Human Cytomegalovirus (HCMV), the prototypic β-herpesvirus, is the most common cause of congenital infections as well as morbidity and mortality in immunocompromised patients. The anti-HCMV drugs currently available have a number of drawbacks (i.e. detrimental side-effects and/or the appearance of drug resistant strains), which limit their clinical usefulness. Therefore, a better understanding of host-virus interactions is important to develop new, safe and effective ways to treat HCMV. HCMV has evolved various strategies to make the host cell more conducive for the replication process, many of these involve modulation of host signalling pathways through proteins or non-coding RNAs. The focus of this thesis is on the regulation of one class of non-coding RNA, microRNAs (miRNA) by HCMV as well as murine CMV (MCMV). miRNAs are short ~22 nucleotide RNA sequences, which negatively regulate the stability and translational efficiency of specific target messenger RNAs (mRNAs). It has been previously shown that three host-encoded miRNAs, miR-199-3p, miR-199-5p and miR-214, are down-regulated in both MCMV and HCMV infected cells. Despite the biological and genomic differences between the two viruses, this down-regulation occurs in both infections, suggesting a possible conserved antiviral role of the miRNAs in mouse and human cells. Consistent with this, miR-199-3p and miR-214 manifest antiviral properties against MCMV and HCMV when over-expressed in vitro. This thesis investigates two hypotheses: 1) CMV down-regulates the expression of these host miRNAs through a mechanism involving viral factors, 2) The down-regulation of miR-199-3p leads to the up-regulation of its targets and this influences the cell in a way that favours some aspect of the viral life cycle. The first part of this project examined the regulation of miR-199-3p, miR-199-5p, and miR-214, which derive from a single primary transcript (pri-miRNA). The down-regulation of all three miRNAs was found to occur at the transcriptional level by 4 hours post infection. The promoter of the miR-199a/214 cluster was therefore cloned into a reporter vector in order to interrogate the factors regulating transcription of pri-miRNA in infection; this was carried out in the murine model based on availability of reagents. The reduction in the pri-miRNA was found to correlate with a decrease in the transcriptional activity of miR-199a/214 promoter in infected cells. Further analysis revealed the presence of a sequence between -421 to -273 relative to the transcription start site (TSS) that was critical for promoter activity. This sequence contains a putative serum response element (SRE), which includes two binding sites for the SRF dimer (serum response factor) and a binding site for a molecule of TCF (ternary complex factor), ELK-1. Initial knock-down studies suggest that these transcription factors are required for basal activity but it remains unknown whether they are involved in the differential expression of miR-199a/214 observed during infection. Another binding site for the transcription factor TWIST-1 was found outside this region, which is known to regulate the miR-199a/214 cluster in other cell types. Western blot analysis showed reduced expression of TWIST-1 in cells infected with HCMV and MCMV infections, by 24 and 48 hours, respectively, suggesting a role of TWIST-1 in regulating miR-199a/214 cluster during these infections. This regulation seems to be dependent on viral gene expression, as a replication deficient viral mutant fails to repress the promoter function and subsequent pri-miRNA production. Taken together, these results suggest an active viral mechanism for transcriptional repression of the miR-199a/214 promoter. To understand the antiviral function of miR-199-3p, the second part of this thesis examined whether miR-199-3p regulates host signalling pathways important for CMV replication and/or the life cycle. A microarray analysis was carried out with samples from cells transfected with miR- 199-3p mimic versus inhibitor. This revealed 198 genes significantly down-regulated by the miRNA. From the 198 genes, Ingenuity pathway analysis (IPA) software identified several host pathways with a potential role in HCMV infection including: PI3K/AKT signalling, the ERK-MAPK cascade, and prostaglandin production. This thesis examined the role of miR-199-3p in regulating the PI3K/AKT pathway in HCMV infection. It was found that miR-199-3p modulates the phosphorylation of the central regulator of PI3K/AKT signalling, AKT. Transfection of miR-199-3p before the infection impedes the complete phosphorylation of AKT, which is known to be required for the immediate early viral gene expression and replication. This provides an explanation for the antiviral function of miR-199-3p, through its ability to modulate AKT phosphorylation. An open question, however, is how the natural down-regulation of miR-199-3p from 24 to 72 hours post infection naturally affects AKT phosphorylation. Several predicted targets of miR-199-3p, such as PIK3CB, ITGA3, and ITGA6 were shown to be up-regulated at these late time points, correlating with the miR-199-3p down-regulation. The interaction of miR-199-3p with target sites in the 3′UTRs of PIK3CB and ITGA3 was validated by luciferase reporter assays and western blotting and qRT-PCR results indicated that protein and mRNA levels of ITGA6 were regulated by miR-199-3p mimic transfection. However, the knock-down of these three targets did not result in a significant decrease of the viral growth, and thus cannot alone explain the antiviral function of miR-199-3p. Overall, this study suggests that the transcriptional repression of miR- 199a/214 is likely a strategy employed by CMV to support its own growth through attenuating the biological effect of miR-199-3p within the host cell.

