Tesi sul tema "303.48/4/09"

Il danno microvascolare è un evento precoce nella sclerodermia (SSc, Sclerosi Sistemica) ed è noto come lo stress ossidativo sia implicato nella patogenesi della malattia. I radicali liberi dell’ossigeno (ROS) sono ampiamente riconosciuti come importanti secondi messaggeri cellulari nell’ambito della parete vascolare e la NADPH ossidasi rappresenta la principale fonte di radicali liberi a questo livello; in particolare NOX 4 è la principale isoforma di NADPH oxidasi espressa nelle cellule muscolari lisce vascolari. Poiché il nostro gruppo ha di recente identificato la presenza di auto anticorpi attivatori diretti contro il recettore del PDGF nel siero dei pazienti sclerodermici, in questa tesi si è voluto verificare quali fossero gli effetti biologici di questi autoanticorpi sulle cellule muscolari lisce di arteria polmonare valutando l’implicazione relativa di NOX4 e dei ROS. I risultati ottenuti mostrano chiaramente che le pareti dei vasi dei pazienti affetti da Sclerodermia sono caratterizzate da un abnorme stress ossidativo e da un aumento dell’espressione di NOX4. Inoltre, in vitro, le immunoglobuline sclerodermiche sono in grado di indurre la produzione di radicali libero dell’ossigeno, la proliferazione, la migrazione e la produzione di collagene 1 (alpha1) nelle cellule muscolari lisce di arteria polmonare attraverso l’interazione con il recettore del PDGF e l’induzione di NOX4. Per concludere, quindi, possiamo affermare che gli autoanticorpi anti-recettore del PDGF costituiscono una nuova fonte di danno nella sclerodermia, contribuendo alla patogenesi delle lesioni vascolari tipiche della malattia.
Microvasculature damage is one of the earliest events in the onset of Scleroderma (SSc, Systemic sclerosis). Oxidative stress has been implicated in the pathogenesis of SSc and reactive oxygen species (ROS) have been recognized as important signaling molecules in the vascular wall. The NADPH oxidase family is the major source of ROS in the vasculature. Between the various existing NADPH oxidase isoformes, NOX4 is the predominant form in vascular smooth muscle cells. Since we have already identified the presence of anti PDGF receptor (PDGFR) stimulatory antibodies in patients with Systemic Sclerosis, in this study we sought to investigate the effects of these autoantibodies on human vascular smooth muscle cells (HPASMC), in order to understand the biological effects and the implication of ROS and NOX4. With this work we were able to show ex vivo that SSc is characterized by excessive oxidative stress and upregulation of NOX4 within the vascular wall. In vitro, Scleroderma immunoglobulins induce ROS production in HPASMC. This effect is mediated through the PDGFR and NOX4. Furthermore PDGF and SSc immunoglobulins mediate NOX4 upregulation which in turns determines a ROS mediated mitogenic and pro-migratory effect on human pulmonary artery smooth muscle cells and a significant induction in collagen I (alpha1) gene expression. In conclusion Anti PDGFR autoantibodies may contribute to the pathogenesis of this disease through their effects on vascular damage typically observed in Scleroderma.

Gliomas are considered among the most puzzling problems of medicine due to the complexity of their anatomic localization, and the lack of a real therapeutic treatment. The main topic of this work concerns the possibility to use bovine herpesvirus type 4 (BoHV-4) as a vector for gene therapy for the treatment of glioma, and that the cAMP-dependent protein kinases (PKA) pathway as a possible target for such therapy. This study is based on a research project that involved both University of Padova and University of Parma. Here BoHV-4 to is shown to infect rat glioma cells in vitro and in vivo, in addition, BoHV-4 can infect human immortalized glioma cells in vitro and human brain tumour primary cell cultures. The study of the relationship between gliomas and the PKA pathway shows a peculiar distribution of the PKA regulatory subunits in glioma cells in mouse, rat and human. The present data suggest that the PKA pathway modulation may be targeted for the treatment of gliomas. The present Study pave the way to the use of a safe and efficient BoHV-4-based vector for the delivery of therapeutic genes or for the targeting of specific abnormal pathways, like the PKA-mediated one, for the treatment of the hopeless high-grade glioma.
