Citotoxicidade e genotoxicidade de nanotubos de carbono

There are many controversies regarding the cyto- and genotoxicity of carbon nanotubes (CNTs). In this work, we discuss that many of the incongruous arguments are probably associated with the poor physical-chemical characterization of the CNT samples used in many publications. This manuscript presents examples of carbon nanostructures observed under high resolution electron microscopy that can be found in typical CNT samples, and shows which roles in cyto- and genotoxicity need to be better investigated. Issues concerning chemical treatment are addressed and examples of misunderstandings that can occur during the studies of cyto- and genotoxicity of CNT samples are given.

Dry spell mitigation to upgrade semi-arid rainfed agriculture : Water harvesting and soil nutrient management for smallholder maize cultivation in Machakos, Kenya

Improvements in on-farm water and soil fertility management through water harvesting may prove key to up-grade smallholder farming systems in dry sub-humid and semi-arid sub-Sahara Africa (SSA). The currently experienced yield levels are usually less than 1 t ha-1, i.e., 3-5 times lower than potential levels obtained by commercial farmers and researchers for similar agro-hydrological conditions. The low yield levels are ascribed to the poor crop water availability due to variable rainfall, losses in on-farm water balance and inherently low soil nutrient levels. To meet an increased food demand with less use of water and land in the region, requires farming systems that provide more yields per water unit and/or land area in the future. This thesis presents the results of a project on water harvesting system aiming to upgrade currently practised water management for maize (Zea mays, L.) in semi-arid SSA. The objectives were to a) quantify dry spell occurrence and potential impact in currently practised small-holder grain production systems, b) test agro-hydrological viability and compare maize yields in an on-farm experiment using combinations supplemental irrigation (SI) and fertilizers for maize, and c) estimate long-term changes in water balance and grain yields of a system with SI compared to farmers currently practised in-situ water harvesting. Water balance changes and crop growth were simulated in a 20-year perspective with models MAIZE1&2. Dry spell analyses showed that potentially yield-limiting dry spells occur at least 75% of seasons for 2 locations in semi-arid East Africa during a 20-year period. Dry spell occurrence was more frequent for crop cultivated on soil with low water-holding capacity than on high water-holding capacity. The analysis indicated large on-farm water losses as deep percolation and run-off during seasons despite seasonal crop water deficits. An on-farm experiment was set up during 1998-2001 in Machakos district, semi-arid Kenya. Surface run-off was collected and stored in a 300m3 earth dam. Gravity-fed supplemental irrigation was carried out to a maize field downstream of the dam. Combinations of no irrigation (NI), SI and 3 levels of N fertilizers (0, 30, 80 kg N ha-1) were applied. Over 5 seasons with rainfall ranging from 200 to 550 mm, the crop with SI and low nitrogen fertilizer gave 40% higher yields (**) than the farmers’ conventional in-situ water harvesting system. Adding only SI or only low nitrogen did not result in significantly different yields. Accounting for actual ability of a storage system and SI to mitigate dry spells, it was estimated that a farmer would make economic returns (after deduction of household consumption) between year 2-7 after investment in dam construction depending on dam sealant and labour cost used. Simulating maize growth and site water balance in a system of maize with SI increased annual grain yield with 35 % as a result of timely applications of SI. Field water balance changes in actual evapotranspiration (ETa) and deep percolation were insignificant with SI, although the absolute amount of ETa increased with 30 mm y-1 for crop with SI compared to NI. The dam water balance showed 30% productive outtake as SI of harvested water. Large losses due to seepage and spill-flow occurred from the dam. Water productivity (WP, of ETa) for maize with SI was on average 1 796 m3 per ton grain, and for maize without SI 2 254 m3 per ton grain, i.e, a decerase of WP with 25%. The water harvesting system for supplemental irrigation of maize was shown to be both biophysically and economically viable. However, adoption by farmers will depend on other factors, including investment capacity, know-how and legislative possibilities. Viability of increased water harvesting implementation in a catchment scale needs to be assessed so that other down-stream uses of water remains uncompromised.