Neurodegenerative diseases are a diverse group of progressive diseases that include Alzheimer’s disease (AD) and prion disease. Although these diseases differ in etiology, they share a number of similarities at the molecular level. For instance, microRNA (miRNA), small RNA molecules that post-translationally regulate gene expression, are often differentially regulated during disease. A previous study identified key miRNA that are dysregulated during prion disease in the hippocampus. Of these miRNA, miR-16-5p is of particular interest, as it has also been found to be dysregulated in AD. The objective of this thesis is to characterize the role of miR-16-5p within hippocampal neurons in order to understand its function during neurodegeneration. It is hypothesized that hippocampal miR-16-5p, given its induction in hippocampal neurons during preclinical disease, plays a role in regulating the dendritic remodeling and synaptic pruning that is the earliest pathological feature of neuronal degeneration in prion disease. To address this hypothesis, primary hippocampal neurons were dissected from embryonic day 18 mice and treated with a lentiviral vector at maturity. This vector either encoded miR-16 or miRZIP-16, causing overexpression or knockdown of miR-16, respectively. Immunoprecipitation of the miRNA-16 enriched RISC complex was then performed, and the co-immunoprecipitated target mRNA was subjected to a whole genome microarray. Analysis of microarray data in Ingenuity Pathway Analysis pinpointed 181 genes involved in neuronal morphology and neurological disease targeted by miR-16. In particular, the MAPK/ERK pathway was targeted at TrkB, MEK1 and c-Raf. This is of interest, as we know that this pathway is disrupted in other neurodegenerative diseases and is directly implicated in neuronal morphology. Subsequent morphological analysis revealed that overexpression of miR-16 in neuronal cells decreased neurite length and branching, consistent with the downregulation of components of the MAPK/ERK pathway. In conclusion, miR-16 targets many mRNA transcripts within the hippocampus that are important members of pathways involved in neuronal development and neurodegeneration, including the MAPK/ERK pathway. / February 2017

Characterized by the severe progressive wastage of skeletal muscle, Duchenne muscular dystrophy (DMD) is a crippling X-linked recessive disease that is caused by the absence of the protein dystrophin. This thesis aimed to critically evaluate the potential of different therapeutic options to combat this disease. Utrophin is a paralogue of dystrophin. The Fiona mouse is an mdx (dystrophin-deficient) transgenic mouse that overexpresses the full-length utrophin protein in skeletal muscle, and various studies have shown that it does not display a dystrophic phenotype. However, these studies have only been performed on sedentary mice. In this work it is demonstrated that utrophin’s protective effect is partially diminished after a sustained period of exercise-induced stress, highlighting for the first time a functional difference between dystrophin and utrophin. This thesis also presents results of two mdx mouse drug trials testing the ameliorative effects of the administration of the drugs GW501516 and C1100, which show that treatment with both drugs results in partial amelioration of the dystrophic phenotype. GW501516 administration results in a beneficial fast-to-slow fibre type switch and an in vivo increase in utrophin protein levels. We have also shown that C1100 treatment results in a significant increase in utrophin A promoter activity in vitro, and the mechanism of action of this drug on this promoter has been deciphered. The global dysregulation of microRNAs in skeletal muscle of mdx and dko (dystrophin- and utrophin-deficient) mice was evaluated by microarray analysis to identify microRNAs involved in the dystrophic pathological cascades. The results of detailed expression analyses of miR-31, miR-206 and miR-503 are presented, and two therapeutically-relevant predicted targets of miR-503 were validated. Overall, this thesis evaluates the potential of different and possibly complementary therapeutic options to combat DMD.

