Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a wide ecological niche and causes a number of diseases in human and animals. It invades mammalian host cells and escapes from the vacuoles prior to replication in the host cell cytoplasm and infecting adjacent cells via actin-based mobility. Phosphoinositide (PIP) metabolism is essential to mammalian cells in signal transduction, actin remodelling, endosome dynamics and membrane trafficking. Modulation of host PIP metabolism by bacteria PIP phosphatases is important for pathogenicity and virulence of many human pathogens. In this study the function of two L. monocytogenes tyrosine and inositol phosphatases LipA and LipB were studied in vitro. The lipA and lipB deletion mutants generated in EGDe and InlA strains were not affected in invasion but were attenuated in intracellular growth in Caco-2 and Hela M cell lines but not in mouse macrophages. Deletion of lipA or lipB did not affect the actin polymerisation but caused reduced plaque number in the plaque assay. The turnover of five PIPs in Hela M cells during L. monocytogenes infection were studied by expression of fluorescent protein tagged domains that specifically recognizes individual PIPs. L. monocytognenes did not affect the metabolism of PI4P, PI(4,5)P2, PI(3,4,5)P3 but co-localised with PI3P at 1.5 hr post-infection and with PI(3,4)P2 at 6 hr to 24 hr post-infection. The PI(3,4)P2 effector protein lamellipodin was discovered to be recruited to actin-associated L. monocytogenes at 4 hr to 24 hr post-infection in Hela M cells. This discovery leads to the hypothesis of a novel mechanism of lamellipodin-dependant cell-to-cell spread. The lipA mutant was found to be attenuated in PI(3,4)P2 recruitment and therefore hypothesized to participate in the proposed lamellipodin pathway by converting PI(3,5)P2 into PI5P, leading to the activation of PI3K and subsequent production of PI(3,4)P2. LipB showed partial localisation at the Golgi complex when over-expressed in Hela M cells, and it was assumed to act mainly as a protein-tyrosine phosphatase. In summary, this study provides some evidence on L. monocytogenes modulating host PIP metabolism by the production of inositol phosphatases. It gives us a better understanding on the intracellular growth of this pathogenic bacterium, and on the interaction between host and parasite.

Syftet med detta examensarbete är att fördjupa förståelsen för musikproduktion och synthprogrammering genom att återskapa två Dua Lipa-låtar och sedan applicera denna kunskap för att omvandla Harry Styles "Watermelon sugar" i Dua Lipa-stil.