L'a(rche)tipico Brian O'Nolan. Comico e riso dalla tradizione al post-

This study aims at analysing Brian O'Nolans literary production in the light of a reconsideration of the role played by his two most famous pseudonyms ,Flann Brien and Myles na Gopaleen, behind which he was active both as a novelist and as a journalist. We tried to establish a new kind of relationship between them and their empirical author following recent cultural and scientific surveys in the field of Humour Studies, Psychology, and Sociology: taking as a starting point the appreciation of the comic attitude in nature and in cultural history, we progressed through a short history of laughter and derision, followed by an overview on humour theories. After having established such a frame, we considered an integration of scientific studies in the field of laughter and humour as a base for our study scheme, in order to come to a definition of the comic author as a recognised, powerful and authoritative social figure who acts as a critic of conventions. The history of laughter and comic we briefly summarized, based on the one related by the French scholar Georges Minois in his work (Minois 2004), has been taken into account in the view that humorous attitude is one of man’s characteristic traits always present and witnessed throughout the ages, though subject in most cases to repression by cultural and political conservative power. This sort of Super-Ego notwithstanding, or perhaps because of that, comic impulse proved irreducible exactly in its influence on the current cultural debates. Basing mainly on Robert R. Provine’s (Provine 2001), Fabio Ceccarelli’s (Ceccarelli 1988), Arthur Koestler’s (Koestler 1975) and Peter L. Berger’s (Berger 1995) scientific essays on the actual occurrence of laughter and smile in complex social situations, we underlined the many evidences for how the use of comic, humour and wit (in a Freudian sense) could be best comprehended if seen as a common mind process designed for the improvement of knowledge, in which we traced a strict relation with the play-element the Dutch historian Huizinga highlighted in his famous essay, Homo Ludens (Huizinga 1955). We considered comic and humour/wit as different sides of the same coin, and showed how the demonstrations scientists provided on this particular subject are not conclusive, given that the mental processes could not still be irrefutably shown to be separated as regards graduations in comic expression and reception: in fact, different outputs in expressions might lead back to one and the same production process, following the general ‘Economy Rule’ of evolution; man is the only animal who lies, meaning with this that one feeling is not necessarily biuniquely associated with one and the same outward display, so human expressions are not validation proofs for feelings. Considering societies, we found that in nature they are all organized in more or less the same way, that is, in élites who govern over a community who, in turn, recognizes them as legitimate delegates for that task; we inferred from this the epistemological possibility for the existence of an added ruling figure alongside those political and religious: this figure being the comic, who is the person in charge of expressing true feelings towards given subjects of contention. Any community owns one, and his very peculiar status is validated by the fact that his place is within the community, living in it and speaking to it, but at the same time is outside it in the sense that his action focuses mainly on shedding light on ideas and objects placed out-side the boundaries of social convention: taboos, fears, sacred objects and finally culture are the favourite targets of the comic person’s arrow. This is the reason for the word a(rche)typical as applied to the comic figure in society: atypical in a sense, because unconventional and disrespectful of traditions, critical and never at ease with unblinkered respect of canons; archetypical, because the “village fool”, buffoon, jester or anyone in any kind of society who plays such roles, is an archetype in the Jungian sense, i.e. a personification of an irreducible side of human nature that everybody instinctively knows: a beginner of a tradition, the perfect type, what is most conventional of all and therefore the exact opposite of an atypical. There is an intrinsic necessity, we think, of such figures in societies, just like politicians and priests, who should play an elitist role in order to guide and rule not for their own benefit but for the good of the community. We are not naïve and do know that actual owners of power always tend to keep it indefinitely: the ‘social comic’ as a role of power has nonetheless the distinctive feature of being the only job whose tension is not towards stability. It has got in itself the rewarding permission of contradiction, for the very reason we exposed before that the comic must cast an eye both inside and outside society and his vision may be perforce not consistent, then it is satisfactory for the popularity that gives amongst readers and audience. Finally, the difference between governors, priests and comic figures is the seriousness of the first two (fundamentally monologic) and the merry contradiction of the third (essentially dialogic). MPs, mayors, bishops and pastors should always console, comfort and soothe popular mood in respect of the public convention; the comic has the opposite task of provoking, urging and irritating, accomplishing at the same time a sort of control of the soothing powers of society, keepers of the righteousness. In this view, the comic person assumes a paramount importance in the counterbalancing of power administration, whether in form of acting in public places or in written pieces which could circulate for private reading. At this point comes into question our Irish writer Brian O'Nolan(1911-1966), real name that stood behind the more famous masks of Flann O'Brien, novelist, author of At Swim-Two-Birds (1939), The Hard Life (1961), The Dalkey Archive (1964) and, posthumously, The Third Policeman (1967); and of Myles na Gopaleen, journalist, keeper for more than 25 years of the Cruiskeen Lawn column on The Irish Times (1940-1966), and author of the famous book-parody in Irish An Béal Bocht (1941), later translated in English as The Poor Mouth (1973). Brian O'Nolan, professional senior civil servant of the Republic, has never seen recognized his authorship in literary studies, since all of them concentrated on his alter egos Flann, Myles and some others he used for minor contributions. So far as we are concerned, we think this is the first study which places the real name in the title, this way acknowledging him an unity of intents that no-one before did. And this choice in titling is not a mere mark of distinction for the sake of it, but also a wilful sign of how his opus should now be reconsidered. In effect, the aim of this study is exactly that of demonstrating how the empirical author Brian O'Nolan was the real Deus in machina, the master of puppets who skilfully directed all of his identities in planned directions, so as to completely fulfil the role of the comic figure we explained before. Flann O'Brien and Myles na Gopaleen were personae and not persons, but the impression one gets from the critical studies on them is the exact opposite. Literary consideration, that came only after O'Nolans death, began with Anne Clissmann’s work, Flann O'Brien: A Critical Introduction to His Writings (Clissmann 1975), while the most recent book is Keith Donohue’s The Irish Anatomist: A Study of Flann O'Brien (Donohue 2002); passing through M.Keith Booker’s Flann O'Brien, Bakhtin and Menippean Satire (Booker 1995), Keith Hopper’s Flann O'Brien: A Portrait of the Artist as a Young Post-Modernist (Hopper 1995) and Monique Gallagher’s Flann O'Brien, Myles et les autres (Gallagher 1998). There have also been a couple of biographies, which incidentally somehow try to explain critical points his literary production, while many critical studies do the same on the opposite side, trying to found critical points of view on the author’s restless life and habits. At this stage, we attempted to merge into O'Nolan's corpus the journalistic articles he wrote, more than 4,200, for roughly two million words in the 26-year-old running of the column. To justify this, we appealed to several considerations about the figure O'Nolan used as writer: Myles na Gopaleen (later simplified in na Gopaleen), who was the equivalent of the street artist or storyteller, speaking to his imaginary public and trying to involve it in his stories, quarrels and debates of all kinds. First of all, he relied much on language for the reactions he would obtain, playing on, and with, words so as to ironically unmask untrue relationships between words and things. Secondly, he pushed to the limit the convention of addressing to spectators and listeners usually employed in live performing, stretching its role in the written discourse to come to a greater effect of involvement of readers. Lastly, he profited much from what we labelled his “specific weight”, i.e. the potential influence in society given by his recognised authority in determined matters, a position from which he could launch deeper attacks on conventional beliefs, so complying with the duty of a comic we hypothesised before: that of criticising society even in threat of losing the benefits the post guarantees. That seemingly masochistic tendency has its rationale. Every representative has many privileges on the assumption that he, or she, has great responsibilities in administrating. The higher those responsibilities are, the higher is the reward but also the severer is the punishment for the misfits done while in charge. But we all know that not everybody accepts the rules and many try to use their power for their personal benefit and do not want to undergo law’s penalties. The comic, showing in this case more civic sense than others, helped very much in this by the non-accessibility to the use of public force, finds in the role of the scapegoat the right accomplishment of his task, accepting the punishment when his breaking of the conventions is too stark to be forgiven. As Ceccarelli demonstrated, the role of the object of laughter (comic, ridicule) has its very own positive side: there is freedom of expression for the person, and at the same time integration in the society, even though at low levels. Then the banishment of a ‘social’ comic can never get to total extirpation from society, revealing how the scope of the comic lies on an entirely fictional layer, bearing no relation with facts, nor real consequences in terms of physical health. Myles na Gopaleen, mastering these three characteristics we postulated in the highest way, can be considered an author worth noting; and the oeuvre he wrote, the whole collection of Cruiskeen Lawn articles, is rightfully a novel because respects the canons of it especially regarding the authorial figure and his relationship with the readers. In addition, his work can be studied even if we cannot conduct our research on the whole of it, this proceeding being justified exactly because of the resemblances to the real figure of the storyteller: its ‘chapters’ —the daily articles— had a format that even the distracted reader could follow, even one who did not read each and every article before. So we can critically consider also a good part of them, as collected in the seven volumes published so far, with the addition of some others outside the collections, because completeness in this case is not at all a guarantee of a better precision in the assessment; on the contrary: examination of the totality of articles might let us consider him as a person and not a persona. Once cleared these points, we proceeded further in considering tout court the works of Brian O'Nolan as the works of a unique author, rather than complicating the references with many names which are none other than well-wrought sides of the same personality. By putting O'Nolan as the correct object of our research, empirical author of the works of the personae Flann O'Brien and Myles na Gopaleen, there comes out a clearer literary landscape: the comic author Brian O'Nolan, self-conscious of his paramount role in society as both a guide and a scourge, in a word as an a(rche)typical, intentionally chose to differentiate his personalities so as to create different perspectives in different fields of knowledge by using, in addition, different means of communication: novels and journalism. We finally compared the newly assessed author Brian O'Nolan with other great Irish comic writers in English, such as James Joyce (the one everybody named as the master in the field), Samuel Beckett, and Jonathan Swift. This comparison showed once more how O'Nolan is in no way inferior to these authors who, greatly celebrated by critics, have nonetheless failed to achieve that great public recognition O’Nolan received alias Myles, awarded by the daily audience he reached and influenced with his Cruiskeen Lawn column. For this reason, we believe him to be representative of the comic figure’s function as a social regulator and as a builder of solidarity, such as that Raymond Williams spoke of in his work (Williams 1982), with in mind the aim of building a ‘culture in common’. There is no way for a ‘culture in common’ to be acquired if we do not accept the fact that even the most functional society rests on conventions, and in a world more and more ‘connected’ we need someone to help everybody negotiate with different cultures and persons. The comic gives us a worldly perspective which is at the same time comfortable and distressing but in the end not harmful as the one furnished by politicians could be: he lets us peep into parallel worlds without moving too far from our armchair and, as a consequence, is the one who does his best for the improvement of our understanding of things.