Micro RNA sind nicht-kodierende dsRNA ~22 Nukleotiden lang, die eine wichtige Rolle in der Entwicklung und Regulation in beinahe allen Eukaryoten spielen. MiRNAs binden target mRNA, was zu einer Blockierung der Proteintranslation führt. Viele Krankheiten sind bekannt, die durch veränderte miRNA Expressionsmuster entweder beeinflusst oder verursacht werden. Demnach könnte eine Manipulation der miRNA-Bildung einen therapeutischen Ansatz darstellen. MiRNA werden im Zytoplasma von längeren haarnadelförmigen prekursor RNA (pre-miRNA) durch das Enzym Dicer freigsetzt. Inhibition dieser Spaltung könnte durch spezifische pre-miRNA-bindende Moleküle erfolgen. Selektive Binder der pre-miRNA als Inhibitoren der miRNA-Reifung können durch testen von Substanzbibliotheken mit Hochdurchsatzscreening (HTS) gefunden werden. Diese Arbeit beschreibt den ersten homogenen Assay der miRNA-Reifung. Eine Fluoreszenzsonde in Form einer pre-miRNA wurde benutzt, die einen 5´-Fluorophor (FAM, Cy3, oder TMR) und einen 3´-Quencher (DABCYL) aufweist. Durch die nahe Nachbarschaft von Fluorophor und Quencher in der nativen Haarnadelstruktur erfolgt Fluoreszenzlöschung. Dicer spaltet diese Struktur effizient, was zur Dissoziation von Fluorophor und Quencher und somit zu einem Fluoreszenzanstieg führt. Der Assay wurde für HTS optimiert. Die ersten Verbindungen wurden auf deren Inhibition der miRNA-Reifung getestet. Mit einem Duplexassay, wobei zwei unterschiedliche pre-miRNA Sonden mit verschiedenen Fluorophoren eingesetzt wurden, konnte etwas spezifische Inhibition gezeigt werden. Der Assay wurde in Zellen durchgeführt und der Fluoreszenzanstieg mit Fluoreszenzmikroskopie detektiert. Somit ist ein Zell-basiertes Screening von Inhibitoren möglich. Eine einfachere Synthese der Sonde mittels in vitro Transkription und anschließender enzymatischen Ligation wurde entwickelt. Verwendung des Assays um hoch selective Inhibitoren der miRNA-Reifung zu entdecken könnte zu therapeutischen Ansätzen führen. / Micro RNAs (miRNAs) are non-coding dsRNAs of ~22 nucleotides that play a vital role in development and regulation in nearly all eukaryotes. MiRNAs bind target mRNA, thus blocking protein translation. Many diseases have been found to be influenced or caused by aberrant expression of miRNAs. A manipulation of miRNA formation may have therapeutic potential. MiRNAs are cleaved from longer hairpin precursor RNA (pre-miRNA) in the cytoplasm by the enzyme Dicer. It might be possible to inhibit this cleavage through specific pre-miRNA binding molecules. Selective binders of pre-miRNA as inhibitors of miRNA maturation can be found by testing large libraries of substances through high throughput screening (HTS) using an appropriate assay. This work describes the first homogenous assay of miRNA maturation. A fluorescent probe in the form of a pre-miRNA containing a 5´-fluorophore (FAM, Cy3, or TMR) and a 3´-quencher (DABCYL) was used. This ‘beacon’ in its native hairpin formation brings the fluorophore and quencher moieties into close proximity, resulting in fluorescence quenching. Dicer efficiently cleaves this structure, leading to dissociation of fluorophore and quencher and thus a fluorescence increase. In the presence of an RNA ligand that blocks cleavage, a lower fluorescence increase is seen. The assay was optimized for HTS. The first compounds were tested for their inhibition of miRNA maturation. Using a duplex assay, with two different pre-miRNA probes each containing a different fluorogenic group, some specific inhibition was shown. The assay was performed in cells using fluorescence microscopy to measure the fluorescence. This would allow for a cell-based screening of inhibitors. A simpler approach of beacon synthesis using in vitro transcription followed by enzymatic ligation was also established. Use of this assay to discover highly selective inhibitors of miRNA maturation may lead to disease therapeutics.

