Дисертації з теми "Production de ROS"

Organisationsförändringar kan medföra risker som innefattar oro, stress och konflikter inom verksamheten. Det är inte ovanligt att ledningen förväntar sig att verksamheten ska fungera som den alltid gjort samtidigt som genomförandearbetet läggs på medarbetarna. Risken finns att en organisationsförändring kan ge konsekvenser som innebär att folk säger upp sig. Denna kandidatuppsats syftar till att ta reda på hur omorganisationen av polismyndigheten som genomfördes den 1 Januari 2015 upplevs av poliser i den ingripande verksamheten. Vi vill skapa förståelse för hur polisens arbete har påverkats i och med organisationsförändringen. Det som gör vår studie unik och egen är att den på ett nyanserat sätt belyser nya aspekter av omorganisationen ur en sociologisk synvinkel, med fokus på polisernas egna känslor och upplevelser. Teorierna vi valt att utgå ifrån behandlar organisationsförändring ur ett strukturellt perspektiv men även på individnivå. Organisationsmodellerna Lean Production, Den byråkratiska organisationsformen och Organisationsförändringsteorin har vi valt att tillämpa för att skapa förståelse för hur poliserna upplever omorganisationens verksamhetsidé i praktiken. Vi har även använt oss av teorierna Alienation och Erkännande för att få en djupare förståelse för hur poliserna upplever att omorganisationen har påverkat deras arbete och arbetsvillkor. Vår sociologiska analys kommer grunda sig i samtliga teorier, vetenskapliga artiklar och den empiriska sammanställningen genom en utförlig diskussion i förhållande till varandra.  Vi har använt oss av ett kvalitativt tillvägagångssätt där den empiriska datan samlades in genom semistrukturerade intervjuer. Den insamlade empirin tillsammans med med våra teoretiska utgångspunkter lade grunden för vår analys samt avslutande reflektioner och slutsatser. Slutsatsen vi kunde dra utifrån polisernas upplevelser av omorganisationen är att det var brist på kommunikation och information under reformens genomförande, vilket i sin tur skapade motstånd och friktioner i relation till polisernas försämrade arbetsvillkor.
Unsuccessful organizational changes can imply risks that include anxiety, stress and conflicts within the agency. While the management expects the agency to function as it always has, it is not uncommon for the implementation work to be imposed on the employees. Therefore, there is a risk that organizational changes can have consequences that results in people resigning. This bachelor's thesis aims to examine how the reorganization of the Swedish police which implemented on January 1, 2015 is experienced by cops in Intervention activities. Furthermore, we also intended to create an understanding for how police work has been affected by this particular organizational change. What makes our study unique is that it in a nuanced way highlights new aspects of the reorganization from a sociological approach directed on the police officers own emotions and experiences related to the organizational change. The theories we have chosen to start from deal with organizational change from a structural perspective, but also at the individual level. The organizational models Lean Production, The Bureaucratic organizational form and The organizational change theory we have chosen to apply to create an understanding of how the police experience the reorganization's business idea in practice. We have also used the theories Alienation and The struggle to Recognition to gain a deeper understanding of how the police experience that the reorganization has affected their work and working conditions. Our sociological analysis will be based on all theories, scientific articles and the empirical compilation through a detailed discussion in relation to each other. Our study had been based on a qualitative approach through which we collected the empirical data by semi-structured interviews. The collected empirics, associated with our theoretical basis, formed the foundation for our analysis as well as our concluding reflections and conclusions. The most comprehensive conclusion we can compose from the police officers personal experiences of the reorganization, is that there was a deficient communication and information related to the reform that constituted resistance and frictions within the agency.

The free radical theory of ageing proposes ageing is the result of macromolecules, damaged by free radicals, accumulating in cells over time. Mitochondria are critical to this theory as they are the primary source of the free radical superoxide. This thesis aims to understand the effect of age and dietary restriction (DR) with regard to mitochondrial protein abundance and superoxide generation. Superoxide production and protein composition was studied from multiple ages in isolated mitochondria from both ad libitum (AL) and DR mice. Superoxide production was assessed by measuring hydrogen peroxide release from multiple electron transport chain (ETC) sites. Mass spectrometry was used to determine the mitochondrial protein composition from mouse liver tissue. Older mice have increased hydrogen peroxide release from ETC complexes I and III. DR has decreased complex I hydrogen peroxide release in brain, skeletal muscle and liver mitochondria analysed at 15 and 24 months old. DR doesn’t prevent but delays the age associated hydrogen peroxide release. Hydrogen peroxide release at the same survival point is not significantly different between AL and DR mice. The liver mitochondrial proteome is affected by age and DR. Fatty acid metabolism protein abundance increases with age whereas amino acid metabolism protein abundance decreased. Superoxide clearance protein abundance is increased in older and DR liver mitochondria. Catalase had increased abundance in DR mitochondria at 15 and 24 months than at 3 or 36 months old. In conclusion hydrogen peroxide release, superoxide clearance protein abundance and fatty acid metabolism protein abundance are increased with ageing. The age associated increase in hydrogen peroxide release is delayed in DR mitochondria possibly due to increased abundance of catalase.

This study presents the first attempt to combine the fields of ultraviolet (UV) photobiology, plant cell wall biochemistry, aerobic methane production and reactive oxygen species (ROS) mechanisms to investigate the effect of UV radiation on vegetation foliage. Following reports of a 17% increase in decomposition rates in oak (Quercus robur) due to increased UV, which were later ascribed to changes in cell wall carbohydrate extractability, this study investigated the effects of decreased UV levels on ash (Fraxinus excelsior), a fast-growing deciduous tree species. A field experiment was set up in Surrey, UK, with ash seedlings growing under polytunnels made of plastics chosen for the selective transmission of either all UV wavelengths, UV-A only, or no UV. In a subsequent field decomposition experiment on end-of-season leaves, a significant increase of 10% in decomposition rate was found after one year due to removal of UV-B. However, no significant changes in cell wall composition were found, and a sequential extraction of carbohydrate with different extractants suggested no effects of the UV treatments on cell wall structure. Meanwhile, the first observations of aerobic production of methane from vegetation were reported. Pectin, a key cell wall polysaccharide, was identified as a putative source of methane, but no mechanism was suggested for this production. This study therefore tested the effect of UV irradiation on methane emissions from pectin. A linear response of methane emissions against UV irradiation was found. UV-irradiation of de-esterified pectin produced no methane, demonstrating esters (probably methyl esters) to be the source of the observed methane. Addition of ROS-scavengers significantly decreased emissions from pectin, while addition of ROS without UV produced large quantities of methane. Therefore, this study proposes that UV light is generating ROS which are then attacking methyl esters to create methane. The study also demonstrates that this mechanism has the potential to generate several types of methyl halides. These findings may have implications for the global methane budget. In an attempt to demonstrate ROS generation in vivo by UV irradiation, radio-labelling techniques were developed to detect the presence of oxo groups, a product of carbohydrate attack by ROS. Using NaB3H4, the polysaccharides of ash leaflets from the field experiment were radio-labelled, but did not show any significant decrease in oxo groups due to UV treatments. However, UV-irradiation of lettuce leaves showed a significant increase in radio-labelling, suggesting increased UV irradiation caused an increase in the production of ROS. The study shows that the use of this radio-labelling technique has the potential to detect changes in ROS production due to changes in UV levels and could be used to demonstrate a link between ROS levels and methane emissions.

It is clear that the voltage-gated proton channel HVCN1 plays an essential role in a range of cell types, in particular immune cells. Previous published work has confirmed the existence of proton channels in both murine and human macrophages. However, the role of HVCN1 in macrophages has not been investigated. Given that the current literature on voltage-gated proton channels in immune cells has found HVCN1 to be involved in several cellular processes (such as the respiratory burst and signalling events) it is important to establish its functional role in macrophages, which are a crucial constituent of the immune system. The aim of my thesis was to investigate the function of voltage-gated proton channels in macrophages with the use of mice with a disrupting mutation within the Hvcn1 gene, which results in HVCN1 loss. In particular, I wanted to address how Hvcn1-/- macrophages responded to LPS activation. I hypothesised that HVCN1 regulates the respiratory burst of macrophages and that it potentially modulates mitochondrial ROS production, and in doing so, may affect several functional aspects of macrophage biology.

Mestrado em Biologia Molecular e Celular
Microglial cells are the resident immune cells of central nervous system (CNS) and the major players in neuroinflammation. These cells are also responsible for surveilling the neuronal microenvironment, and upon injury to the CNS they change their morphology and molecular profile and become activated. Activated status is associated with microglia proliferation, migration to injury foci, increased phagocytic capacity, production and release of reactive oxygen species (ROS), cytokines (pro- or anti-inflammatory) and reactive nitrogen species. Microglia activation is crucial for tissue repair in the healthy brain. However, their chronic activation or deregulation might contribute for the pathophysiology of neurodegenerative diseases. A better understanding of the mechanisms underlying microglial cell activation is important for defining targets and develop appropriate therapeutic strategies to control the chronic activation of microglia. It has been observed an increase in profilin (Pfn) mRNA in microglial cells in the rat hippocampus after unilateral ablation of its major extrinsic input, the entorhinal cortex. This observation suggested that Pfn might be involved in microglia activation. Pfn1 is an actin binding protein that controls assembly and disassembly of actin filaments and is important for several cellular processes, including, motility, cell proliferation and survival. Here, we studied the role of Pfn1 in microglial cell function. For that, we used primary cortical microglial cell cultures and microglial cell lines in which we knocked down Pfn1 expression and assessed the activation status of microglia, based on classical activation markers, such as: phagocytosis, glutamate release, reactive oxygen species (ROS), pro- and anti-inflammatory cytokines. We demonstrated that Pfn1 (i) is more active in hypoxia-challenged microglia, (ii) modulates microglia pro- and anti-inflammatory signatures and (iii) plays a critical role in ROS generation in microglia. Altogether, we conclude that Pfn1 is a key protein for microglia homeostasis, playing an essential role in their activation, regardless the polarization into a pro or anti-inflammatory signature.
As células da microglia são células imunes residentes no sistema nervoso central (SNC) e desempenham um papel importante em processos neuroinflamatórios. Estas células são responsáveis por monitorizar o parênquima neuronal, sendo capazes de responder rapidamente a danos no SNC. Após ativação, a microglia altera a sua morfologia e o seu perfil de expressão de proteínas. O processo de ativação induz a proliferação, migração para a foco da lesão, aumento da capacidade fagocítica, bem como produção e libertação de espécies reativas de oxigénio (EROs), espécies reativas de azoto e citocinas (pro- e anti-inflamatórias). A ativação da microglia é essencial para a reparação de tecidos e a manutenção da homeostasia do SNC. No entanto, a ativação crónica ou a sua desregulação podem contribuir para a patofisiologia de doenças neurodegenerativas. Assim sendo, o estudo dos mecanismos subjacentes à ativação das células da microglia é importante para ajudar a definir e desenvolver estratégias terapêuticas apropriadas para prevenir a sua ativação crónica. Um estudo anterior reportou o aumento dos níveis de RNAm da profilina (Pfn) em células da microglia no hipocampus de ratos após lesão unilateral no córtex entorrinal, sugerindo que a Pfn poderá estar envolvida no processo de ativação da microglia. A Pfn1 é uma proteína de ligação à actina que regula a polimerização do citoesqueleto de actina, sendo importante em diversos processos celulares, incluindo motilidade, proliferação e sobrevivência. Neste trabalho, nós estudamos o papel da Pfn1 na função da microglia. Para tal, utilizamos linhas celulares e células primárias de microglias corticais de rato nas quais reduzimos a expressão da Pfn1 e avaliamos o seu estado de ativação com base em marcadores clássicos de ativação, tais como: fagocitose, libertação de glutamato, produção e libertação de EROs e citocinas pro- e anti-inflamatórias. Nós demonstramos que a Pfn1 (i) se encontra mais ativa após estímulo da microglia por hipoxia, (ii) modula as assinaturas pro- e anti-inflamatória da microglia e (iii) desempenha um papel importante na produção de EROs pela microglia. Nesse estudo concluímos que a Pfn1 é uma proteína importante para o funcionamento da microglia, desempenhando um papel essencial na ativação da microglia, independentemente da polarização pró ou anti-inflamatória.

Tricomoníase é a doença sexualmente transmissível não-viral mais comum no mundo e pode gerar sérias consequências na saúde reprodutiva, câncer e transmissão e aquisição do HIV. Por esta razão, esta infecção resulta em um pesado fardo para os sistemas de saúde pública. O único tratamento aprovado para esta infecção, que consiste nos 5-nitromidazois metronidazol e tinidazol, apresenta efeitos adversos e há uma subestimada taxa de resistência da infecção, atualmente considerada uma doença negligenciada, a estes fármacos. Portanto, há uma necessidade urgente de novas alternativas terapêuticas para a tricomoníase. Chalconas são uma família de moléculas que apresenta várias aplicações biológicas, como atividade contra diversos patógenos, incluindo protozoários patogênicos. Este trabalho apresenta o potencial anti-Trichomonas vaginalis de derivados de chalcona sintetizados e seus efeitos sobre os trofozoítos. Os valores de IC50 dos compostos mais ativos variaram de 27,5 a 76,4 μM, e as moléculas 4’-hidroxichalcona e 3’-aminochalcona apresentaram os valores mais baixos (27,5 e 28,9 μM). Estes dois compostos foram citotóxicos contra a linhagem de células epiteliais vaginais HMVII, consequentemente apresentaram baixos Índices de Seletividade; contudo, ao se utilizar larvas de Galleria mellonella, como modelo de toxicidade in vivo, não foi observada diminuição da viabilidade após o tratamento. As moléculas também não provocaram hemólise em eritrócitos humanos em 1 e 24 horas. Os compostos não induziram significativa produção de espécies reativas de oxigênio (EROs) nos trofozoítos. Neutrófilos humanos apresentaram aumento na produção de EROs quando coincubados com trofozoítos tratados com os compostos. Os resultados indicam que as chalconas são uma família de moléculas com potencial atividade contra T. vaginalis.
Trichomoniasis is the most common non-viral sexually transmitted disease worldwide and can lead to serious consequences in reproductive health, cancer and HIV acquisition. For this reason, this infection results in a heavy burden for public health systems. Current approved treatment, which consists in 5-nitromidazole drugs, metronidazole and tinidazole, present adverse effects and there is underestimate drug resistance data on this parasitic infection, currently considered a neglected disease. Therefore, there is an urgent need for new alternatives for trichomoniasis treatment. Chalcones are a family of molecules that present various biological applications, such as activity against many pathogenic organisms including protozoan pathogens. This study presents the anti-Trichomonas vaginalis potential of synthetized chalcone derivatives and their effects on the trophozoites. IC50 values of the most active compounds ranged from 27.5 to 76.4 μM, and 4’-hydroxychalcone and 3’- aminochalcone presented the lowest values of IC50 (27.5 and 28.9 μM). These two compounds showed cytotoxicity against HMVII vaginal epithelial cells, thus presenting a low Selectivyty Index; however, when Galleria mellonella larvae were used as model for in vivo toxicity no significant decrease in viability after treatment was observed. The chalcones also did not induce hemolysis in human erythrocytes The compounds did not induce significant reactive oxygen species (ROS) production in the trophozoites. Human neutrophils have increased ROS production when exposed to treated trophozoites. Results indicate that chalcones are a family of molecules with potential activity against T. vaginalis.