L’ipotesi alla base di questo lavoro è che vi sia la possibilità di impiegare quale vettore virale per la terapia genica dei gliomi l’herpesvirus bovino di tipo 4 (BoHV-4) e di determinare se la via di trasduzione del segnale mediata dalle protein chinasi AMPc-dipendenti (PKA) possa essere utilizzata come bersaglio terapeutico. Infatti i gliomi sono ritenuti essere uno dei più importanti e stimolanti problemi irrisolti della medicina. Questo sia per la evidente complessità della sede anatomica di insorgenza, sia per il fatto che, nonostante gli enormi sforzi in cui gli scienziati di tutto il mondo si sono profusi non si è ancora riusciti a giungere alla messa a punto di un protocollo curativo realmente efficace. Il progetto di ricerca su cui si è basato il presente studio è il risultato della convergenza di due linee di ricerca preesistenti presso le Università di Padova e di Parma. I risultati conseguiti hanno dimostrato la capacità di BoHV-4 di infettare in vitro e in vivo cellule di glioma di ratto. Inoltre tale capacità è stata confermata in vitro sia su cellule immortalizzate di glioma umano che su colture primarie di tumore cerebrale umano. Per quanto riguarda lo studio della relazione tra PKA e tumori cerebrali, è stato in primo luogo rilevata una distribuzione peculiare delle diverse subunità regolatorie delle medesime che è caratteristica delle cellule di glioma. Inoltre diversi esperimenti suggeriscono che la modulazione di tale via possa essere impiegata per il trattamento dei gliomi oltre che per fini diagnostici. Tutti i risultati ottenuti suggeriscono di proseguire ed ampliare il progetto facendo convergere entrambe le sue linee costituenti in un modello da poter esportare nella pratica clinica nel minor tempo possibile.

Hypocretin 1 and 2 (HCRT, also called Orexin A and B) are neuropeptides released by neurons in the lateral hypothalamus. HCRT neurons widely project to the entire neuroaxis. HCRT neurons have been reported to participate in various hypothalamic physiological processes including cardiovascular functions, wake-sleep cycle, and they may also influence metabolic rate and the regulation of body temperature. HCRT neurons are lost in narcolepsy, a rare neurological disorder, characterized by excessive daytime sleepiness, cataplexy, sleep fragmentation and occurrence of sleep-onset rapid-eye-movement episodes. We investigated whether HCRT neurons mediate the sleep-dependent cardiovascular adaptations to changes in ambient temperature (Ta). HCRT-ataxin3 transgenic mice with genetic ablation of HCRT neurons (n = 11) and wild-type controls (n = 12) were instrumented with electrodes for sleep scoring and a telemetric blood pressure (BP) transducer (DSI, Inc.). Simultaneous sleep and BP recordings were performed on mice undisturbed and freely-behaving at 20 °C, 25 °C, and 30 °C for 48 hours at each Ta. Analysis of variance of BP indicated a significance of the main effects of wake-sleep state and Ta, their interaction effect, and the wake-sleep state x mouse strain interaction effect. BP increased with decreasing Ta. This effect of Ta on BP was significantly lower in rapid-eye-movement sleep (REMS) than either in non-rapid-eye-movement sleep (NREMS) or wakefulness regardless of the mouse strain. BP was higher in wakefulness than either in NREMS or REMS. This effect of sleep on BP was significantly reduced in mice lacking HCRT neurons at each Ta, particularly during REMS. These data suggest that HCRT neurons play a critical role in mediating the effects of sleep but not those of Ta on BP in mice. HCRT neurons may thus be part of the central neural pathways which mediate the phenomenon of blood pressure dipping on passing from wakefulness to sleep.