miRNA sind kurze 21 – 23 nukleotidlange nicht kodierende RNAs endogenen Ursprungs und regulieren auf post-transkriptionaler Ebene die Genexpression. Da aberrante Expressionsmuster der miRNAs im Zusammenhang mit verschiedenen Krankheiten stehen, ist das Interesse groß, Kontrolle über die miRNA-vermittelte Genexpression zu erhalten. Bei Krankheitsbildern, die eine Überexpression der miRNA aufweisen, kann die Inhibition der miRNA Reifung als Therapieansatz dienen. Inhibition kann z. B. durch peptidische Strukturen und durch small molecules, wie Aminoglykoside erfolgen. Ziel dieser Arbeit war die nahezu vollständig festphasenbasierte Synthese von zyklischen Peptiden und Peptid-Aminoglykosid-Konjugaten als potentielle Inhibitoren der miRNA Reifung. Ferner sollte die Guanidinylierung an fester Phase mit verschiedenen Testsubstraten gezeigt werden. In der vorliegenden Arbeit wurden im ersten Teilprojekt zehn zyklische Peptide mit Hilfe eines bisfunktionalen Linkermoleküls in den Seitenketten zweier im Peptid enthaltener Cysteine synthetisiert und isoliert. Basierend darauf wurden neun weitere zyklische Peptide an fester Phase synthetisiert, mit ausgewählten Aminoglykosiden in einer CuAAC gebunden und erfolgreich isoliert. Im zweiten Teilprojekt dieser Arbeit wurde die Guanidinylierung an fester Phase mittels des Goodman’s Reagent gezeigt. In ersten Studien wurden vier Testpeptide an fester Phase guanidinyliert. Im Anschluss wurde die Limitierung dieser Methode geprüft. Dazu wurden Aminoglykoside mittels CuAAC an verschiedene Peptid- und Peptid PNA-Rückgrate geknüpft und guanidinyliert. Nicht für alle Substrate konnte die vollständige Guanidinylierung an fester Phase gezeigt werden. Ein weiteres Teilprojekt zeigte die Funktionalisierung von kommerziell erhältlichen Polymeren für die SPPS in Hinsicht auf fluorophorbasierte „Hochdurchsatz Screening-Methoden“. Dazu wurde ein peptidischer Spacer entworfen, der eine Knüpfungsstelle für Fluorophore mittels CuAAC enthielt. / miRNA are short long non-coding RNAs endogenous origin with a length of 21 – 23 nucleotides. MicroRNAs regulate the gene expression on post-transcriptionally level. Starting in the nucleus, primary transcripts are processed into precursor-miRNAs. Accordingly, the miRNA matures after export into cytosol. Since aberrant expression patterns are related to different diseases, it’s from interest to gain control about miRNA mediated gene expression. Some diseases are related to over expression of miRNA. For that reason, the inhibition of the miRNA maturing is object of research. The inhibition can be resulted from peptide structures or with small molecules like aminoglycosides. Aim of this work was the solid phase synthesis of cyclic peptides derivatives and peptide aminoglycoside conjugates as potential inhibitors of the miRNA maturing. In addition, the guanidinylation on solid phase should be evidenced with different substrates. In the first part of the project ten cyclic peptides were synthesized on solid phase. The cyclization was carried out with a bifunctional linking molecule in the side chains of two cysteines. Based on that nine cyclic peptides were synthesized and elected aminoglycosides were bound with help of CuAAC. The second part of this work showed the guanidinylation on solid phase by using Goodman’s reagent under mild conditions. Four peptides were used for initial studies. Due to the success of this method the limit was evaluated. Therefore, aminoglycosides were bound via CuAAC to different peptide and peptide-PNA backbones. By mischance, not all the chosen substrates were fully guanidinylated on solid phase. A further short project showed the functionalization of commercially available resins for solid phase peptide synthesis in relation to fluorophore based high throughput screening methods. For this purpose, a peptide spacer was devised with a binding site for fluorophores via CuAAC.