Se llevó a cabo un estudio inmunohistoquímico incluyendo un panel de 18 biomarcadores y un estudio molecular en 81 biopsias de cáncer de cuello uterino (59 carcinomas epidermoides y 19 adenocarcinomas cervicales) diagnosticados en el Servicio de Anatomía Patológica del Hospital Clínico Universitario de Valencia, con la finalidad de realizar la caracterización inmunohistoquímica de esta neoplasia invasora y determinar la presencia de ADN-HPV asociado. El estudio inmunohistoquímico se realizó, en tumores cervicales y tejido cervical benigno como control, mediante la técnica de avidina-biotina, interpretado mediante una escala semicuantitativa. Se determinó la presencia y genotipificación de ADN-HPV, en las muestras neoplásicas, utilizando el método PCR-SPF10/LiPA.El tipo histológico más frecuente fue el carcinoma epidermoide moderadamente diferenciado (43.5%) y el adenocarcinoma endocervical (42.1%). La edad promedio de las pacientes fue 50.67±13.62 años. Los hallazgos inmunohistoquímicos fueron los siguientes: los carcinomas cervicales mostraron un índice proliferativo (Ki-67/MIB-1) entre moderado y alto; presentaron inmunoreactividad positiva para las proteínas celulares reguladoras ciclina D1, pRb, p16, p21, p27 y ciclina E igual a 32.1%, 30%, 79.5%, 52.5%, 53.2% y 7.7% respectivamente. Respecto a las proteínas apoptóticas, la reactividad ante el anticuerpo contra p53 fue de 3.9%, MDM-2 (100%), Bax (67.5%) y Bcl-2 (3.7%). Las moléculas de adhesión celular cadherina-E, CD44s y CD44v3 estaban sobrexpresadas con localización preferentemente citoplasmática en el 76.6%, 90.9% y 97.5%. La inmunorespuesta para la ubicuitina fue de 98.7%, citoqueratinas (AE1/AE3) 97,5%, CEA 89.7% y telomerasa 52.6%. Se encontraron diferencias estadísticamente significativas en la inmunoreactividad de los tumores en comparación con los controles a excepción de la reactividad de p53, Bcl-2 y CEA; también en cuanto a la reactividad de p27, MDM-2, citoqueratinas, localización celular de la cadherina-E y CD44v3 en los carcinomas de diferentes grados de maduración y en el caso de los subtipos histológicos de adenocarcinoma cervical para Bax, telomerasa y CD44v3. Hallamos correlación en la inmunoexpresión de 1) Ki-67 con p16 (p=0.003), p21 (p=0.023), p27 p=0.032), telomerasa (p=0.012) y ubicuitina (p=0.007). 2) pRb y ciclina D1 (p=0.027), CEA (p=0.050) y CD44s (p=0.009); p53 con ciclina D1 (p=0.011) y CEA (p=0.015). 3) p16/p21 (p=0.046), p16/p27 (p=0.027), p21/p27 (p=0.003), p27/Bax (p=0.015), p27/Bcl-2 (p=0.039) y p16/MDM-2 (p=0.013). 4) ciclina D1 con p21 (p=0.000), CEA (p= 0.000) y Cadherina-E (p=0.001). 5) Cadherina-E cn CD44s (p=0.000), CD44v3 (p=0.013), con MDM-2 (p=0.000) y ubicuitina (p=0.026); CD44s/CD44v3 (p=0.000); CD44v3 y p16 (p=0.007), ubicuitina (p=0.012), telomerasa (p=0.025) y citoqueratinas (p=0.009). 6) Citoqueratinas con p27 (p=0.022), Bax (p=0.007) y Bcl-2 (p=0.009). 7) Telomerasa con p16 (p=0.004), p27 (p=0.037), MDM-2 (p=0.002), CD44v3 (p=0.025) y Ki.67 (p=0.012). En cuanto a la presencia de ADN-HPV en las muestras neoplásicas, el 96.3% (78/81) fueron positivas; de estos, el 59% (46/78) presentó infección única y el 41% (32/78) infección múltiple. El tipo viral específico más frecuente fue el HPV16 (55.13%) seguido de los HPV18, HPV58 y HPV X, en igual porcentaje (1.28%), así mismo el HPV16 fue el tipo viral más frecuente tanto en carcinomas epidermoides (55.9%) como en adenocarcinomas cervicales (52.6%). No encontramos diferencias estadísticamente significativas en la inmunoexpresión del panel de biomarcadores estudiados entre la infección única y múltiple. El cáncer de cuello uterino asociado a infección por HPV oncogénico se caracteriza porque muestra inmunoreactividad alterada en las principales vías reguladoras del ciclo celular, modulación de las proteínas apoptóticas, inmunolocalización celular anormal de las moléculas de adhesión celular, así como sobrexpresión de CEA y citoqueratinas, intensa degradación de proteínas y activación de la telomerasa. Los tipos histológicos de cáncer de cuello uterino comparten un proceso de carcinogénesis multifactorial en el que está involucrada, comúnmente, la presencia del HPV oncogénico como agente causal necesario para el desarrollo de esta neoplasia. / Molecular and inmunohistochemistry (IHC) studies including eighteen biomarkers were performed in eighty-one cervical cancer biopsies with the aim of performing the IHC characterization and to detect the presence of human papillomavirus (HPV).IHC studies were performed using benign cervical tissue as control of expression and results were evaluated using a semi quantitative scale. AND-HPV presence was detect and genotyped in each neoplasic sample by using SPF10/LiPA method.More frequent cancer histologic subtypes were moderately differentiated epidermoid carcinoma (43,5%) and endocervical adenocarcinoma (42,1%). IHC findings obtained for the tested antibodies are described next. Proliferative index inferred from Ki-67/MIB-1 was between moderate and high. Regulating proteins rendered positive results in different percentages: cyclin D1 (32,1%), cyclin E (7,7%), pRB (30%), p16 (79,5%), p21 (52,5%), p27 (53,2%) and apoptotic proteins: p53 (3,9%), MDM-2 (100%), Bax (67,5%) and Bcl-2 (3,7%). Cellular adhesion molecules: cadherin-E, CD44s and CD44v3 were overexpressed with cytoplasmic localization mainly in 76,6%; 90,9% and 97,5%, respectively. Other tumor related molecules were ubiquitin, cytokeratins (AE1/AE3), CEA and telomerase with 98.7%, 97,5%, 89,7% and 52,6%, respectively.Statistical analysis showed significant differences between tumours and controls in all antibodies tested with the exception of p53, BCL2 and CEA. When combining the antibodies we established strech association in the inmunoexpression of them. Presence of AND-HPV was detected in 96,3% of samples showing single infection the 59% and multiple infection the rest of tumours. The most frequent virus type was HPV16 (55,13%) followed by HPV18, HPV58 and HPV X with the same associated frequency (1,28%). HPV16 was the most frequent virus type in both epidermoid and cervical tumours. It was not possible to establish differences between single and multiple infections related to IHC expression profile.Cervical cancer is characterized by the altered expression of IHC patterns in cellular regulatory pathways affecting apoptotic proteins, cellular cycle, adhesion molecules inmunolocalization, CEA and cytoqueratines overexpression and increase of protein degradation and telomerase activity. Histological subtiypes of cervical cancer share a multifactorial complex process including HPV infection as common tumour agent.