Bone remodelling is a fundamental mechanism for removing and replacing bone during adaptation of the skeleton to mechanical loads. Skeletal unloading leads to severe hypoxia (1%O2) in the bone microenvironment resulting in imbalanced bone remodelling that favours bone resorption. Hypoxia, in vivo, is a physiological condition for osteocytes, 5% O2 is more likely physiological for osteocytes than 20% O2, as osteocytes are embedded deep inside the mineralized bone matrix. Osteocytes are thought to be the mechanosensors of bone and have been shown to orchestrate bone formation and resorption. Oxygen-deprived osteocytes seem undergo apoptosis and actively stimulate osteoclasts. Hypoxia and oxidative stress increase 150-kDa oxygen-regulated protein (ORP 150) expression in different cell types. It is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. It well known that ORP 150 plays an important role in the cellular adaptation to hypoxia, as anti-apoptotic factor, and seems to be involved in osteocytes differentiations. The aims of the present study are 1) to determine the cellular and molecular response of the osteocytes at two different conditions of oxygen deprivation, 1% and 5% of O2 compared to the atmospheric oxygen concentration at several time points. 2) To clarify the role of hypoxic osteocytes in bone homeostasis through the detection of releasing of soluble factors (RANKL, OPG, PGE2 and Sclerostin). 3) To detect the activation of osteoclast and osteoblast induced by condition media collected from hypoxic and normoxic osteocytes. The data obtained in this study shows that hypoxia compromises the viability of osteocytes and induces apoptosis. Unlike in other cells types, ORP 150 in MLO-Y4 does not seem to be regulated early during hypoxia. The release of soluble factors and the evaluation of osteoclast and osteoblast activation shows that osteocytes, grown under severe oxygen deprivation, play a role in the regulation of both bone resorption and bone formation.

Il trasportatore del glutammato GLT-1 e’ responsabile della ricaptazione di glutammato rilasciato a livello sinaptico. Grazie alla sua azione, il glutammato extracellulare e’ tenuto a bassi livelli di concentrazione, evitando fenomeni patologici di eccitotossicita’. Per questa ragione, GLT-1 e’ coinvolto nella patogenesi di numerose malattie. Recentemente e’ stato dimostrato che e’ possibile incrementare l’espressione di GLT-1 somministrando il ceftriaxone (CEF), un comune antibiotico. Recenti studi hanno rivelato che la somministrazione di CEF e’ in grado di alterare l’elaborazione dell’infromazione nervosa e la plasticita’ sinaptica. Date queste premesse, ipotiziamo che il CEF, aumentando l’espressione di GLT-1, alteri l’attivita’ di ampie popolazioni di neuroni. Per dimostrare questo, ci siamo avvalsi di studi elettroncefalografici (EEG), misurando differenze quantitative della potenza dello spettro del segnale EEG prima e dopo il trattamento con CEF. Inoltre abbiamo valutato se le alterazioni EEG si associavano ad alcuni aspetti comportamentali. Per fare questo, abbiamo registrato l’EEG dale cortecce frontali e parietali di ratto, abbiamo inoltre registrato l’EMG ed abbiamo video registrato gli animali. Infine abbiamo studiato l’andamento nel tempo della potenza dello spettro e dell’ativita’motoria per mezzo dell’analisi EMG e delle registrazioni video. Abbiamo scoperto che il CEF riduce la potenza del segnale nelle frequenze theta con un picco a 7-9 Hz e che incrmenta l’attivita’ locomotoria degli animali. Queste alterazioni mostravano un andamento temporale sovrapponibile, indicando un possibile legame tra le alterazioni EEG e l’aumento dell’attivita’ locomotoria. Inoltre, i dati presentati suggeriscono che il CEF e’ in grado di alterare, attraverso la modulazione della trasmissione glutammatergica, l’attivita’ di ampie popolazioni di neuroni.
The glutamate transporter GLT-1 is responsible for clearing synaptically released glutamate from the extracellular space. By this action, it maintains low levels of ambient glutamate, thus preventing excitotoxic damage, and contributes to shape synaptic transmission. GLT-1 dysregulation has been implicated in several diseases. Recently, it has been demonstrated that ceftriaxone (CEF)-induced GLT-1 up-regulation impaired the prepulse inhibition, a simple form of information processing, and determined a strong reduction of the hippocampal mGluR-dependent long-term depression, which was reversed by dihydrokainate, a specific GLT-1 antagonist. Along this line, we hypothesize that CEF, by over-expressing GLT-1, could lead to large-scale dysfunctions involving large populations of neurons. To test this possibility, we verified whether CEF treatment was able to interfere with long-range cortical activity, by measuring the quantitative differences at electroencephalogram (EEG) of rats before and after CEF treatment. In addition, we tested out whether EEG changes could reflect some behavioural modifications. To do this, we performed EEG recordings in left and right fronto-parietal cortices, EMG and video recordings of WKY male rats. We treated the animals with CEF and we studied the time course analysis of EEG power spectra, EMG activity and motion activity detected by video recordings. We found that CEF treatment was associated to a remarkable reduction of theta power (7-9 Hz) and to an increase of motor activity. Those alterations showed a similar time course, indicating a feasible relation between EEG modifications and motor activity changes. In addition, the present results suggest that GLT-1, by controlling the efficacy of the glutamatergic transmission, might also modulate the activity of wide neural circuits.

