miRNAS circulantes como biomarcadores para fibrilação atrial aguda

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Some non-coding RNAs (miRNAs) have been involved in regulatory activity in arrhythmogenesis, targeting genes that contribute to the development of AF. The present study aimed to evaluate the expression of candidate miRNAs in plasma from patients with AF and new-onset AF and its application as future markers for diagnosis and monitoring of disease. miR-21, miR-133a, miR-133b, miR-150, miR-328 and miR-499 were selected as targets in this study through a prior literature review. They were isolated from plas-ma of individuals aged from 20 to 85 years old with AF (n = 17), new-onset AF (n = 5) and without AF (n = 15), where the latter was the control group. The results were ana-lyzed by Real-Time PCR (RT-PCR) with miScript SYBR Green PCR. We observed that miR-21, miR-133b, miR-328 and miR-499 had different levels of expression be-tween the three groups (p <0.05). Increased expression of miR-21 (0.6-fold), miR-133b (1.4-fold), miR-328 (2.0-fold) and miR-499 (2.3-fold) in patients with new-onset AF when compared to AF and control subjects. The miR-133a and miR-150 expression did not differ among the groups. miR-21, miR-133b, miR-328 and miR-499 may be potential biomarkers for AF as well as for new-onset AF, for monitoring and for the di-agnosis. These findings may contribute to the understanding of the process that trig-gers AF and suggest application these molecules as future biomarkers for AF.

Acurácia dos indicadores clínicos do diagnóstico de enfermagem falta de adesão em pessoas vivendo com AIDS

In the early 1990s, a major milestone in the treatment of Acquired Immune Deficiency Syndrome was the development of highly active combination antiretroviral therapy. The great benefit generated by the use of this therapy was prolonging the survival of the people who got this disease, since it is no longer considered fatal, becoming a chronic condition. Despite improvements generated by this therapy, there are still many difficulties to be overcome. One is the patient adherence to their treatment, bringing challenges to services and health professionals. Hence the need for early identification of nursing diagnosis Lack of Accession so that solutions are sought by the nurse with the patient and his family. With this problem, adds to the difficulty of hospital nurses in inferring that diagnosis, especially in identifying their defining characteristics. In this context, the objective was to evaluate the accuracy of clinical indicators of nursing diagnosis Lack of Adherence to antiretroviral treatment for people living with the Acquired Immunodeficiency Syndrome. The research took place in two stages. The first consists of the evaluation of the diagnostic indicators in the study; and second, the diagnostic inference performed by specialist nurses. The first step took place in a referral hospital in the treatment of infectious diseases in the Northeast of Brazil, and data were collected through an instrument for carrying out history and physical examination and analyzed for the presence or absence of the diagnostic indicators. In the second stage, the data were sent to experts, who judged the presence or absence of the diagnosis in the studied clientele. The project was submitted to the Ethics Committee of the Federal University of Rio Grande do Norte, obtaining approval with the General Certificate for Ethics Assessment (CAAE) No 46206215.3.0000.5537. Data were analyzed using descriptive and inferential statistics. Test were used Fisher's exact, chi-square test of Pearson and logistic regression. Since the accuracy of clinical indicators was measured by sensitivity, specificity, predictive values, likelihood ratios. As a result, we identified the presence of diagnosis Lack of Accession on 69% (n = 78) of the study patients. The defining characteristics that showed statistically significant association with the diagnosis studied were: lack of adherence behavior, complications related to development, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. The characteristic with greater sensitivity was missing scheduled appointments and the highest specificity behavior of noncompliance. The logistic regression showed as predictors for the diagnosis Lack of Accession: lack of adherence behavior, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. It was concluded that the identification of clinical indicators accurately enabled a good prediction of the nursing diagnosis Lack of Accession on people living with the Acquired Immune Deficiency Syndrome, helping nurses develop early on strategies for promoting adherence to the use of antiretrovirals.

Acurácia dos indicadores clínicos do diagnóstico de enfermagem falta de adesão em pessoas vivendo com AIDS

