a native-like coordination to the heme iron, the other possessing a misligated endogeneous ligand axially coordinated to the metal, as shown by the following scheme: His18-Fe(III)-Met80 ↔ His18-Fe(III)-X where X is an endogenous ligand. Resonance Raman measurements indicate probably a lysine as the most likely nonnative ligand. In order to determine which role is played by the other two histidines located in the aminoacidic sequence of the protein (H33 and H39), we have produced and characterized the double mutants H26YH33Y, H26YH39K, H33YH39K, which retain only one His in the amino acidic sequence. In particular, the H39K mutation inserts a lysine at position 39, as in the sequence of horse cyt c. Data obtained indicate that neither H33 or His39 induce significant alteration in the protein structure and stability; this highlights the critical role played H 26 for protein stability. The properties of the H26Y mutant of equine cyt c were also investigated. Although showing a close structural similarity with respect to yeast iso-1-cyt c (as revealed by X-ray cristallography), this protein exhibits a higher stability we wish to underline. Recombinant horse ferricyt c produced in our laboratory shows spectral properties and stability practically identical to the native protein, as shown by CD, electronic absorption and high frequency RR data. Interestingly, also the H26Y mutant does not exhibit significant changes with respect to the wt protein, at neutral pH. However, the H26Y mutant undergoes alkaline isomerization at a pH lower than the wt form (pKa: 8.3 vs 9.2), but higher than that of the yeast variant. The observed ∆pKa between the mutant and wt form (∆ (∆Go) = 5.1 KJ/mol) measures the stability decrease induced by the H26 mutation; the same ∆pKa was determined for horse and yeast cyt c, which suggests that the H26 mutation decreases the stability of the two proteins to a similar extent, despite the fact that equine cyt c displays a higher stability. Therefore, the properties of the ionizing group controlling the alkaline transition (i.e. the trigger) are equally affected in both proteins. Hence, the groups responsible for the higher stability of horse cyt c at neutral pH can be excluded from any involvement in the conformational changes associated with the alkaline isomerization of cyt c

Shear layer over floodplain vegetation with a view on bending and streamlining effects

Publisher Copyright: © 2022, The Author(s).Abstract: Shrubby and woody vegetation growing on floodplains profoundly influences hydrodynamic and transport processes in riverine systems. Existing hydrodynamic research is mostly focused on conditions with aquatic plants and rigid model vegetation. To appreciate the different hydrodynamic impacts of submerged floodplain and riverbank vegetation, a novel flume investigation was carried out. We simulated conditions found in riparian environments in terms of vegetation density, plant structure and flexibility, and presence of a grassy understory. Four experimental cases were defined so that vegetation exhibited different degrees of bending and streamlining. Extensive set of velocity measurements allowed reliable description of the double averaged flow. Vegetation morphology, with the flexibility-induced streamlining and dynamic motion controlled the magnitude and distribution of the vegetative drag, shaping the shear penetration within the canopy. The flows were highly heterogeneous, thus calling for spatially averaged approaches for the flow field investigation. The relative importance of dispersive momentum fluxes was high in the canopy bottom region where both Reynolds and dispersive stresses were small. The contribution of dispersive fluxes to momentum transport decreased with increasing reconfiguration. The results revealed the shear layers over floodplain vegetation to be dynamically similar to other environmental flows over porous obstructions. However, the velocity-dependent vegetative drag and deflected height introduced additional complexity in the flow simulation. Altogether our findings implied that accurate description of vegetated floodplain flows can be achieved only when plant morphology and flexibility are appropriately described in drag models. Article highlights: A novel experimental setup with flexible woody plants and grasses was used to model the hydrodynamics of vegetated floodplains.Plant morphology and flexibility controlled the vegetative drag, affecting key shear layer features, including the shear penetration.The spatially heterogeneous flows had higher dispersive stresses at the canopy bottom, where the total fluid stress was small.Peer reviewe