Breast cancer affects 1 in 8 women in the UK, and breast cancer patients often report increased levels of psychological stress. Psychological stress results in an increase in the circulating levels of the stress hormones glucocorticoids and catecholamines. Currently, few molecular mechanisms exist linking the actions of stress hormones and breast cancer progression. However, it has recently been suggested that stress hormones can promote DNA damage through the generation of reactive oxygen/nitrogen species (ROS/RNS). This research aims to explore the effect of stress hormone signalling on breast cancer progression and response to treatment. The generation of ROS/RNS and induction of DNA damage was measured in breast cancer cell lines. Pharmacological inhibition of the glucocorticoid receptor (GR) and inducible nitric oxide synthase (iNOS) was used to negate the effects of stress hormone exposure. Psychological stress, using restraint stress, was induced in a syngeneic mouse model of breast cancer, alongside in vivo inhibition of NOS. DNA damage and repair process were examined in response to the glucocorticoid cortisol in an endocrine therapy resistant cell line, and the effect of exposure to the exogenous glucocorticoid dexamethasone on the efficacy of chemotherapy in breast cancer cells was also explored. Stress hormones were shown to induce the generation of ROS/RNS and promote DNA damage. Specifically, exposure to cortisol produced an increase in nitric oxide (NO) through an iNOSmediated pathway. Inhibition of both the GR and iNOS reduced cortisol-induced DNA damage. In a mouse model of breast cancer, inhibition of NOS significantly reduced primary tumour volume, angiogenic signalling in the primary tumour and metastatic spread in stressed mice. Cortisol increased ROS/RNS and DNA damage in endocrine therapy resistant breast cancer cells compared to parental cells, and deregulated DNA repair processes. The cytotoxic effect of chemotherapy agents was reduced in response to co-treatment with the glucocorticoid dexamethasone, through upregulation of the antioxidant response. In conclusion, this research demonstrates that stress hormones impact tumourigenic progression in breast cancer through the induction of DNA damage, mediated by the release of NO. This data also shows that endocrine resistant breast cancer cells are more responsive to the actions of glucocorticoids on DNA damage and repair. Furthermore, exogenous glucocorticoids can impair the efficacy of chemotherapies, through the generation of ROS/RNS. The role of psychological stress should therefore be considered in the treatment of breast cancer patients, and stress hormone receptor signalling could provide potential therapeutic targets for the treatment of breast cancers.

Cerebral stroke has become one of the leading causes of death and disability worldwide. During an ischemic stroke, oxygen and nutrient deprivation occurs, which combined lead to cell starvation, anoxia, and eventually cell death. However, when blood flow is restored, reperfusion damage occurs resulting in increased cell death through several mechanisms. One of the main reasons behind ischemia/reperfusion damage is oxidative stress due to elevated production of reactive oxygen species (ROS) during reperfusion. There are several proteins and processes that are thought to be involved in elevated oxidative stress and the formation of ROS during reperfusion, among which the NADPH oxidase (Nox) family is suggested to be the main contributor of ROS.To examine this hypothesis, in the present work, we inhibited activity of the Nox2 and Nox4 enzymes during ischemia/reperfusion with the Glucox Biotech AB (Sweden) inhibitors M4, M107, and M114 to evaluate whether reducing Nox activity could reduce the ischemia/reperfusion-induced cell death, hence be used as a potential stroke treatment, the cell viability was measured with MTS after ischemia/reperfusion induction and treatment with the Nox substances. We also examined the gene expression levels of the Nox enzymes Nox2 and Nox4 with qPCR after induced ischemia/reperfusion in the neuroblastoma cell line NB69.Our results showed a decrease in Nox4 gene expression after 1h ischemia/8h reperfusion and an increased expression after 1h ischemia/24h reperfusion in NB69 cells. Treatment with M114 resulted in increased cell viability after 2h ischemia/72h reperfusion. However, the toxic effect of ischemia/reperfusion-induced response was found to be inadequate, as indicated by extensive proliferation and lack of cell death. This unfavorable outcome is suggested to be excess of oxygen in medium, metabolization of L-glutamine, and effects of growth factors in the serum used in cell culture medium during the ischemic phase. Therefore, the cell culture protocol was modified to the use of PBS instead of glucose-free medium under serum-free condition during the ischemia. The altered ischemic conditions resulted in continuous reduction in cell viability at increasing ischemic time points with total cell death at 2h ischemia, suggesting applicable conditions for ischemia/reperfusion studies. Even though a conclusion could not be made about the inhibitors M4, M107, and M114 as the cell viability assay was performed under insufficient conditions; the Nox inhibitors shows high potential as future ischemic stroke treatments, which may help save lives and improve life quality for affected patients.

Under oxidative stress condition, telomerase catalytic subunit can shuttle from the nucleus and localises within mitochondria. hTERT can improve mitochondrial functions and contribute to a decreased oxidative stress suggesting an entirely new function of telomerase in protecting mitochondria and cells under stress. However, there are still many questions about the mechanism and what factors influence the protective function of telomerase. In this study we investigated the kinetic exclusion of hTERT, the catalytic subunit of telomerase, in various cell lines under different oxidative stress conditions. We also used organelle specific hTERT localisation vectors to model hTERT localisation and investigated a correlation between hTERT location, nuclear DNA damage and ROS production. We found that cells excluded endogenous hTERT from the nucleus in a heterogeneous fashion independently of the cell types. Importantly, nuclear DNA damage showed a significant correlation with the localisation of hTERT. Cells where hTERT remained in the nucleus displayed high DNA damage while cells which excluded hTERT from the nucleus displayed no or very low DNA damage. Our results from specific hTERT localisation vectors specified that mitochondrial localisation of hTERT protects the nucleus from DNA damage and did not showed any sign of apoptosis induction while nuclear localisation of hTERT correlated with higher amounts of DNA damage and apoptosis. Moreover, mitochondrial localisation of hTERT decreased mitochondrial ROS generation levels directly after both endogenous and exogenous stress which we interpret as the reason for the prevention of nuclear DNA damage. Additionally, we analysed whether p53 status might influence the protective function of telomerase. Our results in an isogenic cell pair of glioblastoma cells showed that p53 status does not prominently influence the protective function of mitochondrial hTERT under low stress condition. However, nuclear hTERT of cells which contained inactive p53 displayed a significantly higher nuclear DNA damage than cells which contained an active p53 and this became more pronounced when stress levels were increased. We hypothesise that telomerase localisation might possibly interact with p53 when a cancer cell is under stress condition. However, the molecular mechanism for that is unknown. Our results demonstrate a novel link between mitochondrial localisation of hTERT, decrease of mitochondrial ROS generation and the protective capacity of hTERT to nuclear DNA from damage after stress treatments.

The aim of the present study was to evaluate the relationship between AOPP (Advanced oxidation protein products) and bovine neutrophils 'in vitro'. For this purpose AOPP were produced "in vitro" by oxidizing bovine serum albumin with HOCl (hypochlorous acid) and bovine neutrophils were isolated from whole blood of dairy cattles. AOPP-BSA were incubated with freshly isolated bovine neutrophils, unstimulated and stimulated with PMA a strong activator of the respiratory burst. Neutrophils ROS production and viability were measured by luminol-amplified chemiluminescence and by MTT and lactate dehydrogenase assays (LDH), respectively. Results obtained have shown that AOPP-BSA are able to reduce significantly ROS production of PMA stimulated neutrophil and their viability measured by MTT assay, no cell lysis was detected by LDH assay. On the basis of these results, our work has studied if AOPP are able to trigger apoptotic events. For this purpose, caspase 8-9-3 and DNA laddering were used as markers in order to discriminate between the 'intrinsic' and the 'extrinsic' pathway of apoptosis. The results obtained showed that un-stimulated bovine neutrophils incubated with AOPP-BSA show a higher but not significant production of active caspase 8 in comparison with the incubation with BSA. Also caspase 3 display an increase, but not significant, in un-stimulated neutrophils after 6 hours of incubation with AOPP-BSA, respects the incubation with BSA. No differences were obtained for caspase 9 and for DNA laddering. Therefore, in these experimental conditions is possible to conclude that the 'intrinsic' pathway of apoptosis was not involved in the reduced functionality of neutrophils or in their reduced viability, but bovine neutrophils incubated with AOPP-BSA seem to be "accompanied" to the early phases of the 'extrinsic' pathways of apoptosis. In addition, the present work wanted to evaluate the capacity of triggered neutrophils to generate AOPP in vitro. BSA was incubated with un-stimulated and PMA-stimulated bovine neutrophils for 1-2-3 hours and the production of specific markers of protein oxidation such as AOPP, dityrosines and carbonyls was assessed. BSA incubated with stimulated neutrophils presents a significant higher level of AOPP and dityrosines respects the incubation with un-stimulated neutrophils. Carbonyls don't seem to be produced in these condition, at least at the beginning of the incubation. In parallel, BSA incubated with the same concentration of HOCl produced by PMA-stimulated neutrophils, for 1-2-3 hours, presents a higher level of AOPP, dityrosines and carbonyls. Therefore, it's possible to conclude that bovine neutrophils are able to oxidize BSA in vitro and generate chemical and structural modification such as AOPP and dityrosines, in the experimental condition used. However, carbonyls seem to be a non-specific indicator of neutrophils-mediated protein oxidation. The direct exposure of BSA to HOCl couldn't fully mimic the complex events leading to BSA oxidation and AOPP production by activated neutrophils.
Lo scopo del presente studio è stato quello di valutare le relazioni tra AOPP (prodotti avanzati di ossidazione proteica) e i neutrofili di bovino "in vitro". A questo scopo le AOPP sono state generate "in vitro", ossidando l'albumina sierica bovina con HOCl (acido ipocloroso) mentre i neutrofili di bovino sono stati isolati da sangue intero di bovine da latte. Le AOPP-BSA sono state incubate con i neutrofili di bovino appena isolati in condizioni di assenza di stimolo o stimolati con PMA un forte attivatore del "burst" respiratorio. La produzione di ROS da parte dei neutrofili e la loro vitalità , sono state misurate rispettivamente mediante chemiluminescenza amplificata dal luminolo e dai saggi MTT e lattato deidrogenasi (LDH). I risultati ottenuti hanno mostrato che le AOPP-BSA sono in grado di ridurre significativamente la produzione di ROS da parte dei neutrofili stimolati con PMA e la loro vitalità , misurata con il saggio MTT mentre non è stata rilevata lisi cellulare mediante saggio LDH. Sulla base di questi risultati il presente lavoro si è proposto di studiare se le AOPP sono in grado di scatenare eventi apopotici. A questo scopo le caspasi 3, 8 , 9 e la frammentazione del DNA sono stati utilizzati come marker con l'obiettivo di discriminare tra la via intrinseca e quella estrinseca di apoptosi. I risultati ottenuti hanno mostrato che i neutrofili di bovino non stimolati e incubati con AOPP-BSA per 1 ora e 6 ore, presentano una maggiore ma non significativa produzione di caspasi 8 attiva, se comparati con l'incubazione con BSA. Anche la caspasi 3 mosta un incremento, non significativo in neutrofili non stimolati incubati con AOPP-BSA per 6 ore, rispetto all'incubazione con BSA. Non è stata ottenuta alcuna differenza per quanto riguarda la caspasi 9 e la frammentazione del DNA. Tuttavia, in queste condizioni sperimentali è possibile concludere che la via intrinseca dell'apoptosi non è coinvolta nella riduszione della funzionalità  dei neutrofili di bovino o nella loro vitalità  ma i neutrofili di bovino incubati con AOPP-BSA sembrano piuttosto essere 'accompagnati' verso le fasi precoci della via estrinseca dell'apoptosi. Inoltre, il seguente studio ha voluto valutare la capacità  dei neutrofili di bovino attivati di generare AOPP 'in vitro'. La BSA è stata incubata con neutrofili di bovino non stimolati e stimolati con PMA per 1-2-3 ore, ed è stata misurata la formazione di specifici marcatori di ossidazione proteica come le AOPP le ditirosine e i carbonili. La BSA incubata con neutrofili stimolati con PMA, presenta un livello significativamente alto di AOPP e ditirosine rispetto all'incubazione con neutrofili non stimolati. I carbonili invece sembrano non essere prodotti in queste condizioni, almeno nelle fasi inziali dell'incubazione. In parallelo, la BSA incubata con la stessa concentrazione di HOCl prodotta dai neutrofili stimolati, per 1-2-3 ore, presenta livelli più elevato di AOPP, ditirosine e carbonili. Tuttavia è possibile concludere che i neutrofili di bovino sono in grado di ossidare la BSA e generare modificazioni chimiche e strutturali come AOPP e ditirosine nelle condizioni sperimentate. I carbonili invece sembrano non essere un marcatore specifico di ossidazione proteica mediata dai neutrofili. In aggiunta la diretta esposizione della BSA all'HOCl non è in grado di mimare completamente la complessità  degli eventi che portano all'ossidazione della BSA e alla produzione di AOPP da parte dei neutrofili attivati.