Alla luce degli sviluppi normativi e della crescente attenzione alla sicurezza sismica delle strutture, soprattutto dopo gli ultimi terremoti italiani, l'analisi e la verifica del patrimonio edilizio esistente è diventato uno strumento fondamentale per valutare la vulnerabilità sismica, per salvaguardare le vite umane e pianificare interventi strutturali. Il patrimonio edilizio italiano è caratterizzato da elevata complessità ed eterogeneità, sia dal punto di vista architettonico che strutturale, e consiste in strutture costruite in epoche diverse, collocate sia nel centro della città che in periferia, realizzate con diverse tecniche costruttive e caratterizzate da diversi usi. Lo scopo di questa ricerca è quello di individuare una metodologia di verifica facilmente gestibile e adattabile a molti edifici diversi, ma allo stesso tempo in grado di determinare lo stato effettivo della struttura in termini di criticità e carenze strutturali. Per fare ciò si è preso come riferimento il patrimonio dell'Università di Bologna, in particolare 59 plessi universitari quantificabili in circa 470.000 m2 di superficie utile da analizzare. Nel capitolo 1 è presentato lo "stato dell'arte" dei metodi per la valutazione della vulnerabilità sismica degli edifici esistenti. Nel capitolo 2 viene descritto il patrimonio edilizio dell'Ateneo, si analizzano le condizioni ei vincoli operativi che hanno determinato l'individuazione della metodologia utilizzata. Nel capitolo 3 è illustrata nel dettaglio la metodologia e la campagna sperimentale effettuata sui materiali nel processo di acquisizione delle informazioni. Il capitolo 4 descrive un caso studio al fine di comprendere meglio la metodologia sviluppata e le sue potenzialità. Il capitolo 5 presenta i risultati ottenuti sul patrimonio attraverso diversi indicatori di vulnerabilità e, mediante analisi probabilistica e deterministica, definisce una valutazione della vulnerabilità affidabile delle strutture analizzate.
In the light of the recent code developments and of the growing attention given to the seismic safety of structures, especially after the last Italian earthquakes, the analysis and the verification of the existing building heritage has become a fundamental tool to assess the seismic vulnerability, to safeguard human lives and to plan structural interventions. The Italian building heritage is characterized by high complexity and heterogeneity, both from architectural and structural points of view, and consists in structures built in various ages, placed both in the city center and in the outskirt, realized with different structural techniques, characterized by several intended uses. The purpose of this research is to identify a methodology of verification easily manageable and adaptable to many different buildings, but at the same time able to determine the actual state of structure in terms of critical steps and structural deficiencies. In order to develop this methodology, the building heritage of the University of Bologna has taken as a reference. In particolar this building heritage has a overall floor area of approximately 470,000 m2 and consist in 59 buildings. Chapter 1 presents the "state of the art" of the methods for seismic vulnerability assessment of existing buildings. Chapter 2 describes the building heritage of the Athenaeum, analyzes its conditions and the operational constraints that determined the selection and identification of the methodology used. Chapter 3 illustrates in detail the methodology and the experimental campaign performed on the materials in the process of acquiring information on the constructions. Chapter 4 describes a case study in order to better under stand the methodology developed and its potentiality. Chapter 5 presents the results obtained from the overall building heritage through different indicators of vulnerability and, by means of probabilistic and deterministic analysis, defines a reliable vulnerability assessment of the structures analyzed.

