Caratterizzazione del legame di molecole di interesse farmaceutico alla sieroalbumina umana mediante biocromatografia e dicroismo circolare

Negli ultimi anni, un crescente numero di studiosi ha focalizzato la propria attenzione sullo sviluppo di strategie che permettessero di caratterizzare le proprietà ADMET dei farmaci in via di sviluppo, il più rapidamente possibile. Questa tendenza origina dalla consapevolezza che circa la metà dei farmaci in via di sviluppo non viene commercializzato perché ha carenze nelle caratteristiche ADME, e che almeno la metà delle molecole che riescono ad essere commercializzate, hanno comunque qualche problema tossicologico o ADME [1]. Infatti, poco importa quanto una molecola possa essere attiva o specifica: perché possa diventare farmaco è necessario che venga ben assorbita, distribuita nell’organismo, metabolizzata non troppo rapidamente, ne troppo lentamente e completamente eliminata. Inoltre la molecola e i suoi metaboliti non dovrebbero essere tossici per l’organismo. Quindi è chiaro come una rapida determinazione dei parametri ADMET in fasi precoci dello sviluppo del farmaco, consenta di risparmiare tempo e denaro, permettendo di selezionare da subito i composti più promettenti e di lasciar perdere quelli con caratteristiche negative. Questa tesi si colloca in questo contesto, e mostra l’applicazione di una tecnica semplice, la biocromatografia, per caratterizzare rapidamente il legame di librerie di composti alla sieroalbumina umana (HSA). Inoltre mostra l’utilizzo di un’altra tecnica indipendente, il dicroismo circolare, che permette di studiare gli stessi sistemi farmaco-proteina, in soluzione, dando informazioni supplementari riguardo alla stereochimica del processo di legame. La HSA è la proteina più abbondante presente nel sangue. Questa proteina funziona da carrier per un gran numero di molecole, sia endogene, come ad esempio bilirubina, tiroxina, ormoni steroidei, acidi grassi, che xenobiotici. Inoltre aumenta la solubilità di molecole lipofile poco solubili in ambiente acquoso, come ad esempio i tassani. Il legame alla HSA è generalmente stereoselettivo e ad avviene a livello di siti di legame ad alta affinità. Inoltre è ben noto che la competizione tra farmaci o tra un farmaco e metaboliti endogeni, possa variare in maniera significativa la loro frazione libera, modificandone l’attività e la tossicità. Per queste sue proprietà la HSA può influenzare sia le proprietà farmacocinetiche che farmacodinamiche dei farmaci. Non è inusuale che un intero progetto di sviluppo di un farmaco possa venire abbandonato a causa di un’affinità troppo elevata alla HSA, o a un tempo di emivita troppo corto, o a una scarsa distribuzione dovuta ad un debole legame alla HSA. Dal punto di vista farmacocinetico, quindi, la HSA è la proteina di trasporto del plasma più importante. Un gran numero di pubblicazioni dimostra l’affidabilità della tecnica biocromatografica nello studio dei fenomeni di bioriconoscimento tra proteine e piccole molecole [2-6]. Il mio lavoro si è focalizzato principalmente sull’uso della biocromatografia come metodo per valutare le caratteristiche di legame di alcune serie di composti di interesse farmaceutico alla HSA, e sul miglioramento di tale tecnica. Per ottenere una miglior comprensione dei meccanismi di legame delle molecole studiate, gli stessi sistemi farmaco-HSA sono stati studiati anche con il dicroismo circolare (CD). Inizialmente, la HSA è stata immobilizzata su una colonna di silice epossidica impaccata 50 x 4.6 mm di diametro interno, utilizzando una procedura precedentemente riportata in letteratura [7], con alcune piccole modifiche. In breve, l’immobilizzazione è stata effettuata ponendo a ricircolo, attraverso una colonna precedentemente impaccata, una soluzione di HSA in determinate condizioni di pH e forza ionica. La colonna è stata quindi caratterizzata per quanto riguarda la quantità di proteina correttamente immobilizzata, attraverso l’analisi frontale di L-triptofano [8]. Di seguito, sono stati iniettati in colonna alcune soluzioni raceme di molecole note legare la HSA in maniera enantioselettiva, per controllare che la procedura di immobilizzazione non avesse modificato le proprietà di legame della proteina. Dopo essere stata caratterizzata, la colonna è stata utilizzata per determinare la percentuale di legame di una piccola serie di inibitori della proteasi HIV (IPs), e per individuarne il sito(i) di legame. La percentuale di legame è stata calcolata attraverso il fattore di capacità (k) dei campioni. Questo parametro in fase acquosa è stato estrapolato linearmente dal grafico log k contro la percentuale (v/v) di 1-propanolo presente nella fase mobile. Solamente per due dei cinque composti analizzati è stato possibile misurare direttamente il valore di k in assenza di solvente organico. Tutti gli IPs analizzati hanno mostrato un’elevata percentuale di legame alla HSA: in particolare, il valore per ritonavir, lopinavir e saquinavir è risultato