V ANGLICKÉM JAZYCE Immunotherapy of ovarian carcinoma with dendritic cells Anticancer immunotherapy is a therapeutical strategy aimed at elicitation and maintenance of immune responses against cancer cells. In this study we have focused on immunotherapy of ovarian cancer, because it is one of the most common gynaecological tumors with poor prognosis and high mortality. Our immunotherapy protocol involves preparing dendritic cells (DC) from monocytes isolated from patient's peripheral blood, which are subsequently pulsed with irradiated cells of established ovarian cancer cell line. These immature pulsed DC are maturated and subsequently co-cultivated with autologous T lymphocytes. The aim of this study was to demonstrate, that DC are able to elicit specific immune response after addition of suitable mature agens in combination with apoptotic ovarian tumor cells. Our observations indicate that 24 hours are sufficient for induction of tumor cells apoptosis. Additionally, we have shown that DC successfully ingested most of the apoptotic tumor cells after 4 hours of co-incubation. Furthermore, we have found out that ingestion of apoptotic cells by dendritic cells, which are stimulated with polyI:C, inhibits maturation of DC and consequently also production of cytokines IL-12p70, IL-6 and TNF-α. Whereas...

Title: Assessment of the level of condition, basic motor coordination, and football skills between two age categories of young football players. Objectives: Find out possible differences in the level of condition, basic motor coordination, and specific football skills between two age categories of young players (U14 and U15). At the same time, the aim is to determine the effect of different rates of biological growth on fitness performance, basic motor coordination, and football skills. The last goal is to find out the level of association between the indicator of the condition, basic motor coordination and football skills. Methods: The main scientific method of theoretical - empiric character was observation and testing. Research field consisted of the players of category U14 (n=24; Age = 13,79±0,49; Height = 166,1±8,42; Weight = 46,7±8,65) and U15 (n=25; Age = 14,8±0,33; Height = 173,17±7,39; Weight = 61,82±8,52) from the football club FK Motorlet Praha. The level of fitness was measured in tests of a sprint at 10 and 30 meters, 505 agility test, Illinois agility test, long jump, Yo-Yo Intermittent Recovery Test Level 1. The level of basic motor coordination was determined with the Körperkoordinationtest für Kinder test battery (KTK test) containing 4 subtests: balancing backwards, jumping...

Maternally supplied mRNAs encode for necessary developmental regulators that pattern early embryos in many species until zygotic transcription is activated. In Caenorhabditis elegans, post-transcriptional regulatory mechanisms guide early development during embryogenesis. Maternal transcripts remain in a translationally silenced state until fertilization. A suite of RNA-binding proteins (RBP’s) regulate these maternally supplied mRNAs during oogenesis, the oocyte-to-embryo transition, and early embryogenesis. Identifying the target specificity of these RNA-binding proteins will reveal their contribution to patterning of the embryo. We are studying post-transcriptional regulation of maternal mRNAs during oocyte maturation, which is an essential part of meiosis that prepares oocytes for fertilization. Although the physiological events taking place during oocyte maturation have been well studied, the molecular mechanisms that regulate oocyte maturation are not well understood. OMA-1 and OMA-2 are essential CCCH-type tandem zinc finger (TZF) RBP’s that function redundantly during oocyte maturation. This dissertation shows that I defined the RNA-binding specificity of OMA-1, and demonstrated that OMA-1/2 are required to repress the expression of 3ʹUTR reporters in developing oocytes. The recovered sequences from in vitro selection demonstrated that OMA-1 binds UAA and UAU repeats in a cooperative fashion. Interestingly, OMA-1 binds with high affinity to a conserved region of the glp-1 3ʹUTR that is rich in UAA and UAU repeats. Multiple RNA-binding proteins regulate translation of GLP-1 protein, a homolog of Notch receptor. In addition to previously identified RBP’s, we showed that OMA-1 and OMA-2 repress glp-1 reporter expression in C. elegans oocytes. Mapping the OMA-1 dependent regulatory sites in the glp-1 mRNA and characterizing the interplay between OMA-1 and other factors will help reveal how multiple regulatory signals coordinate the transition from oocyte to embryo but the abundance of OMA-1 binding motifs within the glp-1 3ʹUTR makes it infeasible to identify sites with a functional consequence. I therefore first developed a strategy that allowed us to generate transgenic strains efficiently using a library adaptation of MosSCI transgenesis in combination with rapid RNAi screening to identify RBP-mRNA interactions with a functional consequence. This allowed me to identify five novel mRNA targets of OMA-1 with an in vivo regulatory connection. In conclusion, the findings in this dissertation provide new insights into OMA-1 mediated mRNA regulation and provide new tools for C. elegans transgenesis. Development of library MosSCI will advance functional mapping of OMA-1 dependent regulatory sites in the target mRNAs. Extending this strategy to map functional interactions between mRNA targets and RNAbinding proteins in will help reveal how multiple regulatory binding events coordinate complex cellular events such as oocyte to embryo transition and cell-fate specification.