Chemoreception represents one of the most important sensory modality to guarantee animals survival, and it plays a fundamental role also in an insect life. In fact, the detection of food sources and proper sites of ovoposition, the identification of conspecifics for mating or aggregation, the recognition of prey or predators are all behaviors resulting from the activation of different processes after the exposure to the wide range of soluble and volatile chemical molecules in the environment. In this respect, insects present clearly separate senses of taste and olfaction comparable to those of vertebrates, and they represent an excellent experimental model to investigate the complexity of gustative and olfactory systems also thanks to the relative simple organization of their neuronal circuits, as well as the feasibility of the breeding. On the basis of these considerations, aim of my work was to give a contribution on our understanding of chemoreceptive mechanisms. In particular, I investigated two main aspects of chemoreception in insects, divided in two separate sections. Section 1: The spike generator in the labellar taste receptors of the blowfly is differently affected by 4-aminopyridine and 5-hydroxytryptamine In taste chemoreception of invertebrates the interaction of taste stimuli with specific membrane receptors and/or ion channels located in the apical membrane of taste receptor cells results in the generation of a receptor potential which, in turn, activates the ‘encoder’ region to produce action potentials which propagate to the CNS. This study investigates, in the labellar chemosensilla of the blowfly, Protophormia terraenovae, the voltage-gated K+ currents involved in the action potential repolarization and repetitive firing of the neurons by way of the Kv channel inhibitors, 4-aminopyridine and 5-hydroxytryptamine. The receptor potential and the spike activity were simultaneously recorded from the ‘salt’, ‘sugar’ and ‘deterrent’ cells, by means of the extracellular side-wall technique, in response to 150 mM NaCl, 100 mM sucrose and 1 mM quinine HCl, before, 0-10 min after apical administration of 4-AP (0.01–10 mM) or 5-HT (0.1–100 mM). The results show that the receptor potential in all three cells is neither affected by 4-AP nor by 5-HT. Instead, spike activity is significantly decreased, by way of blocking different Kv channel types: an inactivating A-type K+ current (KA) modulating repetitive firing of the cells and responsible for the after hyperpolarization, and a sustained K+ current that resembles the delayed rectifier (DKR) and contributes to action potential repolarization. Section 2: Morphological characterization of the antennal lobes in the Mediterranean fruit fly Ceratitis capitata The medfly (Ceratitis capitata Wied.) is one of the most important pest for horticulture, targeting a great variety of fruit and vegetables species worldwide. Due to its commercial relevance, many studies focused on the development and improvement of control strategies based on olfactory chemoreception. A complete knowledge of the anatomical and functional properties of the olfactory system is still lacking. Aim of this work is to give a morphological characterization based on the three-dimensional reconstruction of the antennal lobes (ALs) in adult medfly brains. In order to reach this goal, we performed unilateral antennal backfills of olfactory receptor neurons (ORNs) in adult males and females by means of the neuronal tracer neurobiotin, revealed by streptavidin-Cy3 or Avidin-Alexafluor 488 conjugated. In association with the anterograde staining, immunohistochemistry was applied in some brains. Confocal stacks acquired from whole-mount specimens were analyzed with the AMIRA software, using the Segmentation tool. Unilateral neurobiotin and immunohistochemical stainings successfully revealed the AL structure of the adult medfly in all the specimens tested. As in other insects, the ALs of C. capitata are organized in glomeruli, more tightly packed in the anterior part than the posterior one. Axons of ORNs innervate a bilateral pair of homologous glomeruli and form a commissure between the two ALs, which is a typical feature of Diptera. We counted systematically a number of 53 glomeruli in each AL studied, with few exception. Our results provide a basis for future investigations on the interactions with host plants of this important agricultural pest.

In linea con uno degli obiettivi strategici dell’Università Cà Foscari di Venezia, ovvero la diffusione e promozione dell’Agenda 2030 e i relativi 17 obiettivi per lo sviluppo sostenibile, questa tesi di laurea magistrale si propone dapprima di esaminare se i principi e valori generali integrati nell’obiettivo 4 dell’Agenda 2030 per lo sviluppo sostenibile siano inclusi o meno nelle politiche di cooperazione multilaterale in materia di istruzione promosse dalla Comunità di Paesi di Lingua Portoghese (CPLP). Successivamente si intende verificare se vi sia un allineamento delle politiche nazionali di istruzione dei nove Paesi Membri della CPLP (Angola, Brasile, Capo Verde, Guinea Bissau, Guinea Equatoriale, Mozambico, Portogallo, San Tomè e Principe, Timor Est) all’obiettivo 4, in particolar modo per quanto concerne il tema della promozione dello sviluppo sostenibile per mezzo dell’istruzione (target 4.7).