maggiore del 95%. Questi risultati sono in accordo con dati presenti in letteratura, ottenuti attraverso il biosensore ottico [9]. Inoltre, questi risultati sono coerenti con la significativa riduzione di attività inibitoria di questi composti osservata in presenza di HSA. Questa riduzione sembra essere maggiore per i composti che legano maggiormente la proteina [10]. Successivamente sono stati eseguiti degli studi di competizione tramite cromatografia zonale. Questo metodo prevede di utilizzare una soluzione a concentrazione nota di un competitore come fase mobile, mentre piccole quantità di analita vengono iniettate nella colonna funzionalizzata con HSA. I competitori sono stati selezionati in base al loro legame selettivo ad uno dei principali siti di legame sulla proteina. In particolare, sono stati utilizzati salicilato di sodio, ibuprofene e valproato di sodio come marker dei siti I, II e sito della bilirubina, rispettivamente. Questi studi hanno mostrato un legame indipendente dei PIs ai siti I e II, mentre è stata osservata una debole anticooperatività per il sito della bilirubina. Lo stesso sistema farmaco-proteina è stato infine investigato in soluzione attraverso l’uso del dicroismo circolare. In particolare, è stato monitorata la variazione del segnale CD indotto di un complesso equimolare [HSA]/[bilirubina], a seguito dell’aggiunta di aliquote di ritonavir, scelto come rappresentante della serie. I risultati confermano la lieve anticooperatività per il sito della bilirubina osservato precedentemente negli studi biocromatografici. Successivamente, lo stesso protocollo descritto precedentemente è stato applicato a una colonna di silice epossidica monolitica 50 x 4.6 mm, per valutare l’affidabilità del supporto monolitico per applicazioni biocromatografiche. Il supporto monolitico monolitico ha mostrato buone caratteristiche cromatografiche in termini di contropressione, efficienza e stabilità, oltre che affidabilità nella determinazione dei parametri di legame alla HSA. Questa colonna è stata utilizzata per la determinazione della percentuale di legame alla HSA di una serie di poliamminochinoni sviluppati nell’ambito di una ricerca sulla malattia di Alzheimer. Tutti i composti hanno mostrato una percentuale di legame superiore al 95%. Inoltre, è stata osservata una correlazione tra percentuale di legame è caratteristiche della catena laterale (lunghezza e numero di gruppi amminici). Successivamente sono stati effettuati studi di competizione dei composti in esame tramite il dicroismo circolare in cui è stato evidenziato un effetto anticooperativo dei poliamminochinoni ai siti I e II, mentre rispetto al sito della bilirubina il legame si è dimostrato indipendente. Le conoscenze acquisite con il supporto monolitico precedentemente descritto, sono state applicate a una colonna di silice epossidica più corta (10 x 4.6 mm). Il metodo di determinazione della percentuale di legame utilizzato negli studi precedenti si basa su dati ottenuti con più esperimenti, quindi è necessario molto tempo prima di ottenere il dato finale. L’uso di una colonna più corta permette di ridurre i tempi di ritenzione degli analiti, per cui la determinazione della percentuale di legame alla HSA diventa molto più rapida. Si passa quindi da una analisi a medio rendimento a una analisi di screening ad alto rendimento (highthroughput- screening, HTS). Inoltre, la riduzione dei tempi di analisi, permette di evitare l’uso di soventi organici nella fase mobile. Dopo aver caratterizzato la colonna da 10 mm con lo stesso metodo precedentemente descritto per le altre colonne, sono stati iniettati una serie di standard variando il flusso della fase mobile, per valutare la possibilità di utilizzare flussi elevati. La colonna è stata quindi impiegata per stimare la percentuale di legame di una serie di molecole con differenti caratteristiche chimiche. Successivamente è stata valutata la possibilità di utilizzare una colonna così corta, anche per studi di competizione, ed è stata indagato il legame di una serie di composti al sito I. Infine è stata effettuata una valutazione della stabilità della colonna in seguito ad un uso estensivo. L’uso di supporti cromatografici funzionalizzati con albumine di diversa origine (ratto, cane, guinea pig, hamster, topo, coniglio), può essere proposto come applicazione futura di queste colonne HTS. Infatti, la possibilità di ottenere informazioni del legame dei farmaci in via di sviluppo alle diverse albumine, permetterebbe un migliore paragone tra i dati ottenuti tramite esperimenti in vitro e i dati ottenuti con esperimenti sull’animale, facilitando la successiva estrapolazione all’uomo, con la velocità di un metodo HTS. Inoltre, verrebbe ridotto anche il numero di animali utilizzati nelle sperimentazioni. Alcuni lavori presenti in letteratura dimostrano l’affidabilita di colonne funzionalizzate con albumine di diversa origine [11-13]: l’utilizzo di colonne più corte potrebbe aumentarne le applicazioni.