Glucocorticoids have long been used in the treatment of acute lymphoblastic leukaemia due to their ability to cause cell cycle arrest and apoptosis of lymphoid cells. However, some patients do not respond to glucocorticoid treatment and the majority, who initially respond, may relapse upon prolonged hormone treatment. The inefficiency of the treatment is mainly attributed to the gradual loss of the cellular sensitivity to glucocorticoid-induced apoptosis. Therefore, the need to understand the molecular mechanisms of resistance/ sensitivity of acute lymphoblastic leukaemia cells to glucocorticoid-induced apoptosis is of vital importance, as this will help to develop better prognostic outcomes and improve glucocorticoids therapy. Several mechanisms have been proposed to explain the evasion of glucocorticoid mediated apoptosis in resistant cells. These include post-translational modifications of GR especially phosphorylation which modulates the GR transcriptional activity, and GR mediated signalling thereby affecting gene expression and hence the balance between pro- and anti-apoptotic Bcl-2 family members. In addition the concentration of components of the energy metabolism pathways (i.e. oxidative phosphorylation and glycolysis) and ROS generation are altered in the acute lymphoblastic leukaemia cells. The hypothesis that differentially phosphorylated GR in the resistant versus sensitive ALL cells modulate GR transcriptional activity and target selectively resulting in diverse pro- or anti-apoptotic Bcl-2 family members' gene expression in the two cell lines was tested. Furthermore, in a similar manner, the possibility that differential GR phosphorylation diversely affected gene expression of GR transcriptional target genes that are components of cellular energy production pathways in resistant versus sensitive cells, altering energy and ROS production levels in distinct ways in the two cell lines was explored. GR was found to be predominantly phosphorylated at S211 in the glucocorticoid-sensitive CEM C7-14, and at S226 in the glucocorticoid-resistant CEM C1-15 cells. Differential GR phosphorylation is presumably an indication of dominant p38 MAPK activity in CEM C7-14 and JNK kinase activity in CEM C1-15, which could lead to adverse gene expression of some pro- and anti-apoptotic Bcl-2 family members and particularly Mcl-1, in the two cell lines. Furthermore, differential GR phosphorylation at S211 and S226 in CEM C7-14 and CEM C1-15 affected the gene expression of the Cytochrome C Oxidase assembly factors Surf-1 and SCO2 as well as the nuclear encoded Cytochrome C Oxidase subunit COX-Va and the mitochondrial encoded COX-I, COX-II and COX-III. This effect was more pronounced in the glucocorticoid-sensitive CEM C7-14 cells, probably due to the fact that GR was predominantly phosphorylated at S211 and hence transcriptionally active in these cells. Moreover, in comparison to the resistant CEM C1-15 cells, the CEM C7-14 cells exhibited higher levels of ROS, increased number of active mitochondria and up-regulated glycolysis upon inhibition of oxidative phosphorylation. Glucocorticoids further reduced ROS levels in the CEM C1-15 cells, and increased the NADH/ NAD+ ratio. In conclusion results presented in this thesis provide evidence that differential GR phosphorylation in resistant versus sensitive to glucocorticoid induced apoptosis cells plays essential role in the regulation of programmed cell death and energy metabolism pathways, offering a potential explanation for the molecular events that determine resistance/sensitivity to glucocorticoid-induced apoptosis in ALL cells.

Endothelial cells are a vital region in the pathophysiology of the vasculature because it is the interface between blood flow and the vessel. One way that the structure of the vessels wall can change is by the accumulation of reactive oxygen species (ROS), which has been correlated to aneurysm formation. Four main ROS sources in endothelial cells are: NADPH oxidase, mitochondria electron transport chain, eNOS uncoupling, and xanthine oxidase. Endothelial cells are an essential component of vasculature that has distinct functions and morphology. The aorta and brain arteries are highly populated by endothelial cells but the morphology and cellular signaling has been shown to be different. This study focuses on the difference between brain and aorta ROS production and how flow affects ROS. Joeseph Moran-Guiati and Jason Kushner provided the brain and aortic endothelial cultures for these studies. NADPH oxidase complex is the main contributor in both cell types but more in brain. Surprisingly, both cell types contain approximately the same number of NOX subunits, suggesting that the difference in ROS production is dependent on how activated these subunits are. Mitochondrial ROS was only significantly generated in brain cells and is verified because brain endothelium contains higher numbers of mitochondria. Both uncoupling of eNOS and xanthine oxidase did not contribute to ROS generation in static cultures. ROS production increased even further in both cell types when cells were exposed to flow and even higher in brain, suggesting that flow effects ROS generation. These results provide useful information in the difference between ROS generation and how it can be harmful in possibly causing intracranial aneurysm formation.

Alzheimer’s disease (AD), the leading cause of dementia worldwide, is characterized by the accumulation of the β-amyloid peptide (Aβ) within the brain. Genetic, biochemical, and behavioral research suggest that physiologic generation of the neurotoxic Aβ peptide from sequential amyloid precursor protein (APP) proteolysis is the crucial step in the development of AD. APP is a single pass transmembrane protein expressed at high levels in the brain and metabolized in a rapid and highly complex fashion by a series of sequential proteases. Aβaccumulates in the brains of elderly individuals due to changes in APP metabolism or Aβ elimination. Particularly, AD brain is characterized by plaques containing beta-amyloid protein surrounded by astrocytes and reactive microglia. Activation of microglia by beta-amyloid initiates production of reactive oxygen species (ROS) by the plasmalemmal NADPH oxidase. The resultant oxidative stress contributes to neurodegeneration in AD. In previous study has been reported from our laboratory and from others that betaamyloid upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia. In the present study we show that CLIC1 is responsible of maintaining the resting membrane potential of microglia cells more hyperpolarized during acute beta-amyloid stimulation. This condition is functional to a more efficient ROS production. It is important to highlight that the specific role of microglia cells is to attach and destroy external bran intruders such as bacteria. In this case the action of the immune competent cells is localized in the infection site. Even if all the toxic products released by the activated microglia are armful for neurons as well, the damaged area results insignificant for the central nervous system in its extend. However, in the case of an over production of aberrant proteins like beta-amyloid, microglia cells recognize the aggregation of misfolded protein as a threat and try to destroy them. Unfortunately beta-amyloid oligomers are present in vast areas of the brain and in particular in the hippocampus region and in the cortex. Thus ROS overproduction become armful for a large amount of neurons resulting in cognitive impairment and eventually in massive neuronal death. Since the synergy between CLIC1 and NADPH ROS production, the chloride channel could represent a valid target to counteract the neurodegenerative process. The aim of the present work is to uncover the molecular mechanism that link CLIC1 to ROS production by showing the importance of CLIC1 chloride current to antagonize the loss of negative charges due to the work of membrane NADPH oxidase.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in models of cardiovascular disease. We previously suggested sphingosine-1-phosphate (S1P) activates volume-sensitive Cl- current (ICl,swell) by ROS-dependent signaling. S1P and its analog, FTY720 (fingolimod), primarily act via G-protein coupled receptors (S1PR; S1PR1-3 in heart), but several intracellular S1P ligands are known. We investigated how these agents regulate ICl,swell. ICl,swell was elicited by bath S1P (500 nM), FTY720 (S1PR1,3 agonist; 10 μM), and SEW2871 (S1PR1 agonist; 10 μM) and was fully inhibited by DCPIB, a specific blocker. These data suggested role of S1PR in activation of ICl,swell. Surprisingly, neither CAY10444 (S1PR3 antagonist; 10 μM) nor VPC23019 (S1PR1,3 antagonist; 13 μM) blocked FTY720-induced ICl,swell. Also, gallein a pan Gbeta-gamma inhibitor, failed to block the S1P-induced current. Moreover, 100 nM FTY720 applied via the pipette evoked a larger, faster activating current than 10 μM bath FTY720. Similarly, 500 nM S1P gave larger, faster activating ICl,swell when added to the pipette than when added in the bath. In contrast to FTY720, bath S1P-induced ICl,swell was blocked by CAY10444, but a 3-fold higher concentration failed to eliminate the response to pipette S1P, and VPC23019 failed to suppress bath and pipette S1P-induced currents. Taken together, inconsistencies in the responses to S1PR agents and the greater sensitivity to pipette than bath S1P and FTY720 support the notion that intracellular ligands rather than sarcolemmal S1PR activated ICl,swell. Next we tested if S1P and FTY720, like osmotic swelling, require both NADPH oxidase and mitochondrial ROS production to evoke ICl,swell. S1P- and FTY720-induced ICl,swell were blocked by rotenone but were insensitive to gp91ds-tat, suggesting only mitochondrial ROS production was needed. One possibility is that S1P and FTY720 elicit ICl,swell by binding to mitochondrial prohibitin-2, an S1P ligand whose knockdown augments mitochondrial ROS productions. These data suggest ICl,swell may be activated by S1P accumulation in ischemia-reperfusion and CHF. Understanding S1P-signaling that elicits ICl,swell may provide insight into electrophysiological mechanisms of cardiac pathology and help identify novel targets for therapy.

La endonucleasa G (ENDOG) és una nucleasa mitocondrial específica d’ADN/ARN que s’expressa de manera ubiqua i sembla que està implicada en processos de recombinació i de degradació de l’ADN independent de caspases. Estudis de genòmica funcional han permès establir una associació entre la hipertròfia cardíaca independentment de la pressió arterial i la manca d’ENDOG. Resultats anteriors publicats pel nostre grup demostraven que la deficiència d’ENDOG indueix una reducció de l’activitat de la cadena respiratòria, un augment en la producció de radicals lliures d’oxigen (ROS) i de la mida dels cardiomiòcits in vivo i in vitro. No obstant, encara no està clara la senyalització que comporta aquest creixement anormal dels miòcits en absència d’ENDOG. Degut a que el cor respon a la pèrdua de cardiomiòcits induint hipertròfia en les cèl•lules restants, creiem que l’augment de mida dels cardiomiòcits en els cors Endog-/-podria ser degut a un número de cèl•lules menor. En aquest estudi demostrem que els cors neonatals de ratolins Endog-/-tenen menys cardiomiòcits i són de major mida respecte als cors de tipus salvatge, així com una expressió disminuïda de gens implicats en la replicació de l’ADN i la divisió cel•lular que s’associa a una reduïda capacitat proliferativa dels cardiomiòcits Endog-/- durant el desenvolupament del cor. També demostrem que ENDOG és necessària per la proliferació in vitro de cèl•lules de rosegadors i humanes, incloses línies cel•lulars tumorals, en les quals la manca d’ENDOG restringeix la capacitat per generar tumors in vivo. L’augment en la producció de ROS en cèl•lules deficients en ENDOG suggereix que aquestes podrien estar implicadesen el rol d’ENDOG en el creixement i la proliferació cel•lular, ja que la neutralització de ROS redueix el creixement anormal dels cardiomiòcits neonatals i es recupera parcialment la proliferació en cèl•lules de rosegadors i humanes amb una expressió reduïda d’ENDOG in vitro. Per aquesta raó, ens hem centrat en la identificació dels mediadors i reguladors que influeixen en la producció de ROS en absència d’ENDOG. Les mitocòndries aïllades de cors Endog-/- tenen una capacitat reduïda per sintetitzar ADNmit i els fibroblasts embrionaris Endog-/-, amb un número de còpies d’ADNmit per mitocòndria menor, no poden recuperar els nivells normals d’ADNmit, el que suggereix una disminució de la replicació d’ADNmit degut a la manca d’ENDOG. A més, la falta d’ENDOG no dificulta la proliferació de cèl•lules que no tenen ADNmit (cèl•lules Rho(0)), confirmant la seva funció en la replicació de l’ADNmit per damunt dels efectes observats sobre la proliferació cel•lular. En conjunt, els nostres resultats demostren que ENDOG és necessària per la replicació de l’ADNmit influint en la producció mitocondrial de ROS i la senyalització dependent de ROS per regular la proliferació i el creixement en cardiomiòcits, així com en altres tipus cel•lulars, contribuint al desenvolupament dels teixits i el creixement tumoral.
La endonucleasa G (ENDOG) es una nucleasa mitocondrial específica de ADN/ARN que se expresa de forma ubicua, y que parece estar implicada en procesos de recombinación y de degradación del ADN independiente de caspasas. Estudios de genómica funcional han permitido establecer una asociación entre la hipertrofia cardiaca independiente de la presión arterial y la falta de ENDOG. Resultados anteriores publicados por nuestro grupo demostraban que la deficiencia en ENDOG induce una reducción de la actividad de la cadena respiratoria, un aumento en la producción de radicales libres del oxígeno (ROS) y en el tamaño de los cardiomiocitos in vivo e in vitro. Sin embargo, todavía no se ha esclarecido la señalización que conduce a este crecimiento anormal de los miocitos en ausencia de ENDOG. Debido a que el corazón responde a la pérdida de cardiomiocitos induciendo la hipertrofia en las células restantes, pensamos que el aumento de tamaño de los cardiomiocitos en los corazones Endog -/- podía ser debido a un menor número de células. En este estudio demostramos que los corazones neonatales de ratones Endog -/- tienen un menor número de cardiomiocitos y de mayor tamaño respecto a los corazones de tipo salvaje, así como una expresión disminuida de genes implicados en la replicación del ADN y la división celular que se asocia a una reducida capacidad proliferativa de los cardiomiocitos Endog -/- durante el desarrollo del corazón. También demostramos que ENDOG es necesaria para la proliferación in vitro de células de roedores y humanas, incluidas líneas celulares tumorales, en las que la falta de ENDOG restringe su capacidad para generar tumores in vivo. El aumento en la producción de ROS en células deficientes en ENDOG sugiere que éstas podrían mediar en el rol que ENDOG ejerce sobre el crecimiento y la proliferación celular, puesto que la neutralización de ROS reduce el crecimiento anormal de los cardiomiocitos neonatales y recupera parcialmente la proliferación en células de roedores y humanas con una reducida expresión de ENDOG in vitro. Por esta razón, nos hemos centrado en la identificación de los mediadores y reguladores que influyen en la producción de ROS en ausencia de ENDOG. Las mitocondrias aisladas de corazones Endog -/- tienen una reducida capacidad para sintetizar ADNmit y los fibroblastos embrionarios Endog -/-, con un menor número de copias de ADNmit por mitocondria, no pueden recuperar los niveles normales de ADNmit, lo que sugiere una disminución de la replicación del ADNmit debido a la falta de ENDOG. Además, la falta de ENDOG no dificulta la proliferación de células que carecen de ADNmit (células Rho(0)), confirmando su función en la replicación del ADNmit por encima de los efecto observados sobre la proliferación celular. En conjunto, nuestros resultados demuestran que ENDOG es necesaria para la replicación del ADNmit influyendo en la producción mitocondrial de ROS y la señalización dependiente de ROS para regular la proliferación y el crecimiento en cardiomiocitos, así como en otros tipos celulares, contribuyendo al desarrollo de los tejidos y el crecimiento tumoral.
Endonuclease G (ENDOG) is a mitochondrial DNA/RNA-nuclease ubiquitously expressed, which may be involved in DNA recombination and caspase-independent DNA degradation. Functional genomics established an association between blood pressure-independent heart hypertrophy and the lack of ENDOG. Our previous results showed that ENDOG deficiency induces a reduction of the respiratory chain activity, increases oxygen free radical production (ROS) and cardiomyocyte enlargement in vivo and in vitro. However, the signaling leading to abnormal myocyte growth in the absence of ENDOG was not elucidated. Because the heart responds to the loss of cardiomyocytes by inducing hypertrophy of the remaining cells, we speculated that the increased cardiomyocyte size in Endog-/- hearts could be due to a reduced number of cells. Indeed, we found that the hearts of neonatal ENDOG deficient mice had fewer and bigger cardiomyocytes than wild type hearts with a decreased expression of genes involved in DNA replication and cell division, which means a reduction in cardiomyocyte proliferation during heart development. We also found that ENDOG is required for cell proliferation of other rodent and human cells, including tumor cell lines, in which ENDOG deficiency restrain their capacity to generate tumors in vivo. Increased ROS production in ENDOG deficient cells suggested that ROS could mediate ENDOG roles in cell growth and proliferation. ROS scavenging reduced abnormal growth of neonatal cardiomyocytes and partially recovered the proliferation of rodent and human cells with reduced ENDOG expression in vitro. For this reason, we focused on identifying the mediators and regulators influencing ROS production in the absence of ENDOG. Isolated Endog-/- cardiac mitochondria had reduced capacity to synthesize mtDNA and Endog-/- embryonic fibroblasts with a decreased mtDNA copy number were unable to regain normal mtDNA levels, which suggested us reduced mtDNA replication due to lack of ENDOG. ENDOG deficiency could no further slow division in cells lacking mtDNA (Rho(0) cells) confirming that its function in mtDNA replication is upstream of the effects on cell proliferation. Together, our results show that ENDOG is required for mtDNA replication influencing mitochondrial ROS production and ROS-dependent signaling to control proliferation and growth in cardiomyocytes and other cell types, contributing to tissue development and tumor growth.