What Happened to Participation? Urban Development and Authoritarian Upgrading in Cairo's Informal Neighbourhoods

Participation appeared in development discourses for the first time in the 1970s, as a generic call for the involvement of the poor in development initiatives. Over the last three decades, the initial perspectives on participation intended as a project method for poverty reduction have evolved into a coherent and articulated theoretical elaboration, in which participation figures among the paraphernalia of good governance promotion: participation has acquired the status of “new orthodoxy”. Nevertheless, the experience of the implementation of participatory approaches in development projects seemed to be in the majority of cases rather disappointing, since the transformative potential of ‘participation in development’ depends on a series of factors in which every project can actually differ from others: the ultimate aim of the approach promoted, its forms and contents and, last but not least, the socio-political context in which the participatory initiative is embedded. In Egypt, the signature of a project agreement between the Arab Republic of Egypt and the Federal Republic of Germany, in 1998, inaugurated a Participatory Urban Management Programme (PUMP) to be implemented in Greater Cairo by the German Technical Cooperation (Deutsche Gesellschaft für Technische Zusammenarbeit, GTZ) and the Ministry of Planning (now Ministry of Local Development) and the Governorates of Giza and Cairo as the main counterparts. Now, ten years after the beginning of the PUMP/PDP and close to its end (December 2010), it is possible to draw some conclusions about the scope, the significance and the effects of the participatory approach adopted by GTZ and appropriated by the Egyptian counterparts in dealing with the issue of informal areas and, more generally, of urban development. Our analysis follows three sets of questions: the first set regards the way ‘participation’ has been interpreted and concretised by PUMP and PDP. The second is about the emancipating potential of the ‘participatory approach’ and its ability to ‘empower’ the ‘marginalised’. The third focuses on one hand on the efficacy of GTZ strategy to lead to an improvement of the delivery service in informal areas (especially in terms of planning and policies), and on the other hand on the potential of GTZ development intervention to trigger an incremental process of ‘democratisation’ from below.

Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Entwicklung einer Enzymimmunoelektrode zur Bestimmung von Atrazin in Wasser

ZusammenfassungIn der vorliegenden Arbeit ist eine Enzymimmunoelektrode zur Bestimmung von Atrazin in Wasser entwickelt worden. Die Motivation war, einen Immunoassay zu entwickeln, der ohne die speziellen Geräte, wie einen EIA-Reader, durchgeführt werden konnte. Dafür müssen drei Änderungen vorgenommen werden. Es muß das Detektorsystem EIA-Reader zur Meßwerterfassung ersetzt werden, und damit muß die nur im EIA-Reader verwendbare Mikrotiterplatte ausgetauscht werden. Als drittes muß der Immunoassay dem neuen Detektorsystem angepaßt werden. Eine pH-Elektrode wurde anstelle des EIA-Readers benutzt. Als Enzym, das eine pH-Änderung induziert, wurde Lactamase ausgewählt. Als Festphase wurden anstelle der Mikrotiterplatte Polystyrolmikropartikel (PSMP) verwendet. Die Entwicklung der Enzymimmunoelektrode erfolgte in drei Schritten: Entwicklung des Immunoassays für Atrazin unter Verwendung von Lactamase, Übertragung auf die Festphase PSMP und Einsatz der pH-Elektrode als Detektorsystem. Zuerst wurden Tracer mit dem Enzym Lactamase hergestellt. Als Haptene wurden 2-Chlor-4-(isopropylamino)-6-[(1-carboxypent-5-yl)amino]-s-Triazin (iPr/Cl/C6), Di-Chloratrazin und Di-Chlorsimazin verwendet. Es wurden unterschiedliche Testmittelpunkte im Immunoassay erreicht, (iPr/Cl/C6 I50 = 1.22µg/L; Dichloratrazin I50 = 0.27µg/L; Di-Chlorsimazin I50 = 0.12µg/L). Aufgrund der nur mäßigen Stabilität der Tracer unter Verwendung der Di-Chlorderivate wurde auf deren Verwendung bei der Entwicklung der Immunoelektrode verzichtet.Im zweiten Schritt erfolgte die Übertragung auf PSMP. Die Verwendung der PSMP hatte außer einer Verbesserung des Testmittelpunktes auf 1.00µg/L noch den Vorteil, daß die benötigten Mengen an Antikörper verringert werden konnten.Danach wurde die pH-Elektrode als Signalwandler zur Bestimmung des Atrazins eingesetzt. Unter Verwendung der pH-Elektrode konnte der bisher niedrigste Testmittelpunkt (I50 = 0.005µg/L) zur Bestimmung von Atrazin erreicht werden.

Povertà: sfida educativa e responsabilità sociale. Il ruolo dell'educazione verso una società più giusta, equa e solidale

The thesis focuses on the link between education and poverty. The first part of the work investigates these concepts through a multidisciplinary study (History, Anthropology, Sociology, Psychology, Pedagogy) to show the complexity of the phenomena, and analyzes poverty considering it as an educational challenge. The second part presents the outcomes of a qualitative research about the role and the power that education has in the fight against poverty. The interviews and the focus groups with teachers and educators who work with the poor in several Italian cities and abroad (Denver and Los Angeles, Israel and Palestine), and the observations of the educational work done in some schools and services considered the "best practices" highlight the importance to re-educate our society, that is impoverished by the crisis of welfare state and the weakness of social networks. The final chapter is dedicated to a reflection on social justice, solidarity and sobriety, as pillars for Social Pedagogy in a society that cannot close its eyes to the inequalities that it generates.

Design, Synthese und biologische Evaluierung von Pyrrolcarboxamiden mit Nucleobasen- oder tricyclischer Aromatenfunktion

Ausgehend von den Naturstoffen Netropsin und Distamycin A, antitumoraktiven Pyrrolcarboxamiden, die selektiv an AT-reiche Sequenzen in der kleinen Rinne (Minor-Groove) der DNA binden, sollten neue Nucleobasen- bzw. Interkalator-gekoppelte Derivate (letztere werden als „Combilexine“ bezeichnet) synthetisiert und biologisch evaluiert werden. Unter Zuhilfenahme quantenchemischer AM1-Rechnungen sollten Struktur-Wirkungs-Beziehungen abgeleitet werden. Als Grundgerüst diente die Mono- bzw. Bispyrrolcarboxamid-Einheit mit C-terminaler N,N-Dimethyl-1,3-diaminopropan-Seitenkette, die die ebenfalls basische Amidinstruktur der Leitsubstanzen imitieren sollte. Variationen erfolgten ausschließlich am N-terminalen Ende. Hierbei wurden zunächst Adenin-, Thymin- und Uracil-alkancarbonsäuren mit variabler Kettenlänge synthetisiert und über verschiedene Amidkupplungsverfahren an die Aminofunktion des Pyrrolcarboxamid-Grundgerüstes geknüpft. In Analogie hierzu folgte die Synthese von Combilexinen mit Acridon, (Nitro-)Naphthalimid und Iminostilben als Interkalatorkomponenten. Im 3. synthetischen Teil der Arbeit wurden Carbonsäure- und Sulfonylchloride des Interkalators Acridin und des Interkalators und Photosensibilisators Anthrachinon über die aliphatischen Linker ß-Alanin und -Aminobuttersäure an das Pyrrolcarboxamidgrundgerüst gebunden. Testungen von Verbindungen aller 3 Serien auf Zytotoxizität beim National Cancer Institute, USA, und DNA-Bindestudien und Topoisomerase-Hemmtests im Laboratory of Pharmacology, INSERM in Lille, Frankreich, schlossen sich an. Bei allen Verbindungen mit mindestens 3 Carboxamid-Funktionen zeigte sich gute bis ausgezeichnete DNA-Bindung; einige wiesen Topoisomerase II - Hemmung auf. Beide Parameter korrelierten allerdings nicht mit der Zytotoxizität, was vor allem an der mangelhaften Zellmembranpermeation einiger Verbindungen aufgrund zu geringer Lipophilie liegen dürfte. Quantenchemische Rechnungen ergaben ebenfalls wenige Gesetzmäßigkeiten. Ein elektronenarmer N-terminaler Rest (wie im Falle des hochpotenten Iminostilben-Derivates) scheint aber die Zytotoxizität einer Substanz ebenso wie zunehmende Linkerlänge zu begünstigen. Eine Ausnahme bilden hier die Anthrachinonderivate. Die drei zytotoxisch aktivsten Vertreter dieser Gruppe besitzen als Linker ß-Alanin, was eine aus der sonst bei Minor-Groove-Bindern üblichen Kurvature herausragende Konformation zur Folge hat. Diese ermöglicht vermutlich eine besonders gute Interaktion mit der DNA.