The Politics of Purchasing Power: Feminist Tourism and Women's Development in Nepal

My purpose in this essay is to explore how ideas about women and development are created and circulated at the moment of consumption of wares produced at a women's development project in Nepal. I analyze the project as an example of the ways that women's development is an object of material and discursive consumption. Artifacts produced and sold by Nepali women, and purchased by tourists from the "first world," become part of an international exchange of power, money, and meaning. Based on a survey of consumers and ethnographic observations, I conclude that feminist tourists forge relations with disempowered "Others" through the pleasurable activity of an alienated market transaction. Consumers of crafts produced at a women's development project assume a position of empowerment and enlightenment, ready to help out their "women" counterparts through their support of an enterprise with circular logic: within the industry of development (although not necessarily for feminist tourists themselves), at least one of the central projects of development is the development project itself. At the same time, feminist tourists locate themselves outside the oppressive structures and ideologies affecting their "third-world sisters." This is a relation of sympathy and imagined empathy, with no sense of differential location within systems of oppression. They fail to examine or articulate the global link between their own purchasing power and local living conditions of Maithil women; the connection is effectively built out of the discourse.

RESILIENT WOMEN, METISTIC SCIENTISTS: A MULTIPLE CASE STUDY OF HOW WOMEN NEGOTIATE THEIR SITUATEDNESS IN SCIENCE FIELDS

The concept of feminist metistic resilience postulates that the voiceless, the marginalized and the minority in societies employ strategies in order to turn tables in their favor. This study presents a qualitative analysis of how women, considered to be the minority, negotiate their situatedness in science fields in order to effect change in their lives or that of the society and why they become successful. By “situatedness,” I refer to the everyday life of women as they live and encounter people, society and culture, especially, the life of women who have transcended the culturally stipulated role of women and are excelling in a male dominated field. The study, in different dimensions, conceptualizes the reason for the fewer number of women in science; looks at how scientific methods and practices inhibit the development of women in science; and, finally, interrogates the question of objectivity in science. It becomes apparent, through feminist metistic resilience, that women become successful when they accept conventional practices in scientific arrangements and structures. They accept the practices by embracing and not questioning structures and arrangements that have shaped the field of science and by shifting shapes and assuming different forms in order to adapt to conditions they encounter. Apart from adapting and shape shifting, the women also become successful through environmental and social influences. My analysis suggests that more women can be encouraged to pursue science when women practicing science begin to question structures and arrangements that have shaped the practice of science over the centuries. The overall findings of the research provide implications for policy makers, educators and feminist researchers.

An Innovative Approach to Impacting Student Academic Achievement and Attitudes: Pilot Study of the HEADS UP Virtual Molecular Biology Lab

Introduction: The Virtual Molecular Biology Lab is an innovative, computer-based educational program designed to teach advanced high school biology students how to create a transgenic mouse model in a simulated laboratory setting. It was created in an effort to combat the current decrease in adolescent enthusiasm for and academic achievement in science and science careers, especially in Hispanic students. Because studies have found that hands-on learning, particularly computer-based instruction, is effective in enhancing science achievement, the Virtual Lab is a potential tool for increasing the number of Hispanic students that choose to enter science fields. [See PDF for complete abstract]

Alveolar derecruitment and collapse induration as crucial mechanisms in lung injury and fibrosis.