Alors que les castes d'insectes sociaux présentent d'énormes différences de longévité - quelques mois pour les ouvrières contre des décennies pour les reines - ma thèse vise à comprendre comment la bioénergétique mitochondriale soutient la longévité remarquable des reines, en particulier chez la fourmi noire des jardins, Lasius niger. Mes résultats montrent qu’il ne s'agit pas seulement de la gestion du stress oxydant (théorie radicalaire), mais plutôt d’un cumul de facteurs. Ces résultats, basées sur plusieurs approches, mettent en lumière la capacité des reines à optimiser la production d'énergie (mesure de l’AEC), leur taux métabolique (calorimétrie indirecte), et l’investissement dans la maintenance mitochondriale (microscopie électronique et protéomique), apportant un éclairage nouveau sur le paradoxe longévité/reproduction des reines. L’exploration de ces mécanismes ouvre la voie à une compréhension plus approfondie des fondements évolutifs de la longévité de ces insectes
While social insect castes exhibit huge differences in longevity, i.e. months for workers vs. decades for queens, my thesis uncovers how mitochondrial bioenergetics underpins the queen's remarkable longevity in black garden ant, Lasius niger. Contrary to conventional theories, my results reveal that it's not merely about oxidative stress management (by measuring oxidative stress markers such as the aconitase/fumarase ratio and antioxidant enzyme activities like catalase and glutathione), but rather a sophisticated interplay of factors. My findings, based on several approaches, highlight the queen's ability to optimize energy production (AEC), metabolic rate (indirect calorimetry), and invest in mitochondrial maintenance (electron microscopy and proteomics), providing crucial insights into the enigmatic paradox of the queen's longevity and reproduction. By exploring these mechanisms, we pave the way for deeper exploration into the evolutionary underpinnings of insect longevity

O status oxidativo do espermatozoide atua sobre ele de diferentes formas, desde a capacitação até a fecundação do oócito. No entanto, as espécies reativas de oxigênio (EROs) podem ser benéficas ou prejudiciais dependendo do contexto celular. Tendo em vista a baixa proteção antioxidante do sêmen criopreservado, associado às sucessivas manipulações que antecedem a fecundação in vitro, entender como esta célula se comporta em um ambiente oxidante e os impactos deste sobre o embrião é de suma importância. Com isto, o objetivo deste trabalho foi propor um modelo dose-dependente para o estudo do estresse oxidativo sobre o espermatozoide e possível impacto no desenvolvimento embrionário. Para isto, no experimento 1, palhetas de sêmen criopreservado de touros (n=5) da raça Nelore foram submetidas à incubação por 1 hora a 38,5 ºC e 5% CO2, com doses crescentes de peróxido de hidrogênio (0; 12,5; 25; e 50 µM). Ao final da incubação, os parâmetros de motilidade foram avaliados pelo sistema Computer Assisted System Analysis (CASA). No experimento 2, foram escolhidas duas doses de peróxido de hidrogênio com base nos resultados do experimento 1: alta (50 µM), baixa (12,5 µM) e também um controle (0 µM). Os espermatozoides foram incubados com as respectivas doses de peróxido de hidrogênio por 1 hora, e ao final da incubação foram utilizados para a fecundação in vitro (D=0). Neste experimento, além das análises do CASA, foram feitas avaliações do status oxidativo (CellROX® green e 2'-7'diacetato de diclorofluoresceína - DCFH), do potencial mitocondrial (JC-1), da cromatina (LA) e da capacitação espermática (clortetraciclina). Os embriões foram avaliados com relação à taxa de clivagem rápida (30 horas pós-inseminação), taxa de clivagem (D=3), taxa de desenvolvimento (D=5) e taxa de blastocisto (D=8). Para análise estatística foi utilizado o modelo de regressão polinomial, considerando p≤0,05. Tanto no experimento 1 quanto no experimento 2 houve detrimento dose-dependente do peróxido de hidrogênio sobre o padrão de movimento e de porcentagem de células móveis. Houve aumento dose-dependente da porcentagem de células positivas para CellROX® células capacitadas e positivas para o LA. Com relação às taxas de clivagem e de blastocisto, houve diminuição da porcentagem de embriões clivados e de blastocistos, conforme a dose de peróxido de hidrogênio foi aumentada. Referente à taxa de desenvolvimento embrionário, houve bloqueio das estruturas em 2-4 células. Nestas condições, o espermatozoide quando exposto a um ambiente oxidante, apresenta alterações no padrão de motilidade, no status oxidativo e na capacitação, sendo que estas interferem de forma negativa no desenvolvimento embrionário, desde o início da clivagem até a formação do blastocisto.
Oxidative status may influence spermatozoa by distinct mechanisms, from capacitation to oocyte fertilization. However, reactive oxygen species (ROS) could be beneficial or harmful depending on cellular context. Due to the low levels of antioxidant enzymes of cryopreserved semen, associated to successive manipulations prior to in vitro fertilization (IVF), it is momentous to understand the mechanism involved on sperm status in such conditions and the further impact on embryo development. The present study aimed to assess the possible impact of a dose-dependent model for sperm oxidative stress on embryo development. In experiment 1, straws from five Nelore bulls were subjected to a 1 hour incubation at 38,5 ºC and 5% CO2, with increase doses of hydrogen peroxide (0; 12,5; 25; e 50 µM). At the end of incubation period, motility parameters were evaluated by Computed Assisted System Analysis (CASA). Based on the results of the experiment 1, experiment 2 was designed to study a high (50 µM) and a low (12,5 µM) dose of hydrogen peroxide and also a control (0 µM). Sperm samples were incubated with each dose for 1 hour and subsequently used for in vitro fertilization (D=0). Samples were analyzed by CASA, oxidative status (CellROX® green and 2'-7' diclorofluorescein diacetate - DCFH), mitochondrial potential (JC-1), chromatin (LA) and sperm capacitation status (chlortetraciclin). Embryos were evaluated based on fast cleavage rate (30 hours pos-insemination), cleavage rate (D=3), development rate (D=5) and blastocyst rate (D=8). Statistical analysis was performed by polynomial regression model, considering significant a p≤0,05. A dose-dependent deleterious effect of hydrogen peroxide was observed on most motility variables evaluated by CASA, including the percentage of motile cells. Similarly, a dose-dependent increase was observed on the percentages of positive cells for CellROX®, capacitated sperm and also for LA. A decrease on the percentage of cleaved embryos and blastocyst was observed as hydrogen peroxide increased. Interestingly, a blockage was detected during the 2-4 cell stage. In these conditions when exposed to oxidative environment, sperm may present disabled motility characteristics, oxidative status and premature capacitation and such abnormalities result on impaired embryo development, from the first cleavage to blastocyst.

Automated guided vehicles (AGVs) are the key equipment of flexible production systems and an important means for realizing a modern logistics system that meets the demands of Industry 4.0. AGVs are used from the mid 50th to delegate monotonous work of delivering products from the human to the automated device. In the long run, the usage of AGVs brings huge benefits to the manufacturing companies. But the purchase and installation of these devices significantly increase operational costs. This fact halts small and medium-sized enterprises from adopting this technology on their shop floors. The idea of this thesis work is to design and create a device that can be retailed at a significantly lower price without compromising flexibility and functional properties, to be used by smaller businesses. For this mater are used more affordable parts that can bring the cost down of a final product. This work describes the process of developing a differential drive mobile platform under the control of the robotic operating system. The process includes the development of a virtual model; selection of required components and investigation of their compatibility; development of chassis, suspension, and gear system; development of a hardware interface to interact with hardware components; configuration of different algorithms of control, cartography, and navigation; evaluation of the device. The research method is used in this work is design and creation due to the necessity of creating a physical prototype. The budget specification for the project was set to 50000 SEK and the desired payload capacity was set to 100kg. The work has resulted in the creation of a prototype of the AGV. The cost of the project is 20595 SEK. The evaluation of a prototype resulted in a maximum towing force of 300N. The load capacity is limited by the mobile base is 400kg. Safety sensors are not used in this project as the device was meant to operate in a controlled environment. The work also gives an evaluation of the Gmapping algorithm in case of using the laser scanner (RPlidar A1) and two algorithms of navigation stack: TrajectoryPlannerROS and DWA planner. The final prototype is evaluated to support an autonomous movement within a controlled environment.