Orientador: Marcus Antônio Rossi Feliciano / Resumo: Objetivou-se avaliar e descrever as características ultrassonográficas (modo B, Doppler e elastrográfica) de tecidos materno-fetais (pulmão, fígado, rins, placentônio e cérvix) em ovelhas gestantes de parto natural ou prematuro induzido; verificar diferenças de achados entre os tipos de parto e, correlacionar com os valores obtidos dos sinais específicos do parto e impedância do muco vaginal, visando predizer a proximidade do processo de parturição. Trinta ovelhas Santa Inês adultas foram selecionadas e divididas em dois grupos: Parto Normal-GPN (n=15); e Parto Prematuro Induzido-GPPI (n=15). Os animais foram avaliados a cada 12 horas até o momento da parturição (última semana gestacional), as ovelhas pertencentes ao GPN a partir do 143º dia de gestação e as do GPPI, a partir do 135º dia de gestação (dia correspondente à indução do parto com betametasona e antiprogestágeno aglepristona). Foram avaliadas as estruturas pulmonares, renais e hepática dos conceptos, placêntonio e cérvix, obtendo-se as caraterísticas ultrassonográficas convencional e Doppler (placentônio e artéria uterina), elastografia quantativa (velocidade de cisalhamento – m/s), além dos batimentos cardíacos fetais, impedância do muco vaginal e, presença de sinais específicos e comportamentais do parto (edema vulvar, isolamento, ingurgitamento de úbere e temperatura vulvar). O nível de significância utilizado foi de 5% (p < 0,05) para todos os testes estatísticos realizados. A temperatura vulvar média foi maior... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate and describe the ultrasonographic characteristics (B mode, Doppler and elastographic) of maternal-fetal tissues (lung, liver, kidneys, placentonium and cervix) in pregnant ewes with normal and induced preterm parturition; to verify differences of findings between the types of lambing and to correlate with the values obtained from specific signs of lambing and impedance of vaginal mucus, aiming to predict the proximity of the parturition process. Thirty adult Santa Inês sheep were selected and divided into two groups: Parturition at term-GPN (n=15); and Induced pre-term parturition-GPPI (n=15). The animals were evaluated every 12 hours until the moment of parturition (last gestational week), the ewes belonging to the GPN from the 143rd day of gestation and those of the GPPI from the 135th day of gestation (day corresponding to the induction of lambing with betamethasone and antiprogestin aglepristone). Pulmonary, renal and hepatic structures of the concepts,, placentonium and cervix were evaluated, obtaining the conventional ultrasound and Doppler ultrasound characteristics (placentonium and uterine artery), quantitative elastography (shear velocity - m / s), in addition to fetal heart rate, impedance of vaginal mucus and presence of specific and behavioral signs of lambing (vulvar edema, isolation, udder engorgement and vulvar temperature). The significance level used was 5% (p <0.05) for all statistical tests performed. Mean vulvar tempe... (Complete abstract click electronic access below) / Doutor

This thesis explores the development of individuality in Nigerian books for young adults, Akata Witch and Akata Warrior by Nnedi Okorafor. For that, the literary concepts of young adult literature and the heroic journey (monomyth) is used. This thesis aims to prove that the heroic journey framework may be applied to young adult literature, primarily concerning the formation of identity resulting a certain correlation between the two types of narrative. Based on the description of concepts of literary theory, such as the Other, described by Robyn McCallum, and the individual constitutive elements of the heroic journey, in the theoretical part, the analysis from both vantage points and subsequent correlation between them is described in the practical part. The two books that are analysed are described from the point of three major concepts of young adult literature and all twenty six steps of the heroic journey, based on Campbell and other literary theorists. Based on this analysis, it is concluded that there is a certain correlation between the two narrative frameworks. It is especially similar concerning the growth of individuality of its protagonists. Despite there being a correlation among the two types of narrative, young adult literature and the heroic journey, certain elements do not have...