The Lipa Temporary Reception Centre is a transit camp, located in the North-West part of Bosnia and Herzegovina, for only single men who cross the Balkan Route to enter the European Union territory through the Croatian border. This thesis aims to describe the life inside the camp, combining an analysis of Lipa architecture with the experiences of the people who transited through there. A focus on space and relationships will then drive this thesis, reflecting on how the transit camp features and stylistic decisions affected people's experiences: discussing how places and individuals mutually influenced each other in such a context. More specifically, it will highlight the broad political implications that led to the opening of migrant reception centres like Lipa and discuss their hypothetical temporary nature, studying the roles played by European Union Institutions and non-governmental organizations within the field. This research is the outcome of ethnographic fieldwork conducted inside the Lipa Temporary Reception Centre from November 8th until December 19th and from the investigation of the existing literature regarding the design of camps and the Balkan Route.

Lipid droplets (LDs) are organelles with many functions in cells and numerous protein interactors facilitate their biogenesis, maintenance, and turnover. The mammalian lipase responsible for LD turnover during lipophagy, LipA, has two candidate homologs in Arabidopsis: MPL1 and LIP1. One or both of these plant homologs may function in a similar manner to mammalian LipA, providing an LD breakdown pathway. To test this hypothesis, wild type (WT) Arabidopsis plants, MPL1 over-expressing (OE) mutants, and T-DNA insertion mutants of MPL1 (mpl1) and LIP1 (lip1) were examined for LD phenotypes in normal conditions and in environments where LD numbers are known to fluctuate. Plants to be imaged by confocal microscopy were exposed to heat stress and wounding to increase LD accumulation, senescence was induced in leaves to deplete lipids, and LDs were imaged throughout the day/night period to observe their diurnal regulation. The mutation of both MPL1 and LIP1 lead to an increase in LDs within the leaf mesophyll cells, although the spatial distribution of the LDs differed between the two mutants. mpl1 mutants had disrupted diurnal regulation of their LDs, but lip1 mutants did not. Alternately, lip1 mutants retained LDs during dark-induced senescence, and mpl1 mutants did not. Together these results suggest that MPL1 and LIP1 are likely both important for LD dynamics; however they appear have roles in different aspects of LD accumulation and turnover.