Recombinant expression of molluscan hemocyanin (KLH) substructures in a prokaryotic system: E. coli

The recombinant expression of 19 different substructures of KLH in the prokaryotic sys-tem E. coli has been successfully achieved: each one of the eight single FUs a to h of both isoforms, KLH1 and KLH2, two substructures consisting of two consecutive FUs (KLH1-bc and KLH1-gh) as well as a cDNA encompassing KLH1-abc. All recombinant proteins, fused to an N-terminal 6xHis tag, have successfully been detected by immuno precipitation using monoclonal α-His-antibodies and polyclonal α-KLH1- and α-KLH2-antibodies. One exception remained: SP-KLH2-a, which was not detected by the α-His-antibodies. This allows speculations as to whether the coexpressed signal peptide can lead, at one hand, to the secretion of the recombinant protein, and on the other to the simultaneous cut-off of the leader peptide, which results in the splitting off of even more N-terminal 6xHis tag, leading to failed recognition by the appropriate antibodies. The comparison of native KLH with recombinantly expressed prokaryotic (E. coli) and eukaryotic (Sf9 insect cells) KLH was done using FU-1h. The weak detection by the polyclonal α-KLH1-antibodies of both recombinantly expressed proteins showed that the native protein was the best recognized. For the prokaryotic one, both the denaturation applied for solubilisation of the bacterial inclusion bodies and the inability of bacterial cells to add N-linked glycosylation, are the reason for the poor hybridization. In contrast, KLH1-h expressed in eukaryotic insect cells is likely to be glycosylated. The incubation with the α-KLH1-antibodies resulting in the same weak detection, however, revealed that the linked carbohydrate side chains are not those expected. The establishment of SOE-PCR, together with further improvement, has enabled the generation of a clone encompassing the complete subunit KLH1-abcdefgh. The se-quence analysis compared to the original KLH1 sequence showed, however, that the resulting recombinant protein is defective in two histidines, required for the copper bind-ing sites in FU-1b and FU-1d and in three disulfide bridges (FU-1a, FU-1b and FU 1g). This is due to polymerase-related nucleotide exchanges, resulting in a changed amino acid sequence. Nevertheless, all eight potential N-glycosylation sites are present, leading to the speculation that the recombinant protein can in theory be fully glycosylated, which is the most important aspect for the clinical applicability of recombinant KLH as an im-munotherapeutic agent. The improvement of this method elaborated during the present work indicates bright prospects for the future generation of a correct cDNA sequence encoding for the complete KLH2 subunit.

In vitro generation and characterization of acute myeloid leukemia-reactive CD8 + cytotoxic T-lymphocyte clones from healthy donors