Nanoparticles (NPs) are particulate structures of various shapes and different composition with size ranging 1 and 100 nm. They are divided in NPs of natural origin (produced by combustion as in volcanoes), NPs of anthropogenic origin (produced by diesel engines or industrial incinerators) and artificial NPs (obtained through nanotechnology). These structures possess unique and innovative physical and chemical properties, dependent on their nanoscale dimensions and especially on the high ratio surface area/volume, that give to the NPs a new chemical reactivity and new optical, magnetic, catalytic and electrochemical properties (Sanvincens et al., 2008). In the last decades, these characteristics have made the NPs of considerable interest in technological development and wide used in medicine and diagnostics (Sanvincens et al., 2008), in biotechnology (Abu-Salah et al., 2010; Karn et al., 2009) and in cosmetics, food and materials (Liu et al., 2009). However, the increasing exposure to nanoscale particles requires studies that characterize the properties and potential cytotoxic effects. Although they are applied in a vast number of fields that seem to be destined to increase, their behavior inside the cell remains undetermined. It seems that NPs may lead to in vitro alteration of gene expression and cell death and that they are able to induce DNA damage both directly and indirectly, causing oxidative stress and inflammatory responses (Singh et al., 2009). Although many suppositions have been made about the possible harmful effects of nanoparticles on the body, it is not clear yet what is the exact mechanism by which these nanostructures interact with cells and subcellular structures. My Ph.D. work is part of the project funded by ECSIN (European Centre for the Sustainable Impact of Nanotechnology) that aims to assess the toxicity induced by nanostructures produced at industrial scale. In particular, a research was carried out on in vitro cytotoxicity of commercial Ludox® nanoparticles (a trademark product of W. R. Grace & Co) in human cell systems. These colloidal amorphous silica NPs are widely used in various industrial fields, such as in the production of printer's inks and paints, in textile industry, and in food industry for the fining of drinks. In particular the formulations that have been used are AS30 and SM30, 20 and 7 nm in diameter respectively. First of all, the two aqueous solutions of nanoparticles were characterized in collaboration with the Department of Chemistry, University of Padua, by measuring the ζ,potential, an indicator of the stability of colloidal suspension, by analyzing the form and the size with the transmission electron microscope, and finally with the analysis of the diameter by dynamic light scattering. It was also investigated the interaction of nanoparticles with the components of cell culture medium and serum proteins: studies of spectroscopy and analysis by dynamic light scattering have shown that even at low concentrations (0.01 mg/ml), Ludox® NPs aggregate in presence of even small percentages of serum (3%). The interaction with serum proteins resulting in large aggregates takes place immediately after preparing the solution of NPs in medium with serum and increases with incubation time. This phenomenon does not occur when the NPs are retained in aqueous suspension or in culture medium without serum. Because of the many toxicological studies conducted on cultured lung fibroblasts (Mroz et al., 2007; Foldbjerg et al., 2010) and the high risk of exposure to nanoparticles in the lungs (Gwinn et al., 2006; Nel et al., 2006), I selected a human cell line, CCD-34 Lu, derived from neonatal lung fibroblasts, and two human tumoral cell lines, A549 from a lung cancer and fibrosarcoma's cells HT-1080, were selected for this work. Initially the exposure's effects to various concentrations of Ludox® nanoparticles on cell viability were tested using the MTS colorimetric assay and the clonogenic assay. Cell viability was measured by incubating the cells in culture medium supplemented with 3% of serum for different times (24, 48 and 72 h) and for only 2 h in absence of serum to avoid the aggregation phenomena. The results showed that the two tested silica NPs give a dose and time-dependent toxicity in all the three cell lines. In addition, it was found that NPs with smaller diameter and greater surface air (Ludox SM30®) have generally a higher cytotoxic activity in agreement with literature studies (Lin et al., 2006; Napierska et al., 2009). Probably the smaller NPs can more easily penetrate the membranes and, at the same weight, they are also administered to the cells in a bigger amount than the AS30 NPs. The results of cell viability tests were compared by treating the cells in presence or in absence of serum in culture medium for a short time (2 h): both the clonogenic and the MTS assays showed that cells have a higher viability when the treatment occurs in medium with 3% of serum. I hypothesize that the NPs, forming reversible and unstable aggregates with serum proteins, are less toxic, probably because they are unable to penetrate the cell membrane because of their larger size. Finally, the normal cell line CCD-34 Lu was more sensitive to treatment with NPs than the two tumor cell lines, which show a significant decrease in cell viability only at doses that resulted almost lethal to normal cells (~0.02 mg/ml). Given the many clinical and experimental evidences that nanoparticles can damage cells and cause toxic effects (Nel et al., 2006), the production of reactive oxygen species (ROS) by cells after incubation with Ludox® AS30 and SM30 was analyzed. In agreement with the results of viability tests, it was observed that the normal line CCD-34 Lu produces high levels of ROS at concentrations of NPs at which the tumor cell lines were unaffected (~0.03mg/ml). For all the three cell lines, however, it was found a dose-dependent production of ROS after 2 h of incubation in culture medium without serum. Previous data have shown that the formation and accumulation of reactive oxygen species may cause serious damages to cells and are able to induce double breaks to DNA (Mroz et al., 2007; Mroz et al., 2008). For this reason, the induction of DSBs (double strand breaks) has been analyzed by the presence of foci of the phopshorylated form of the histone H2AX (γH2AX) in the nucleus of treated cells. The phosphorylation of this histone is necessary for the signalling of the damage and the consequent recruitment of proteins of DNA repair in the breaking point. The histone H2AX phosphorylation is not exclusively induced by the presence of DNA double strand breaks, but it is highly correlated to them, as demonstrated by several studies of induction of oxidative stress and exposure to ionizing radiation (Hamer et al., 2003). The results obtained in this work have revealed that only the fibrosarcoma human cell line HT-1080 was positive for the presence of foci after treatment with Ludox® nanoparticles AS30 and SM30, at concentrations of 0.02-0.04-0.06 mg/ml, whereas CCD-34 Lu and A549 cells were negative for all times and doses of treatment analysed. To assess whether the treatment with Ludox® NPs induces cell death by apoptosis, the cells were analysed by means of fluorescence microscopy after staining with the nuclear dye DAPI to detect the nuclear morphology and the presence of apoptotic bodies. An increase of apoptotic index was found following the treatment with nanoparticles, especially those with a smaller diameter (SM30, 0.04 mg/ml), and mainly in tumour cell line HT-1080. CCD-34 Lu cells are proved negative instead, in agreement with data showing that the normal fibroblasts does not meet to apoptosis but present different ways of response to cytotoxic activity of various agents. These data were then confirmed in the two cancer lines through the fluorimetric assay of caspase-3 activation, a cistein-protease involved in the initial stages of apoptosis. Finally to assess the possible genotoxic effects caused by incubation with Ludox® NPs, the gene expression alteration is assessed through Agilent®'s kit "Whole Human Genome Oligo Microarray". With DNA microarray technology it is possible to measure the expression levels of thousands of genes simultaneously, using the basic principles of hybridization of nucleic acids. A typical microarray experiment is divided into four distinct phases: 1) marking of the sample; 2) hybridization on a solid support; 3) image acquisition; 4) extraction of raw data and statistical analysis of measured values. The signal intensity detected in each spot of the array is ultimately an indirect measure of the concentration of that target (in this case mRNA) in the cell. Through the microarray analysis it is possible therefore to understand not only which genes are expressed in the examined conditions, but also if their expression is altered compared to the control sample (Kronick, 2004). The preliminary results achieved in this work relate to the A549 cell line incubated with Ludox® nanoparticles AS30 and SM30 at a concentration of 0.02 mg/ml, with a treatment of 2 h without serum, followed by a recovery in complete medium for 3 h (for SM30 and AS30 NPs) or 22 h (only for AS30 NPs). The number of genes whose expression is significantly altered compared to the control is higher in the sample treated with SM30 NPs (354 genes) compared to the sample treated with AS30 NPs (222 at 3 h after the end of treatment and 118 at 22 h). In both cases a greater number of altered genes are over-expressed in relation to those under-expressed when compared with the control sample. Furthermore, the level of gene expression is more altered when the analysis is conducted after 3 h of incubation in normal medium compared to 22 h. Investigating the expression levels of the main altered genes and the cellular pathways in which they are involved, it was observed that the main altered pathways are cell cycle control, the ways regulated by p53, the signaling pathway of the MAPK and the organization of the cell cytoskeleton. Although the study of gene expression profiles has revealed an alteration of the expression of genes in the cell cycle after treatment with Ludox® NPs, cell cycle analysis by flow cytometry in all the three cell lines examined did not bring out any change due to the NPs in any of the treatment conditions studied. The results obtained during my Ph.D. thesis constitute a preliminary study conducted in vitro on human cells about the cytotoxic effects caused by Ludox® silica nanoparticles, which are a model of commercial nanoparticles. In recent decades the NPs have found an increasingly wide use in various fields, from construction to textiles and food, imposing at the same time the need to provide exhaustive information for an assessment of the impact of nanomaterials on human health and the consequent regulation of their use.
Le nanoparticelle (NP) sono strutture particolate, di varia forma e di diversa composizione, con dimensioni comprese tra 1 e 100 nm. Si distinguono in NP di origine naturale (prodotte da combustioni come nei vulcani), NP di origine antropogenica (prodotte da motori diesel o inceneritori industriali) e NP artificiali (ottenute attraverso le nanotecnologie). Queste strutture possiedono proprietà  fisico-chimiche innovative uniche, dipendenti dalle loro dimensioni nanometriche e soprattutto dall'elevato rapporto area superficiale/volume, che conferiscono una nuova reattività  chimica e nuove proprietà  ottiche, magnetiche, catalitiche ed elettrochimiche (Sanvincens et al., 2008). Queste caratteristiche hanno reso le NP negli ultimi decenni di notevole interesse nello sviluppo tecnologico e di largo impiego in campo medico-diagnostico (Sanvincens et al., 2008), in campo biotecnologico (Abu-Salah et al., 2010; Karn et al., 2009) e nell'industria cosmetica, alimentare e dei materiali (Liu et al., 2009). Tuttavia, la crescente esposizione a particelle nanometriche necessita di studi che ne caratterizzino le proprietà e i potenziali effetti citotossici. Nonostante esse siano applicate in un numero così vasto di campi che sembra essere destinato ad aumentare, il loro comportamento all'interno della cellula resta ancora da accertare; sembra che possano indurre in vitro alterazione dell'espressione genica e morte cellulare e che siano in grado di causare danni al DNA sia in modo diretto che indiretto, inducendo stress ossidativi o risposte infiammatorie (Singh et al., 2009). Sebbene si siano fatte numerose ipotesi sui possibili effetti dannosi delle NP per l'organismo, tuttavia non è ancora chiaro quale sia l'esatto meccanismo con il quale queste nanostrutture interagiscano con le cellule e con le strutture subcellulari. Questo lavoro di Dottorato di Ricerca si colloca all'interno del progetto promosso e finanziato da ECSIN (European Centre for the Sustainable Impact of Nanotechnology) volto a valutare la tossicità  indotta da nanostrutture prodotte a livello industriale. In particolare è stata condotta un'indagine in vitro sulla citotossicità  di nanoparticelle commerciali Ludox® (prodotto a marchio registrato della W. R. Grace & Co) in sistemi cellulari umani. Queste NP colloidali di silice amorfa sono ampiamente utilizzate in vari campi industriali, ad esempio nella produzione di inchiostri per la stampa e vernici, nell'industria tessile e in quella alimentare per la chiarificazione di bevande. In particolare sono state utilizzate le formulazioni AS30 e SM30, rispettivamente di 20 e 7 nm di diametro. Prima di tutto le due soluzioni acquose di nanoparticelle sono state caratterizzate in collaborazione con il Dipartimento di Scienze Chimiche dell'Università  di Padova, attraverso la misura del potenziale ζ,, indicatore della stabilità  della sospensione colloidale, attraverso analisi al microscopio elettronico a trasmissione per visualizzarne forma e dimensione, e infine con la misura del diametro mediante analisi al dynamic light scattering. Inoltre si è indagato sulla possibile interazione delle nanoparticelle con le componenti del terreno di coltura cellulare e con le proteine del siero: studi di spettrofotometria e analisi al dynamic light scattering hanno dimostrato che anche a concentrazioni basse (0,01 mg/ml), le NP Ludox® aggregano in presenza anche di piccole percentuali di siero (3%). L'interazione con le proteine del siero con conseguente formazione di aggregati di dimensioni maggiori avviene subito dopo la preparazione della soluzione di NP in terreno con siero e aumenta con l'aumentare del tempo di incubazione. Questo fenomeno non si verifica quando le NP vengono mantenute in sospensione acquosa o in terreno di coltura privo di siero. Dati i numerosi studi di tossicità  condotti su colture di fibroblasti polmonari (Mroz et al., 2007; Foldbjerg et al., 2010) e l'alto rischio di esposizione alle nanoparticelle a livello polmonare (Gwinn et al., 2006; Nel et al., 2006), per questo lavoro sono state selezionate una linea cellulare umana, CCD-34 Lu, derivata da fibroblasti di polmone neonatali, e due linee umane tumorali: A549, di carcinoma polmonare, e HT-1080, di fibrosarcoma. Gli effetti dell'esposizione a varie concentrazioni di nanoparticelle Ludox® sulla vitalità  cellulare sono state innanzitutto analizzati mediante il saggio colorimetrico MTS e il saggio clonogenico, dopo incubazione a diversi tempi: 24, 48 e 72 h in terreno di coltura addizionato al 3% di siero. La vitalità  cellulare è stata misurata anche incubando le cellule per 2 h in assenza di siero, nella condizione in cui le NP non vanno incontro a fenomeni di aggregazione. I risultati ottenuti hanno evidenziato innanzitutto che le due NP di silice saggiate danno una tossicità  dose e tempo dipendente in tutte e tre le linee cellulari. Inoltre, è stato verificato che le NP di diametro inferiore e aerea superficiale maggiore (Ludox® SM30) possiedono generalmente una maggiore attività  citotossica in accordo con studi di letteratura (Lin et al., 2006; Napierska et al., 2009), probabilmente perché, essendo più piccole, possono penetrare più facilmente nelle membrane e inoltre, a parità  di peso, ne vengono somministrate alle cellule un numero maggiore rispetto alle NP AS30. Sono stati poi confrontati i risultati dei saggi di vitalità  cellulare trattando le cellule in presenza di siero nel terreno di coltura o in assenza di siero per tempi brevi (2 h): sia il saggio clonogenico che il test MTS hanno messo in evidenza che le cellule hanno una vitalità  superiore quando il trattamento con le NP Ludox® avviene in terreno con il 3% di siero. Questo probabilmente è determinato dal fatto che le NP, formando aggregati reversibili ed instabili con le proteine del siero, risultano meno tossiche, probabilmente perché non sono in grado di penetrare nella membrana cellulare date le maggiori dimensioni. Infine, la linea cellulare normale CCD-34 Lu è risultata più sensibile al trattamento con le NP rispetto alle due linee tumorali, che mostrano un calo significativo della vitalità  cellulare solo a dosi di NP che risultano pressoché letali per la linea normale (~0,02 mg/ml). Date le numerose evidenze sperimentali e cliniche che le nanoparticelle possono causare danni a livello cellulare e avere effetti tossici (Nel et al., 2006), in questo lavoro è stata analizzata la produzione di specie reattive dell'ossigeno (ROS) da parte delle cellule, come conseguenza dell'incubazione con Ludox® AS30 e SM30. In accordo con i risultati dei test di vitalità , si è osservato che la linea normale CCD-34 Lu produce alti livelli di ROS a concentrazioni di NP a cui le linee cellulari tumorali risultano insensibili (~0,03 mg/ml). Per tutte e tre le linee cellulari prese in esame si è comunque riscontrata una produzione di ROS dose-dipendente dopo 2 h di incubazione in terreno di coltura in assenza di siero. Dati precedenti hanno dimostrato che la formazione e l'accumulo di specie reattive dell'ossigeno possono causare notevoli danni a livello cellulare e sono in grado di indurre doppie rotture a livello del DNA (Mroz et al., 2007; Mroz et al., 2008). Per questo motivo è stata analizzata tramite immunofluorescenza l'induzione di DSBs (double strand breaks) al DNA per mezzo di un marcatore di tale lesione, ovvero i foci dell'stone H2AX fosforilato. La fosforilazione di questa variante istonica è necessaria per la segnalazione del danno e il conseguente reclutamento di proteine di riparazione del DNA nel sito di rottura. La fosforilazione dell'istone H2AX non è esclusivamente indotta dalla presenza di doppie rotture al DNA, ma è altamente correlabile ad esse, come dimostrato da diversi studi di induzione di stress ossidativo e di esposizione a radiazioni ionizzanti (Hamer et al., 2003). I risultati ottenuti in questo lavoro hanno evidenziato che solo la linea cellulare di fibrosarcoma umano HT-1080 è risultata positiva per la presenza di foci di riparazione dopo trattamento con nanoparticelle Ludox® AS30 e SM30, alle concentrazioni di 0,02-0,04-0,06 mg/ml, mentre le cellule CCD-34 Lu e A549 sono risultate negative per tutti i tempi e le dosi di trattamento analizzate. Per valutare poi se il trattamento con NP Ludox® induce morte cellulare per apoptosi, tutte e tre le linee cellulari prese in esame sono state analizzate al microscopio a fluorescenza dopo fissazione con il colorante nucleare DAPI per evidenziare la morfologia nucleare e l'eventuale presenza di corpi apoptotici. Si è così potuto evidenziare un aumento dell'indice apoptotico in seguito al trattamento con nanoparticelle soprattutto per quelle di diametro inferiore (SM30, 0,04 mg/ml), principalmente nella linea cellulare tumorale HT-1080 e in misura minore anche nella linea cellulare A549. Le cellule CCD-34 Lu sono invece risultate negative a conferma di dati riportati in letteratura che dimostrano che questa linea di fibroblasti polmonari umani normali non va incontro ad apoptosi ma presenta differenti modalità  di risposta all'attività  citotossica di diversi agenti. Questi dati sono stati poi confermati nelle due linee tumorali tramite il saggio fluorimetrico di attivazione della caspasi 3, una cistein-proteasi coinvolta nelle fasi iniziali dell'apoptosi. Per valutare poi gli eventuali effetti genotossici causati dall'incubazione con NP Ludox® si è valutata l'alterazione dell'espressione genica tramite il kit della Agilent® 'Whole Human Genome Oligo Microarray'. Con la tecnologia dei microarray a DNA è possibile misurare il livello di espressione di migliaia di geni contemporaneamente, sfruttando i principi di base dell'ibridazione degli acidi nucleici. Un tipico esperimento di microarray si divide in quattro fasi distinte: 1) marcatura del campione; 2) ibridazione sul supporto solido; 3) acquisizione dell'immagine; 4) estrazione dei dati grezzi ed analisi statistica dei valori misurati. L'intensità del segnale rilevata in ogni spot dell'array è in definitiva una misura indiretta della concentrazione di quel target (in questo caso RNA messaggero) nella cellula. Tramite l'esperimento di microarray è possibile quindi capire non solo quali sono i geni espressi nelle condizioni esaminate, ma anche se la loro espressione è alterata rispetto al campione di controllo (Kronick, 2004). I risultati preliminari raggiunti in questo lavoro riguardano la linea cellulare A549 incubata con nanoparticelle Ludox® AS30 e SM30 alla concentrazione di 0,02 mg/ml, con un trattamento di 2 h senza siero, seguito poi da un ripristino in terreno completo di 3 h (per le NP AS30 e SM30) o di 22 h (solo per le NP AS30). Il numero di geni la cui espressione risulta significativamente alterata rispetto al controllo è più alto nel campione trattato con le NP SM30 (354 geni) rispetto al campione trattato con le NP AS30 (222 dopo 3 h dalla fine del trattamento e 118 dopo 22 h). In entrambi i casi comunque un numero maggiore di geni alterati risulta sovra-espresso rispetto a quelli sotto-espressi, se confrontati con il campione di controllo. Inoltre, il livello di espressione genica risulta maggiormente alterato quando l'analisi viene condotta dopo 3 ore di incubazione in terreno normale rispetto a 22 h. Indagando i livelli di espressione dei principali geni alterati e le vie metaboliche cellulari in cui questi sono coinvolti, si è potuto osservare che i principali pathways alterati sono il controllo del ciclo cellulare, le vie regolate da p53, la via di signalling delle MAPK e la regolazione dell'organizzazione del citoscheletro cellulare. Nonostante lo studio dei profili di espressione genica abbiano messo in evidenza un'alterazione dell'espressione di geni del ciclo cellulare dopo trattamento con NP Ludox®, l'analisi del ciclo cellulare tramite citofluorimetria a flusso in tutte e tre le linee cellulari prese in esame non ha portato in evidenza alcuna alterazione imputabile alle NP in nessuna delle condizioni di trattamento studiate. I risultati di questo lavoro costituiscono uno studio preliminare condotto in vitro degli effetti citotossici provocati in cellule umane dalle nanoparticelle di silice Ludox®, che costituiscono un modello di nanoparticelle commerciali. Le NP negli ultimi decenni hanno trovato un impiego sempre più vasto in svariati campi, dall'edilizia al settore tessile ed alimentare, imponendo allo stesso tempo l'esigenza di fornire informazioni esaurienti per una valutazione dell'impatto dei nanomateriali sulla salute umana e una conseguente regolamentazione del loro utilizzo.