In the early 1990s, a major milestone in the treatment of Acquired Immune Deficiency Syndrome was the development of highly active combination antiretroviral therapy. The great benefit generated by the use of this therapy was prolonging the survival of the people who got this disease, since it is no longer considered fatal, becoming a chronic condition. Despite improvements generated by this therapy, there are still many difficulties to be overcome. One is the patient adherence to their treatment, bringing challenges to services and health professionals. Hence the need for early identification of nursing diagnosis Lack of Accession so that solutions are sought by the nurse with the patient and his family. With this problem, adds to the difficulty of hospital nurses in inferring that diagnosis, especially in identifying their defining characteristics. In this context, the objective was to evaluate the accuracy of clinical indicators of nursing diagnosis Lack of Adherence to antiretroviral treatment for people living with the Acquired Immunodeficiency Syndrome. The research took place in two stages. The first consists of the evaluation of the diagnostic indicators in the study; and second, the diagnostic inference performed by specialist nurses. The first step took place in a referral hospital in the treatment of infectious diseases in the Northeast of Brazil, and data were collected through an instrument for carrying out history and physical examination and analyzed for the presence or absence of the diagnostic indicators. In the second stage, the data were sent to experts, who judged the presence or absence of the diagnosis in the studied clientele. The project was submitted to the Ethics Committee of the Federal University of Rio Grande do Norte, obtaining approval with the General Certificate for Ethics Assessment (CAAE) No 46206215.3.0000.5537. Data were analyzed using descriptive and inferential statistics. Test were used Fisher's exact, chi-square test of Pearson and logistic regression. Since the accuracy of clinical indicators was measured by sensitivity, specificity, predictive values, likelihood ratios. As a result, we identified the presence of diagnosis Lack of Accession on 69% (n = 78) of the study patients. The defining characteristics that showed statistically significant association with the diagnosis studied were: lack of adherence behavior, complications related to development, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. The characteristic with greater sensitivity was missing scheduled appointments and the highest specificity behavior of noncompliance. The logistic regression showed as predictors for the diagnosis Lack of Accession: lack of adherence behavior, missing scheduled appointments, failure to achieve results, and exacerbation of symptoms. It was concluded that the identification of clinical indicators accurately enabled a good prediction of the nursing diagnosis Lack of Accession on people living with the Acquired Immune Deficiency Syndrome, helping nurses develop early on strategies for promoting adherence to the use of antiretrovirals.

Winter lovers: engajamento e participação no universo de game of thrones da internet

The formation of groups acquired new features with the arrival of the internet. The links before straitened between family, work groups and close friends today reach long distances through online communities. These communities represent groups that have affinities and common interests, and use the community space to discuss these. Examples of these communities are those related to franchise Game of Thrones, a literary phenomenon that has expanded by various media, including the social, television, and communities. This report aims to present the work steps and the theoretical reflection, necessary for the achievement of a final product in file format, which aimed to measure the engagement and participation of GOT fans on the Internet, especially in two Facebook GOT communities during the fifth season of the series aired on HBO.

microRNA Regulation of Cellular Immunity

Immunity is broadly defined as a mechanism of protection against non-self entities, a process which must be sufficiently robust to both eliminate the initial foreign body and then be maintained over the life of the host. Life-long immunity is impossible without the development of immunological memory, of which a central component is the cellular immune system, or T cells. Cellular immunity hinges upon a naïve T cell pool of sufficient size and breadth to enable Darwinian selection of clones responsive to foreign antigens during an initial encounter. Further, the generation and maintenance of memory T cells is required for rapid clearance responses against repeated insult, and so this small memory pool must be actively maintained by pro-survival cytokine signals over the life of the host.

T cell development, function, and maintenance are regulated on a number of molecular levels through complex regulatory networks. Recently, small non-coding RNAs, miRNAs, have been observed to have profound impacts on diverse aspects of T cell biology by impeding the translation of RNA transcripts to protein. While many miRNAs have been described that alter T cell development or functional differentiation, little is known regarding the role that miRNAs have in T cell maintenance in the periphery at homeostasis.

In Chapter 3 of this dissertation, tools to study miRNA biology and function were developed. First, to understand the effect that miRNA overexpression had on T cell responses, a novel overexpression system was developed to enhance the processing efficiency and ultimate expression of a given miRNA by placing it within an alternative miRNA backbone. Next, a conditional knockout mouse system was devised to specifically delete miR-191 in a cell population expressing recombinase. This strategy was expanded to permit the selective deletion of single miRNAs from within a cluster to discern the effects of specific miRNAs that were previously inaccessible in isolation. Last, to enable the identification of potentially therapeutically viable miRNA function and/or expression modulators, a high-throughput flow cytometry-based screening system utilizing miRNA activity reporters was tested and validated. Thus, several novel and useful tools were developed to assist in the studies described in Chapter 4 and in future miRNA studies.

In Chapter 4 of this dissertation, the role of miR-191 in T cell biology was evaluated. Using tools developed in Chapter 3, miR-191 was observed to be critical for T cell survival following activation-induced cell death, while proliferation was unaffected by alterations in miR-191 expression. Loss of miR-191 led to significant decreases in the numbers of CD4+ and CD8+ T cells in the periphery lymph nodes, but this loss had no impact on the homeostatic activation of either CD4+ or CD8+ cells. These peripheral changes were not caused by gross defects in thymic development, but rather impaired STAT5 phosphorylation downstream of pro-survival cytokine signals. miR-191 does not specifically inhibit STAT5, but rather directly targets the scaffolding protein, IRS1, which in turn alters cytokine-dependent signaling. The defect in peripheral T cell maintenance was exacerbated by the presence of a Bcl-2YFP transgene, which led to even greater peripheral T cell losses in addition to developmental defects. These studies collectively demonstrate that miR-191 controls peripheral T cell maintenance by modulating homeostatic cytokine signaling through the regulation of IRS1 expression and downstream STAT5 phosphorylation.