La polykystose rénale autosomique dominante (PKRAD) est une maladie génétique rénale qui se manifeste par le développement de kystes au rein. Elle résulte de mutations dans le gène PKD1/polycystine-1 (PC-1), contenant un motif coiled-coil et dans le gène PKD2/PC-2 dont les fonctions restent à élucider. À partir d’un chromosome artificiel bactérien, le gène Pkd1 murin (BAC-Pkd1) a été modifié afin de générer 4 lignées de souris transgéniques délété du motif coiled-coil Pkd1Δcoiled-coil et 3 lignées contrôles Pkd1TAG possédant 1-35 copies du transgène. Ces 2 transgènes ont un profil d’expression identique à l’endogène et le niveau dépend du nombre de copies. Alors que les souris Pkd1TAG développent la PKRAD de sévérité proportionnelle au niveau d’expression, les lignées Pkd1Δcoiled-coil en sont épargnées. Ces résultats démontrent l’importance du motif coiled-coil dans la maladie. Les souris Pkd1Δcoiled-coil croisées par Pkd1-/- (létale à la naissance), Pkd1-/- ; Pkd1Δcoiled-coil survivent après la naissance et permettent d’analyser in vivo les interactions, la signalisation et le rôle physiologique du motif coiled-coil. Ces souris Pkd1-/- ; Pkd1Δcoiled-coil avec une copie du transgène présentent des kystes rénaux et meurent ~2 semaines alors que celles à hautes copies n’ont aucun phénotype comme les Pkd1TAG. Les résultats génétiques et biochimiques démontrent que Pc-1Δcoiled-coil est hypomorphe. Bien que Pc-1Δcoiled-coil subit son clivage autoprotéolytique, l’analyse du transport intracellulaire de Pc1Δcoiled-coil montre un délai de maturation. Alors qu’in vitro le trafic Pc-1/Pc-2 dépend du motif coiled-coil pour leur interaction, in vivo une interaction Pc-1Δcoiled-coil/Pc-2 est détectée, suggérant un site d’interaction distinct. Nos études in vivo démontrent que le motif coiled-coil de Pc-1 joue un rôle clé dans sa maturation et l’existence d’un nouveau partenaire de Pc-1 pour son transport intracellulaire. / Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic disorder that is characterize by the formation of bilateral renal cysts and leads to kidney failure. It results from mutations in the PKD1/polycystin-1(PC-1) gene, containing a coiled-coil motif, and in the PKD2/PC-2 gene, the functions of which remain to be elucidated. From a bacterial artificial chromosome, the murine Pkd1 gene (Pkd1-BAC) was modified in order to obtain 4 transgenic mouse lines deleted from it coiled-coil motif (Pkd1Δcoiled-coil) and 3 Pkd1TAG control lines possessing 1-35 copies of the transgene. These two transgenes have an expression profile identical to the endogenous gene and their expression depends on the number of copies. While Pkd1TAG mice develop ADPKD with a severity proportional to the level of expression, the Pkd1Δcoiled-coil lines are spared. These results demonstrate the importance of the coiled-coil motif in the disease. Pkd1Δcoiled-coil mice crossed by Pkd1-/- (lethal by birth), Pkd1-/-; Pkd1Δcoiled-coil survive after birth and allow the interactions, signaling and physiological role of the coiled-coil motif to be analyzed in vivo. These Pkd1-/-; Pkd1Δcoiled-coil mice with one copy of the transgene show kidney cysts and die ~ 2-weeks while high-copy ones have no phenotype as opposed to Pkd1-/-; Pkd1TAG who eventually develop cyst after 1 year. The genetic and biochemical results demonstrate that Pc-1Δcoiled-coil is hypomorphic. Although Pc-1Δcoiled-coil undergoes its autoproteolytic cleavage, analysis of the intracellular transport of Pc-1Δcoiled-coil shows a delay in maturation. While Pc-1 and Pc-2 appear to interact in vitro through their coiled-coil motif and co-transport, a Pc-1Δcoiled-coil -Pc-2 interaction is conserved in the kidney, suggesting a distinct mechanism of interactions in vivo. Thus, our results show that the coiled-coil motif of Pc-1 in vivo is involved in the maturation independently of Pc-2, highlighting the existence of a critical partner in intracellular transport.

No description available.