Pierce s Disease (PD) of grapevines is caused by the bacterium Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important diseases in many plants. A characteristic symptom of PD is leaf scorching, with regions of chlorosis progressing into necrotic zones at the peripheral margins of infected leaves. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been hypothesized as the main cause of PD symptom development; however, Xf virulence mechanism has not been elucidated. The analysis of Xf Temecula 1 secretome revealed a putative lipase/esterase (PD1703) that was abundantly secreted in the bacterial culture supernatant, and was characterized as a protein ortholog of the cell wall degrading enzyme LipA of Xanthomonas strains. The LipA was secreted and associated with a biofilm filamentous network and additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LipA in leaf regions was positively associated with PD symptoms and inversely correlated with bacterial titer. The lipase/esterase was found to elicit a hypersensitive response in grapevine and was regulated by quorum-sensing signaling, which is known to modulate bacterial pathogenesis. We propose that Xff pathogenesis is caused by LipA secretion mediated by OMV cargos, and its release and accumulation in leaf margins leads to the observed PD symptoms development. / A Doença de Pierce (PD) em videiras (Vitis vinifera L.) é causada pela bactéria Xylella fastidiosa (Xf), uma gama-proteobactéria responsável por diversas doenças em plantas economicamente importantes. Um sintoma característico da PD é a queimadura, caracterizado por zonas de clorose progredindo em necrose nas margens da lâmina foliar. O processo resultante da oclusão dos elementos do xilema por células bacterianas e pelo biofilme associados às mesmas, bem como o consequente bloqueio do fluxo da seiva, têm sido hipotetizado como a principal causa dos sintomas comumente observados na PD. Embora tal hipótese seja suportada por algumas evidências, o mecanismo de virulência de Xf não foi totalmente compreendido. A análise do secretoma de Xf Temecula 1 revelou que uma lipase/esterase (PD1703) é abundantemente secretada in vitro. Esta proteína foi caracterizada como ortóloga à proteína LipA presente em bactérias do grupo das Xanthomonas e funcionalmente caracterizada como degradante da parede celular neste grupo. A proteína LipA foi associada à matriz extracelular filamentosa e análises proteômicas adicionais revelaram sua presença nas vesículas de membrana externa. O acúmulo da proteína LipA em folhas de plantas infectadas foi positivamente associado aos sintomas da PD e inversamente correlacionado com o título bacteriano. LipA induziu resposta de hipersensibilidade em videiras e foi regulada por sinalização célula-célula, mecanismo conhecido por modular a patogênese bacteriana. Com base em tais evidências, propomos que a secreção da proteína LipA mediada pelas vesículas de membrana externa e sua liberação e acúmulo em margens foliares, onde leva ao desenvolvimento dos sintomas comumente observados na doença, seja um mecanismo essencial à patogênese de X. fastidiosa na PD. / Doutor em Genética e Bioquímica

Min tanke med detta arbete var att hitta nya sätt att både skriva och producera låtar på, genom att använda mig av referenslåtar som inspirationskälla. Jag fokuserade på tre olika beståndsdelar från referenslåtarna, som sedan implementerades i tre nyskapade demolåtar. I låt 1 fokuserade jag på basen roll. I låt 2 fokuserade jag på solosångens relation till körsång och i låt 3 fokuserade jag på elpianot Fender Rhodes. När jag analyserade referenslåtarna så tog jag med mig vissa principer från respektive referenslåt och implementerade i mina egna låtar. Detta resulterade i att jag bland annat upptäckte nya spelsätt, sound och produktionstekniker att inspireras av till mina tre nyskrivna demolåtar. Att jobba med referenslåtar upplevde jag var ett bra startskott för att komma igång med låtskrivarprocessen. Samtidigt kunde jag stundtals känna mig begränsad och låst till referenslåtarna. Trots detta så var det en väldigt lärorik metod som hjälpte mig at vidga mina vyer och möjliggjorde för mig att skapa tre demolåtar som jag kanske inte annars hade skrivit.

The aim of this thesis is to analyze and evaluate the prerequisites tourism and the tourism potential in the defined area of the Pelhřimov district. The theoretical part focuses on the basic terminology of tourism, regional development and theoretical determination of preconditions for tourism development. In the practical part, the prerequisites of the defined area are analyzed and evaluated from the tourism point of view. This is followed by the evaluation method of tourism potential. Based on the analyses, several recommendations for the use and connection of local attractions are suggested with the aim to promote local tourism in the defined area.