Idiopathic pulmonary fibrosis (IPF) and bleomycin-induced pulmonary fibrosis are associated with surfactant system dysfunction, alveolar collapse (derecruitment), and collapse induration (irreversible collapse). These events play undefined roles in the loss of lung function. The purpose of this study was to quantify how surfactant inactivation, alveolar collapse, and collapse induration lead to degradation of lung function. Design-based stereology and invasive pulmonary function tests were performed 1, 3, 7, and 14 days after intratracheal bleomycin-instillation in rats. The number and size of open alveoli was correlated to mechanical properties. Active surfactant subtypes declined by Day 1, associated with a progressive alveolar derecruitment and a decrease in compliance. Alveolar epithelial damage was more pronounced in closed alveoli compared with ventilated alveoli. Collapse induration occurred on Day 7 and Day 14 as indicated by collapsed alveoli overgrown by a hyperplastic alveolar epithelium. This pathophysiology was also observed for the first time in human IPF lung explants. Before the onset of collapse induration, distal airspaces were easily recruited, and lung elastance could be kept low after recruitment by positive end-expiratory pressure (PEEP). At later time points, the recruitable fraction of the lung was reduced by collapse induration, causing elastance to be elevated at high levels of PEEP. Surfactant inactivation leading to alveolar collapse and subsequent collapse induration might be the primary pathway for the loss of alveoli in this animal model. Loss of alveoli is highly correlated with the degradation of lung function. Our ultrastructural observations suggest that collapse induration is important in human IPF.

Coupled human and natural system dynamics as key to the sustainability of Lake Victoria's ecosystem services

East Africa’s Lake Victoria provides resources and services to millions of people on the lake’s shores and abroad. In particular, the lake’s fisheries are an important source of protein, employment, and international economic connections for the whole region. Nonetheless, stock dynamics are poorly understood and currently unpredictable. Furthermore, fishery dynamics are intricately connected to other supporting services of the lake as well as to lakeshore societies and economies. Much research has been carried out piecemeal on different aspects of Lake Victoria’s system; e.g., societies, biodiversity, fisheries, and eutrophication. However, to disentangle drivers and dynamics of change in this complex system, we need to put these pieces together and analyze the system as a whole. We did so by first building a qualitative model of the lake’s social-ecological system. We then investigated the model system through a qualitative loop analysis, and finally examined effects of changes on the system state and structure. The model and its contextual analysis allowed us to investigate system-wide chain reactions resulting from disturbances. Importantly, we built a tool that can be used to analyze the cascading effects of management options and establish the requirements for their success. We found that high connectedness of the system at the exploitation level, through fisheries having multiple target stocks, can increase the stocks’ vulnerability to exploitation but reduce society’s vulnerability to variability in individual stocks. We describe how there are multiple pathways to any change in the system, which makes it difficult to identify the root cause of changes but also broadens the management toolkit. Also, we illustrate how nutrient enrichment is not a self-regulating process, and that explicit management is necessary to halt or reverse eutrophication. This model is simple and usable to assess system-wide effects of management policies, and can serve as a paving stone for future quantitative analyses of system dynamics at local scales.

How narcissistic self-construction sabotages their relationships: Accuracy and bias in partner perception

Trapped by their superordinate goal to maintain a grandiose self, narcissists navigate their relationships wishing to re-assure themselves of their specialness and superiority, using strategies bound to fail. Dyadic analyses of diary data (14 days, 83 couples) showed that narcissists focus on their partners’ shortcomings, becoming experts at detecting negative forms of support, while simultaneously minimizing their partner’s positive intentions. Partners of narcissists, in contrast, appear to have a positive bias in ongoing relationships and seem to compensate for some of the narcissists’ shortcomings. Narcissists’ focus on their own selfconstruction purposes also extends to their parenting roles. In a retrospective study, narcissistic mothers were found to demand superior performance, desiring to show-off their child, yet concurrently attributing their child’s success to themselves. They also exercise psychological control and role-reversal, requiring the child to cater to their needs, and to admire them, while simultaneously oblivious to their child’s needs and desires.