The productivity advances generated from lean manufacturing are self-evident. Plants that adopt lean are more capable of achieving high levels of quality, shorter lead times, and less waste in the system. While it seems logical that higher levels of productivity and quality, as is common in lean companies, should result in positive financial performance, the research community has failed to establish the financial profitability of lean. Those researchers who have studied the financial returns issue report varying results. The goal of this research was to determine if a connection exists between lean and financial success and to discover why so many researchers are finding mixed results. Information Velocity (IV) was theorized to provide the solidifying link between lean and financial performance. Measured by combining the environmental volatility with a company's leanness, IV measures how fast a company can transmit information from the market into a customer-satisfying product in the hands of the consumer. This study analyzed over 530 publicly-traded manufacturing companies to validate the following hypotheses: 1) there is a positive relationship between leanness and financial returns, 2) there is a negative relationship between environmental volatility and financial returns, and 3) there is a positive relationship between IV and financial returns. Regression models were run in various combinations to determine the effect of lean, environmental instability, environmental unpredictability, and IV on financial performance indicators such as return on sales (ROS), return on assets (ROA), and quarter-closing stock price. The outcome of this study showed that financial rewards do result from lean, which positively affected financial performance in almost all scenarios. Environmental instability always negatively correlated with financial returns, and IV mostly shows a positive effect, but with mixed results. Lastly, IV does not explain why researchers find mixed results on the profitability measures of lean. The results of this thesis highlight the significance of implementing lean manufacturing, especially in a dynamic environment. As the instability in the environment increases, profitability decreases. Therefore, an increase in leanness by boosting inventory turns can compensate for the volatility and create enhanced productivity measures and financial results.

Nutrition plays an important role in modulating livestock species immunity. Therefore, this thesis aimed at evaluating the effects of different dietary molecules used in animal nutrition on mammalian and avian immunity. Both, in vitro functional analyses and OMIC technologies (proteomics and miRNAomics) were implemented herein for an integral characterization of the molecules’ impact on the animals’ immune response. Specifically, in this thesis the in vitro impact of the n-6 conjugated linoleic acid (CLA), citrus pectin (CP), and porcine milk exosomes and n-3 polyunsaturated fatty acids (PUFA) on bovine, chicken and porcine mononuclear cells immune response was evaluated, respectively. In the first study, the in vitro activity of CLA on bovine monocytes apoptosis and immune activities, including chemotaxis, phagocytosis, killing capability, and extracellular reactive oxygen species (ROS) production was assessed. Anti-apoptotic effects and an increase in extracellular ROS production during experimental pro-inflammatory conditions were observed, only when using the mixture of the two main isomers of CLA in equal proportions (50:50). The present results demonstrated for the first time that CLA does have immunomodulatory effects on some functions of bovine monocytes in vitro and that the CLA (50:50) mixture is more effective than the CLA isomers individually. The proteomics analysis performed on bovine peripheral blood mononuclear cells (PBMC) revealed that CLA (50:50) mixture does modulate bovine PBMC proteome, supporting the antiapoptotic and immunomodulatory effects observed in the previous in vitro study on bovine monocytes, and propose a potential cytoprotective role of CLA (50:50) mixture against oxidative stress. In the second study, the in vitro activity of CP on chicken monocytes viability, apoptosis, chemotaxis and phagocytosis was assessed. The study demonstrated for the first time that CP inhibits monocytes’ chemotaxis and phagocytosis in vitro, suggesting a potential anti-inflammatory activity. The proteomics analysis carried out on chicken PBMC provided a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro reduction of phagocytosis and chemotaxis is associated with changes in proteins related to the actin cytoskeleton. In the third study, the in vitro activity of porcine milk exosomes on porcine monocytes viability, apoptosis, chemotaxis, phagocytosis, killing capability and extracellular ROS production was assessed. Milk exosomes were successfully purified from sows’ milk and characterized using their size, concentration, morphology, and exosome protein markers. This study reported for the first time that porcine milk exosomes can be internalized by porcine monocytes in vitro and that they can modulate the cell's immune response, by decreasing their chemotaxis and phagocytosis; and increasing their ROS production under resting and pro-inflammatory conditions. The proteomics analysis performed on porcine PBMC demonstrated for the first time that porcine milk exosomes can modulate porcine PBMC proteome in vitro. Moreover, the gene ontology (GO) functional analyses revealed that porcine milk exosomes enrich biological processes related to innate immune-related processes and exosome uptake processes, supporting the immunomodulatory effects and the exosome internalization observed in the previous in vitro study. In the last study, the in vitro activity of the n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on porcine monocytes viability, apoptosis, chemotaxis, phagocytosis, and intracellular, extracellular and total ROS production was assessed. The results of the study showed that DHA and EPA at the highest concentration (200 µM) decreased porcine monocytes' viability. In addition, it was reported for the first time that DHA and EPA can exert differential in vitro immunomodulatory effects in pigs, by dampening monocytes' chemotaxis and potentiating their intracellular oxidative burst, respectively. The proteomics and miRNAomics analyses were not performed for this study. Instead, a first glance on the results from the bioinformatic analyses of the miRNAomics data of all the rest of the studies is presented herein. In conclusion, this thesis provides both, a phenotypical and molecular characterization of the in vitro impact of these dietary molecules on bovine, porcine and chicken immune responses.

This work enables accelerated fluid recovery in oil and gas reservoirs by automatically controlling fluid height and bottomhole pressure in wells. Several literature studies show significant increase in recovered oil by determining a target bottomhole pressure but rarely consider how to control to that value. This work enables those benefits by maintaining bottomhole pressure or fluid height. Moving Horizon Estimation (MHE) determines uncertain well parameters using only common surface measurements. A Model Predictive Controller (MPC) adjusts the stroking speed of a sucker rod pump to maintain fluid height. Pump boundary conditions are simulated with Mathematical Programs with Complementarity Constraints (MPCCs) and a nonlinear programming solver finds a solution in near real-time. A combined rod string, well, and reservoir model simulate dynamic well conditions, and are formulated for simultaneous optimization by large-scale solvers. MPC increases cumulative oil production vs. conventional pump off control by maintaining an optimal fluid level height.

Les polynucléaires neutrophiles représentent 50-70% des leucocytes sanguins et possèdent un rôle majeur dans la défense de l'organisme contre les pathogènes. Le Ca2+ est un second messager qui joue un rôle primordial dans le chimiotactisme, la phagocytose, la dégranulation et la production de formes réactives de l'oxygène (FRO) afin de neutraliser l'agent pathogène. Dans ces cellules, l'influx calcique de type SOCE est essentiel pour l'homéostasie calcique. Il est peu étudié en raison du manque d'outils pharmacologiques spécifiques d'où l'importance dans un premier temps de chercher de nouvelles molécules. Les cellules T Jurkat dont le SOCE est largement caractérisé servent de modèle pour la caractérisation initiale de ces molécules. Le 2-APB est parmi les molécules les plus largement utilisées dans la caractérisation du SOCE en raison de sa double activité sur le SOCE avec une potentialisation à [1-10 μM] et une inhibition à [> 20 μM]. En revanche, ce produit manque de spécificité et agit sur d'autres cibles cellulaires comme les récepteurs à l'inositol (1,4,5)-trisphosphate (InsP3Rs). La 1ère étape est de sélectionner à partir d'analogues commerciaux du 2-APB (Methoxy-APB, Dimethoxy-APB, Cyclic-APB, Benzothienyl-APB, Thienyl-APB et MDEB), des composés plus spécifiques et également plus efficaces que la molécule mère. Deux molécules se sont distinguées : le MDEB comme uniquement potentialisant du SOCE et le Benzothienyl-APB comme un puissant inhibiteur. En revanche, tous les analogues du 2-APB inhibent les InsP3Rs à l'exception du MDEB qui semble plus spécifique du SOCE. L'effet du MDEB sur le courant calcique, ICRAC, a été étudié grâce à la technique du patch-clamp. Il augmente d'environ 4 fois l'amplitude de ICRAC par rapport à celle enregistrée dans les cellules contrôle. Par ailleurs, le MDEB ralentie l'inactivation rapide de ICRAC due au Ca2+. Sur le plan physiologique, le MDEB à des concentrations croissantes inhibe la synthèse de l'IL-2 dans les cellules Jurkat stimulées et ceci malgré son effet potentialisant du SOCE. Cette activité est liée à son effet pro-apoptotique dans les cellules Jurkat stimulées. Le MDEB et le Benzothienyl-APB caractérisés dans la 1ère partie nous ont servi d'outils potentiels afin d'étudier le SOCE des cellules PLB-985 différenciées en cellules proches de neutrophiles. Le SOCE a été induit soit par un traitement des cellules avec la thapsigargine (Tg) soit de manière physiologique avec les peptides fMLF et le WKYMVm deux chimioattractants, ligands des récepteurs aux peptides formylés FPR et FPRL1 respectivement. En plus, le SOCE induit par la Tg est modulable par le 2-APB, potentialisé par le MDEB et inhibé par le Benzothienyl-APB. La phagocytose des levures par les cellules PLB-985 différenciées ainsi que la production de FRO intraphagosomales ont été inhibées par le MDEB et le Benzothienyl-APB. Les FRO extracellulaires ont été également inhibées par Benzothienyl-APB en revanche à cause de la forte interférence du MDEB avec la technique de mesure nous n'avons pas pu étudier ses activités. En conclusion, le MDEB et le Benzothienyl-APB sont de nouveaux outils pharmacologiques potentialisant ou inhibant le SOCE des leucocytes, qui nous permettront dans l'avenir une meilleure compréhension de l'entrée calcique et ses rôles dans ces cellules.

Il lavoro di questa tesi riguarda principalmente la progettazione, simulazione e test di laboratorio di tre versioni successive di schede VME, chiamate Read Out Driver (ROD), che sono state fabbricate per l'upgrade del 2014 dell'esperimento ATLAS Insertable B-Layer (IBL) al CERN. IBL è un nuovo layer che diverrà parte del Pixel Detector di ATLAS. Questa tesi si compone di una panoramica descrittiva dell'esperimento ATLAS in generale per poi concentrarsi sulla descrizione del layer specifico IBL. Inoltre tratta in dettaglio aspetti fisici e tecnici: specifiche di progetto, percorso realizzativo delle schede e test conseguenti. Le schede sono state dapprima prodotte in due prototipi per testare le prestazioni del sistema. Queste sono state fabbricate al fine di valutare le caratteristiche e prestazioni complessive del sistema di readout. Un secondo lotto di produzione, composto di cinque schede, è stato orientato alla correzione fine delle criticità emerse dai test del primo lotto. Un'indagine fine e approfondita del sistema ha messo a punto le schede per la fabbricazione di un terzo lotto di altre cinque schede. Attualmente la produzione è finita e complessivamente sono state realizzate 20 schede definitive che sono in fase di test. La produzione sarà validata prossimamente e le 20 schede verranno consegnate al CERN per essere inserite nel sistema di acquisizione dati del rivelatore. Al momento, il Dipartimento di Fisica ed Astronomia dell'Università di Bologna è coinvolto in un esperimento a pixel solamente attravers IBL descritto in questa tesi. In conclusione, il lavoro di tesi è stato prevalentemente focalizzato sui test delle schede e sul progetto del firmware necessario per la calibrazione e per la presa dati del rivelatore.

Le potentiel de production d'acide a été évalué pour les résidus miniers provenant de deux haldes à stériles de l'ancienne mine Eustis (1865-1939), de la région de Sherbrooke (Québec, Canada). Les mécanismes de contamination par le drainage minier acide ont aussi été étudiés pour ce site. La mine Eustis a généré plus de 74 000 tonnes de stériles acidogènes riches en fer, soufre, cuivre, zinc et plomb. L'exploitation est localisée sur le flanc d'une vallée fluviale drainée par le ruisseau Eustis qui se jette ultimement dans la rivière Massawippi. La superficie et le volume de deux haldes à stériles faiblement minéralisés ont été évalués lors des travaux de terrain. Une vaste campagne d'échantillonnage et d'analyse des eaux de surface et souterraines a été réalisée afin d'identifier les sources de contamination et leur qualité en fonction des conditions météorologiques. Des déversoirs triangulaires ont été installés afin de mesurer les débits les plus importants et de calculer les débits massiques des éléments analysés. Au terme de cette étude, diverses solutions de restauration ont été proposées. Ces solutions combinent le captage et la neutralisation des eaux contaminées, le détournement des eaux propres et le recouvrement imperméable des déblais et des puits de mine."--Résumé abrégé par UMI.

A presente tese analisa o processo de apropriação privada dos rios de São Paulo e sua participação na produção do espaço da cidade, aprofundando aspectos relativos ao desenvolvimento social, político e econômico, desde sua fundação no século XVI até o início do século XX. Partindo das obras de canalização dos rios Tamanduateí, Tietê e Pinheiros, a pesquisa realiza um recuo histórico até o momento em que os rios e córregos de São Paulo se constituíam como um bem comum e sua principal característica era o uso de suas águas e terras. As diversas atividades relacionadas aos rios e córregos, nos primeiros séculos da ocupação, caracterizam-se pela convivência entre o consumo imediato, a utilização de mão de obra cativa e a obtenção de renda através do trabalho livre, em um momento em que a economia de São Paulo era tímida e a poluição dos rios já era percebida. Durante o século XIX, a partir da cultura do café e da imigração, estabeleceu-se uma economia baseada no trabalho livre assalariado e na valorização da propriedade fundiária. Na cidade de São Paulo, o crescimento populacional e a insuficiência da distribuição de água e esgotamento, associados ao significado econômico da propriedade e a disponibilidade de mão de obra, passaram a representar a possibilidade de valorização do capital a partir do estabelecimento de condições gerais de produção. Os rios de São Paulo foram então incorporados ao processo de provisão de infraestruturas e redes de serviços urbanos. Esse processo de acumulação de riqueza, baseado na expropriação da terra e da água, transformou os rios de São Paulo em recursos econômicos e engendrou um espaço que se caracteriza pela sobreposição do domínio particular sobre o domínio comum.
This thesis analyzes the process of private appropriation of the São Paulo and his participation in the production of the city\'s space, Aspects related to social, political and economic development, From its foundation in the 16th century to the beginning of the 20th century. Starting from the Pipelines of the Tamanduateí, Tietê and Pinheiros rivers, the research Historical retreat until such time as the rivers and streams of São Paulo were constituted as a common good and its main characteristic Was the use of its waters and lands. The various activities Rivers and streams, in the first centuries of occupation, are characterized By the coexistence between the immediate consumption, the use of labor And income through free labor, at a In which the economy of São Paulo was timid and the pollution of the rivers was already Perceived. During the nineteenth century, from the culture of coffee and immigration, An economy based on free wage labor was established And in the valuation of land ownership. In the city of São Paulo, the Population growth and the insufficient distribution of water and depletion, Associated with the economic significance of the property and Labor market began to represent the possibility of Capital appreciation based on the establishment of general conditions of production. The rivers of São Paulo were then incorporated into the Provision of urban services infrastructures and networks. This process Of accumulation of wealth, based on the expropriation of land and water, Transformed the São Paulo rivers into economic resources and spawned A space that is characterized by the overlapping of the particular domain On the common domain.