Recentes estudos têm revelado o envolvimento de microRNAs (miRNAs) no controle da hipertrofia cardíaca e na função do miocárdio. Ainda, várias pesquisas têm demonstrado que o consumo de dieta rica em gordura pode induzir hipertrofia e remodelamento cardíaco. No presente estudo, investigou-se o efeito de dietas contendo diferentes porcentagens de gordura na expressão do miRNA-22, um miRNA que está diretamente envolvido na regulação da morfologia e da função cardíaca e um importante mediador da hipertrofia e falência cardíaca deflagradas por diferentes estímulos. Para isso, camundongos C57BL/6 machos, com idade entre 4 e 5 semanas, foram alimentados com uma dieta controle (10% das calorias provenientes de lipídeos) ou dietas hiperlipídicas (HF) contendo 45% de kcal de gordura (HF45%) e 60% de kcal de gordura (HF60%) por 10 ou 20 semanas. A dieta HF60% promoveu um aumento do peso corpóreo, aumento dos níveis de glicose, insulina, leptina, colesterol total e triglicérides e induziu intolerância a glicose. As dietas HF promoveram remodelamento cardíaco, conforme evidenciado pelo aumento no diâmetro transverso dos cardiomiócitos e deposição de colágeno. A análise de sequenciamento de RNAs demonstrou que as dietas ricas em gordura induziram padrões distintos de expressão de miRNAs no coração, incluindo o miRNA-22. Análise de bioinformática identificou a caveolina-1 como potencial alvo do miRNA-22 e seus níveis encontraram-se aumentados no grupo HF60% tratado por 20 semanas. Considerando que o miRNA-22 está envolvido no desenvolvimento da hipertrofia cardíaca e falência do coração, é possível que algumas destas alterações estruturais e funcionais cardíacas induzidas pela dieta rica em gordura sejam, ao menos em parte, influenciadas pelo aumento da expressão deste miRNA. Entretanto estudos funcionais são necessários para determinar a contribuição do miRNA-22 para os efeitos promovidos pela dieta rica em gordura no coração. / Recent studies have revealed the involvement of microRNAs (miRNAs) in the control of cardiac hypertrophy and myocardial function. In addition, several reports have demonstrated that high fat (HF) diet induces cardiac hypertrophy and remodeling. In the current study, we investigated the effect of diets containing different percentages of fat on the miRNA-22 expression, which is a miRNA involved in the control of the cardiac morphology and function and an important mediator of cardiac hypertrophy and heart failure triggered by different stimuli. To address this question, 4-week-old male C57Bl/6 mice were fed with a low fat diet (10 kcal% fat) or high fat diets (HF), containing 45 kcal% fat (HF45%) and 60 kcal% fat (HF60%) for 10 and 20 weeks. HF60% diet promoted an increase on body weight, fasting glycemia, insulin, leptin, total cholesterol, triglycerides and induced glucose intolerance. HF feeding promoted cardiac remodeling, as evidenced by increased cardiomyocyte transverse diameter and interstitial fibrosis. RNA sequencing analysis demonstrated that HF feeding induced distinct miRNA expression patterns in the heart, including miRNA-22. Bioinformatics analysis identified caveolin-1 as a potential target of miRNA-22 and its levels were increased in HF60% group treated for 20 weeks. Considering that miRNA-22 is involved in the development of cardiac hypertrophy and heart failure, it is possible that some of the cardiac structural and functional alterations induced by high fat diet are, at least in part, influenced by the increased expression of this miRNA. However functional studies are needed to determine the contribution of miRNA-22 in the effects promoted by high fat diet in the heart.