The thesis entitled “-Keto phosphonates from tartaric acid in the total synthesis of ()-4-epi-gabosine A, ()-dihydrokawain-5-ol, ()-bengamide E and Identification of MPK-09: A small molecule that restores the wild type function of mutant p53.’’ demonstrates the utility of -keto phosphonate derived from tartaric acid as a building block in the synthesis of bioactive natural products small molecules of therapeutic importance. The thesis is divided into three sections. Section I of the thesis deals with the utility of new -keto phosphonate 1, derived by desymmetrization of bis-Weinreb amide of L-(+)-tartaric acid (Scheme 1). Scheme 1: (a) MeP(O)(OMe)2, n-BuLi, THF, –78 ˚C, 1.5 h, 91%. A series of functionalized aldehydes were used in the Horner-Wadsworth-Emmons type reaction of phosphonate 2. Under the optimized condition, a series of aldehydes including aryl, aliphatic aldehydes with chiral centers next to the aldehyde functionality underwent facile olefination to yield the , unsaturated ketones in good yields (Scheme 2). Scheme 2. (a) Cs2CO3, aldehyde, iPrOH, rt, 1 h. Application of the synthesized unsaturated ketones in the synthesis of various molecular architectures of therapeutic importance was undertaken. Application of this strategy in the total synthesis of (+)-4-epi-gabosine A and (+)-dihydrokawain-5-ol is delineated. The -keto amides were reduced with good diastereoselectivity with NaBH4 to afford the -hydroxy amide which was transformed to the diene. Ring closing metathesis of the diene led to the cyclohexenone which on deprotection gave ()-4-epi-gabosine A (Scheme 3). The key reactions during the synthesis are Horner-Wadsworth-Emmons reaction, stereoselective reduction and ring closing metathesis as shown in Scheme 3. Scheme 3: Stereoselective total synthesis of ()-4-epi-gabosine A. Dihydrokawain-5-ol 11 is a unique 6-alkyl-5-hydroxy-5,6-dihydropyran-2-one isolated from the methanol extracts of the kava plant (Piper mythisticum), a Polynesian shrub of the pepper family and exhibits promising biological activities. Stereoselective total synthesis of this natural product from tartaric acid is described in this section. Key features of the synthesis include the elaboration of the unsaturated ketone obtained from the phosphonate to the allylic alcohol 8 and further elaboration to the natural product 11 as depicted in Scheme 4. Scheme 4: Total synthesis of (+)-dihydrokawain-5-ol. (Part of this work has been communicated). Section II of the thesis deals with the enantiospecific total synthesis of ()-bengamide E. Bengamide E was isolated from the jaspidase sponges by the Crew research group. Enantiospecific total synthesis of bengamide E was accomplished in 8.5% overall yield in a linear sequence of 10 steps starting from the bis-(dimethylamide) unit of tartaric acid as chiral pool precursor (Scheme 5). Key features of the synthesis includes combination of the addition of 1, 3-dithian-2-yllithium, stereoselective reduction and Horner-Wadsworth-Emmons reaction. Scheme 5: Enantiospecific total synthesis of bengamide E. (This work has been published: Metri, P. K.; Schiess, R.; Prasad, K. R. Chem. Asian. J. 2013, 8, 488). Section III of the thesis deals with MPK-09 and a series of novel lactones, structurally similar to bioactive styryllactones synthesized from tartaric acid and studied their in vitro ability to inhibit the growth and induce apoptosis in human tumor cell lines. It was found that MPK-09 a small molecule in the series is more cytotoxic towards cancer cell lines harboring p53 mutation E285K and R273C. Ectopic expression of p53 plasmids harboring various hotspot mutations, demonstrated that MPK-09 was more effective on the tested p53 mutants compared to the wt p53 harboring cells. The restoration of p53 function was further corroborated by the transactivation of its pro-apoptotic signaling pathways as shown by the induction of p21 and Bax protein expression by western analysis. It was demonstrates that the activity was due to the restoration of the wild type conformation of mutant p53. (Part of this work is published: Metri, P. K.; Naz, S.; Kondaiah, P.; Prasad, K. R. ACS Chem. Biol. 2013, 8, 1429).