Transforming energy systems to fulfill the needs of a low-carbon economy requires large investments in renewable electricity production (RES-E). Recent literature underlines the need to take a closer look at the composition of the RES-E investor group in order to understand the motives and investment processes of different types of investors. However, existing energy policies generally consider RES-E investments made on a regional or national level, and target investors who evaluate their RES-E investments according to least-cost high-profit criteria. We present empirical evidence to show that RES-E investments are made by a heterogeneous group of investors, that a variety of investors exist and that their formation varies among the different types of renewable sources. This has direct implications for our understanding of the investment process in RES-E and for the study of motives and driving forces of RES-E investors. We introduce a multi-dimensional framework for analyzing differences between categories of investors, which not only considers to the standard economic dimension which is predominant in the contemporary energy literature, but also considers the entrepreneurship, innovation-adoption and institutional dimensions. The framework emphasizes the influence of four main investor-related factors on the investment process which should be studied in future research: motives, background, resources and personal characteristics.

Highlights

► The RES-E investor group is heterogeneous. ► Investors with no traditional background within electricity production make the majority of RES-E investments in Sweden. ► Different types of RES-E investors invest in different renewables. ► A standard economic perspective is not sufficient to understand emerging RES-E investors. ► Motives, background, resources and personal characteristics of RES-E investors matter.

NYEL - Nya investerare i förnybar elproduktion

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Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior
This work aimed to evaluate the effects of inclusion of bakery waste (BW) in sheep diets on intake, apparent digestibility, balance of nitrogen compounds and ruminal parameters, and determine the carbohydrates and nitrogenous fractions of food and diets. Five levels of corn replacement by BW (0, 25, 50, 75 and 100%) were studied, using five male lambs with live weight of 30 kg, in a Latin square 5 X 5 design. The experimental diets were composed of concentrate and hay Tifton 85 (Cynodon spp) in a forage:concentrate ratio of 60:40. To determine the intake and digestibility were collected samples of food, orts and feces. Urine was collected in buckets for a period of 24 hours to determination of nitrogen balance. Data were interpreted with to variance analysis and regression, using the t test at 5% significance. The BW present higher value for the non fiber carbohydrate and protein fraction with fast ruminal degradation (A+B1), which brought a greater proportion of these fractions in diets replacing 100% of corn by the BW and greater timing of energy supply and protein in the rumen. There wasn t effect (P>0.05) levels of substitution of intake, digestibility of nutrients, balance of nitrogen compounds and weight gain. Different levels of substitution didn t affect (P>0.05) pH values and concentrations of volatile fatty acids, but for the concentration of ammonia nitrogen (N-NH3) was found linear reduction (P<0.05) with the level of replacement, in which each 1% BW promoted reduction of 0,11 mg/dL in the concentration of N-NH3, which may be related to increased ruminal availability of energy, which allows greater use of ammonia for microbial growth. The addition of BW caused a reduction of 51,15% in the cost of ration. It was concluded that the BW can replace the corn in concentrate rations for sheep.
Objetivou-se, com este estudo, avaliar os efeitos da inclus?o do res?duo de panifica??o (RP) na dieta de ovinos, sobre o consumo, a digestibilidade aparente, o balan?o de compostos nitrogenados e os par?metros ruminais, e determinar as fra??es de carboidratos e nitrogenadas dos alimentos e das dietas. Foram estudados cinco n?veis de substitui??o do milho pelo RP (0; 25; 50; 75 e 100%), utilizando-se cinco cordeiros machos, com peso m?dio de 30 kg, distribu?dos segundo um delineamento em Quadrado Latino 5 x 5. As dietas experimentais foram compostas de concentrado e feno de capim-Tifton 85 (Cynodon spp), numa rela??o volumoso:concentrado de 60:40. Para a determina??o do consumo e digestibilidade aparente foram coletadas amostras dos alimentos, sobras e das fezes. Foram realizadas coletas de urina por um per?odo de 24 horas para a determina??o do balan?o de compostos nitrogenados. Os resultados foram interpretados de acordo com a an?lise de vari?ncia, e regress?o, utilizando-se o teste t a 5% de signific?ncia. O RP destacou-se pelos elevados valores das fra??es de carboidratos n?o fibrosos e de prote?na de r?pida fermenta??o ruminal (A+B1), o que conferiu maior propor??o dessas fra??es nas dietas com a substitui??o de 100% do milho pelo o RP e maior sincronismo da disponibilidade de energia e prote?na no r?men. N?o houve efeito (P>0,05) dos n?veis de substitui??o sobre o consumo, digestibilidade dos nutrientes, balan?o de compostos nitrogenados e ganho de peso. Os diferentes n?veis de substitui??o n?o afetaram (P>0,05) o pH do l?quido ruminal e as concentra??es dos ?cidos graxos vol?teis, por?m para a concentra??o de nitrog?nio amoniacal (N-NH3) foi observada redu??o linear (P<0,05) em fun??o dos n?veis de substitui??o, em que cada 1% de RP promoveu redu??o de 0,11 mg/dL na concentra??o de N-NH3, o que pode estar relacionada ao aumento na disponibilidade de energia no r?men, que possibilita maior utiliza??o da am?nia para o crescimento microbiano. A adi??o de RP causou redu??o de at? 51,15% no custo da ra??o concentrada. Concluindo-se que o RP pode substituir totalmente o milho nas ra??es concentradas de ovinos.

Development and environment. Bioethics role in a framework of global environmental changesThe article intends to demonstrate how the dominant development model drives the planet to environmental unsustainable limits. Since the 1950s the development model has been led by economic growth without considering other dimensions as social or environmental. Maddison (2005) explains both production and population have been significantly increased in the last century; however, at the same time regional blocs’ gaps have augmented, persisting poverty levels and malnutrition in some countries.However, production and consumption levels have risen triggering an energetic and environmental crisis. These levels have become unsustainable and are driven the world to catastrophic scenarios. In this context, bioethics rises to give people’s guidance concerning personal, social and naturebehavior. Anyway, behavior changes should happen in the shortest term since this will shape the future of the planet and humanity
El artículo tiene por objetivo mostrar cómo el modelo de desarrollo dominante ha llevado al planeta a límites insostenibles del medio ambiente. El modelo de desarrollo desde la década de los cincuenta ha estado conducido por el crecimiento económico, sin considerar otras dimensiones como las ambientales y/o sociales. Maddison muestra que tanto la producción, como la población han aumentado considerablemente en el último siglo, pero, a la vez, las brechas han aumentado cada vez más en los bloques regionales, persistiendo los niveles de pobreza y malnutrición en algunos países. Sin embargo, los niveles de producción y consumo han aumentado y ocasionado la crisis energética y ambiental. Estos niveles se han vuelto insostenibles y están llevando al mundo a escenarios catastróficos. Es en este contexto donde la bioética aparece para dar algunas orientaciones sobre cómo deben conducirse los individuos con relación a la vida personal, social y a la naturaleza. Pero estos cambios en la conducta deben ocurrir en el plazo más inmediato ya que ello determinará el futuro del planeta y de la humanidad

To compete, manufacturing companies need production systems that quickly can respond to changes. To handle change drivers such as volume variations or new product variants, reconfigurability is advocated as a competitive means. This implies an ability to add, remove, and/or rearrange the structure of the production system to be ready for future changes. Still, it is not clear how the production system design process can capture and support the de-sign of reconfigurable production systems. Therefore, the objective of this thesis is to increase the knowledge of how to support the design of reconfig-urable production systems. Reconfigurability could be defined by a number of reconfigurability char-acteristics including convertibility, scalability, automatibility, mobility, modularity, integrability, and diagnosability. In eight case studies, reconfigu-rability characteristics in production system design were studied in order to investigate reconfigurability needs, knowledge, and practice in manufactur-ing companies. In three of the case studies reconfigurable production sys-tems were studied to identify the links between change drivers and reconfig-urability characteristics. In the remaining five case studies, reconfigurability in the production system design processes was addressed in terms of needs, prerequisites, and consideration. Based on the literature review and the case studies, support for reconfigu-rable production system design is suggested including two parts. The first part comprises support for analyzing the need for reconfigurability. Based on relevant change drivers the need for reconfigurability must be identified to enable selection of right type and degree of reconfigurability for each specif-ic case of application. A comprehensive view of the reconfigurability charac-teristics is presented and links between change drivers and reconfigurability characteristics are described. The characteristics are divided into critical characteristics, that lead to a capacity or functionality change of the produc-tion system, and supporting characteristics, that reduce system reconfigura-tion time but do not necessarily lead to a modification of functionality or capacity of the production system. The second part provides support in how to consider reconfigurability in the production system design process. A holistic perspective is crucial to design reconfigurable production systems and therefore constituent parts of a production system are described. Accord-ing to their character physical, logical, and human reconfiguration must be considered through the whole production system design process.

The genomes of diverse organisms are predicted to contain 20 – 30% membrane protein encoding genes and more than half of all therapeutics target membrane proteins. However, only 2% of crystal structures deposited in the protein data bank represent integral membrane proteins. This reflects the difficulties in studying them using standard biochemical and crystallographic methods. The first problem frequently encountered when investigating membrane proteins is their low natural abundance, which is insufficient for biochemical and structural studies. The aim of my thesis was to provide a simple method to improve the production of recombinant proteins. One of the most commonly used methods to increase protein yields is codon optimization of the entire coding sequence. However, our data show that subtle synonymous codon substitutions in the 5’ region can be more efficient. This is consistent with the view that protein yields under normal conditions are more dependent on translation initiation than elongation. mRNA secondary structures around the 5’ region are in large part responsible for this effect although rare codons, as well as other factors, also contribute. We developed a PCR based method to optimize the 5’ region for increased protein production in Escherichia coli. For those proteins produced in sufficient quantities several additional hurdles remain before high quality crystals can be obtained. A second aim of my thesis work was to provide a simple method for topology mapping membrane proteins. A topology map provides information about the orientation of transmembrane regions and the location of protein domains in relation to the membrane, which can give information on structure-function relationships. To this end we explored the split-GFP system in which GFP is split between the 10th and 11th β-strands. This results in one large and one small fragment, both of which are non-fluorescent but can re-anneal and regain fluorescence if localized to the same cellular compartment. Fusing the 11th β-strand to the termini of a protein of interest and expressing it, followed by expression of the detector fragment in the cytosol, allows determination of the topology of inner membrane proteins. Using this strategy the topology of three model proteins was correctly determined. We believe that this system could be used to predict the topology of a large number of additional proteins, especially single-spanning inner membrane proteins in E. coli. The methods for efficient protein production and topology mapping engineered during my thesis work are simple and cost-efficient and may be very valuable in future studies of membrane proteins.

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Tesis para optar al grado de Ingeniero Comercial, Mención Economía
De acuerdo a la teoría del crecimiento económico, el cambio tecnológico es de una relevancia primordial para el desarrollo económico de los países. Es así como se aplican muchas políticas para fomentar la innovación con el objetivo de contribuir al crecimiento. Una de ellas son las leyes de propiedad intelectual, las que permiten a los innovadores internacionalizar los beneficios de sus invenciones; de esta forma incentivan la generación de contenido que de otra manera se perder a dada su naturaleza de bien público. Sin embargo, a su vez, la información es un elemento fundamental en el proceso de innovación; actúa como elemento detonante y facilitador del proceso de creación intelectual, por lo que la restricción del acceso a la información ocasionada por las leyes de propiedad intelectual es contraproducente para el desarrollo de nuevas ideas productivas. En este trabajo desmenuzamos el proceso de innovación y revisamos distintas formas que existen para conservar los incentivos sin la necesidad de restringir el acceso a la informaci ón pública como lo hacen las leyes de propiedad intelectual. Indicamos que un país como Chile puede beneficiarse de leyes de propiedad mas laxas que fomenten el uso de la información en los esfuerzos de innovación. Ejemplificamos con el fenómeno del Software Open Source, que instituciones mas permisivas junto con un uso fructífero de las nuevas tecnologías de comunicación de redes pueden resultar en esfuerzos colaborativos de innovación que disminuyan los costos tanto en términos monetarios como en términos de incertidumbre.

Tough market situation from one side and global competition from another side are persuading companies to search for new manufacturing concepts and try to stay competitive. But “how” to consider “new” manufacturing systems is still a big question mark.This thesis aims to analyze reconfigurability as an enabler for competitiveness in manufacturing systems. The frame of work in this study is “Reconfigurable Manufacturing System” or briefly RMS. In first chapter, some background about reconfigurability has been stated. Then it will continue with research questions, delimitations and expected results.Then the research methodology and challenges for applying RMS have been stated. This chapter explains researchers’ method for data reviewing and data collection. Another focus area in this thesis is SME (Small and Medium size Enterprises). So this report tries also to examine reconfigurability challenges in SMEs. There is a big gap between “ideal” production system and “designing” of this ideal production system. So this thesis tried to increase the knowledge about design of reconfigurable manufacturing systems.In empirical study chapter two case studies have been analysed and as a result a list of challenges for implementing reconfigurable system has been proposed. Then some solutions and methods are proposed in order to answer to challenges. This solutions and methods are then discussed and evaluated.Finally, in last chapter, challenges and prerequisites for implementing reconfigurable manufacturing system in general and for SMEs in specific have been stated. This chapter was ended by expressing future works.