How to prevent overdiagnosis.

Overdiagnosis is the diagnosis of an abnormality that is not associated with a substantial health hazard and that patients have no benefit to be aware of. It is neither a misdiagnosis (diagnostic error), nor a false positive result (positive test in the absence of a real abnormality). It mainly results from screening, use of increasingly sensitive diagnostic tests, incidental findings on routine examinations, and widening diagnostic criteria to define a condition requiring an intervention. The blurring boundaries between risk and disease, physicians' fear of missing a diagnosis and patients' need for reassurance are further causes of overdiagnosis. Overdiagnosis often implies procedures to confirm or exclude the presence of the condition and is by definition associated with useless treatments and interventions, generating harm and costs without any benefit. Overdiagnosis also diverts healthcare professionals from caring about other health issues. Preventing overdiagnosis requires increasing awareness of healthcare professionals and patients about its occurrence, the avoidance of unnecessary and untargeted diagnostic tests, and the avoidance of screening without demonstrated benefits. Furthermore, accounting systematically for the harms and benefits of screening and diagnostic tests and determining risk factor thresholds based on the expected absolute risk reduction would also help prevent overdiagnosis.

Emerging Identities in Colonial Tunisia: "Alliancist" and Zionist Representations in Tunis prior to World War I

By 1900 the Jewish community of Tunisia witnessed the emergence of new competing identities: “assimilationist” of the Alliance Israelite Universelle, termed “Alliancist,” and Zionist. Strikingly, two members of the same family in Tunis, Raymond Valensi, President of the AIU Regional Committee, and Alfred Valensi, President of the Zionist Federation, led the struggle for their separate causes. In his discussion of identity in the modern world, Homi Bhabha asks, "How do strategies of representation or empowerment come to be formulated in the competing claims of communities…where, despite shared histories of …discrimination, the exchange of values, meanings and priorities…may be profoundly antagonistic…?" It is in this context that the claims of the Alliance and Zionism will be examined prior to World War I, based on the Archives of the AIU and on such secondary sources as the indispensable work of Paul Sebag. The tensions between the Alliancists and Zionists continued until the outbreak of World War II, as the French-speaking Jews of Tunisia sought to define their individual and collective identities.

The Political Logic of Disintegration: Seven Lessons from the Soviet Collapse. CEPS Essay (No. 1), September 2012

In short, the European Union, as we know it, no longer exists. The very foundations on which it was built are eroding. Shared memories of the Second World War have faded away – half the 15- and 16-year-olds in German high schools do not know that Hitler was a dictator, while a third believe that he protected human rights. The collapse of the Soviet Union has stripped away the geopolitical rationale for European unity. The democratic welfare state that was at the heart of the post-war political consensus is under siege by, among other things, sheer demographics. And the prosperity that bolstered the European project’s political legitimacy is vanishing. More than six out of ten Europeans believe that the lives of today’s children will be more difficult than those of people from their own generation. Against this background, how unthinkable is the EU’s disintegration? Should Europeans make the mistake of taking the Union for granted? Should they assume that the Union would not collapse because it should not collapse? Here, Europe’s capacity to learn from the Soviet precedent could play a crucial part. For the very survival of the EU may depend on its leaders’ ability to manage a similar mix of political, economic and psychological factors that were in play in the process of the Soviet collapse. The game of disintegration is primarily a political one driven much more by the perceptions and misperceptions of the political actors than simply by the constellation of the structural factors – institutional and economic.

The Political Response of Firms to the 1992 Single Market Program: The Case of the German Automobile Industry Pamela Camer. CES Germany & Europe Working Papers, No. 03.1, 1994

This paper examines the political responses of German automobile firms to the 1992 Single Market initiative. I argue that the decision by firms to try to influence EC policies depends on the perceived economic impact of the single market and ,the market alternative open to firms, while the decision on how to lobby depends on the size of the finn and the institutional and strategic environment in which a firm operates. I use this framework to explain why German automobile firms were slow in responding the single market initiative and why, when they did choose to lobby, the firms pursued different political strategies. The research suggests that we should not limit our studies to the political activity of trade associations and sectors, but should also examine the political strategies and activities of individual firms. It also suggests that, as integration efforts in Europe proceed, there is likely to be increased activity by individual firms and national associations because European trade associations may not be able to agree on specific EC policy proposals.