Donor-derived CD8+ cytotoxic T lymphocytes (CTLs) eliminating host leukemic cells mediate curative graft-versus-leukemia (GVL) reactions after allogeneic hematopoietic stem cell transplantation (HSCT). The leukemia-reactive CTLs recognize hematopoiesis-restricted or broadly expressed minor histocompatibility and leukemia-associated peptide antigens that are presented by human leukocyte antigen (HLA) class I molecules on recipient cells. The development of allogeneic CTL therapy in acute myeloid leukemia (AML) is hampered by the poor efficiency of current techniques for generating leukemia-reactive CTLs from unprimed healthy donors in vitro. In this work, a novel allogeneic mini-mixed lymphocyte/leukemia culture (mini-MLLC) approach was established by stimulating CD8+ T cells isolated from peripheral blood of healthy donors at comparably low numbers (i.e. 10e4/well) with HLA class I-matched primary AML blasts in 96-well microtiter plates. Before culture, CD8+ T cells were immunomagnetically separated into CD62L(high)+ and CD62L(low)+/neg subsets enriched for naive/central memory and effector memory cells, respectively. The application of 96-well microtiter plates aimed at creating multiple different responder-stimulator cell compositions in order to provide for the growth of leukemia-reactive CTLs optimized culture conditions by chance. The culture medium was supplemented with interleukin (IL)-7, IL-12, and IL-15. On day 14, IL-12 was replaced by IL-2. In eight different related and unrelated donor/AML pairs with complete HLA class I match, numerous CTL populations were isolated that specifically lysed myeloid leukemias in association with various HLA-A, -B, or -C alleles. These CTLs recognized neither lymphoblastoid B cell lines of donor and patient origin nor primary B cell leukemias expressing the corresponding HLA restriction element. CTLs expressed T cell receptors of single V-beta chain families, indicating their clonality. The vast majority of CTL clones were obtained from mini-MLLCs initiated with CD8+ CD62L(high)+ cells. Using antigen-specific stimulation, multiple CTL populations were amplified to 10e8-10e10 cells within six to eight weeks. The capability of mini-MLLC derived AML-reactive CTL clones to inhibit the engraftment of human primary AML blasts was investigated in the immunodeficient nonobese diabetic/severe combined immune deficient IL-2 receptor common γ-chain deficient (NOD/SCID IL2Rγnull) mouse model. The leukemic engraftment in NOD/SCID IL2Rγnull was specifically prevented if inoculated AML blasts had been pre-incubated in vitro with AML-reactive CTLs, but not with anti-melanoma control CTLs. These results demonstrate that myeloid leukemia-specific CTL clones capable of preventing AML engraftment in mice can be rapidly isolated from CD8+ CD62L(high)+ T cells of healthy donors in vitro. The efficient generation and expansion of these CTLs by the newly established mini-MLLC approach opens the door for several potential applications. First, CTLs can be used within T cell-driven antigen identification strategies to extend the panel of molecularly defined AML antigens that are recognizable by T cells of healthy donors. Second, because these CTLs can be isolated from the stem cell donor by mini-MLLC prior to transplantation, they could be infused into AML patients as a part of the stem cell allograft, or early after transplantation when the leukemia burden is low. The capability of these T cells to expand and function in vivo might require the simultaneous administration of AML-reactive CD4+ T cells generated by a similar in vitro strategy or, less complex, the co-transfer of CD8-depleted donor lymphocytes. To prepare clinical testing, the mini-MLLC approach should now be translated into a protocol that is compatible with good manufacturing practice guidelines.

Numerical simulation of interdigitated back contact hetero-junction solar cells

The goal of this thesis is the application of an opto-electronic numerical simulation to heterojunction silicon solar cells featuring an all back contact architecture (Interdigitated Back Contact Hetero-Junction IBC-HJ). The studied structure exhibits both metal contacts, emitter and base, at the back surface of the cell with the objective to reduce the optical losses due to the shadowing by front contact of conventional photovoltaic devices. Overall, IBC-HJ are promising low-cost alternatives to monocrystalline wafer-based solar cells featuring front and back contact schemes, in fact, for IBC-HJ the high concentration doping diffusions are replaced by low-temperature deposition processes of thin amorphous silicon layers. Furthermore, another advantage of IBC solar cells with reference to conventional architectures is the possibility to enable a low-cost assembling of photovoltaic modules, being all contacts on the same side. A preliminary extensive literature survey has been helpful to highlight the specific critical aspects of IBC-HJ solar cells as well as the state-of-the-art of their modeling, processing and performance of practical devices. In order to perform the analysis of IBC-HJ devices, a two-dimensional (2-D) numerical simulation flow has been set up. A commercial device simulator based on finite-difference method to solve numerically the whole set of equations governing the electrical transport in semiconductor materials (Sentuarus Device by Synopsys) has been adopted. The first activity carried out during this work has been the definition of a 2-D geometry corresponding to the simulation domain and the specification of the electrical and optical properties of materials. In order to calculate the main figures of merit of the investigated solar cells, the spatially resolved photon absorption rate map has been calculated by means of an optical simulator. Optical simulations have been performed by using two different methods depending upon the geometrical features of the front interface of the solar cell: the transfer matrix method (TMM) and the raytracing (RT). The first method allows to model light prop-agation by plane waves within one-dimensional spatial domains under the assumption of devices exhibiting stacks of parallel layers with planar interfaces. In addition, TMM is suitable for the simulation of thin multi-layer anti reflection coating layers for the reduction of the amount of reflected light at the front interface. Raytracing is required for three-dimensional optical simulations of upright pyramidal textured surfaces which are widely adopted to significantly reduce the reflection at the front surface. The optical generation profiles are interpolated onto the electrical grid adopted by the device simulator which solves the carriers transport equations coupled with Poisson and continuity equations in a self-consistent way. The main figures of merit are calculated by means of a postprocessing of the output data from device simulation. After the validation of the simulation methodology by means of comparison of the simulation result with literature data, the ultimate efficiency of the IBC-HJ architecture has been calculated. By accounting for all optical losses, IBC-HJ solar cells result in a theoretical maximum efficiency above 23.5% (without texturing at front interface) higher than that of both standard homojunction crystalline silicon (Homogeneous Emitter HE) and front contact heterojuction (Heterojunction with Intrinsic Thin layer HIT) solar cells. However it is clear that the criticalities of this structure are mainly due to the defects density and to the poor carriers transport mobility in the amorphous silicon layers. Lastly, the influence of the most critical geometrical and physical parameters on the main figures of merit have been investigated by applying the numerical simulation tool set-up during the first part of the present thesis. Simulations have highlighted that carrier mobility and defects level in amorphous silicon may lead to a potentially significant reduction of the conversion efficiency.