The studies described in this dissertation collectively demonstrate that miR-191 has a profound role in the maintenance of T cells at homeostasis in the periphery. Importantly, the manipulation of miR-191 altered immune homeostasis without leading to severe immunodeficiency or autoimmunity. As much data exists on the causative agents disrupting active immune responses and the formation of immunological memory, the basic processes underlying the continued maintenance of a functioning immune system must be fully characterized to facilitate the development of methods for promoting healthy immune function throughout the life of the individual. These findings also have powerful implications for the ability of patients with modest perturbations in T cell homeostasis to effectively fight disease and respond to vaccination and may provide valuable targets for therapeutic intervention.

MicroRNA expression and function in neonatal hypoxic ischaemic encephalopathy

Hypoxic ischaemic encephalopathy (HIE) is a devastating neonatal condition which affects 2-3 per 1000 infants annually. The current gold standard of treatment - induced hypothermia, has the ability to reduce neonatal mortality and improve neonatal morbidity. However, to be effective it needs to be initiated within the therapeutic window which exists following initial insult until approximately 6 hours after birth. Current methods of assessment which are relied upon to identify infants with HIE are subjective and unreliable. To overcome this issue, an early and reliable biomarker of HIE severity must be identified. MicroRNA (miRNA) are a class of small non-coding RNA molecules which have potential as biomarkers of disease state and potential therapeutic targets. These tiny molecules can modulate gene expression by inhibiting translation of messenger RNA (mRNA) and as a result, can regulate protein synthesis. These miRNA are understood to be released into the circulation during cellular stress, where they are highly stable and relatively easy to quantify. Therefore, these miRNAs may be ideal candidates for biomarkers of HIE severity and may aid in directing the clinical management of these infants. By using both transcriptomic and proteomic approaches to analyse the expression of miRNAs and their potential targets in the umbilical cord blood, I have confirmed that infants with perinatal asphyxia and HIE have a significantly different UCB miRNA signature compared to UCB samples from healthy controls. Finally, I have identified and investigated 2 individual miRNAs; both of which show some potential as classifiers of HIE severity and predictors of long term outcome, particularly when coupled with their downstream targets. While this work will need to be validated and expanded in a new and larger cohort of infants, it suggests the potential of miRNA as biomarkers of neonatal pathological conditions such as HIE.

IMMUNE-ANGIOGENESIS MECHANISMS ASSOCIATED WITH PORCINE PREGNANCY SUCCESS AND FAILURE

Spontaneous fetal loss (25-40%) leading to decrease in litter size is a significant concern to the pork industry. A deficit in the placental vasculature has emerged as one of the important factors associated with fetal loss. During early pig pregnancy, the endometrium becomes enriched with immune cells recruited by conceptus-derived signals including specific chemokine stimuli. These immune cells assist in various aspects of placental development and angiogenesis. Recent evidence suggests that microRNAs (miRNAs: small non-coding RNAs that regulate gene expression) regulate immune cell development and their functions. In addition, intercellular communication including exchange of biomolecules (e.g. miRNAs) between the conceptus and endometrium regulate key developmental processes during pregnancy. To understand the biological significance of immune cell enrichment, regulation of their functions by miRNAs and transfer of miRNAs across the maternal fetal-interface, we screened specific sets of chemokines and pro- and anti-angiogenic miRNAs in endometrial lymphocytes (ENDO LY), endometrium, and chorioallantoic membrane (CAM) isolated from conceptus attachment sites (CAS) during early, gestation day (gd)20 and mid-pregnancy (gd50). We report increased expression of selected chemokines including CXCR3 and CCR5 in ENDO LY and CXCL10, CXCR3, CCL5, CCR5 in endometrium associated with arresting CAS at gd20. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5, and CCR5) in endometrium and CAM. We report for the first time significant differences for miRNAs involved in immune cell-derived angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between ENDO LY associated with healthy and arresting CAS. Significant differences were also found in endometrium and CAM for some miRNAs (miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, we confirm that placenta specific-exosomes contain proteins and 14 select miRNAs including miR-126-5P, miR-296-5P, miR-16, and miR-17-5P that are of relevance to early implantation events. We further demonstrated the bidirectional exosome shuttling between porcine trophectoderm cells (PTr2) and porcine aortic endothelial cells (PAOEC). PTr2-derived exosomes were able to modulate the endothelial cell proliferation that is crucial for the establishment of pregnancy. Our data unravels the selected chemokines and miRNAs associated with immune cell-regulated angiogenesis and reconfirm that exosome mediated cell-cell communication opens-up new avenues to understand porcine pregnancy.