The Dangers and Inanity of (Euro-) Nationalism: From Communitarianism to Cosmopolitanism. Egmont Paper No. 77, April 2015

INTRODUCTION In the current times of multifaceted crisis, nationalism looks, more than ever, like a positive and necessary feeling. It seems both natural and indispensable if we are to have viable political and social institutions that meet the needs and preferences of all citizens. The following paper contests this vision. Its criticism of nationalism is directed not only at its national forms, but also at any defence of collective identity based on the same model, such as the various forms of European nationalism. Furthermore, the same overriding criticism can be made of different kinds of nationalism, regardless of their more or less open and progressive political content. In order to ground our argument theoretically and practically, we will try to show that nationalism is always potentially harmful to individual rights, and unnecessary for the maintenance of a just social and political system. We will thus oppose any acritical defence of the intrinsic value of a specific community and the belief in its artificial homogeneity. The historical construction of a supposedly homogeneous community, and the insistence on its values, which are perceived as superior and binding, facilitate the absorption of the individual into the collective. As we will explain further in more details, this holistic approach is typical of communitarian approaches. In that respect, it does not really matter whether they appeal to passion or to reason, to some irrational binding features of the community or to more rational political aspects of a common identity. The main problem in nationalism is not the emotion it can trigger, it is not even its reliance on particular values. What makes nationalism problematic is, firstly, that it tends to overlook the intrinsically divisive and contradictory nature of individual and collective interests in unjust societies; secondly, that it attributes an intrinsic superiority to a particular community over others; and thirdly, that it sees politics as a means to promote the interests, values or identity of that community. As an alternative, we will very briefly advocate a cosmopolitan approach that grounds political legitimacy in a demanding approach to individual freedom, rather than in a shared collective identity. However, even if only briefly, we will also carefully distinguish our own vision of cosmopolitanism from those commonly put forward. Frequently, cosmopolitan perspectives entangle their identity frameworks with concrete political projects, without clearly explaining how the latter derive from the former. Our approach to cosmopolitanism, on the other hand, is, first and foremost, a critical vision of all communitarian postulates according to which politics should be based on some form of collective identity. Thus, we insist on the conceptual distinction between a general stance on identity issues and the more practical political ideology one stands for. In a subsequent step, we link this cosmopolitan framework with a progressive approach to individual rights. Because of our demanding approach to individual freedom, our cosmopolitanism goes hand in hand with a revival of identity-free sovereignty. It is therefore distinct from the severe condemnation of sovereignty often found in most mainstream cosmopolitan positions. Finally, instead of the frequent confusion found in public discourses and in the literature between ideals and reality, our position acknowledges the deep gulf separating these two dimensions. It therefore sketches out very general strategic principles to bring normative ideals closer to political reality.

Climate change in mountain regions: how local communities adapt to extreme events

This paper examines how local communities adapt to climate change and how governance structures can foster or undermine adaptive capacity. Climate change policies, in general, and disaster risk management in mountain regions, in particular, are characterised by their multi-level and multi-sectoral nature during formulation and implementation. The involvement of numerous state and non-state actors at local to national levels produces a variety of networks of interaction and communication. The paper argues that the structure of these relational patterns is critical for understanding adaptive capacity. It thus proposes an expanded concept of adaptive capacity that incorporates (horizontal and vertical) actor integration and communication flow between these actors. The paper further advocates the use of formal social network analysis to assess these relational patterns. Preliminary results from research on adaptation to climate change in a Swiss mountain region vulnerable to floods and other natural hazards illustrate the conceptual and empirical significance of the main arguments.

Highly accurate RNA and DNA sequencing: Application to longstanding questions in aging and cancer

Thesis (Ph.D.)--University of Washington, 2016-06

Why many eligible individuals choose not to go on welfare

This article investigates why many eligible for welfare do not participate. We show that on-the-job wage-rising potential is the key factor motivating nonparticipation. Although individuals with very low earnings and little wage-rising potential are typically welfare recipients, those with good wage-rising potential may choose to work, participate in old age, or never participate. Nonparticipation remains the best choice for eligible individuals with large wage-rising potential even if universal old-age social security is available. We will also apply this model to a comprehensive welfare system in Hong Kong.