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-11-08T12:07:50Z No. of bitstreams: 1 LeanePerimRodriguesDissertacao2017.pdf: 2016012 bytes, checksum: e7e5d2c10971d922d61bf00b6b50f5da (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2017-11-08T12:08:24Z (GMT) No. of bitstreams: 1 LeanePerimRodriguesDissertacao2017.pdf: 2016012 bytes, checksum: e7e5d2c10971d922d61bf00b6b50f5da (MD5) / Made available in DSpace on 2017-11-08T12:08:24Z (GMT). No. of bitstreams: 1 LeanePerimRodriguesDissertacao2017.pdf: 2016012 bytes, checksum: e7e5d2c10971d922d61bf00b6b50f5da (MD5) Previous issue date: 2017-03-20 / Myelofibrosis is a hematological disease inserted in the group of myeloproliferative neoplasias. It has as main characteristic fibrosis of the bone marrow, consequence of a variety of histological changes presented in the medullary microenvironment. The pathophysiology of myelofibrosis involves the activation of signal transduction pathways and nowadays several mutations such as JAK2V617F, CalR and MPL have been associated with this process. Recent studies have shown that microvesicles produced by body cells may be associated with the cellular communication process. These microvesicles carry in their content proteins, lipids and RNA capable of regulating diverse cellular processes. The literature shows that several miRNAs present in the microvesucular content can regulate the hematopoiesis of normal stem cells and also of compromised progenitors, having an important role in the pathogenesis of some acquired hematological neoplasias such as myeloproliferative diseases. With the objective of investigating the presence of specific miRNAs in microvesicles excreted in the peripheral blood plasma of patients with myelofibrosis, microvesicles were isolated from the plasma by ultracentrifugation and by means of molecular biology techniques it was possible to validate the presence of these microbes. Real-time PCR assays were performed to evaluate the expression of specific miRNAs (miR-146b, miR-221, miR-143 and miR-150) in the microvesic contents. In order to analyze the functional activity of MVs, the cell migration assay was performed, aiming at the progression of pathology and tumor invasiveness. The results show that there is no increase in the microvesicular concentration in peripheral blood when comparing the group of patients with the control group. The data found show that miR-146b and miR-150 are differentially expressed in patients with myelofibrosis, being little expressed. However, miR-221 and miR-143 didn???t show amplification in the technique, which leads to the conclusion that these miRNAs are specific to the medullary microenvironment or aren???t released in the microvesicular content. Regarding the migration test, the result shows that there was no influence of the microvesicles in the process of cell proliferation and migration and it can be considered that the microvesicles isolated in our study may not present this role because they are not exclusively of the bone marrow environment. As future prospects, assays with microvesicles removed from the bone marrow can be performed. / A Mielofibrose ?? uma doen??a hematol??gica inserida no grupo de neoplasias mieloproliferativas. Possui como principal caracter??stica a fibrose da medula ??ssea, consequ??ncia de uma variedade de mudan??as histol??gicas apresentadas no microambiente medular. A fisiopatog??nese da Mielofibrose envolve a ativa????o de vias de transdu????o de sinais e nos ??ltimos anos v??rias muta????es como a JAK2V617F, a CalR e a MPL foram associadas a doen??a. Estudos recentes demonstraram que microves??culas produzidas por c??lulas do organismo podem estar associadas ao processo de comunica????o celular. Estas microves??culas transportam em seu conte??do prote??nas, lip??dios e RNA capazes de regular variados processos celulares. A literatura mostra que diversos miRNAs presentes no conte??do microvesucular podem regular a hematopoiese de c??lulas-tronco normais e tamb??m de progenitores comprometidos, tendo um papel importante na patog??nese de algumas neoplasias hematol??gicas adquiridas como as doen??as mieloproliferativas. Com objetivo de investigar a presen??a de miRNAs espec??ficos em microves??culas excretadas no plasma de sangue perif??rico de pacientes portadores de mielofibrose, microves??culas foram isoladas do plasma por m??todo de ultracentrifuga????o e por meio de t??cnicas de biologia molecular foi poss??vel confirmar a presen??a destas. Ensaios com PCR em tempo real foram realizados afim de avaliar a express??o de miRNAs espec??ficos (miR-146b, miR-221, miR-143 e miR-150) no conte??do das microves??culas e para analisar a atividade funcional das M.Vs foi realizado o ensaio de migra????o celular que visa avaliar progress??o da patologia e a invasividade tumoral. Os resultados obtidos mostram que n??o h?? aumento da concentra????o microvesicular em sangue perif??rico quando comparado o grupo de pacientes com o grupo controle. Os dados encontrados mostram que o miR-146b e o miR-150 apresentam-se diferencialmente expressos em pacientes com mielofibrose, com pouca express??o. J?? o miR-221 e o miR-143 n??o apresentaram detec????o na t??cnica, o que o que leva a concluir que estes miRNAs s??o pr??prios do microambiente medular ou n??o s??o liberados no conte??do microvesicular. Quanto a ensaio de migra????o o resultado mostra que n??o houve influ??ncia das microves??culas no processo de prolifera????o e migra????o celular e pode ser considerado que as microves??culas isoladas em nosso estudo podem n??o apresentar este papel por n??o serem exclusivamente do ambiente medular. Como perspectivas futuras, ensaios com microves??culas retiradas da medula ??ssea poder??o ser realizados.