Circulating miRNAs: Potential Novel Biomarkers for Hepatopathology Progression and Diagnosis of Schistosomiasis Japonica in Two Murine Models

BACKGROUND: Schistosomiasis remains a major public health issue, with an estimated 230 million people infected worldwide. Novel tools for early diagnosis and surveillance of schistosomiasis are currently needed. Elevated levels of circulating microRNAs (miRNAs) are commonly associated with the initiation and progression of human disease pathology. Hence, serum miRNAs are emerging as promising biomarkers for the diagnosis of a variety of human diseases. This study investigated circulating host miRNAs commonly associated with liver diseases and schistosome parasite-derived miRNAs during the progression of hepatic schistosomiasis japonica in two murine models.

METHODOLOGY/PRINCIPAL FINDINGS: Two mouse strains (C57BL/6 and BALB/c) were infected with a low dosage of Schistosoma japonicum cercariae. The dynamic patterns of hepatopathology, the serum levels of liver injury-related enzymes and the serum circulating miRNAs (both host and parasite-derived) levels were then assessed in the progression of schistosomiasis japonica. For the first time, an inverse correlation between the severity of hepatocyte necrosis and the level of liver fibrosis was revealed during S. japonicum infection in BALB/c, but not in C57BL/6 mice. The inconsistent levels of the host circulating miRNAs, miR-122, miR-21 and miR-34a in serum were confirmed in the two murine models during infection, which limits their potential value as individual diagnostic biomarkers for schistosomiasis. However, their serum levels in combination may serve as a novel biomarker to mirror the hepatic immune responses induced in the mammalian host during schistosome infection and the degree of hepatopathology. Further, two circulating parasite-specific miRNAs, sja-miR-277 and sja-miR-3479-3p, were shown to have potential as diagnostic markers for schistosomiasis japonica.

CONCLUSIONS/SIGNIFICANCE: We provide the first evidence for the potential of utilizing circulating host miRNAs to indicate different immune responses and the severity of hepatopathology outcomes induced in two murine strains infected with S. japonicum. This study also establishes a basis for the early and cell-free diagnosis of schistosomiasis by targeting circulating schistosome parasite-derived miRNAs.

Mechanismen kleiner regulatorischer RNAs in Dictyostelium discoideum: Charakterisierung und Anwendung

Die RNA-Interferenz (RNAi) ist ein in Eukaryoten weit verbreiteter Mechanismus, der die transkriptionelle oder posttranskriptionelle Stilllegung von Genen beschreibt. Die Spezifität wird dabei durch die Sequenz einer kleinen, nicht-kodierenden RNA gewährleistet. Diese RNA leitet einen Effektorkomplex, dessen Zentrum immer von einem Argonautenprotein gebildet wird, üblicherweise zu einer komplementären mRNA. In der Folge kommt es zum Abbau des Transkripts oder zur Inhibierung der Translation. Aktuelle Veröffentlichungen konnten zudem das Aktivitätsprofil der Argonautenproteine beträchtlich erweitern: Im Zellkern vorkommende Argonautenproteine wurden beispielsweise mit Spleißvorgängen, der Promotorkontrolle von Genen und der DNA-Reparatur in Verbindung gebracht. In den letzten Jahren konnten weitreichende Kenntnisse bezüglich der Kontrolle einiger transposabler Elemente durch RNAi sowie der Biogenese kleiner regulatorischer RNAs in Dictyostelium discoideum und anderen Organismen gewonnen werden. Ein Fokus dieser Arbeit lag zunächst auf der Charakterisierung des Argonautenproteins AgnB und der Identifikation von Interaktionspartnern. Es konnte gezeigt werden, dass AgnB zumindest partiell im Zellkern der Amöbe lokalisiert und dort vermutlich regulatorische Aufgaben wahrnimmt. Gestützt wurde diese Annahme durch die massenspektrometrische und Western Blot basierte Detektion nukleärer Bindungspartner. Weiterführende Analysen konnten AgnB zudem als positiven Regulator für drei entwicklungsregulierte Gene beschreiben und so die Verbindung zum Prozess der RNA activation in der Amöbe herstellen. Identifizierte posttranslationale Modifikationen könnten diesbezüglich die Aktivität des Argonauten steuern. Mit Hilfe von Crosslink-RNA-Immunopräzipitation und anschließender Hochdurchsatz-sequenzierung oder der Northern Blot basierten Auswertung konnte eine Assoziation von AgnB und der Class I RNAs gezeigt werden. Diese Klasse umfasst nicht-kodierende RNAs mit einer Länge von etwa 42 bis 65 Nukleotiden und wurde bisher nicht als Substrat für die RNAi-Maschinerie in D. discoideum angesehen. Ein weiterer Teil dieser Arbeit beschäftigte sich mit dem Einfluss von AgnA und AgnB auf die Promotorbereiche des D. discoideum Retrotransposons DIRS-1. Im Verlauf der Untersuchungen konnte beobachtet werden, dass die Anordnung entgegengesetzt operierender DIRS-1 Promotor-sequenzen für die Stilllegung eines Reportergens ausreichend war. Darauf aufbauend konnte ein DIRS-1 basiertes knockdown System etabliert werden. Mit Hilfe dieses Systems konnten die Proteinmengen ausgewählter Zielgene so effektiv reduziert werden, dass die entsprechenden Stämme den Phänotyp des korrespondierenden knockout Stammes zeigten. Darüber hinaus war es möglich die Proteinreduktion zu modulieren, um so beispielsweise dosisabhängige Effekte zu registrieren. Vorangegangene Arbeiten zur Biogenese von micro (mi)RNAs konnten das RNA-bindende Protein RbdB als eine Hauptkomponente für die Prozessierung maturer miRNAs identifizieren. Der miRNA defiziente RbdB- Stamm wurde in dieser Arbeit zur Identifikation putativer miRNA Ziele genutzt. Dazu wurde sowohl das Transkriptom des D. discoideum Wildtyps als auch des rbdB knockout Stammes in hohem Durchsatz sequenziert, um so differentiell exprimierte Gene zu detektieren. Vielversprechende Kandidaten wurden mittels qRT-PCR verifiziert. Dabei wurde unter anderem ein putativer Transkriptionsfaktor als miRNA target identifiziert, der eine Vielzahl weiterer Gene regulieren könnte. Abschließend konnte in dieser Arbeit gezeigt werden, dass RbdB ebenfalls für die Generierung von small interfering (si)RNAs aus strukturierten Loci von Bedeutung ist. Dies weist auf mindestens zwei unterschiedliche Mechanismen zur siRNA Prozessierung in D. discoideum hin.