Detta kandidatexamensarbete är en förstudie utfört på Cementa AB:s fabrik i Slite, Gotland, och grundar sig i att Cementa AB, Sveriges enda tillverkare av cement, står inför en omställning till helhetskonceptet Industri 4.0 som bygger på tekniker och principer för automation och digitalisering. Fabriken står även inför en hållbarhetsomställning, då branschen bidrar med stora koldioxidutsläpp genom sin tillverkning. Målet med kandidatexamensarbetet är att analyseravar Cementa AB står inför en Industri 4.0 omställning och även undersöka om det har någon koppling till deras hållbarhetsarbete. För att kunna avgöra utgångsläget för fabriken har studien konstruerat och använt sig av en mognadsmodell som bedömer vart företaget står inför en framtida omställning. Mognadsmodellen baseras på fem olika mognadsmodeller och innehåller dimensionerna Produktion, Teknik, Strategi, R&amp;D, HR, Ledarskap, Kultur och Standardisering. Undersökningen av företagets mognad genomfördes med hjälp av en enkät, som distribuerades till fabrikens alla avdelningar. Svarsfrekvensen var på 82%. Enkätens frågor har svarsalternativ som är bedömda med poäng där lägst poäng innebär frånvaro av Industri 4.0 och högsta poäng innebär mognad inom Industri 4.0. Till varje dimensions mognad beräknades ett mognadsindex. Resultatet av enkäten analyserades med avseende på vardera dimensions avvikande mognadsobjekt för att belysa styrkor samt se potentialen inom varje dimension. Dimensionerna med lägst mognadsindex var HR och Strategi. Fabrikens totala mognad inom Industri 4.0 beräknas till nivån Existens som innebär att företaget har ett påbörjat arbete inom Industri 4.0 men saknar strategi, målbild och plan för vidare arbete. För att ytterligare belysa fabrikens möjligheter konstrueras en lista med de frågor som har störst potential. I denna lista belyses dimensionerna Strategi och Ledarskap som mest frekvent återkommande och med det anses arbetet med strategi och ledning fundamental för omställningens existens. Detta gäller även kopplingen till hållbarhetsarbetet, då ett tydligt fokus på hållbarhetsaspekterna kopplat med Industri 4.0-teknologier ger synergieffekter som hjälper varandra. Studien rekommenderar företaget i ett första skede att initiera en tvärfunktionell arbetsgrupp på ledningsnivå som har som uppgift att driva omställningen och fokuserar i ett första stadie på strategi och ledning. Sedan rekommenderar studien också att företaget i valfri ordning utvecklar enkäten för vidare uppföljning och kartläggning, jämföra sig med liknande fabriker, etablera samarbeten mellan företag, akademi och leverantörer, utforma en Roadmap för företaget och dess avdelningar samt fortsätta digitalisera verksamhetens system och strukturer. / This bachelor thesis is a case study that has been carried out at Cementa AB’s factory in Slite, Gotland, and targeted the Cementa AB’s future adaption to the concept Industry 4.0 which is based on techniques and principles for automation and digitalization. The factory is also facing a sustainability change, as the industry contributes to large carbon dioxide emissions through its manufacturing. The aim of the bachelor’s degree project is to analyze where Cementa AB is facing an Industry 4.0 transition and also to investigate whether it has any connection to their sustainability work. To be able to determine the starting position for the factory, the study has constructed and used a maturity model that assesses where the company is facing a future transition. The maturity model is based on five different maturity models and contains the dimensions Production, Technology, Strategy, R&amp;D, HR, Leadership, Culture and Standardization. The study of the company's maturity was conducted by means of a survey, which was distributed to all the departments of the factory. The response rate was 82%. The survey's questions have response alternatives that are assessed with points where the lowest score means Absence of Industry 4.0 and highest score means Maturity within Industry 4.0. For each dimension maturity, a maturity index was calculated. The results of the survey were analyzed with respect to the different maturity objects of each dimension in order to highlight strengths and see the potential within each dimension. The dimensions with the lowest maturity index were HR and Strategy. The factory's total maturity in Industry 4.0 is calculated to the Existence level, which means that the company has started work in Industry 4.0 but lacks strategy, goals and plans for further work. To further clarify the factory's possibilities, a list of the issues with the greatest potential is constructed. In this list, the dimensions Strategy and Leadership are highlighted as the most frequent recurring, and with this the work with strategy and leadership is considered fundamental to the existence of the transition. This also applies to the link to sustainability work, as a clear focus on the sustainability aspects linked to Industry 4.0 technologies produces synergies that help each other. The study recommends the company, in a first stage, to initiate a cross-functional working group at management level which has the task of driving the transition and focuses in a first stage on strategy and management. The study also recommends that the company in any arrangements develops the survey for further follow-up and mapping, compares itself with similar factories, establishes collaborations between companies, academia and suppliers, design a roadmap for the company and its departments, and continue to digitize the company's systems and structures.