The development of Nano Drug Delivery Systems (NDDS) is a promising approach for developing intelligent therapeutic systems, which will bring significant advances in the diagnosis and treatment of disease, with the challenges to maximize therapeutic activity and to minimize undesirable side effects. The aim of this thesis was focused on the design of two kind of self-assembled nanocarrier for the delivery of hydrophobic drugs: Hyaluronan based nanohydrogel and pNIPAAm based micelles that both show hydrophobic internal domains and a surrounding hydrophilic shell. To achieve such self-assembled nanostructures, amphiphilic polymers were synthesized and extensively characterized. Hyaluronan (HA) and the thermosensitive di-block copolymer of methoxy poly(ethylene glycol)-b-(N-isopropylacrylamide)-co-(2-azidoethyl) methacrylate (mPEG-b-p(NIPAAm)-co-AzEMA) constituted the starting polymers on which riboflavin 2',3',4',5'- tetrabutyrate (Rfv), due to its interesting chemical-physical proprieties and due to its particularly biocompatibility, was conjugated as hydrophobic moiety via azide-alkyne click chemistry reaction. The HA-Prop derivatives were synthesized in order to provide the polymer of alkyne groups and make it versatile for “click” reaction. In this way, the azido-hexyl derivative of riboflavin tetrabutyrate was synthesized and covalently coupled to the propargyl derivative of hyaluronic acid by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC), due to the high efficacy and selectivity of this kind of reaction. Sterile self-assembled NHs were obtained in one-step by thermal treatment in autoclave of the aqueous dispersion of the HA-c-Rfv polymers. Size and polydispersity of the obtained NHs resulted be influenced from the Mw and from the degrees of derivatization of the starting Hyaluronan. Micellar nano-assemblies composed of thermosensitive amphiphilic block polymers were formed by heating the aqueous solutions of the resulting poly(NIPAAm) block copolymer, in order to obtain temporal control of the release of the encapsulated drugs. For this purpose, mPEG-b-p(NIPAAm)-co-AzEMA block copolymer was synthesized by free radical polymerization and subsequently, a propargyl derivative of riboflavin (Rfv-Prop) was synthesized and covalently coupled to the azide modified diblock copolymers by Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC). The nanosystems thus formed were intended to be used for the delivery of hydrophobic drugs, thus taking advantages of the lipophilic core represented by riboflavin. Moreover, the potential π-π stacking interactions between the aromatic rings of the riboflavin in the core of such nanocarriers, may bring more stable nanostructures able to provide increased loading capacity. Highly hydrophilic and biocompatible nanocarriers based on polysaccharide hydrogels (nanohydrogels, NHs) were shown to be promising systems for drug delivery applications. Inspired by these emerging and promising drug carriers for therapeutic applications, in this work we aimed to develop self-assembled hydrogel nanoparticles based on amphiphilic derivative of hyaluronic acid (HA). For this purpose, new HA-Riboflavin (HA-c-Rfv) derivatives were synthetized by “click” Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction, requiring a previous HA derivatization with alkyne moieties and Riboflavin modification with azide groups. The resulting amphiphilic product was able to form nanohydrogels in aqueous environments by suitable treatments, in particular by an innovative method by using an autoclave cycle. Various HA molecular weights (Mw) and derivatization degrees of the starting polymers have been used in order to assess the effect of different parameters on the NHs formation. The derivative HA220-c-Rfv (Mw 220kDa, 40% of propargylic portions and 40% of Rfv moieties), was chosen as the most interesting NHs forming system; NHs were 150-200 nm in size and showed a ζ-potential in the range -40 / -50 mV, depending on the experimental conditions adopted. NHs resulted to be stable in water and at physiological pH. Moreover, by the addition of a suitable cryoprotectant, the NHs suspension can be freeze-dried and recovered by re-suspension in water. The developed system is intended to be used for the delivery of hydrophobic drugs, such us dexamethasone, piroxicam and paclitaxel, used as model drugs. Drug encapsulations were performed by hydrating drugs film with polymers aqueous suspensions, followed by an autoclave treatment, resulting in a high encapsulation efficiency (EE%). Moreover, the HA-propargyl backbone with 60% of propargylic portions and partially linked to Rfv, was capable to react with other molecules bearing an azide group, opening the route to a wide spectrum of functionalization opportunities: in this direction, PEG-N3 have been tested as model molecule for the NHs. Micellar nano-assemblies composed of thermosensitive amphiphilic block polymers are formed spontaneously above their