Poor reproductive performance is a major challenge for the bovine dairy industry, with implications for profitability and animal welfare. Early pregnancy diagnosis and accurate oestrus detection can improve reproductive performance through efficient herd management. However currently available methods do not allow this. Circulating microRNAs (miRNAs) have been proposed as non-invasive biomarkers of reproductive status in humans. The hypothesis for this work was that differentially expressed miRNAs in plasma will be detectable during early pregnancy / oestrus, which may provide novel potential biomarkers. Using sequencing and PCR-based platforms I successfully identified and validated increases in miR-26a and the miR-26a / miR-205 ratio as early as Day 8 of pregnancy (max. 7.5-fold) in the plasma of pregnant compared to non-pregnant heifers. These miRNAs are known regulators of immunity, angiogenesis and metabolism, however their specific roles in early pregnancy remain to be investigated. I also identified small but significant increases in the levels of miR-125b, let-7f, miR-145 and miR-99a-5p at oestrus, when compared with the luteal phase of the cycle. These miRNAs have been previously shown to regulate the follicular to luteal transition in the bovine ovary. Finally, I provide a validated high-throughput resource which can help identify potential global biomarkers of tissue function, as shown for the liver-enriched miR-802 in the present results. The findings of this work may be useful in the development of diagnostic methods for early pregnancy and oestrus, and pave the way for the functional characterisation of these miRNAs in bovine reproduction.

Background: Extensive research has characterized the embryonic development of a four-chambered heart in mammals. After birth, mammalian cardiomyocytes undergo a transition characterized by a final cell cycle with nuclear division (karyokinesis) in the absence of cytoplasmic division (cytokinesis), generating mature binucleated cardiomyocytes. Downregulation of pro-proliferative signaling and epigenetic changes permanently ‘lock’ cardiomyocytes out of the cell cycle, and nearly all subsequent growth is accomplished via cellular hypertrophy. Before this transition, cardiomyocytes exhibit robust proliferative potential, but afterward are unable to divide. Rationale & Hypothesis: Recent evidence suggests that non-coding RNAs influence early neonatal cardiac development and hypertrophy. We hypothesize that transient expression of regulatory miRNAs may impact the neonatal heart’s transition from proliferation to hypertrophy. Results: Cardiac mRNA and miRNA were systematically analyzed using microarrays to identify targets that were transiently and significantly changing after birth. Through our analysis we identified three primary ontogenies significantly changing: metabolism, extracellular matrix remodeling, and cell cycle regulation. Global analysis of micro-RNA expression patterns during perinatal heart development identified miR-205 as a novel candidate for modulating cardiomyocyte maturation. We observed miR-205 expression undergoing a 20-fold increase from 1-day postpartum (1D) to 5D, returning to prenatal levels by 10D. It is expressed in cardiomyocytes of the epicardium, the primary location of fetal cardiomyocyte proliferation. MiR-205 targets two important cell cycle regulators: Pten phosphatase of the PI3K/AKT pathway, and Yap1 in the Hippo pathway. Both pathways have proven to be essential for proper heart development. Previous research showed that germline deletion of miR-205 results in death at 5D. To define its role in the heart, we generated an αMHC-Cre postnatal miR-205 cardiac-specific deletion mouse model. Systematic characterization of miR-205-/- hearts confirmed miR-205’s interaction with Pten and Yap1 by western blot and immunohistochemistry. Postnatal miR-205-/- hearts exhibit Hippo pathway dysregulation, increased cardiomyocyte number, more actively cycling cardiomyocytes beyond 7D, and no difference in binucleation. We also generated a DOX-inducible cardiac-specific miR-205 over-expression mouse model. Perinatal miR-205OE hearts expedited the transitional period, with more cardiomyocytes present at 5D and no difference at 14D. These hearts show increased Hippo signaling immediately after birth, suggesting compensatory mechanisms to ensure sufficient cardiomyocyte number. Conclusions: Our data strongly supports miR-205 as a regulator of cardiomyocyte maturation in the neonatal heart, by promoting the neonatal cardiomyocyte transition from hyperplastic to hypertrophic growth. In turn, miR-205’s antiproliferative properties originate in part from suppressing the expression of Pten and Yap1.

MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.