Étude de l'expression des microARNs et des enzymes de synthèse des corticostéroïdes dans le développement pulmonaire

Le syndrome de détresse respiratoire du nouveau-né (SDR) est l’une des pathologies les plus fréquentes dont souffrent les bébés prématurés. Le SDR est causé par un déficit dans la synthèse du surfactant pulmonaire en raison de l’immaturité du poumon lors d’une naissance prématurée. Plusieurs éléments régulent le développement pulmonaire notamment les stéroïdes sexuels et les corticostéroïdes. Le sexe est aussi un élément régulateur du développement pulmonaire. En effet, les garçons sont plus atteints que les filles par le SDR. Ce dimorphisme sexuel est attribué aux androgènes. Le traitement anténatal aux glucocorticoïdes est prescrit aux femmes qui sont à risque d’accoucher prématurément. En effet, les corticostéroïdes favorisent la maturation pulmonaire anténatale. Également, il a été démontré que les microARNs sont primordiaux pour le développement pulmonaire. Ceci nous a conduit à étudier l’impact des androgènes sur le profil d’expression des microARNs lors de la transition du stade canaliculaire au stade sacculaire (jour gestationnel (JG)17.0 au JG18.0), période qui coïncide avec la montée de la synthèse et de la sécrétion du surfactant chez la souris. Tout d’abord, nous avons étudié la stabilité des gènes de normalisation (snoRNAs) afin de quantifier les microARNs par qPCR. Cette analyse a été effectuée avec 3 logiciels différents et sur plusieurs stades du développement notamment de la période pseudoglandulaire jusqu’au stade alvéolaire chez les deux sexes. On a identifié les meilleures combinaisons de gènes de normalisation les plus stables pour chaque stade du développement étudié ainsi que pour la période couvrant tous les stades étudiés. Ensuite nous avons analysé à GD17.0 et GD18.0 le profil d’expression des microARNs chez des fœtus mâles dont les mères ont été traitées au flutamide (anti-androgènes pure). Les résultats ont montré que 43 microARNs matures sont modulés par les androgènes à GD17.0 et 35 microARNs à GD18.0. Pour certains microARNs, nous avons identifié des cibles potentielles qui sont inversement modulées par les androgènes par rapport aux microARNs. Ces cibles sont impliquées dans plusieurs processus biologiques tels que le métabolisme des lipides et la prolifération cellulaire ainsi que dans des fonctions moléculaires tels que la liaison des facteurs de transcription. Des expériences de validation ont été effectuées par qPCR. Nos résultats ont montré que les androgènes régulent des processus qui peuvent être impliqués dans la maturation pulmonaire via la régulation des microARNs. En plus de l’intérêt porté aux androgènes dans la maturation pulmonaire, nous avons analysé l’expression d’enzymes de synthèse des corticostéroïdes dans le poumon fœtal humain. L’expression de l’enzyme 21-hydroxylase a été étudiée par qPCR et par immunobuvardage. Également la localisation de l’ARNm de cette enzyme clé de la synthèse des glucocorticoïdes, a été effectuée par hybridation in situ. L’ARNm de CYP21A2 a été détecté par qPCR dans les 34 échantillons analysés et dont les âges variaient entre 17 et 40 semaines de grossesse. Aucune corrélation, avec l’âge gestationnel ou le sexe, n’a été observée. Des niveaux significatifs de la protéine 21-hydroxylase ont été détectés dans nos échantillons. Nous avons investigué l’expression d’autres enzymes impliquées dans la voie de synthèse des glucocorticoïdes notamment CYP11B1, CYP11B2 et CYP17A1. Les ARNm des gènes CYP11B1, CYP11B2 n’ont pas été détectés dans nos échantillons, contrairement à CYP17A1 dont l’ARNm a été détecté dans tous nos tissus fœtaux analysés. La protéine de la 17α-hydroxylase a été détectée à de faibles niveaux. Nos résultats d’hybridation in situ ont montré que l’expression de CYP21A2 est localisée presqu’exclusivement dans l’épithélium pulmonaire distal. Nos résultats suggèrent que les produits de la 21-hydroxylase agiront via une action intracrine sur l’épithélium distal en activant le récepteur des glucocorticoïdes (GR). L’activation du récepteur des minéralocorticoïdes (MR) ne semble pas dépendre de produits de la 21-hydroxylase en raison des quantités importantes d’aldostérone circulante.