CMC. For these types of polymers, the aqueous solubility properties depend on temperature, and micellar nanostructures are formed by self-assembly above their lower critical solution temperature (LCST) in aqueous media. For this purpose, poly N-isopropylacrylamide (pNIPAAm) block-copolymers were synthesized by free radical polymerization using a polyethylene glycol based macroinitiator. Upon dissolution in aqueous solvents and heating above the LCST, these polymers are able to form micelles. To stabilize these micelles, functional groups facilitating π-π stacking interactions were introduced into the polymers. For this purpose, riboflavin as aromatic moiety was coupled to the polymer backbone by azide-alkyne “click chemistry” reaction. Two block-copolymers, methoxy poly(ethylene glycol)-b-(N-isopropylacrylamide)-co-(2-azidoethyl) methacrylate (mPEG-b-p(NIPAAm)-co-AzEMA) and the corresponding derivative methoxy poly(ethylene glycol)-b-(N-isopropylacrylamide)-co-(2-azidoethyl) methacrylate-riboflavin (mPEG-b-p(NIPAAm)-co-AzEMA-Rfv), were synthesized and compared in terms of physico-chemical properties, micelle size, drug retention and release. Micelles were formed by heating the polymeric aqueous solution from 0 to 50°C, and paclitaxel (PTX) was encapsulated by mixing a concentrated drug solution in ethanol with the polymer solution in phosphate buffer followed by heating. Three different feed PTX loadings (feed drug loading concentration of 5, 10 and 20%) were tested in micelles of both block-copolymers. Upon introduction of riboflavin in the polymeric backbone, a lower critical micelle temperature was obtained by (26°C for mPEG-b-p(NIPAAm)-co-AzEMA and 24° C for the corresponding riboflavin containing polymer, respectively). An average size of ~50 nm for the mPEG-b-p(NIPAAm)-co-AzEMA and ~90 nm for the mPEG-b-p(NIPAAm)-co-AzEMA-Rfv micelles in water was observed respectively for empty micelles, which increased with ~20 nm in phosphate buffered saline. Drug loading resulted in an increase of the micelle size by approximately 10-20 nm (from 60 to 75 nm for mPEG-b-p(NIPAAm)-co-AzEMA based micelles, and from 150 to 170 nm for mPEG-b-p(NIPAAm)-co-AzEMA-Rfv based ones). Increased encapsulation efficiency (EE%) and drug loading (DL%) were obtained for micelles containing riboflavin; almost 100% encapsulation of PTX was found for a feed PTX loading of 5% . Nevertheless, drug release resulted be faster for the Rfv-derivative backbone based micelle, likely due to their lower polymer density. Overall, this novel Rfv containing system is very promising in bringing advantages with regard to drug loading, warranting further investigations in tunability of drug release profiles for further application in stimuli-responsive anticancer therapy.

It is difficult for policymakers to predict the behavior of people in response to a water rationing policy. The public may not necessarily behave as expected or in accordance with market rules or policy mandates. In this research, I will ask whether people were responsive to a summer 2011 City of Saskatoon legal restrictions to reduce their outdoor water consumption due to reduced capacity at the water treatment plant resulting from excessive solids in the river water. I will try to explore the policy response - which can be expressed as a reduction of outdoor water consumption in 2011 in response to the water mandate - while holding constant other factors, including environmental variables (temperature and rainfall), socio-economic factors (income and education level), lot size, and an annual downward trend in water consumption that appeared in many North American cities during the past two decades. Monthly water consumption data for the period from 2004 to 2012 for the City of Saskatoon were analyzed to detect if there is a policy response from the water mandate during June and July 2011. Regression analysis with water consumption as the dependent variable and lot size, temperature, rainfall, education index, income, consumption trend, and policy as independent variables was conducted to test whether there is a policy response in the Saskatoon water records, holding other factors relevant to water consumption constant. Results showed there was a statistically significant reduction in Saskatoon water consumption during June and July 2011 as a result of the water rationing mandate, with considerable variations through different neighborhoods. In addition, there is a positive relationship between water consumption and lot size and a reduction in water consumption over the research period from 2004 to 2012. The policy response varied widely across neighborhoods, and there was relationship between policy and annual income per capita, and household size; households with more income per capita are less responsive to the policy while bigger household sizes showed more policy responsiveness. Key words: City of Saskatoon, water rationing, water policy, water mandate, outdoor water use.