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This research analyzes and reflects on the paper of the University as locus of the research and the scientific production, of the point of view of the process of evaluation practised for the competent instances in Brazil. The raised hypothesis is that it does not have convergence between the evaluation criteria used by the Regulating Agency and the Universities. As theoretical landmark, the study establishes in the elements and concepts contemplated for the Science of Sciences - Scientometry -, as study object, in the practised process of evaluation and, as method, in the bibliographical analysis of area documents, normative documents and reports of evaluation, what has allowed to analyze and to understand the process of evaluation and its implications. As result, expects to have explicitated the convergence degree between the criteria of evaluation practised by the instances above indicated e, thus, to contribute with subsidies to the politics of management of the activities of research and scientific production in the University. The PUC-Campinas consisted as experimental field of test and application of the results.
Este trabalho analisa e reflete sobre o papel da Universidade como locus da pesquisa e da produ??o cient?fica, do ponto de vista do processo de avalia??o praticado pelas inst?ncias competentes no Brasil. A hip?tese levantada ? a de que n?o h? converg?ncia entre os crit?rios de avalia??o utilizados pela Ag?ncia Reguladora e as Universidades. Como marco te?rico, o estudo funda-se nos elementos e conceitos contemplados pela Ci?ncia das Ci?ncias Cientometria -, como objeto de estudo, no processo de avalia??o praticado e, como m?todo, na an?lise bibliogr?fica dos documentos de ?rea, documentos normativos e relat?rios de avalia??o, o que permitiu analisar e compreender o processo de avalia??o e suas implica??es. Como resultado, espera-se haver explicitado o grau de converg?ncia entre os crit?rios de avalia??o praticados pelas inst?ncias acima indicadas e, assim, contribuir com subs?dios ? pol?tica de gest?o das atividades de pesquisa e de produ??o cientifica na Universidade. A PUC-Campinas constituiu-se como campo experimental de teste e aplica??o dos resultados.

El objetivo de esta investigación es poner en valor el pode del agua en la transformación del territorio, en el desarrollo de la economía, en la creación de las instituciones y en la formación de la cultura, y contribuir a recuperar el recurso hídrico como objeto central de la política local. Para tal fin se ha aplicado el método de investigación histórica y el de las ciencias sociales, que incluye el análisis de datos estadísticos para la obtención de información. Se ha tomado como unidad de análisis territorial uno de los cuatro Oasis de la provincia de Mendoza, Argentina: el Oasis Sur, que abarca los departamentos de San Rafael y General Alvear, cuyo desarrollo productivo hoy en día se encuentra en crisis.
Fil: Mercado, Iara Luciana. Universidad Nacional de Cuyo. Facultad de Ciencias Políticas y Sociales.

Um estudo de distribuição temporal, proporção sexual e densidade de caranguejos do gênero Uca foi realizado no estuário dos rios Piraquê-Açu e Piraquê-Mirim, Santa Cruz, Espírito Santo, Brazil. A população de caranguejos presentes em 06 áreas dividias em 90 quadrados de 0.75 m² foi coletada mensalmente durante o período de Julho/2012 à Maio/2013. Os caranguejos foram coletados por escavação manual das tocas, identificados e sexados. A temperatura do ar e da água variou entre 21 a 27°C e 22 a 28°C, respectivamente, enquanto que a salinidade variou de 21 a 35. Um total de 5.391 espécimes foi coletado, sendo 2.823 machos e 2.568 fêmeas (sendo 40 ovadas). A densidade média para a população de Uca sp. (subgênero leptuca) variou de 18,01 ± 1,29 (Maio/13) a 62,22 ± 1,51 ind.m-² (Novembro/12), para Uca thayeri de 3,67 ± 0,63 (Julho/12) a 15,29 ± 1,44 ind.m-² (Janeiro/13) e para a população Uca maracoani de 0,83 ± 0,27 ind.m-² (Julho/13) a 10,31 ± 0,73 (Novembro/12). A proporção sexual foi 1:1 para cada espécie estudada. A análise de correspondência canônica indicou que houve influência dos fatores ambientais na densidade dos caranguejos. Entretanto, as variáveis abióticas não foram limitantes para a distribuição das espécies estudadas. A produção secundária para a população de Uca sp. (subgênero leptuca) variou de 7,34 para 50,89 g PSLC m-1 ano-1, para Uca thayeri de 12,10 para 27,65 g PSLC m-1 ano-1 e para a população Uca maracoani de 1,70 para 8,83 g PSLC m-1 ano-1. A análise de regressão multivariada indicou que para a espécie Uca sp. (subgênero leptuca) a quantidade clorofila-a está relacionada com a produtividade desta espécie e para as demais espécies nenhuma das variáveis abióticas estudadas estaria relacionada com sua produtividade.
The temporal distribution, sex ratio and density of the fiddler crab Uca spp. was studied in populations living in the Piraquê-Açu and Piraquê-Mirim estuaries, Santa Cruz, Espírito Santo State, Brazil. Ninety 0.75 m² in 06 areas were used to obtain monthly samples from July 2012 to May 2013. The crabs were collected manually through excavation of burrows, identified and sexed. Air and water temperatures varied from 21 to 27°C and 22 to 28°C, respectively, and the salinity from 21 to 35. A total of 5,391 fiddler crabs were collected, with 2,823 males and 2,568 females (40 ovigerous). The average densities for the three species found were 18,01 ± 1,29 (May/13) to 62,22 ± 1,51 ind.m-² (November/12), 3,67 ± 0,63 (July/12) to 15,29 ± 1,44 ind.m-² (January/13) for Uca thayeri and 0,83 ± 0,27 ind.m-² (July/13) to 10,31 ± 0,73 (November/12) for Uca maracoani. Sex ratio (male:female) was 1:1. Canonical correspondence analysis showed that abiotic factors can influence crab density, but do not limit species distribution. The average secondary production of the Uca sp. population ranged from 7.34 to 50.89 g PSLC m-1 y-1, for Uca thayeri ranged between 12.10 to 27.65 g PSLC m-1 y-1 and for Uca maracoani from 1.70 to 8.83 g PSLC m-1 y-1. The multiple regression shows the chlorophyll-a may influence Uca sp. (subgênero leptuca) productivity. For the Uca thayeri and Uca maracoani species not a single abiotic factor tested had an influence in productivity.

Le biodiesel, consid??r?? comme une solution pour remplacer le p??trodiesel est un sujet de recherche mondial. Un des principaux probl??mes associ??s au d??veloppement industriel du biodiesel est la source de mati??re premi??re ainsi que le proc??d?? de transformation. Ainsi, la pr??sente ??tude a pour but de trouver une source de mati??re premi??re durable pour la production industrielle de biodiesel et de d??terminer le proc??d?? de transformation de la mati??re premi??re, le plus appropri??, ainsi que les meilleures conditions op??ratoires. Durant la premi??re ??tape de ce projet, une source de mati??re premi??re durable a ??t?? s??lectionn??e : les microalgues. Le proc??d?? de transformation ??tudi?? est la transest??rification enzymatique. L???huile d???olive, huile ayant une composition en acides gras similaires ?? celle de l???huile de la microalgue Chlorella protothecoides, a ??t?? choisie pour effectuer les r??actions. Pendant la deuxi??me ??tape, le proc??d?? de standardisation de la r??action (bior??acteur de 5 mL) consistait ?? faire varier : le type de catalyseur (lipases de Candida antarctica (Novozym?? 435) et de Thermomyces lanuginosus (TL I150)), la concentration du catalyseur (7 ?? 14 % m/mhuile), la temp??rature de r??action (25 ?? 50 ??C) et le ratio molaire alcool:huile (3:1 ?? 4:1) ; la vitesse d???agitation ??tant de 150 rpm pour toutes les r??actions. Des techniques d???optimisation telle que la preincubation de l???enzyme ont ??t?? ??galement essay??es. Le rendement en esters alkyliques de la r??action de transest??rification enzymatique de l???huile en fonction du temps est la variable de contr??le pour toutes les r??actions. La standardisation des variables du proc??d?? a ??t?? faite en fonction de la r??duction du temps de r??action et du rendement en esters alkyliques. Un rendement ??lev?? en esters alkyliques de 92 % (m/m) a ??t?? obtenu sous les conditions op??ratoires suivantes : une concentration de catalyseur (TL I150) de 7 % (m/mhuile), une temp??rature de r??action de 25 ??C, un ratio molaire alcool:huile de 3:1 et un temps de r??action de 4 h ; la lipase a ??t?? preincub??e pendant 6 h avant la r??action de transest??rification. Le temps de r??action, un des param??tres importants lors du proc??d?? de standardisation des variables, a ??t?? r??duit de 24 ?? 4 h. Un autre param??tre significatif de la r??action est la temp??rature : une temp??rature de 25 ??C a ??t?? utilis??e; cette temp??rature de r??action est faible et rend le proc??d?? au niveau industriel plus attrayant.

La production des matières vitreuses, ensemble d'espèces siliceuses renfermant une phase vitreuse, connait un développement remarquable au Proche-Orient au cours de l’Age du bronze, plus particulièrement au cours du IIe millénaire av. J. -C. Notre thèse apporte un éclairage nouveau sur le sujet au travers de l'études du matériel de deux sites levantins, celui de Ras Shamra-Ougarit, capitale du royaume cananéen du même nom, et celui de son port principal implante à Minet El-Beida. Localisée sur la côte nord-syrienne, la cité florissante d'Ougarit occupe alors une position privilégiée au carrefour des échanges internationaux, entre l'Égée, l'Égypte, l’Anatolie, la Palestine, la Syrie intérieure et la Mésopotamie. Le matériel étudie provient des fouilles archéologiques réalisées depuis la fin des années 20 jusqu'à aujourd'hui. L'ensemble constitue un corpus de référence de près de 20. 000 pièces, l'un des plus importants du Proche-Orient. Le matériel, date du IIe millénaire av. J. -C. (soit des périodes du bronze moyen et du bronze récent), se répartit ainsi : près de 18. 000 objets en « faïence », 1000 en verre, 266 en « bleu égyptien » et 26 en céramique argileuse à Glacure. Le catalogue exhaustif de ce corpus, en grande partie inédit, est présenté dans le tome II de la thèse qui comprend deux volumes (textes et illustrations). Le tome 1 (constitue de deux volumes) de la thèse est consacré à l'analyse du matériel. L'approche typologique, stylistique, et technique {cf. Plusieurs programmes de recherche archéométrique réalisés au laboratoire de recherche des musées de France à Paris) du matériel a pour objectif premier la définition de l'origine de la production, approche particulièrement complexe dans le cas d'une cite comme Ougarit caractérisée par son cosmopolitisme culturel. Dans un second temps, la dimension sociale de ces produits de luxe est abordée à la lumière des données archéologiques et textuelles.

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Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico
We seek consistency empirical and analytical rigor to the concept of touristification in the course of the contemporary arrangements of subjectivity and urbanity stations through a landscape of social (dis)encounters between residents and foreigners living in the capital of Rio Grande do Norte, Natal. In the last three decades the city has become a major destination country for national and international tourism, making tourism an important monitor of choreography in search of synchrony between cities and subjectivities, orchestrated by the cyclical crises and the relentless battle for systemic capitalistic expansion and survival. We proceed with an ethnographic and cartographic inspiration in Ponta Negra, where the waterfront redevelopment in 2000 conducted by the government, influenced the implementation of various establishments, services and practices related to the construction of Natal s major industry hub of tourism and entertainment. We proposed an arch-genealogy of touristification inspared on Michel Foucault analytical perspective, increased by authors in confluence or in different theoretical and methodological approaches, among which stand out Karl Marx, Gilles Deleuze and Felix Guattari, Michael Hardt and Antonio Negri. The reflections that initially turned to the advancement of the sphere of consumerism as the issue capable of articulating the developmental trajectory of the capitalist system and the practice of tourism, promoting some effects (un)desirable in the conflictual dynamics of the condition of the district of Ponta Negra, territory each again (re) produces the designs to meet the consumption by affluent portion of the population and foreign, encountering social exclusion and inclusion differentiated a growing trend. We go forward with an analysis of the "liberation of desire" phenomenal features of the process of producing subjective, providing a breakdown of the recent world s financial crisis initially, but proved social and political crisis also, through a plot derived from the operation of real estate speculation, which Natal is mainly caught through touristification, showing the outlines of a generalized crisis in establishing a new order buoyed by the emergence of a context "biopolitical" since it is a major route that uses the capital to survive and expand, while guiding the process of "strategic beautification" held in Ponta Negra, problematized by us. We conclude by assessing the appropriateness of proposing on further analysis to explore the establishment of a "new social ontology in terms of interference touristification ongoing and the constitution of a "new order" local/global, away from that characterize a system's capitalist overcoming, try to give emphasis in the current stage of the radicalization of a capitalistic utopia
Buscamos consist?ncia emp?rica e rigor anal?tico para o conceito de turistifica??o, atrav?s dos arranjos urbanos e subjetivos contempor?neos postos por uma paisagem social de (des)encontros entre os moradores e os estrangeiros que convivem na capital do Rio Grande do Norte, Natal. Nas ?ltimas tr?s d?cadas a cidade transformou-se em um dos principais destinos do pa?s para o turismo nacional e internacional, fazendo do turismo um importante analisador da coreografia em busca de sincronia entre cidades e subjetividades, orquestrada pelas crises c?clicas e sist?micas da incessante batalha por sobreviv?ncia e expans?o capital?stica. Procedemos com uma pesquisa etnogr?fica e de inspira??o cartogr?fica no bairro de Ponta Negra, cuja reurbaniza??o da orla no ano de 2000 conduzida pelo poder p?blico, influenciou a implanta??o de diversos estabelecimentos, servi?os e pr?ticas vinculadas ? constru??o do maior p?lo da ind?stria do turismo e do entretenimento em Natal. Propusemos uma arque-genealogia da turistifica??o inspirada na anal?tica de Michel Foucault, acrescida por autores em conflu?ncia ou de distintas orienta??es te?ricometodol?gicas, entre os quais ganham destaque Karl Marx, Gilles Deleuze e F?lix Guattari, Michael Hardt e Antonio Negri, nas reflex?es que se voltaram inicialmente para o avan?o da esfera do consumismo enquanto inst?ncia capaz de articular a trajet?ria de desenvolvimento do sistema capitalista e a pr?tica do turismo, promovendo alguns efeitos (in)desej?veis na din?mica conflitiva das condi??es de exist?ncia do bairro de Ponta Negra, territ?rio que cada vez mais se (re)produz para atender aos des?gnios do consumo por parcela abastada da popula??o e estrangeira, encontrando na exclus?o social e na inclus?o diferenciada uma tend?ncia crescente. Avan?amos com uma an?lise dos desejos de libera??o , aspectos fenom?nicos do processo de produ??o de subjetividades, fornecendo um detalhamento da recente crise mundial inicialmente financeira, mas que se revelou social e pol?tica, atrav?s de um enredo derivado do funcionamento da especula??o imobili?ria, ao qual Natal se encontra implicado principalmente atrav?s da turistifica??o, evidenciando os contornos de uma crise generalizada no estabelecimento de uma nova ordena??o balizada pela emerg?ncia de um contexto biopol?tico , visto ser uma das principais vias que o capital recorre para sobreviver e se expandir, ao mesmo tempo orientador do processo de embelezamento estrat?gico ocorrido em Ponta Negra, por n?s problematizados. Finalizamos avaliando a pertin?ncia de se propor em an?lises posteriores a constitui??o de uma nova ontologia social face ?s interfer?ncias da turistifica??o em curso e a composi??o de uma nova ordem (g)local, que longe de caracterizar uma supera??o do sistema capitalista, trate de por em evid?ncia a atual etapa de radicaliza??o de uma utopia capital?stica