MicroRNA-induced polarization of microglia and macrophages after focal cerebral ischemia

Der ischämische Schlaganfall ist nicht nur die zweithäufigste Todesursache weltweit, sondern auch eine der Hauptursachen für körperliche Beeinträchtigungen im Erwachsenenalter. Das Ausmaß der durch den Schlaganfall hervorgerufenen Gewebeschädigung ist stark durch das Immunsystem geprägt. Die im Zentralnervensystem (ZNS) ansässigen Mikroglia und die aus dem Blutsystem infiltrierenden Makrophagen sind die Schlüsselzellen der lokalen und systemischen Entzündungsantwort nach dem ischämischen Schlaganfall. Sowohl Mikroglia als auch Makrophagen spielen in der Entwicklung der Gewebeschädigung eine duale Rolle. Zum einen phagozytieren sie Zelltrümmer und unterstützen neuroregenerative Prozesse, zum anderen sind diese Zellen in der Lage den Zustand der Gewebsschädigung zu verschlimmern und einer Regeneration des ZNS entgegenzuwirken. Die Polarisierung der Mikroglia/Makrophagen hin zu verschiedenen Phänotypen ist abhängig von der jeweiligen Phase der Gewebeschädigung. Der destruktive, proinflammatorische Phänotyp (M1) steht dabei dem protektiven, antiinflammatorischen Phänotyp (M2) gegenüber. Die Notwendigkeit einer zielgerichteten Regulierung der polarisierten Mikroglia/Makrophagen zum protektiven M2-Phänotyp wurde bereits mehrfach im Zusammenhang mit der Behandlung von neurodegenerativen Erkrankungen erwähnt. In der vorliegenden Dissertation soll die immunregulierende und neuroprotektive Wirkung der microRibonukleinsäure-124 (miRNA-124) in Bezug auf die Polarisierung von Mikroglia/Makrophagen zu verschiedenen Zeitpunkten nach Verschluss der Arteria cerebri media (ACM) im Gehirn von Mäusen gezeigt werden. Zu diesem Zweck wurde die liposomierte miRNA-124 zu einem frühen Zeitpunkt (Tag 2) und zu einem späten Zeitpunkt (Tag 10) nach Verschluss der ACM verabreicht. Die Behandlung mit der miRNA-124 zu einem frühen Zeitpunkt resultierte dabei in einem signifikanten Anstieg in der Anzahl der M2-positiven Mikroglia/Makrophagen im Vergleich zur Kontrollgruppe. Gleichzeitig nahm die Anzahl der M1-positiven Mikroglia/Makrophagen signifikant ab. Im Wesentlichen resultierte die Behandlung mit der miRNA-124 zu beiden Zeitpunkten in einem geringeren Verhältnis von proinflammatorischen (M1) zu antiinflammatorischen (M2) Mikroglia/Makrophagen. Zu den weiteren Erkenntnissen einer frühzeitigen Behandlung im Rahmen dieser Dissertation gehören: (i) eine signifikante Zunahme des neuronalen Überlebens, das zudem positiv mit der Anzahl der M2-positiven Mikroglia/Makrophagen korreliert, (ii) eine verbesserte funktionelle Erholung, welche in Verbindung mit den veränderten neuroinflammatorischen Ereignissen steht und (iii) ein signifikant verkleinertes Läsionsareal, welches durch reaktive Astrozyten zum gesunden Gewebe hin abgegrenzt wird. Die Ergebnisse dieser Dissertation zeigen, dass die Verabreichung von miRNA-124 eine neue Möglichkeit zur Regulierung der Immunantwort und der Neuroprotektion im Rahmen der Behandlung des ischämischen Schlaganfalls darstellt.