Fluorescence close to dielectric interfaces

The aim of the work was to study the correlation between the orientation and excited-state lifetimes of organic dyes close to dielectric interfaces. For this purpose, an experimental setup was designed and built, guiding the light through a prism in total internal reflection geometry. Fluorescence intensities and lifetimes for an ensemble of dye molecules were analyzed as a function of the excitation and detection polarizations. Working close to the total internal reflection angle, the differences between polarization combinations were enhanced. A classical electromagnetic model that assumes a chromophore as a couple of point-like electrical dipoles was developed. A numerical method to calculate the excitation and emission of dye molecules embedded in a multilayer system was implemented, by which full simulation of the time resolved fluorescence experiments was achieved. Free organic dyes and organic dyes covalently bound to polyelectrolyte chains were used. The polymer functionalization process avoided aggregation and provided control over the dyes position, within a few nanometers to the interface. Moreover, by varying the pH, the polymer chains could be deposited on different substrates with different conformations and the resulting fluorescence characteristics analyzed. Initially the fluorescence of organic dyes embedded in a polymer matrix was studied as a function of the distance between the fluorophores and the polymer-air interface. The non-radiative decay rate, vacuum decay rate and the relative angle between the excitation and emission dipoles of the chromophores could be determined. Different free organic dyes were deposited onto different dielectric spacers, as close as possible to the air-dielectric interface. Surprisingly, the fluorescence characteristics of dyes deposited onto polyelectrolyte layer were in good agreement with theoretical predictions of dyes in a polymer matrix, even when the layer was only 2 nm thick. When functionalized chains were deposited at low pH, on top of a polyelectrolyte spacer, the fluorescence had the characteristics of emitters embedded in a polymer matrix as well. Surface deposition at high pH showed an intermediate behaviour between emitters embedded in polymer and on top of the surface, in air. In general, for low pH values, the chains are deposited on a substrate in a train-like conformation. For high pH values, the chains are deposited in a loop-like conformation. As a consequence at low pH the functionalized polymer strongly interdigitates with the polyelectrolyte chains of the spacer, bringing most of the dyes inside the polymer. Thus, the fluorophores may experience the polymer as surrounding environment. On the other hand, for high pH values the dye-loaded chains adsorbed have a conformational arrangement of dense loops that extend away from the surface. Therefore many fluorophores experience the air as surrounding environment. Changing the spacer from polyelectrolyte to negatively charged silane produced contradictory results for lifetimes and intensities. The fluorescence intensities indicated the behaviour of emitters embedded in a polymer matrix, regardless of the pH value. On the other hand, for low pH values, the excited-state lifetimes showed that the emitters behaved as in air. For higher pH values, an intermediate behaviour between fluorophores located within and above of a dielectric film was observed. The poor agreement between theoretical and experimental data may be due to the simplified model utilized, by which the dipoles are assumed either in one side or in the other with respect to a geometrical air-dielectric interface. In the case when the dielectric film is constituted by the functionalized polymer chains themselves, reality is more complex and a different model may apply. Nevertheless, possible applications of the technique arise from a qualitative analysis.

Da Laodice I a Laodice III: l'orizzonte politico delle regine seleucidi

Questo studio indaga la tradizione storiografica sulle regine seleucidi di III secolo a.C. - da Laodice I fino all’ascesa al trono di Laodice III - alla luce delle testimonianze documentarie. L’intento è stabilire l’orizzonte politico dell’azione delle regine nel momento meno conosciuto e più buio della storia del regno di Siria. Il III secolo seleucidico è infatti un periodo caratterizzato da incertezza nella ricostruzione evenemenziale e nell’analisi delle dinamiche sociopolitiche, soprattutto a causa dell’interesse relativo e limitato delle fonti storiografiche antiche a noi giunte relativamente alle vicende della monarchia seleucidica precedenti l’avvento di Antioco III il Grande. In questo panorama rappresenta un’eccezione il caso di Laodice I, che è al centro di un rilevante numero di testimonianze. Tenendo conto della differente natura delle fonti e della complessità del contesto evenemenziale, non sempre ricostruibile, è stata studiata la figura di Laodice I nell’intento di comprendere il paradigma di regalità femminile stabilito dalle azioni della regina, e quindi l’influenza di tale paradigma sulla generazione seleucide successiva. La ricerca si articola in tre sezioni, legate agli eventi principali del III secolo seleucide che vedono coinvolta la regina. La prima parte è dedicata all’attività di Laodice I dopo il secondo matrimonio di Antioco II con la tolemaica Berenice. La seconda parte riguarda il ruolo di Laodice nei complessi eventi che seguirono la morte del marito Antioco II nel 246 e nella Terza Guerra Siriaca, che vide la regina a fianco del figlio Seleuco II contrapporsi a Tolemeo III e Berenice Sira. La terza sezione si occupa dell’azione di Laodice I nella Guerra Fraterna al fianco di Antioco Ierace contro Seleuco II e Laodice II. Nella conclusione si riflette sull’influenza delle azioni politiche di Laodice I sulla scelta di Antioco III di sposare la cugina Laodice III, prima principessa del Ponto entrata nella dinastia regnante.

Kinematic models of interseismic deformation from inversion of GPS and InSAR measurements to estimate fault parameters and coupling degree

We have used kinematic models in two Italian regions to reproduce surface interseismic velocities obtained from InSAR and GPS measurements. We have considered a Block modeling, BM, approach to evaluate which fault system is actively accommodating the occurring deformation in both considered areas. We have performed a study for the Umbria-Marche Apennines, obtaining that the tectonic extension observed by GPS measurements is explained by the active contribution of at least two fault systems, one of which is the Alto Tiberina fault, ATF. We have estimated also the interseismic coupling distribution for the ATF using a 3D surface and the result shows an interesting correlation between the microseismicity and the uncoupled fault portions. The second area analyzed concerns the Gargano promontory for which we have used jointly the available InSAR and GPS velocities. Firstly we have attached the two datasets to the same terrestrial reference frame and then using a simple dislocation approach, we have estimated the best fault parameters reproducing the available data, providing a solution corresponding to the Mattinata fault. Subsequently we have considered within a BM analysis both GPS and InSAR datasets in order to evaluate if the Mattinata fault may accommodate the deformation occurring in the central Adriatic due to the relative motion between the North-Adriatic and South-Adriatic plates. We obtain that the deformation occurring in that region should be accommodated by more that one fault system, that is however difficult to detect since the poor coverage of geodetic measurement offshore of the Gargano promontory. Finally we have performed also the estimate of the interseismic coupling distribution for the Mattinata fault, obtaining a shallow coupling pattern. Both of coupling distributions found using the BM approach have been tested by means of resolution checkerboard tests and they demonstrate that the coupling patterns depend on the geodetic data positions.