Aggregation of microtubule-associated protein tau is involved in the pathology of several neurodegenerative diseases, including Alzheimer’s disease. The affibody TP4 has been shown to inhibit this aggregation process, and its target-binding positions were previously grafted onto a dimeric affibody scaffold, creating the sequestrin seqTP4. This project constitutes a part of the affinity maturation process of seqTP4, using two different bacterial display methods. An error-prone PCR library was first expressed on Staphylococcus carnosus cells for selection of variants with improved tau-binding properties, resulting in a library of 1.4×107 transformants. Flow cytometric tests indicated difficulties in the setup due to nonspecific interactions, and whereas several different approaches to alleviate the problems were investigated, two cell sorting attempts were ultimately unsuccessful. Subcloning of seqTP4 and the library to an Escherichia coli surface display vector resulted in functional surface expression of seqTP4 on E. coli JK321 and BL21 cells, and a BL21 library size of 1.6×109 transformants. An initial flow cytometric test of this library indicates the presence of improved tau-binding variants, paving the way for future cell sorting. / Aggregering av mikrotubuli-associerat protein tau är involverad i patologin av flera neurodegenerativa sjukdomar, däribland Alzheimers sjukdom. Affibodymolekylen TP4 har visat sig inhibera denna aggregeringsprocess, och överföring av dess målbindande positioner till ett dimeriskt affibodyprotein har tidigare gett upphov till seqTP4, en så kallad sequestrin. Detta projekt utgör ett led i processen att affinitetsmaturera seqTP4, med hjälp av två olika metoder för presentation av proteiner på ytan av bakterieceller. Ett error-prone PCR-bibliotek uttrycktes först på ytan av Staphylococcus carnosus-celler för selektion av varianter med ökad affinitet för tau, vilket resulterade i ett bibliotek av 1.4×107 transformanter. Flödescytometriska tester tydde på svårigheter i detta upplägg på grund av ospecifika interaktioner, och emedan flera olika angreppssätt för att förmildra dessa problem undersöktes, misslyckades slutligen två cellsorteringsförsök. Omkloning av seqTP4 och biblioteket till en vektor för ytpresentation på Escherichia coli resulterade i funktionellt ytuttryck av seqTP4 på E. coli JK321- och BL21-celler, och ett BL21-bibliotek bestående av 1.6×109 transformanter. Ett första flödescytometriskt test av detta bibliotek tyder på närvaron av varianter med förbättrad förmåga att binda tau, och vägen ligger nu relativt öppen för cellsortering.

<p> The age at which a juvenile female reaches sexual maturity can be modulated by a variety of environmental and social factors. Experiments described in this thesis were designed to enhance the current understanding of the relationships among three variables that influence the onset of sexual maturation in female mice (Mus musculus), including: [1] exposure to dietary phytoestrogens during development, [2] variations in prenatal androgens, and [3] the presence or absence of genetically-unrelated males after weaning. For the first time, age at onset of male-induced female puberty was investigated using non-invasive behavioural and fertility measures. Through enzyme immunoassay procedures, daily output of urinary creatinine, 17P-estradiol, and progesterone was profiled in developing females that were either isolated or exposed to adult males. Uterine and ovarian tissue was also measured in such females, and male exposure was observed to increase reproductive tissue mass and was influenced by prior androgen exposure in interaction with diet and male presence. Male-exposed females fed a diet containing phytoestrogens immediately became sexually receptive when housed directly with males, and they conceived earlier than females in other conditions. Females with longer anogenital distance, which reflects higher in utero androgen exposure, displayed more escape attempts and aggressive posturing in the direct presence of males, especially when they had been housed near males and fed the phytoestrogen-containing diet. Urinary 17P-estradiol was substantially reduced in females raised on the phytoestrogenfree diet. Urinary output of progesterone was not strongly influenced by diet. Maleexposed females ' output of progesterone and 17P-estradiol was more dynamic in comparison to that of isolated females. The size of this effect depended on diet, prior androgen exposure, and whether urinary steroid measures were adjusted by urinary creatinine. Urinary creatinine was elevated by the low phytoestrogen diet and reduced by male exposure. These data suggest that dietary phytoestrogens and in utero androgen exposure interact with presence or absence of males in determining the age at onset of sexual maturity in developing females. </p> <p> A final experiment was designed to examine two components of adult male urine, preputial gland emissions and unconjugated estrogens, that have been posited to act on females to advance reproductive maturation. Intact and preputialectomized males were compared in their output of urinary creatinine, 17~-estradiol, and testosterone, and in their influence on reproductive tissue in juvenile females. Lack of preputial glands did not hinder the capacity of males to induce uterine and ovarian growth in females. Male urinary creatinine was reduced by exposure to juvenile females. Creatinine-adjusted 17~estradiol and testosterone were greater in female-exposed males, regardless of whether the preputial glands were present. Based on these findings and those reported elsewhere, it is probable that male excreted urinary steroids are important in regulating reproductive changes in developing females exposed to males. </p> / Thesis / Doctor of Philosophy (PhD)