Generation and Characterisation of a Canine EGFP-HMGA2 Prostate Cancer In Vitro Model

The architectural transcription factor HMGA2 is abundantly expressed during embryonic development. In several malignant neoplasias including prostate cancer, high re-expression of HMGA2 is correlated with malignancy and poor prognosis. The let-7 miRNA family is described to regulate HMGA2 negatively. The balance of let-7 and HMGA2 is discussed to play a major role in tumour aetiology. To further analyse the role of HMGA2 in prostate cancer a stable and highly reproducible in vitro model system is precondition. Herein we established a canine CT1258-EGFP-HMGA2 prostate cancer cell line stably overexpressing HMGA2 linked to EGFP and in addition the reference cell line CT1258-EGFP expressing solely EGFP to exclude EGFP-induced effects. Both recombinant cell lines were characterised by fluorescence microscopy, flow cytometry and immunocytochemistry. The proliferative effect of ectopically overexpressed HMGA2 was determined via BrdU assays. Comparative karyotyping of the derived and the initial CT1258 cell lines was performed to analyse chromosome consistency. The impact of the ectopic HMGA2 expression on its regulator let-7a was analysed by quantitative real-time PCR. Fluorescence microscopy and immunocytochemistry detected successful expression of the EGFP-HMGA2 fusion protein exclusively accumulating in the nucleus. Gene expression analyses confirmed HMGA2 overexpression in CT1258-EGFP-HMGA2 in comparison to CT1258-EGFP and native cells. Significantly higher let-7a expression levels were found in CT1258-EGFP-HMGA2 and CT1258-EGFP. The BrdU assays detected an increased proliferation of CT1258-HMGA2-EGFP cells compared to CT1258-EGFP and native CT1258. The cytogenetic analyses of CT1258-EGFP and CT1258-EGFP-HMGA2 resulted in a comparable hyperdiploid karyotype as described for native CT1258 cells. To further investigate the impact of recombinant overexpressed HMGA2 on CT1258 cells, other selected targets described to underlie HMGA2 regulation were screened in addition. The new fluorescent CT1258-EGFP-HMGA2 cell line is a stable tool enabling in vitro and in vivo analyses of the HMGA2-mediated effects on cells and the development and pathogenesis of prostate cancer.

Producción de microtubérculos de papa (Solanum tuberosum L.), cultivar Burren en biorreactores económicos de inmersión temporal

El estudio de microtuberización del cultivar de papa Burren se realizó en el Laboratorio de Cultivo de Tejidos de la Universidad Nacional Agraria entre los meses de junio a diciembre del 2015. Se estudiaron las fases de multiplicación y formación de microtubérculos en Biorreactores Económicos de Inmersión Temporal (BEIT). En la fase de multiplicación se evaluó la formación de plantas completas cuando se sembraron 70 microesquejes en cinco variantes de medios de cultivo. Se empleó un diseño de Bloques Completos al Azar con arreglo unifactorial con réplicas conformadas por 2 BEIT, y para definir las diferencias estadísticas entre las medias (ANDEVA) de los tratamientos se realizó la prueba de rangos múltiples de Duncan-Waller ( = 0.05). En la formación de plantas se evaluaron la longitud de plantas, número de hojas y número de brotes que se les realizó la prueba de medias de diferencia mínima significativa con α = 0.05, se calculó el porcentaje de supervivencia. Como resultado se obtuvo en las variantes de medios de cultivo que contenían 0.20 mg l-1 de GA3 con 0.50 mg l-1 de BAP y 0.10 mg l-1 de GA3 con 1 mg l-1 de BAP, se obtuvieron medias respectivas en número de entrenudos de 6.32-5.7 y en número de hojas por planta de 7.37-6.93. Adiciones de sacarosa de 80 g l-1 y 110 g l-1 favorecieron la formación de microtubérculos de peso fresco con medias entre 0.69-0.75 gramos. La adición de sacarosa entre 80 g l-1 y 120 g l-1 no se registró diferencias estadísticas significativas entre las medias de las variables diámetro y longitud de microtubérculos. Con microtubérculos con diámetro mayor a los 10 mm se obtuvieron medias en longitud de planta, número de hojas y número de brotes de 21, 16.35 y 5.65 cm respectivamente y con diámetros entre 8-10 mm las medias respectivas fueron de 16.37, 11.35 y 5.35 cm.