Mechanismen der Resistenz und Sensitivierung von menschlichen malignen Melanomzellen gegenüber DNA-alkylierenden Zytostatika

Das metastasierende maligne Melanom ist durch eine geringe p53-Mutations-Rate und eine hohe Resistenz gegenüber Chemotherapie mit alkylierenden Agenzien wie Fotemustin (FM) und Temozolomid (TMZ) gekennzeichnet. In der vorliegenden Arbeit wurde die Rolle von p53 in der Resistenz von malignen Melanomzellen gegenüber FM untersucht und Möglichkeiten zur Sensitivierung von Melanomzellen gegenüber TMZ und FM aufgezeigt.rnAusgangspunkt war die Beobachtung, dass p53 Wildtyp (p53wt) Melanomzellen resistenter gegenüber FM sind als p53 mutierte (p53mt) Zellen. In der vorliegenden Arbeit wurde gezeigt, dass eine FM-Behandlung in p53wt Zellen eine Stabilisierung von p53 und eine Induktion des p53-Zielproteins p21 bewirkte. Mithilfe einer p53wt Zelllinie, welche einen p53 Knockdown trägt, konnte gezeigt werden, dass p53 für die geringe Apoptose-Rate nach FM-Behandlung verantwortlich ist. Eine Untersuchung der Interstrang-Crosslink (ICL)-Reparaturkapazität zeigte, dass p53mt Zellen im Gegensatz zu p53wt Zellen nicht in der Lage sind, FM-induzierte ICL zu reparieren. Dies ging mit einer im Vergleich zu p53wt Zellen starken DNA-Schadensantwort einher. Die Gene für die Proteine DDB2 und XPC wurden als durch FM regulierte DNA-Reparatur-Gene identifiziert, deren Induktion p53-abhängig und lang anhaltend (bis zu 144 h) erfolgt. Da XPC Knockdown-Zellen sensitiver als ihre Kontrollzellen gegenüber FM reagierten, konnte die biologische Relevanz von XPC bei der ICL-Reparatur bestätigt werden. Anhand von Xenograft-Tumoren wurde gezeigt, dass FM auch in situ eine Induktion von DDB2 und XPC auslöst. Die Beobachtung, dass DNA-Reparatur-Gene nach FM-Behandlung hochreguliert werden, liefert eine Erklärung für das schlechte Ansprechen von Melanomen auf eine Therapie mit ICL-induzierenden Chemotherapeutika.rnDes Weiteren befasste sich die vorliegende Arbeit mit Möglichkeiten zur Sensitivierung von Melanomzellen gegenüber den Chemotherapeutika TMZ und FM. In diesem Zusammenhang wurde Valproinsäure (VPA), ein in der Epilepsie-Therapie verwendetes Medikament und Histondesacetylase (HDAC)-Hemmer, bezüglich der chemosensitivierenden Wirkung untersucht. Zunächst konnte der in der Literatur häufig beschriebene stabilisierende Effekt von VPA auf „wildtypisches“ p53-Protein und destabilisierende Effekt auf mutiertes p53-Protein bestätigt werden. Zwei der vier untersuchten Zelllinien konnten mithilfe von VPA gegenüber TMZ sensitiviert werden, während nur eine der vier untersuchten Zelllinien gegenüber FM sensitiviert werden konnte. VPA begünstigt die Induktion von Apoptose, während der Effekt auf die Induktion von Nekrose nur gering ausfiel. Eine Wirkung von VPA auf die Aktivität des Resistenz-vermittelnden Enzyms O6-Methylguanin-DNA-Methyltransferase (MGMT) wurde nicht beobachtet. Zudem wurde ausgeschlossen, dass die Sensitivierung gegenüber TMZ und FM, welche S-Phase abhängige Gentoxine sind, auf einer VPA-induzierten Erhöhung der Proliferation beruht. Mithilfe einer Zelllinie, welche stabil dominant-negatives FADD (Fas-associated death domain) exprimiert, konnten keine Hinweise auf eine Beteiligung des extrinsischen Apoptose-Signalwegs an der VPA-vermittelten Sensitivierung gewonnen werden. Gleichzeitig wurde gezeigt, dass VPA keine Induktion der niedrig exprimierten Procaspase-8 verursachte. Mithilfe eines PCR-Arrays wurden transaktivierende und –reprimierende Effekte von VPA auf die Genexpression gezeigt, wobei das proapoptotische Protein BAX (Breakpoint cluster-2-associated x protein) als ein in der Sensitivierung involviertes Kandidatengen identifiziert wurde. Obwohl eine vollständige Aufklärung der dem Sensitivierungseffekt von VPA zu Grunde liegenden Mechanismen nicht erbracht werden konnte, zeigen die in dieser Arbeit erlangten Beobachtungen einen vielversprechenden Weg zur Überwindung der Resistenz von Melanomzellen gegenüber DNA-alkylierenden Zytostatika auf.rn