The role of miRNAs in stroke

Stroke is currently one of the leading causes of death and disability worldwide. Despite recent advances in the treatment of stroke there is a major unmet clinical need for novel therapeutics for intervention. miRNAs are small coding RNAs which act to post-transcriptionally inhibit expression of genes. Emerging evidence has supported the view that miRNAs play an important role in the development and progression of ischaemic stroke, although understanding remains relatively poor. This research uses several models to investigate the effects of miRNAs in the context of stroke in vivo and in vitro, as well as assessment of patient serum samples in order to identify biomarkers for stroke. miR-29b was found to be significantly upregulated in SHRSP rat brain peri-infarct at 72h following stroke, and downregulated in ischaemic core at 24h and 72h following stroke, whilst miR-29c was significantly downregulated in remainder tissue at 24h following stroke and in infarct at 72h following stroke. The upreglation of miR-29b at 72h corresponded to a significant downregulation of miR-29 target genes MMP2, MMP9 and TGF-β1 in peri-infarct tissue at 72h following stroke. Modulation of miR-29b and miR-29c was achieved in a rat neuronal cell line but suppression of genes of interest was not observed following oxygen glucose deprivation. Several candidate miRNAs were then identified by microRNA Openarray analysis in stroke patient serum samples. Validation of these miRNAs was not demonstrated in the population studied, but assessment of these miRNAs in rat serum and isolated exosomes demonstrated that several of these miRNAs were significantly altered in SHRSP rats following stroke. Finally miR-21 was demonstrated to be significantly upregulated in SHRSP rat peri-infarct following stroke. This was associated with a change in miR-21 localization as determined by in situ hybridization. Modulation of miR-21 via the use of CAG-miR-21 mice demonstrated no difference in infarct size as measured by T2 -weighted MRI scan nor was any difference present in behavioural tests versus wild type. KO of miR-21 resulted in a reduction of survival rate compared with wild type. This thesis demonstrates that miR-29 and miR-21 are modulated following stroke in animal models, and these are potential candidates for therapeutic intervention in the future. Analysis of clinical samples has illustrated difficulties in the identification of serum miRNA profiles and suggests that looking at the exosomal component of serum may provide better information regarding miRNA profiles after stroke.

The role of microRNA 21 in the development of in-stent restenosis

Coronary heart disease is a major cause of morbidity and mortality worldwide. Percutaneous coronary intervention (PCI) has become the most widely used method of coronary artery revascularisation. The use of stents to hold open atherosclerosis induced arterial narrowing has significantly reduced elastic recoil and acute vessel occlusion following balloon angioplasty. However, bare metal stents have been associated with in-stent restenosis attributed to vascular smooth muscle cell (VSMC) hyperplasia and excessive neointimal formation. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath. The development of drug eluting stents has significantly reduced the incidence of in-stent restenosis (ISR). Unfortunately the antiproliferative medications used not only inhibit VSMC proliferation but also re-endothelialisation of the stented vessel. In addition, the drug impregnated polymer coating has been associated with a chronic inflammatory response within the vessel wall predisposing patients to stent thrombosis. Thus the identification of novel therapies which promote vessel healing without excessive proliferative or inflammatory response may improve long term outcome and reduce the need for repeated revascularisation. MicroRNAs (miRs) are short (18-25 nucleotide) non-coding RNAs acting to regulate gene expression. By binding to the 3’untranslated region of mRNA they act to fine tune gene expression either by mRNA degradation or translational repression. Originally identified in coordinating tissue development microRNAs have also been shown to play important roles coordinating the inflammatory response and in numerous cardiovascular diseases. MiR-21 has been identified in human atherosclerotic plaques, arteriosclerosis obliterans and abdominal aortic aneurysms. In addition, its up regulation has been documented in preclinical models of vascular injury. This study sought to identify the role of miR-21 in the development of ISR. Utilising a small animal model of stenting and in vitro techniques, we sought to investigate its influence upon VSMC and immune cell response following stenting. 19 The refinement of a murine stenting model within the Baker laboratory and the electrochemical dissolution of the metal stent from within harvested vascular tissues significantly improved the ability to perform detailed histological analysis. In addition, identification of miRNAs using in situ hybridisation was achieved for the first time within stented tissue. Neointimal formation and ISR was significantly reduced in mice in which miR-21 had been genetically deleted. In addition, neointimal composition was found to be altered in miR-21 KO mice with reductions in VSMC and elastin content demonstrated. Importantly, no difference in re-endothelialisation was observed. In vitro analysis demonstrated that VSMCs from miR-21 KO mice had both reduced proliferative and migratory capacity following platelet derived growth factor stimulation. Molecular analysis revealed that these differences may, at least in part, be due to de-repression of programmed cell death 4 (PDCD4). PDCD4 is a known miR-21 target within VSMCs implicated in the suppression of proliferation and promotion of apoptosis. Unfortunately, initial attempts at antimiR mediated knockdown of miR-21 in vivo, failed to produce a similar change in the suppression of ISR. Furthermore, a significant alteration in macrophage polarisation state within the neointima of miR-21 WT and KO mice was noted. Immunohistochemical staining revealed a preponderance of anti-inflammatory M2 macrophages in KO mice. Analysis of bone marrow derived macrophages from miR-21 KO mice demonstrated an increased level of the peroxisome proliferation activating receptor-γ (PPARγ) which facilitates M2 polarisation. Importantly, significant alterations in numerous pro-inflammatory cytokines, which also have mitogenic effects, were also found following genetic deletion of miR-21. In Summary, this is the first study to look at miRs in the development of ISR. MiR-21 plays an important role in the development of ISR by influencing the proliferative response of VSMCs and modulating the immune response following stent deployment. Further attempts to modulate miR-21 expression following PCI may reduce ISR and the need for repeat revascularisation while also reducing the risk of stent thrombosis.